{"paper_id":"5ed08024-08bf-490d-b790-82d42c819691","body_text":"Endometriosis is a prevalent disease that is typically associated with chronic pelvic\npain or infertility. The precise mechanism for establishing and developing this\ndisease has yet to be fully established. However, several authors have defined an\nessential and irrefutable role for the immune system in the pathophysiology of\nendometriosis ( Herington  et al. ,\n2011 ).\nAntinuclear antibodies (RNP, ScL-70, SS-B, SS-A, and Sm) represent a heterogeneous\ngroup of antibodies that are associated with several autoimmune manifestations and\nimmunological diseases, such as Lupus, systemic sclerosis, polymyositis, arthritis,\nand Sjögren’s syndrome ( Alspaugh  et\nal. , 1976 ;  Sharp  et\nal. , 1976 ;  Moroi  et\nal. , 1980 ).\nMoreover, various reports have attempted to connect endometriosis to several\nautoimmune conditions (e.g., asthma, Lupus, allergies, hypothyroidism, chronic\nfatigue syndrome, rheumatoid arthritis, and fibromyalgia) ( Alspaugh  et al. , 1976 ;  Sharp  et al. , 1976 ), and some reports have\nidentified associations between endometriosis and autoimmune manifestations ( Sharp  et al. , 1976 ;  Moroi  et al. , 1980 ;  Herington  et al. , 2011 ;  Borrelli, 2015 ).\nA well-designed cohort study evaluated the co-occurrence of endometriosis and some\nautoimmune manifestations (associated with multiple sclerosis, Lupus, and\nSjögren’s syndrome), and these authors described a slight association between\nunselected endometriosis cases and autoimmune manifestations ( Nielsen  et al. , 2011 ).\nMoreover, some autoimmune diseases like Lupus and antiplatelet antibodies are related\nto endometriosis (FerrariSouza  et al. , 2023;  Loyau  et al. , 2023 ). The link between\nendometriosis and the immune system is extensively studied to find a cause-effect\nrelationship to explain the development of this disease and the different phenotypes\n(superficial, deep, ovarian). This link (endometriosis and immune system) is also\nfundamental to investigating and developing a new therapeutic option, targeting the\nimmunomodulation of those endometriosis patients ( Yang  et al. , 2023 ).\nTherefore, considering that the precise intensity of the autoantibodies detected in\ncases of endometriosis has not been well studied or adequately described, the\npresent study was designed to evaluate the mean intensity of five different\nantibodies against the extractable nuclear antigen (ENA: RNP, ScL-70, SS-B, SS-A,\nand Sm) in infertile patients with endometriosis.\n\nA prospective case-control study included 43 patients with endometriosis and 46\nhealthy controls. Cases were defined as patients who underwent laparoscopy for\nendometriosis or infertility investigation whose findings were proven (laparoscopy\nand biopsy) endometriosis according to the ASRM classification. Controls were\nfertile patients who underwent laparoscopy for tubal ligation (contraception)\nwithout peritoneal endometriosis. All subjects were recruited from the ambulatory of\nendometriosis at the Hospital de Clínicas de Porto Alegre between 2016-2021.\nExclusion criteria for both groups were evidence or suspicion of any autoimmune\ndisease or clinical manifestations related to these diseases, other findings in the\nlaparoscopy such as cysts or malignancies, and partners with male factor were also\nnot included in this study. Patients were all negative for anti-thyroid antibodies,\nhad normal serum PRL and TSH levels, and had regular menses.\nA serum sample was collected at the time of anesthesia induction. A multiplex\nsemi-quantitative immunofluorescence assay was performed to analyze five\nENA-autoantibodies (Sm, RNP, Scl-70, SS-A, SS-B) in the same reaction using a\ncommercial kit (Quantaplex INOVA Diagnostics Inc., San Diego, CA) with a Luminex\nflow cytometer. The prevalence of autoantibodies and their intensity were compared\nbetween both groups. The mean fluorescence intensity (MIF) was calculated for each\nantibody according to the manufacturer’s instructions. According to pre-established\nguidelines developed for Lupus patients, MIFs greater than 20 UL were considered\npositive. Fisher’s exact test or the chi-square and Student’s t or Mann-Whitney\ntests were applied, with significant results demonstrating  p  values\n<0.05 (IC95%). Multivariable analysis was done using linear or binary regression\nanalysis.\nThe project was approved by the Hospital de Clínicas de Porto Alegre ethics\ncommittee, where the research was conducted (IRB equivalent).\n\nThe baseline characteristics of patients included in this paper are the following:\nThe mean age was similar between groups (cases 32.0±4.4  vs .\ncontrols 33.0±5.5;  p =0.374). Endometriosis patients had\nlower BMI values than controls (23.7±4.3  vs .\n26.3±4.1,  p =0.006). However, the BMI values were not\nassociated with the presence of specific antibodies in the overall sample\n( p >0.05) or among patients with endometriosis\n( p >0.05).\nThe overall prevalence of anti-ENA antibodies in this sample was 19.1% (17/89),\nsimilar in the cases and controls (20.9% x 17.4%;  p =0.877). No\ncases were demonstrating positive anti-Scl-70 or anti-SS-B antibodies in the study\ngroup. Anti-RNP antibodies were similarly detected in 2.2% of cases and 2.3% of\ncontrols ( p =1.000); anti-SS-A antibodies were detected in 14.6% of\nthe sample (14.0% cases x 15.2% controls;  p =1.000); and anti-Sm\nantibodies were detected in 5.6% of the sample (9.3% x 2.2%, respectively;\n p =0.193).\nMoreover, the MIF values were similar between cases and controls for anti-RNP and\nanti-SS-A antibodies (RNP: 7.7±6.2 x 6.7±2.8,\n p =0.316; SS-A: 21.9±56.5 x 24.6±71.4,\n p =0.522, respectively). In addition, the prevalence of anti-ENA\nantibodies among patients with minimal endometriosis (GI) was 27.5% (8/29), whereas\nthat among patients with mild endometriosis (GII) was 7.1% (1/14)\n( p >0.05).\nHowever, the anti-Sm MIF values were higher in the endometriosis group\n(16.0±10.8 x 11.7±3.4,  p =0.007).\n\nThe current study was the first to describe an association between peritoneal\nendometriosis and anti-Sm autoantibodies. The originality and value of this report\nis the inclusion of a homogeneous group of endometriosis patients (only peritoneal,\nminimal/mild stage patients), as the central problem of most papers regarding\nendometriosis and the immune system involves the inclusion criteria applied and,\nconsequently, the comparison between the experimental and control groups.\nOur report states that endometriosis is linked to some autoimmune diseases, primarily\nLupus ( Nielsen  et al. ,\n2011 ). Moreover, the Sm (Smith) antigen is a saline-soluble, non-histone\nglycoprotein, which is not dependent on DNA or RNA for its antigenicity. Antibodies\nto the Sm antigen are considered to be a specific serologic marker due to their high\ndegree of specificity for systemic Lupus (SLE). In addition, these antibodies are\nobserved in up to 30% of SLE patients and have been associated with the activity of\nthis disease.\nThe most robust and convincing study connecting endometriosis to certain connective\ndiseases described a population-based finding with a modestly (less than 60%)\nincreased risk of Lupus among endometriotic patients ( Nielsen  et al. , 2011 ). Our findings are in\nagreement with these authors and demonstrated that although auto-Sm antibodies were\nhigher in patients with endometriosis, this increase was limited and observed in\nless than 10% of the studied subjects.\nRecently, our group also confirmed this finding, demonstrating a link between\nendometriosis and Lupus ( Ferrari-Souza  et\nal. , 2023 ).\nAnti-DNA antibodies are a group of nonspecific antibodies that are associated with\nseveral connective diseases and manifestations. Furthermore, the presence of these\nantibodies may be linked to some of the clinical and laboratory findings of\ndeterioration in these diseases ( Kavanaugh &\nSolomon, 2002 ). The link between the immune system and endometriosis is\nunequivocal; however, most published papers have only explored associations with\nparticular immune pathways, cytokines, or cellular functions ( Eisenberg  et al. , 2012 ). Furthermore, the main\nflaw of these previous studies is their need for a physiological explanation or the\ncomprehension of background factors that link the findings to endometriosis.\nIn the current study, we found that infertile patients with minimal/mild\nendometriosis generally had higher levels of Sm autoantibodies. These complexes can\nlikely disturb connective (collagen IV) tissue and vascular compartments (laminin\nand heparin sulfate) ( Hahn, 1998 ), thereby\npromoting cellular invasion and neoangiogenesis in this group of patients. The\nimmunological pathway related to anti-DNA antibodies involves T Helper cells and, in\nsome cases, is also B cell dependent. In addition, specific cytokines and HLA class\nII molecules may also be involved ( Hahn,\n1998 ).\nAutoimmunity seems to be associated with endometriosis, as several papers have\ndocumented this phenomenon ( Lucena & Cubillos,\n1999 ;  Maeda  et al. ,\n2002 ;  Ulcova-Gallova, 2005 ).\nHowever, the study and control groups of all papers differ and are highly\nheterogeneous. Moreover, the prevalence of anti-DNA antibodies should not be the\nmain factor for comparing our findings to those of other studies; instead, it is\nmost important to describe a rational and well-defined immunologic pathway and to\nstudy why specific dysfunctions are related to the characteristics and grade of\nendometriosis.\nFurthermore, certain autoimmune conditions, such as Lupus and antiplatelet\nantibodies, are connected to endometriosis ( Ferrari-Souza  et al. , 2023 ;  Loyau  et al. , 2023 ). The relationship between\nendometriosis and the immune system is extensively researched to elucidate a\ncause-andeffect connection, explaining the disease’s development and its various\nphenotypes (superficial, deep, ovarian). This connection is pivotal for exploring\nand creating therapeutic approaches that target immunomodulation in individuals with\nendometriosis.\nOur results are in agreement with the idea that mild/ minimal endometriosis is an\nactive immunological disease, even more so than moderate or severe endometriosis,\nwhich could explain why treatments such as immune modulation and IVF protocols could\nincrease pregnancy rates in these patients ( Ulcova-Gallova, 2005 ).\nHowever, our research has several limitations. First, we included only minimal/mild\nendometriosis, preventing our results from being extrapolated to all endometriosis\nstages and sub-types. Second, it was impossible to perform a temporal evaluation or\nconnection between endometriosis and the positivity of these autoantibodies.\nConsequently, our results indicate that only an association and a functional test\nare required to understand better and elucidate the precise immune mechanisms\ninvolved.\n\nIn conclusion, we demonstrated for the first time that the prevalence of anti-ENA\nantibodies among patients with endometriosis GI/II is similar to that of the healthy\nfemale population (19%). However, anti-Sm antibodies were modestly elevated in the\nstudied infertile patients with peritoneal minimal/mild endometriosis. Thus, this\nstudy introduces a new perspective in terms of understanding the intensity of\nanti-Sm antibodies in endometriosis (understanding the Th2/T Helper and NK\ninteraction) and the utility of these antibodies for treatment purposes.","source_license":"public-domain-us","license_restricted":false}