The association between primary ovary insufficiency and endometriosis: an exploration of autoimmune etiology and comorbidity

An autoimmune etiology for endometriosis has been proposed in multiple studies, though definitive evidence and classification remain to be shown. Primary ovary insufficiency (POI) is another female reproductive disorder of which a subset of diagnoses have confirmed autoimmune etiology. Given the frequency of association between autoimmune disorders, this study aims to explore whether there is an association between endometriosis and POI diagnosis. We identified a cohort of POI patients using electronic medical records from the University of Utah and Intermountain Healthcare, then evaluated the cohort for endometriosis diagnosis. For the 613 patients in this cohort, we expected 6.49 patients to have endometriosis, and we observed 10 (p = 0.165). However, after manual review of their charts for confounding factors-including endometrioma diagnosis and surgical procedures involving the ovaries-only 6 of these patients were included in the results. With 6 out of 613 patients, our observed risk of co-diagnosis with endometriosis and POI was even lower than we initially found. As such, this study shows no significant association between the two conditions. However, we highlighted a gap in understanding of endometriosis and its complex relationship with the immune system. Further studies should be carried out to determine whether endometriosis can be classified as an autoimmune disorder.

THE ASSOCIATION BETWEEN PRIMARY OVARY INSUFFICIENCY AND ENDOMETRIOSIS: AN EXPLORATION OF AUTOIMMUNE ETIOLOGY AND COMORBIDITY by Madeline E. Brague A Senior Honors Thesis Submitted to the Faculty of The University of Utah In Partial Fulfillment of the Requirements for the Honors Degree in Bachelor of Arts In The School of Biological Sciences Approved: ______________________________ Corrine K. Welt, MD Professor and Chief, Division of Endocrinology, Metabolism and Diabetes, University of Utah School of Medicine _______ Naina Phadnis, PhD Departmental Honors Liaison _____________________________ Fred Adler, PhD Director, School of Biological Sciences _____________________________ Monisha Pasupathi, PhD Dean, Honors College August 2024 Copyright © 2024 All Rights Reserved ii ABSTRACT An autoimmune etiology for endometriosis has been proposed in multiple studies, though definitive evidence and classification remain to be shown. Primary ovary insufficiency (POI) is another female reproductive disorder of which a subset of diagnoses have confirmed autoimmune etiology. Given the frequency of association between autoimmune disorders, this study aims to explore whether there is an association between endometriosis and POI diagnosis. We identified a cohort of POI patients using electronic medical records from the University of Utah and Intermountain Healthcare, then evaluated the cohort for endometriosis diagnosis. For the 613 patients in this cohort, we expected 6.49 patients to have endometriosis, and we observed 10 (p = 0.165). However, after manual review of their charts for confounding factors—including endometrioma diagnosis and surgical procedures involving the ovaries—only 6 of these patients were included in the results. With 6 out of 613 patients, our observed risk of co-diagnosis with endometriosis and POI was even lower than we initially found. As such, this study shows no significant association between the two conditions. However, we highlighted a gap in understanding of endometriosis and its complex relationship with the immune system. Further studies should be carried out to determine whether endometriosis can be classified as an autoimmune disorder. iii TABLE OF CONTENTS ABSTRACT ii INTRODUCTION 1 METHODS 6 RESULTS 8 DISCUSSION 10 CONCLUSIONS 14 REFERENCES 15 1 INTRODUCTION Endometriosis is a reproductive disorder characterized by the implantation of endometrium-like tissue outside of the uterus, often causing severe pain and reducing or eliminating fertility. Though it can be found in extra-pelvic locations as far as the nasal septum, central nervous system, and thorax, the majority of cases occur in the peritoneum (Andres et al., 2019). There are three forms of pelvic endometriosis, and patients identified to have this condition typically present with all three: superficial peritoneal endometriosis, deep infiltrating endometriosis, and ovarian endometrioma (Bulun, 2022). Despite affecting between 5 to 15% of women, the pathogenesis remains largely debated; the ambiguity surrounding its etiology is exacerbated by the fact that the severity of symptoms does not always correlate with extent of endometriosis, and the most likely mechanism for pathological implantation of endometrial glands and stromal cells— retrograde menstruation—occurs in up to 90% of women (Eisenberg et al., 2012). Retrograde menstruation is the expulsion of endometrium through the uterine tubes and into the peritoneum during regular menstruation (Bulun, 2022). Multiple studies on the etiology of endometriosis have indicated that all three pelvic presentations “are consequences of repeated episodes of ovulation followed by retrograde menstruation,” demonstrating that retrograde menstruation is a critical step in the development of endometriosis (Bulun, 2022, p. 535). However, because only a subset of women present with this condition, retrograde menstruation cannot be the only prerequisite. DNA analysis of ectopic endometriotic stromal cells reveals that these explants do not contain mutations and are therefore genetically identical to normal endometrial cells, 2 however, they displayed significant differences in epigenetic presentation (Bulun, 2022). As endometriosis must therefore involve other clinical abnormalities to negatively affect the epigenetics of these endometrial cells, the diagnosis is better characterized as a symptom complex affecting pelvic tissues rather than one singular defect (Bulun et al., 2019). In fact, defects in the immune system may also be prerequisite to endometriosis development, as the perpetuation of endometriotic explants in the peritoneum indicates a pathological failure of the immune system in clearing these tissues (Bulun et al., 2019). Specifically, women with endometriosis show consistent decreased cytotoxicity of peritoneal natural killer (NK) cells that correlates directly with severity of disease (Nothnick, 2001). As NK cells are critical in the destruction of inappropriate tissue growth, this dysfunction of the immune system demonstrates a possible mechanism of pathogenesis following retrograde menstruation (Nothnick, 2001). Women with endometriosis also demonstrate chronic elevated activation of peritoneal macrophages, and the compounds secreted by these macrophages—particularly fibroblast growth factor in conjunction with tumor necrosis factor—have been shown to increase endometrial cell proliferation in vitro, suggesting that endometriosis involves dysfunction of multiple cellmediated immune pathways (Zeller et al., 1987; Hammond et al., 1993). It is important to differentiate an inappropriate immune response from an autoimmune response, as these findings alone do not show autoimmune etiology for endometriosis. However, endometriosis demonstrates many of the classification criteria of an autoimmune disease, including tissue damage, polyclonal B lymphocyte activation, T lymphocyte immunological abnormalities, association with other autoimmune diseases, familial occurrence, potential environmental and genetic contributions, and altered 3 apoptosis (Nothnick, 2001). In some studies, ELISA, Western blot, and immunodiffusion analysis of peritoneal sera have shown endometrial autoantibodies to be prevalent in women with endometriosis (Mathur et al., 2000). In contrast, earlier studies failed to demonstrate these results (Meek et al., 1988; Switchenko et al., 1991). A more robust argument for the role of autoantibodies in endometriosis lies in the significant reduction of IgG, IgM, and IgA autoantibodies and subsequent increase in fertility in endometriosis patients treated with danazol, suggesting the role of endometrial autoantibodies in pathogenesis (El-Roeiy et al., 1988). Most relevant to this study is the proposed increased prevalence of autoimmune disorders in patients with endometriosis. One meta-analysis of 26 observational population-based studies indicated increased risk of comorbidity of endometriosis and autoimmune diseases including systemic lupus erythematosus, Sjörgen’s syndrome, rheumatoid arthritis, autoimmune thyroid disorder, celiac disease (CD), multiple sclerosis (MS), inflammatory bowel disease (IBD), and Addison’s disease (Shigesi et al., 2018). One limitation noted in this data review was that despite over two dozen studies supporting the association, only a few of them were of high enough quality to indicate significance. Four studies in particular showed strong association between endometriosis and autoimmune disease. One prospective cohort study in the USA showed association with lupus (Harris et al., 2015). Two retrospective cohort studies in Denmark showed association, one with IBD and the other with MS (Jess et al., 2011; Nielsen et al., 2011). One final retrospective cohort study in Sweden showed association with CD (Stephansson et al., 2011). Restricting analysis to only include surgically confirmed endometriosis patients increased the association with IBD (Jess et al., 2011). As 4 laparoscopic removal and biopsy of the tissue is considered to be the gold standard of endometriosis diagnosis, this finding strengthens the association between the diseases (Hebert, 2023). Given the evidence associating endometriosis with autoimmune etiology and comorbidities, the purpose of this study is to evaluate for a potential link between endometriosis diagnosis and autoimmune-based primary ovary insufficiency (POI). POI is another reproductive disorder that is characterized by the cessation of menses before age 40 accompanied by high serum gonadotropin levels, particularly follicle stimulating hormone (FSH) (Welt, 2007). A majority of cases remain poorly understood in their etiology, however, a subset (4%) of these diagnoses show strong evidence for autoimmune-related causation (Welt, 2007; Hoek et al., 1997). Specifically, POI associated with adrenal autoimmunity shows strong links to autoimmune oophoritis, or ovarian inflammation (Welt, 2007). POI demonstrates strong association with Addison’s disease due the cross-reactivity of adrenal autoantibodies with steroid-producing cells in the ovary (Hoek et al., 1997). Due to the theorized comorbidity of endometriosis with autoimmune disorders and the autoimmune etiology of a subset of POI patients, a possible association may be shown between POI diagnosis and endometriosis. Some evidence for this link lies in one study’s increased reporting of Addison’s disease in endometriosis patients; though still very rare, the number of endometriosis patients with Addison’s disease increased 25-fold from the expected population number (Gemmill, 2009). Taken together with the meta-analysis of endometriosis and autoimmune disorders, it is worth exploring whether the possible autoimmune response that results from endometriosis correlates with diagnosis of POI. 5 It should be noted that another explanation for the relationship between the two diseases is a function of treatment or scarring of the ovaries from endometriosis lesions. These structural issues can damage the ovary and cause ovarian dysfunction that resembles POI. However, in the current study, our goal is not to capture the iatrogenic relationship between POI and endometriosis. Rather, we aim to demonstrate a correlation between endometriosis diagnosis and comorbidity with autoimmune-based POI. 6 METHODS This study was conducted using the combined electronic medical records (EMRs) from the University of Utah and Intermountain Healthcare; in total, these two institutions serve approximately 85% of Utah residents. We identified a cohort of women age ≤40 in Utah diagnosed with POI using International Classification of Disease (ICD)-9 and ICD-10 codes (256.3, 256.31, and 256.39; and E28.3, E28.31, E28.39, E28.310, and E28.319, respectively), EMR text indicating diagnosis, or lab results consistent with POI diagnosis (elevated FSH > 20 IU/L or AMH, 0.08 ng/mL). The group of identified patients was verified by chart review by a medical or reproductive endocrinologist. The cohort of POI patients was then similarly analyzed for identifiers of endometriosis diagnosis using ICD-9 and ICD-10 codes (ICD 9: 617.0-9 and ICD-10: N80.1-9). After identifying patients with both conditions, we reviewed their charts for method of diagnosis, age at diagnosis for each condition, location of endometriosis, evidence of ovarian manipulation, diagnosis of endometrioma, and etiology of POI diagnosis. We defined ovarian manipulation as any procedure deliberately excising or ablating ovarian tissue. We also searched for any notes regarding the recommended tests for POI, including the fragile X permutation, karyotype abnormalities, primary adrenal insufficiency, or adrenal cortex autoantibodies. Lastly, charts were manually reviewed for additional autoimmune diagnoses, though these had already been similarly analyzed using ICD-9/10 codes. 7 To calculate the relative risk (RR) of endometriosis in our cohort, we first determined the population rates for each 5-year birth population group represented by the POI cases and birth place (Utah or outside of Utah). We defined the rate as total endometriosis cases within a population group divided by total population group size. The expected cases for each population group was calculated using each population rate. We calculated total expected cases for the cohort to be the sum of all expected cases of each population group, then determined the RR for our cohort to be the ratio of observed number of cases to expected number of cases. As described in our previous studies, approximate 95% confidence intervals and exact hypothesis tests of the null hypothesis (RR = 1.0) were constructed assuming that the number of endometriosis cases found among the population groups follows a Poisson distribution (Verrilli et al., 2023). Following collection of data, we also calculated the mean ages of diagnosis for endometriosis and POI, the standard deviations for these ranges, and average time between diagnoses. 8 RESULTS We found 613 patients with confirmed POI diagnosis. Based on the calculated population rates, we expected 6.49 cases of endometriosis and observed 10, demonstrating no significant increase in the relative risk (RR) of endometriosis (RR = 1.5419; 95% confidence intervals 0.7394, 2.8357; p = 0.165). The minimum age at endometriosis diagnosis was 24, and the minimum at POI diagnosis 28; the maximum ages for endometriosis and POI diagnoses were 35 and 40, respectively (Table 1). The mean age at endometriosis diagnosis was 27±2.95 (range 24-35), and the mean at POI diagnosis was 35.7±3.52 (28-35) (Table 1). The two conditions were thus diagnosed 8.7±3.65 years apart on average (Table 1). Chart review of these 10 patients showed that no etiology for POI was identified after testing for the common causes. 9 out of 10 were laparoscopically diagnosed with endometriosis. The most common sites of endometriosis growth were the rectouterine pouch (n=3), uterosacral ligament (n=2), bowel (n=2), and ovaries (n=3) (Table 2). Three patients had surgical ovarian manipulation: removal of endometrial tissue and drainage of benign ovarian cyst (n=1), removal of a left endometrioma (n=1), and drainage of bilateral endometriomas (n=1) (Table 2). Four patients were diagnosed with endometrioma via ultrasound (Table 2). Reviewing the charts for other autoimmune diseases indicated diagnosis of autoimmune thyroiditis in one patient, with a few other conditions being briefly noted in some charts but never confirmed with ICD-9/10 codes, surgical indication, or lab results (Table 3). 10 DISCUSSION Our goal was to demonstrate an association between endometriosis diagnosis and POI diagnosis based on potential shared autoimmune etiology. This data does not show a significant association between the two conditions. Further refining of these results both affirms their validity and underscores the lack of association demonstrated in our cohort. Therefore, we can conclude that the null hypothesis should not be rejected. As they were diagnosed 8.7 years apart on average, it is highly likely that patients with endometriosis experience symptoms and therefore receive the diagnosis before cessation of menses from POI; thus, it is unlikely that the POI cohort contains a significant number of undiagnosed endometriosis patients whose symptoms were masked or eliminated by POI onset. We therefore conclude that we did not miss any patients based on time of diagnosis. Nine out of ten of the patients identified to have both conditions were diagnosed with endometriosis laparoscopically. As mentioned above, this procedure is considered the gold standard for definitive diagnosis of endometriosis (Hebert, 2023). However, we took note of any procedure that physically removed or altered the ovarian tissue, as it could be a confounding factor to the association of endometriosis with POI. If the ovary were to suffer trauma from excision, ablation, or scarring, any ensuing dysfunction could be mistaken as comorbid POI due to similarity of symptoms, such as reduced follicle (Welt, 2007). As the purpose of this study is to evaluate common etiology between endometriosis and POI specifically through autoimmune disease, such cases would be confounders. With these considerations in mind, it should be stated that laparoscopic 11 diagnosis helped ensure the quality of our results, but it was necessary to further evaluate these surgical procedures to maintain the narrow focus of our study. Similarly, we reviewed these patients’ charts for diagnosis of endometrioma because of the increased risk of damage to the ovary (Nisolle and Donnez, 1997). An endometrioma, or “chocolate cyst,” is currently understood as an abnormal cyst found in the ovaries resulting from invagination of the ovarian cortex that contains accumulated menstrual debris (Nisolle and Donnez, 1997). These cysts and their fluid have multiple characteristics that uniquely damage healthy ovarian tissue, including: high levels of free iron (exceeding 100 mmol/L), which produce reactive oxygen species (ROS) that can lead to oxidative stress; high amounts of ROS, which were shown to induce DNA mutation in epithelial cells in vitro; and high levels of lactose dehydrogenase, which indicate tissue damage (Yamaguchi et al., 2008). Surgical removal of endometriomas presents even more danger to ovarian reserve: one study found that over 50% of laparoscopically removed endometriomas contained primordial follicles, and they theorized that the unique structure of these cysts makes their removal much more difficult without removing and damaging healthy follicles (Garcia-Velasco and Somigliana, 2009). In fact, another study found that laparoscopic treatment of bilateral endometriomas carried a 2.4% risk of POI following surgery (Busacca, 2006). Given all of these factors, we felt it necessary to note both endometrioma diagnosis and any associated surgical procedure. Four patients were diagnosed with an endometrioma; two patients had surgical treatment of the endometriomas, and one patient had bilateral surgical treatment. These patients should be excluded from our results, as endometriomas and these treatments can contribute to POI independent of any shared 12 autoimmune etiology. Additionally, there was one patient who had superficial endometriosis removed from the ovary and a non-endometriotic cyst drained. These procedures may have caused structural damage that contributed to POI development. Removing these charts from our data yielded 5 patients with endometriosis out of the 613 in the POI cohort. This refined number not only reveals even less association between the diseases, but it even falls below the expected number of 6.49. Lastly, as our proposed link between endometriosis and POI hinges on autoimmune dysfunction, we reviewed the charts for any additional diagnoses of autoimmune disease, with special attention to those previously associated with endometriosis or POI in the literature (Shigesi, 2018). The only confirmed autoimmune disease in our patients was autoimmune thyroiditis (n=1). Interestingly, none of the patients had Addison’s disease, which is the autoimmune disease most strongly associated with POI (Hoek, 1997). As none of the 10 patients had specified adrenal insufficiency, we can be confident that their POI diagnoses were not autoimmune-related. Our study has limitations. One such limitation is that our analysis hinged on ICD9/10 codes for endometriosis diagnosis, so any diagnosis that was noted in the EMRs without the codes would not have by identified by our methods. This could occur if, for example, patients were diagnosed in a facility outside of the networks we used Another limitation of this study is that the existing literature on endometriosis and autoimmunity largely hinges on studies from the late 1980s and early 1990s. More research is needed to solidify or reject the association between endometriosis and autoimmunity, both on its own designation and its comorbidity with other autoimmune disorders. 13 As this study utilized a cohort of POI patients, its scope is limited in elucidating patterns of autoimmunity specific to endometriosis. Conducting a similar study with a cohort of endometriosis patients, especially with a larger sample size, would be more effective in exploring the proposed associations between endometriosis and autoimmune conditions. Such a study design would be needed to evaluate conclusions of aforementioned meta-analyses that have attempted to show these relationships. 14 CONCLUSIONS In summary, this study showed no association between POI and endometriosis when iatrogenic causes were removed. As not all POI cases are autoimmune-based and endometriosis is not confirmed to have autoimmune etiology, this outcome is consistent with current understandings of these diseases. Future studies might include identifying a cohort of endometriosis patients and then studying comorbid autoimmune diseases using ICD-9/10 codes, particularly those outlined by previous studies as having strong associations with endometriosis. 15 REFERENCES Andres, M. P., Arcoverde, F. V. L., Souza, C. C. C., Fernandes, L. F., Abrão, M. S., & Kho, R. M. (2020). EXTRAPELVIC endometriosis: A systematic review. Journal of Minimally Invasive Gynecology, 27(2), 373–389. https://doi.org/10.1016/j.jmig.2019.10.004 Bulun, S. E., Yilmaz, B. 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