Endometriosis and inflammatory immune responses: Indian experience

Abstract

Endometriosis is a public health disorder affecting ~ 247 million women globally and ~ 42 million women in India. Women with endometriosis suffer from dysmenorrhea, chronic pelvic pain, dyspareunia, dyschezia, fatigue, depression, and infertility leading to significant socioeconomic impact and morbidity. The etiology of endometriosis is not understood well even after 100 years of research. Currently, there is no permanent cure for endometriosis. The inflammatory immune response is one of the important features of etiopathogenesis of endometriosis and therefore understanding the inflammatory immune response would lead to a better understanding of this enigmatic disorder and may also lead to biomarker discovery for diagnosis of endometriosis. We investigated the autoimmune etiology of endometriosis in the Indian population. Using the proteomics approach, anti-endometrial antibodies (AEAs) were detected in Indian women with endometriosis [anti-endometrial antibodies - tropomyosin 3 (TPM3), stomatin-like protein2 (SLP-2), and tropomodulin 3 (TMOD3)]. The studies on AEAs provided a better understanding of autoimmune mechanisms in endometriosis. All three subtypes of endometriosis; superficial peritoneal, ovarian endometrioma, and deep infiltrating endometriosis were reported in Indian women. In this review, we discuss our experiences of the inflammatory immune response, autoimmunity, comorbidities, and clinical phenotypes in women with endometriosis in India.

1. Introduction

Endometriosis is a complex, multifactorial, disease defined as the presence of endometrium-like tissue (the innermost lining of the uterus) outside the uterine cavity.¹ It leads to chronic inflammatory reactions resulting in the development of scar tissue (adhesions, fibrosis) within the pelvic cavity and also at the extra pelvic locations. It is estimated that endometriosis affects ~10% of women of reproductive age, which infers to ~ 247 million girls and women globally and ~ 42 million girls and women in India.²

Endometriosis manifests mainly between menarche and menopause, but the disease has been described in pre-menarcheal girls, post-menopausal women. Asian women were reported to have a higher prevalence of endometriosis than Caucasians³ with a higher frequency of moderate-to-severe endometriosis amongst Asians.⁴ A slightly increased incidence of endometriosis was seen in Japanese and other Asian populations, with a lower incidence among women of African descent, compared to women of Caucasian descent.^5–8 These observations strongly suggest the necessity to examine the variability in disease presentation in different populations. Endometriosis is found in one out of three women with infertility, 70% of women with unexplained infertility, 30% of adolescent girls with secondary dysmenorrhea, 60% of women with chronic pelvic pain, and 30% of women with adenomyosis.^1,2,5,6 Endometriosis leads to substantial adverse health impacts ranging from reduced quality of life to fatigue, depression, anxiety, and infertility. There is a comparable economic burden of endometriosis with other chronic conditions such as diabetes mellitus, Crohn’s disease, rheumatoid arthritis.¹

Endometriosis is a heterogeneous condition with distinct lesion phenotypes: superficial peritoneal endometriosis (SUP) cystic ovarian endometriosis, or endometrioma (OMA), and deep infiltrating endometriosis (DIE).¹ Endometriosis located on the superficially intra-pelvic region of the peritoneum is considered SUP, whereas ovarian endometrioma or endometriotic cysts visible on laparoscopy are categorized as OMA.¹ DIE is considered when the endometriotic lesions extend > 5 mm beneath the peritoneum.⁹

2. Clinical Phenotypes Of Endometriosis In India

India’s population size is more than 1.4 billion; contributing to nearly 18% of the global population and might have large numbers of diagnosed and undiagnosed cases of endometriosis. Despite this, there is under representation of Indian women in the global clinical and genomic databases on endometriosis. We recruited one of the largest cohorts of endometriosis representing different ethnic and geographical populations in India. A total of 500 women with surgical and histological confirmation of endometriosis [Stage I-II (n = 183) and Stage III-IV (n = 317)] were recruited from Maharashtra, Goa, West Bengal states of India from 2012 to 2015.¹⁰ Ovarian endometrioma (OMA) was the most common subtype of endometriosis (58%), followed by deep infiltrating endometriosis (DIE) [24%] and superficial peritoneal endometriosis (SUP) [15%] in Indian women.¹⁰ The mean age of women with endometriosis was 32.5 ± 5.9 years. The majority of women (88%) in our cohort were married and the majority were unemployed (59%). Sixty-six percent of women were suffering from infertility either primary or secondary. A history of miscarriage was reported in 33% of women who ever conceived. Seventy-seven percent of women reported dysmenorrhea, 23% had dyspareunia, 34% suffered from chronic pelvic pain, and 14% were found to be suffering from dyschezia. Comorbidities were reported in 13% of endometriosis patients, which included type 2 diabetes mellitus, thyroid disorders, heart disease, rheumatoid arthritis, asthma, and tuberculosis. Thyroid dysfunction was the most commonly reported comorbidity in endometriosis patients in our study cohort.¹⁰

Family history with the diagnosis of endometriosis in the mother, sister, or grandmother of the proband, was reported in West Bengal and Assam region of India.¹⁰ Also, there was an increased incidence of DIE in women recruited from West Bengal and Assam regions of India.¹⁰ We also reported an unusual presentation of endometriosis from Maharashtra state in India. Endometriosis-associated endometrioid subtype of ovarian cancer, endometriosis-associated with uterine didelphys, cervical endometriosis were some of the unusual cases reported from Maharashtra.

We established a national network and recruited a large cohort of women with endometriosis (Figure 1). All three subtypes of endometriosis; SUP, OMA, and DIE are reported from all over India (Figure 2). Different endometriotic lesions were documented in the Indian cohort of endometriosis (Figure 3). Women who conceived spontaneously or naturally after laparoscopic surgery were followed up during the 1^st, 2^nd, 3^rd trimester, and postpartum period for documentation of pregnancy outcomes at KGMU Lucknow, Government Medical College, SAT Hospital Trivandrum, and Spectrum Clinic and Endoscopy center Kolkata.¹¹ In our cohort of 204 women in the age group of 20–35 years with endometriosis-associated infertility, we reported DIE as the most common lesion (40%), followed by OMA (31%) and SUP (29%). A majority (83%) of the women had regular menstrual cycles. Dysmenorrhea was the most common pain symptom (82%) followed by pain during sexual intercourse (dyspareunia) [44%] and chronic pelvic pain (15%).¹¹

Figure 1.

Map of India showing centers using the World Endometriosis Research Foundation-Endometriosis Phenome and Biobanking harmonisation Project (WERF-EPHect) modified endometriosis patient questionnaire (EPQ), standard surgical form (SSF), and standard operating procedures (SOPs) for endometriosis research

Figure 2. Representative laparoscopic images of endometriosis sub-phenotypes and adhesions.

(A) Endometriosis on the surface of the left ovary; (B) Left endometrioma; (C) Endometrioma on left ovary with filmy adhesions; (D) DIE on both uterosacral ligaments; (E) DIE with dense adhesions on uterus, ovary, bowel, and left uterosacral ligament; (F) Web adhesions [UT-uterus, POD-pouch of douglas, OV-ovary, BO-bowel]

Figure 3. Representative laparoscopic images of endometriotic lesions.

(A) Red lesions on the right broad ligament; (B) Blue/ black lesions on the left broad ligament; (C) Brown lesion; (D) Yellow lesion; (E) Vascular lesions; (F) Allen-Masters peritoneal defect on the right broad ligament

We classified the women with endometriosis as having single lesion types, two lesion types, and three lesion types. We observed that 46% of women in our study had a single lesion type, 38 % of women had two lesion types and 16 % of women had all three endometriotic lesion types. We also observed that there was a regional difference in the presentation of women with endometriosis according to the lesion types. Obese women appeared to be more commonly diagnosed with superficial peritoneal lesions whereas the highest proportion of deep infiltrating lesions were diagnosed among underweight women. Women recruited in eastern India had the highest proportion of superficial peritoneal lesions (41%). Ovarian endometrioma was most commonly seen in the southern Indian population (40%%), followed by the north Indian population (33%). The highest proportion of deep infiltrating lesions was observed in women recruited in the northern region of India (83%). The majority of women with endometriosis (69%) conceived naturally after surgery. We found that half of the women with single lesion type conceived naturally after surgery. Most of these women who conceived naturally after surgery had superficial peritoneal lesions. We demonstrated that women diagnosed with superficial peritoneal endometriosis and only one type of endometriotic lesion are more likely to conceive after surgery.¹¹

These differences in incidence and disease presentation in different regions of India could be due to ethnic differences and/or environmental factors. Evidence is emerging on the role of genetic and environmental risk factors responsible for the development of this complex condition.^{1,2,5,6,12,13,14} The cause of this puzzling condition remains unclear even though the disease was described more than 150 years ago.¹⁵ Several theories were suggested for the etiology of endometriosis which includes; retrograde menstruation,¹⁶ coelomic metaplasia,^17,18 induction theory,¹⁹ lymphatics and vascular metastasis,^20,21 neonatal uterine bleeding,²² epigenetics,^23,24 genetics,^5,25–27 hormones,²⁸ autoimmunity,^29,30 and inflammation.³¹ However, no single theory can explain all subtypes and all locations of endometriosis. In this review, we attempt to discuss our experience of investigating immune-inflammatory response in Indian women with endometriosis, biomarkers, clinical presentation of endometriosis, clinical, and genetic risk factors of endometriosis.

3. Immunological Factors In Endometriosis

Immunological factors in the pathogenesis of endometriosis are extensively studied and reviewed.^32,33 Both the arms of immunity, i.e., cellular and humoral are involved in the pathogenesis of endometriosis. The immune dysfunctions involved in endometriosis are shown in Figure 4. In women without endometriosis, both innate and adaptive immune mechanisms work towards clearance of the menstrual debris, and help in the initiation of immune cell infiltration and resulting in tissue repair. Women whose immune system fails to generate the required immune response to the refluxed menstrual debris are more susceptible to developing endometriosis.³³ However, whether immune dysfunction is the cause or consequence of endometriosis is still not clear. In women with endometriosis, inflammatory changes happen at both the local and the systemic levels, however, the underlying mechanisms are largely unknown.

Figure 4. Schematic presentation of the immune dysfunction involved in endometriosis.

(A) The female reproductive anatomy in healthy women and those with endometriosis. Endometriotic lesions are indicated as brown areas; endometriotic lesions contain epithelial cells (pink) and stromal cells (yellow), nerve fiber (blue), blood vessels (red). EMSC contributes to lesion establishment. (B1) Immune cell population involved during lesion development in ectopic locations; (B2) Immune cells involved in lesion maintenance; (C) Inflammatory mechanisms involved in the pathogenesis of endometriosis

4. Cellular Immune Response

The implantation of retrogradely shed endometrial cells that results in the development of endometriosis could be due to abnormalities in the cell-mediated immune response that prevents the removal of the retrograde menstrual debris.³⁴ Neutrophils, macrophages, natural killer (NK) cells, and dendritic cells (DCs) are the cell populations of the innate immune system predominantly involved in endometriosis pathophysiology. Increased neutrophil infiltration has been reported in systemic circulation and peritoneal fluid of women with endometriosis as compared to women without endometriosis.^35,36 Symons et al., reported the presence of neutrophils within endometriotic lesions and further showed that endometriotic lesions have a microenvironment that may impact neutrophil recruitment and function.³³ The study also demonstrated that systemic circulating neutrophils in women with endometriosis displayed different transcriptomic profiles as compared to the neutrophils from women without endometriosis.³³

Macrophages are the key components of the innate immune system. Macrophages were significantly raised in the peritoneal fluid and eutopic endometrium of women with endometriosis.^37,38 Endometrial macrophages with lower expression of CD163 (wound-healing marker) were reported in women with endometriosis as compared to women without endometriosis.^37,39 Increased CCL2 levels were observed in the endometrium of women with endometriosis.⁴⁰ Hogg et al. showed that endometriotic lesion-resident macrophages were derived from the eutopic endometrium and there was an infiltration of large peritoneal macrophages (LpM) and monocytes.⁴¹ They further demonstrated that the presence of endometriosis stimulated monocytes and expansion of CCR2+ LpM. It was also observed that reduction of eutopic endometrial macrophages was associated with small endometriotic lesions.⁴¹ On the contrary, inhibition of monocyte recruitment significantly reduced LpM and increased the number of lesions. These findings are suggesting a role of monocyte-derived macrophages in controlling endometriosis disease progression.

In endometriosis, a decrease in NK cell activity may be responsible for diminished clearance of retrogradely shed menstrual tissue from the peritoneal cavity and thus promote the development of endometriotic implants. It is established that NK cell activity was suppressed by the local peritoneal microenvironment and NK cells from women with endometriosis showed aberrant expression of various activating and inhibitory receptors compared to healthy women.^42,43,44 Patients with endometriosis had reduced NK cells cytotoxicity.⁴⁵ However, there was no difference in lymphocyte-mediated cytotoxicity and NK cell activity in baboons with and without endometriosis.⁴⁶ The autoimmunity in endometriosis could be due to the defects in two subclasses of NK cells.⁴⁷ NKT cells kill the cell target and secrete cytokines (IL-4 and IL-10) which are involved in the regulation of autoimmunity.⁴⁸ CD16/CD56 NK cells kill autologous dendritic cells (DCs) presenting self-antigens to autoreactive T cells. The failure of NK cells in removing autologous DCs that express the endometrial self-antigens could permit their presentation to autoreactive T cells thus leading to the development of autoantibodies.⁴⁷ The inability of NK cells to remove autologous endometrial cells may facilitate the establishment of endometriotic lesions. It has also been proposed that NK cells in the endometrium may attack the embryo implantation site and autoantibodies may play a role in this process.⁴⁹

Dendritic cells (DCs) were detected in the peritoneal fluid of women with endometriosis; However, their absolute numbers were not significantly altered in women without endometriosis.³⁶ Currently, there is limited information on the role of DCs in endometriosis and future studies should focus on research to understand the role of DCs in endometriosis.

In women with endometriosis, immune dysfunction causes viable endometrial cells to remain in the peritoneum⁵⁰ where their growth is facilitated by the endometrial mesenchymal stem cells shed from the perivascular regions of the endometrium.⁵¹ Consequently, mesothelial cells in the peritoneum or the endometriotic tissue facilitate the adhesion of endometrial cells and contribute to lesion establishment^52,53 (Figure 4A). The growing endometriotic lesion cause changes in the extracellular milieu by stimulating angiogenesis, nerve development and produces chemoattractant molecules engaging increased number and activity of macrophages⁴¹ and natural killer cells with less phagocytic and cytotoxic activity,⁵⁴ (Figure 4B1). An increase in B cells, mast cells, Th17, Th1, eosinophils, and neutrophils cause an increase in the production of pro-inflammatory molecules NFkB, IL-1β, TNF- α, which promotes fibrosis adhesion and help in lesion maintenance (Figure 1 and 4B2).⁵⁵

DNA methylation modifications in the estrogen and progesterone receptor genes enhance the expression of estrogen receptor β (ER-β) while reducing progesterone receptor B (PR-B) expression (Figure 4C). Augmented ER-β stimulates the expression of genes that translate pro-inflammatory molecules, such as interleukins and COX2. COX2 promotes inflated production of prostaglandin E2 (PGE2) and induces aromatase activity. The very low levels of PR-B in stromal cells of endometriotic lesions do not yield progesterone-induced paracrine factors which are required to stimulate the 17-beta hydroxysteroid dehydrogenase type 2 (17βHSD) enzyme expression. This persistent reduced expression of 17βHSD which converts E2 to E1 (estrone) causes an increase in estrogen (E2) levels⁵⁶ and contributes to inflammation and growth of the lesion. Furthermore, ER-β interrelates with inflammasome to initiate IL-1β which helps in cell adhesion.⁵⁷ ER-β also acts along with apoptosis signal-regulating kinase-1 to diminish its activity and block TNF-α-mediated apoptosis.⁵⁸ Altogether the various cellular components form a network that synchronizes for a continued chronic inflammatory environment that supports increased proliferation or decreased apoptosis of the endometriotic lesions^59,60 and angiogenic factors which help in the growth of the lesion.⁶¹ The inflammatory environment prevents immune cells from clearing ectopic lesions. The endometriotic lesions contain endometrium-like glands and stroma, infiltration of immune cell infiltrates, vascularization with blood vessels, and nerve fibers innervation. Women with heavier and longer duration of menstrual flow are more prone to develop endometriosis,⁶² as there is the persistent presence of menstrual debris causing overload and subsequent immune dysfunction.³³

5. Genetics And Inflammatory Response In Endometriosis

The endometriosis GWA meta-analysis identified 14 genome-wide significant loci in European and Japanese women with endometriosis.⁶³ Out of these risk loci, rs10167914 SNP located on chromosome 2q13 is placed within an inflammatory rich gene part of the genome. This suggested that variation in this part of the genome may be associated with abnormal inflammatory response detected in women with endometriosis. The detailed analysis of the endometriosis risk region nearby the sentinel SNP (rs10167914) showed the identification of 21 individual transcripts. Of these 21 transcripts, six were part of the IL1 family, further three denoted coding genes related to inflammation, four genes represented either novel coding, or pseudogenes and the remaining eight were non-coding RNA transcripts. Alteration in the IL-1 family has been suggested as an attractive point to study the effect of SNP rs10167914 on endometriosis risk to understand the consequences of inflammation on endometriotic lesions.⁶⁴ Combining the evidence from the genetic analyses of endometriosis in humans and non-human primates (rhesus macaques) identified non-hormonal target Neuropeptide S receptor 1 (NPSR1). In the mouse model of endometriosis, there was a significant reduction in abdominal pain as well as inflammatory cell infiltrate on treatment with NPSR1 inhibitor SHA 68R that blocked NPSR1-mediated signaling, pro-inflammatory TNF-α release, and monocyte chemotaxis in vitro.⁶⁵ Further research is needed to explore the non-hormonal treatment for endometriosis.

6. Autoimmunity In Endometriosis

Although endometriosis has not been classified as an autoimmune disease, there are several resemblances with autoimmune disorders such as rheumatoid arthritis, Crohn’s disease, and psoriasis. These resemblances include increased cytokines, reduced cell apoptosis, and abnormalities of T and B-cells.⁶⁶ Inflammatory disease process has been suggested for the pathogenesis of endometriosis which suggests a role of the immune system.^67,68 It is hypothesized that in women with endometriosis there is a diminished immune response resulting in insufficient removal of endometrial cells thereby leading to the development of endometriosis. For the establishment of ectopic endometrial tissue, the defective immune response was proposed as one of the mechanisms.⁶⁷ More than 40 years ago, Weed and Arquembourg suggested that shed menstrual tissue in ectopic locations could stimulate an autoimmune response resulting in infertility.⁶⁹ Their argument was based on the detection of complement C3 and IgG in endometrium and a matching decrease in complement level in endometriosis patients. This increased endometrial autoimmunity could be due to a higher immune response to normal self-antigens either due to the genetic predisposition to autoimmunity or due to an excess of the endometrial auto-antigens present in the peritoneal cavity shed during each cycle of retrograde menstruation. In fact, endometriosis fulfils the basic criteria for autoimmune disease such as polyclonal B-cell activation, abnormalities in T and B-cell function, tissue damage, female preponderance, familial occurrence, co-occurrence with other autoimmune disorders, and multi-organ involvement.^47,70 Suppression of T cell-mediated immunity to autologous endometrium was reported in rhesus macaques having spontaneous endometriosis.³⁴ Similarly decreased cytotoxic activity of peripheral blood lymphocytes against autologous endometrial cells was demonstrated in endometriosis patients.⁷¹ Association of plasma cells and increased production of B lymphocyte stimulator protein was observed in endometriosis patients.⁷² Using the microarray and ingenuity pathway analysis, Hever and Co-workers observed the association of the immune response genes with endometriosis.⁷²

7. Associated Autoimmune Disorders And Cancer In Endometriosis

There is evidence that immune disorders increase the risk of developing endometriosis. Increased incidence of autoimmune inflammatory diseases, hypothyroidism, fibromyalgia, chronic fatigue syndrome, allergies, and asthma were observed in endometriosis patients as compared to the general female population.⁷³ Family history of cancer was reported in more than 50% of women with endometriosis as whereas in controls it was reported in only 11.5 %.⁷⁴ In women with endometriosis, there was an increased familial risk of breast cancer.⁷⁵ A recent systematic review and meta-analysis reported a relationship between endometriosis and autoimmune conditions such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjögren’s syndrome (SS), autoimmune thyroid disorders (ATD), celiac disease (CLD), inflammatory bowel disease (IBD), multiple sclerosis (MS), and Addison’s disease.⁷⁶ These relationships between endometriosis and autoimmune conditions suggest the necessity to increase awareness amongst the clinicians to suspect co-occurrence of endometriosis and autoimmune disorders when either is detected. However, it is still unclear whether comorbidities are an effect or cause of the disease. A recent study suggested that endometriosis is more severe in women with concomitant autoimmune disease and autoimmune disease might be considered a risk factor for severe endometriosis.⁷⁷

8. Role Of Anti-Endometrial Antibodies (Aeas) In Female Infertility

Various investigators have explored the role of circulating antibodies in the pathogenesis of endometriosis. Autoantibodies against carbohydrate epitopes (such as Thomsen-Friedenreich antigen),⁷⁸ α2-HS glycoprotein,⁷⁹ laminin-1,⁸⁰ α-enolase,⁸¹ PDIK1L,⁸², and Syntaxin 5⁸³ have been reported. Autoantibodies against IGF-2 mRNA-binding protein 1 (IMP1) were significantly elevated, in women with endometrioma compared to healthy controls.⁸⁴ Serum Anti-α-enolase antibodies also were significantly higher and the sensitivity and specificity were comparable to CA125 values, and together have high diagnostic value.⁸¹ Anti-laminin-1 antibodies were also found in women with endometriosis.^80,85 Several reviews on biomarkers for endometriosis showed that different antibodies were reported to be elevated in women with endometriosis, but only AEA and interleukin-6 (IL-6) could be useful for the diagnosis of endometriosis. However, there are limitations of AEAs and IL-6 assays as their accuracy still cannot replace the standard surgical diagnosis^86,87,88 supporting earlier systematic reviews^89,90 and clinical guidelines.⁹¹

There are various AEAs reported to be associated with female infertility. Saparik and colleagues investigated the correlation between the AEAs and pregnancy outcomes. They reported levels of alpha-enolase AEAs higher in tubal factor infertility leading to IVF treatment failure.⁹² Subit and co-workers reported the presence of AEA in the sera of women with endometriosis affected fecundity and thus affected the success of an IUI cycle. They further reported lower clinical pregnancy rates in women with endometriosis and there was five times increased clinical pregnancy in women with double IUI cycle.⁹³ AEAs have also been reported to be associated with higher miscarriage rates.⁹⁴ Increased risk of miscarriage was associated with organspecific⁹⁵ and non-organ specific auto-antibodies.⁸⁰ Association of IgG anti-laminin-1- Abs was demonstrated in infertile women with endometriosis.⁹⁶ Anti-Laminin 1- Abs is also known to cause infertility and recurrent spontaneous abortions in animals.⁸⁰ The same group also reported the presence of IgG anti-laminin-1- Abs in women with recurrent spontaneous abortions and associated with subsequent negative pregnancy outcomes.⁹⁷ Therefore, screening of AEAs may also be considered in addition to anti-phospholipids, anti-nuclear, anti-adrenal, anti-thyroid, and anti-ovarian antibodies for the investigation of infertility associated with endometriosis.²⁹ The studies on AEAs provided a better understanding of autoimmune mechanisms in endometriosis.

9. Aeas In Indian Women With Endometriosis

Indian population is heterogeneous in terms of ethnicity, culture, religion, and economy. There is also an urban to rural divide in the availability of the modern health care infrastructure. With the rising incidence and awareness, endometriosis is one of the major benign gynecological disorders in Indian women leading to infertility and chronic pelvic pain. The diagnosis and classification of endometriosis require direct visualization on laparoscopy or laparotomy. The availability of gynecological laparoscopic surgeons in India is mainly restricted to the major cities leading to difficulty in determining the true burden of disease and increasing the load of undiagnosed cases in rural India. For addressing the burden of endometriosis, we established an ‘Endometriosis Working Group (EWG) at ICMR-National Institute for Research in Reproductive Health (NIRRH), Mumbai in 2003. The team members of EWG included gynecological laparoscopic surgeons, infertility specialists, clinicians, public health experts, biomedical scientists, and program managers. A national consultation meeting was held in 2003 at ICMR-NIRRH, Mumbai to identify the endometriosis research priorities. Following were the research priorities identified: 1) Investigating the role of autoimmunity in Indian women with endometriosis; 2) understanding the etiology of endometriosis; 3) clinical presentation and characteristics of endometriosis in Indian women and 4) biomarkers for early diagnosis of endometriosis.

We investigated the levels of anti-endometrial antibodies (AEA) [IgG and IgM type] in sera of forty women with endometriosis aged 20–43 years. Endometriosis was classified as rAFS stage I n = 09, stage II n = 08, stage III n = 11, stage IV n = 12. Thirty women in the age group of 20–39 years without evidence of endometriosis were recruited as controls.⁹⁸ In this study, around 85% of endometriosis cases were infertile. Sera from women with and without endometriosis were screened by western blot and immunohistochemistry. Out of forty women with endometriosis, sera of 13 endometriosis patients tested positive for IgG isotype (33%), also 10/27 IgG negative endometriosis patients were positive for IgM isotype (37%).⁹⁸ We detected multiple endometrial antigens of 30–160 kDa in sera of Indian women with endometriosis. However, amongst the various endometrial antigens, 30 and 45 kDa were immunodominant antigens in women with IgG and IgM AEAs. The AEA reactivity was observed to be independent of the phase of the menstrual cycle. The majority of the IgG and IgM AEA positive women were suffering from ovarian endometrioma (OMA). On immunohistochemistry, the reactivity was seen in the luminal epithelium, glandular epithelium, and stroma suggesting the possible role of AEA in the failure of implantation leading to infertility.⁹⁸

Further, we used the approach of two-dimensional proteomics to discover novel endometrial antigens using AEA positive sera of endometriosis patients that showed reactivity mainly to 30 and 45 kDa on 1D western blot. Using two-dimensional western blot and matrix-assisted laser desorption/ionization time-of-flight/mass spectrometry with MASCOT analysis, we detected endometrial antigens, tropomyosin 3 (TPM3), stomatin-like protein 2 (SLP2), and tropomodulin 3 (TMOD3).²⁹

Stomatin like Protein 2 is involved in the organization of the peripheral cytoskeleton.⁹⁹ Stomatin-like protein 2 has been reported in the proliferation of endometrial stromal cells and differentiation during decidualization in both mice and humans.¹⁰⁰ Studies have reported the involvement of SLP-2 in cell growth regulation and cell adhesion in human esophageal squamous cell carcinoma (ESCC), lung cancer, laryngeal cancer, and endometrial adenocarcinoma.¹⁰¹ Proliferation, migration, and adhesion are hallmarks of endometriosis disease process thus SLP2 may be responsible for the development of endometriosis. Therefore, one could speculate that autoantibodies to SLP2 might have a role in the pathogenesis of endometriosis. Autoimmune responses to Tropomyosin are reported in ulcerative colitis, myasthenia gravis, Behcet’s disease and autoimmune hepatitis.^102,103

Tropomyosins (TPM) are a family of proteins expressed in muscle as well as non-muscle cells. In muscle, TPM mediates contractions and in non-muscle cells, and is involved in the regulation of cellular contraction. Cytoskeletal protein such as Laminin has also been suggested to be involved in early reproductive processes and endometriosis-related infertility.⁹⁶ Therefore, it is possible that TPM3 could also have functions similar to other cytoskeletal proteins.

Tropomodulins belong to a conserved family of TPM-regulated proteins involved in the capping of the actin filaments.¹⁰⁴ The association of Tropomodulin in patients with ophthalmopathy with autoimmune thyroid disorders was reported.¹⁰⁵ These cytoskeletal proteins are involved in cellular functions like proliferation, apoptosis, cell motility, adhesion, receptor function, and second messenger pathways.^106,107 The other cytoskeletal proteins such as alpha-actinin, Ezrin, and talin were reported to be de-expressed in endometriosis and endometrioid carcinoma as compared to the normal endometrium.¹⁰⁸ The study also reported significant changes in the integrity of endometriotic and endometrioid cancer tissues as compared to normal endometrium suggesting the disruption of the individual cells from the parent histology possibly due to loss of cell adhesion.¹⁰⁸

10. Indigenously Developed Elisas For Diagnosis Of Endometriosis

The epitopes for three identified proteins (TPM3, SLP2 and TMOD3) were predicted using several sequence and structure-based epitope prediction methods. ELISAs were established for specific epitopes and validated in an independent cohort of women with endometriosis [ n = 50, rAFS stages I-II (n = 18, age in years, 22 – 43), rAFS stages II-IV (n = 32, age in years, 21 – 42).²⁹ There were significant differences in AEA levels of TPM3, SLP2 and TMOD3 in women with endometriosis as compared to controls. We observed higher sensitivity and specificity of serum AEAs of TPM3a (61%, 93%),TPM3c(44%, 93%),TPM3d (78%, 89%), SLP2a (50%, 96%), SLP2c (61%, 93%), TMOD3b (61%, 96%), TMOD3c (78%, 93%), and TMOD3d (78%, 96%) than CA-125 (21%, 89%) for early detection of endometriosis.²⁹

For large-scale validation of biomarkers, we expanded the network of gynaecological laparoscopic surgeons across India to recruit endometriosis patients from different ethnic and geographical backgrounds.³⁰ We recruited endometriosis and controls from Western, Eastern, North Eastern, and Central Indian populations. Multicentre validation study included 133 women with minimal–mild endometriosis [rAFS stage I-II (n = 133)], age in years 32.6 ± 6.3 as mean ± SD] and 104 women without endometriosis [age in years 32.13 ±.6.03 as mean ± SD]. The minimal to mild endometriosis group was further subdivided into ultrasound negative (n = 80) and ultrasound positive (n = 53).³⁰ Using individual peptide ELISA, sera from women with endometriosis showed higher mean reactivity against all 11 peptides as compared to disease controls and healthy controls. Our data also suggested the ELISA using a cocktail of epitopes was better than individual epitopes for diagnosis of endometriosis.¹⁰⁹ We further observed that mean serum AEA levels of SLP2, TMOD3, and TPM3 epitopes) were significantly higher in early endometriosis (Stage I-II) compared to stage III-IV endometriosis.¹¹⁰ Our study reported higher sensitivity and diagnostic accuracy of serum AEA against the eleven epitopes of SLP2, TPM3, and TMOD3 compared to CA-125, CA19-9, α-enolase, PDIK1L, and Syntaxin 5 for early diagnosis of endometriosis.³⁰ We reported higher sensitivity of 6 biomarkers (anti-TMOD3b-autoAb, anti-TMOD3c-autoAb, anti-TMOD3d-autoAb, anti-TPM3a-autoAb, anti-TPM3c-autoAb, and anti-TPM3d-autoAb) for diagnosis of stage I-II endometriosis. Further, we demonstrated an increase in sensitivity, specificity, and diagnostic accuracy than individual biomarkers using the logistic regression models suggesting the utility of a panel of 6 biomarkers for early detection of endometriosis.³⁰

11. Potential Therapeutic Strategies Targeting Immune Response/Autoimmunity

There is an association between endometriosis and alterations of the local and systemic immune system.¹¹¹ Several studies demonstrated that there is infiltration of immune cells in peritoneal fluid,^112,113 activations of macrophages,^114,115,116 abnormal lymphocyte responses,^117,118,119 decreased natural killer (NK) cell cytotoxicity,^120,121 excess pro-inflammatory, and regulatory cytokine productions.^122,123 Downregulation of NK cell cytotoxicity could be due to the consistent levels of inhibitory cytokines in the peritoneal fluid, or to an increased presence of several inhibitory NK cell receptors.¹¹¹ Functional and genetic studies showed that killer immunoglobulin-like receptors (KIRs), leukocyte immunoglobulin-like receptor subfamily B member (LILRB), and Natural killer G2 (NKG2) family of receptors recognizing MHC class I molecules may be involved in the lysis of endometriotic target cells.¹²⁴ The checkpoints for the removal of endometriotic cells identified so far are KIR2DL1 receptor, LILRB1 receptors, NKG2A receptor.¹²⁴ Future, therapeutic strategies could be focused on blocking these checkpoints. Several studies reported aberrant regulation/activation of the complement system in women with endometriosis.^116–118 The complement inhibition may be explored as a therapeutic approach for endometriosis.¹¹¹

12. Conclusion

Endometriosis still remains an enigmatic disorder without a permanent cure with unique immunological alterations. The cellular immune response is mainly associated with immune escape, adhesion, invasion, migration, angiogenesis, and proliferation. The presence of anti-endometrial antibodies is another consequence of immune dysfunction. These immunological alterations are associated with subfertility in women with endometriosis. The inflammation associated with endometriosis is responsible for the formation of adhesions. Sometimes these adhesions can be severe and may lead to significant morbidity in women with endometriosis as they are responsible for infertility, chronic pelvic pain, dyspareunia, frozen pelvis, and bowel obstruction. The clinicians should be aware of the association of comorbidities of autoimmune disorders in women with endometriosis and the treatment of coexisting endometriosis and autoimmune disease needs to be managed with a multidisciplinary approach. Large scale prospective cohort studies are required to investigate whether endometriosis is a consequence of autoimmune disorders or a risk factor for development of autoimmune disorders. Studies should also be focused on understanding the overlap of biological pathways between endometriosis and associated autoimmune disorders. The information generated from these studies may lead to the development of novel therapies for the management of endometriosis.

Data Availability Statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.