{"paper_id":"0e17373f-2b53-45be-9362-d628cb6f9a37","body_text":"Endometriosis is defined as endometrial tissue outside of the uterus. It is a debilitating, complex, and underdiagnosed condition, impacting approximately 10%-15% of reproductive-aged women worldwide. Endometriosis is associated with chronic pain, infertility, diminished well-being from invasive and delayed diagnostics, and limited treatment options. It is reported to cause approximately 23,000 hospitalizations and cost approximately $9 billion annually due to reduced quality of life and productivity. Endometriosis also occurs spontaneously in rhesus macaques (Macaca mulatta), and similar negative impacts are seen in research colonies. Despite extensive research in both species, much remains unknown about its etiopathogenesis, especially regarding uncommon presentations. This retrospective observational cohort study evaluated clinical findings from 68 rhesus macaques diagnosed with endometriosis at the Emory National Primate Research Center and characteristics associated with their endometriosis lesions. Animals were categorized into 4 endometriosis subtypes: pelvic, abdominal, extra-abdominal, and invasive. Prevalence and descriptive statistics were calculated for reproductive history, physical exam findings, and other observations related to an endometriosis diagnosis. In addition, immunohistochemical analysis was performed on lesions from a subset of 27 macaques and 6 controls to evaluate variation in mean staining intensity and distribution (±SEM) for estrogen receptor α (ERα), progesterone receptor (PR), and CD10 antibodies. Clinical data corroborated established literature by demonstrating common findings associated with endometriosis. It provided novel information by examining the relationship and patterns of these findings in uncommon presentations of endometriosis. Analysis of mean staining intensities (±SEM) revealed significant differential expression of ERα and PR in abdominal endometriosis compared with extra-abdominal and invasive subtypes, as well as autologous eutopic uterine tissue. This study provides important data that may begin to unravel some of the underlying mechanisms by which endometriosis behaves in varied ways.\nEvaluating Subtypes of Endometriosis in Rhesus Macaques (Macaca mulatta) Using Clinical Findings and Immunohistochemistry\nIntroduction\nEndometriosis is a painful and debilitating disease defined by the presence of endometrial tissue outside of the uterus.1–19,20 It occurs spontaneously in species that menstruate, including humans and other primate species such as apes, macaques, baboons, vervets, and DeBrazza’s monkeys.7,9,10,12,13,17,21,22 Although considered a benign, inflammatory disease,1–6,12,14–16,19 endometriosis sometimes presents with characteristics of malignancy, such as dysregulated cell growth, abnormal tissue morphology, invasion into other tissues, and migration to lymph nodes and distant organs.1,2,10,12,16,17,22 Described as “enigmatic” or as a “missed disease” due to its complex nature,6,15 endometriosis lesions do not have a definitive etiopathogenesis.1–6,9,10,15,16 Studies in rhesus macaques and humans have provided generally accepted predisposing factors and rationales for the condition related to reproductive history, hormonal influences, and lymphatic and vascular “metastasis.”1–10,12,13,15–19,22,23 However, even with improved understanding of the condition, questions remain about the behavior, severity, and extent of the varying presentations of endometriosis lesions and their impact on the disease’s overall clinical presentation. This creates an opportunity for further research to better understand this condition.\nRhesus macaques (Macaca mulatta) are often used as models for human diseases such as endometriosis9,13,17,21,20,23–26 due to their 95% similar genetic homology and relatively shorter lifespans compared with humans.20,23,26,27 Studies have described features of endometriosis in both human and rhesus populations, showing many shared characteristics between the species. Both species show similar relative age ranges affected, nonspecific clinical signs, difficulty diagnosing the condition, and symptomatic rather than curative treatment.1–12,15–19,23,24 Furthermore, rhesus macaques in research populations are greatly impacted by the condition, as endometriosis has been reported in up to 30% of female rhesus macaques at some institutions.7,21–23 Given their similarities to humans, established use and availability in research, and the frequency of occurrence in their own populations, rhesus macaques are an ideal model for studying spontaneous endometriosis.\nA retrospective analysis of endometriosis cases in rhesus macaques at the Emory National Primate Research Center (ENPRC) was performed. Using immunohistochemistry (IHC) and clinical data, we sought to evaluate subtypes of endometriosis lesions in rhesus macaques according to the location and characteristics of the lesions: pelvic, abdominal, extra-abdominal, and invasive endometriosis subtypes. We evaluated antemortem clinical information and postmortem findings across the study population and endometriosis subtypes. In addition, IHC analysis using CD10, estrogen receptor α (ERα) and progesterone receptor (PR) antibodies was performed to evaluate histologic samples of endometriosis lesions for characteristics associated with subtypes of endometriosis. To our knowledge, patterns in clinical history and the variability in expression of these IHC markers have not been evaluated for the subtypes of endometriosis. We hypothesize that there will be variability in the expression of IHC markers and trends in clinical findings based on endometriosis subtypes. This study aims to help better understand endometriosis, especially regarding its less common presentations.\nMaterials and Methods\nAnimals.\nThis study used the Replacement, Reduction, and Refinement (3Rs) principle of reduction by conducting hypothesis-driven research with tissue samples stored from animals necropsied from 2010 to 2024 (Figure 1). Archived tissues were selected from rhesus macaques at ENPRC based on the inclusion criteria of an endometriosis diagnosis at necropsy. The 68 subjects were females aged 6.8-26.9 years at the time of necropsy, with a mean age of 18 years. Animals were housed at the ENPRC Field Station (Lawrenceville, GA) or Main Station (Atlanta, GA), where they were assigned to breeding protocols or studies related to infectious disease, neuroscience, or behavior. Animals were housed in standard caging, runs, or compound-style enclosures depending on social dynamics or their roles in respective studies. Standard enrichment was supplied such as climbing structures, forage boards, and toys. Rhesus macaques were fed a standard commercial primate diet (Lab Diet 5037 Monkey Diet Jumbo, Brentwood, MO). They were supplemented daily with a quarter of a fresh orange and varying enrichment items, including rice cakes, peanut butter, yogurt, and fresh vegetables in accordance with ENPRC standard operating procedures. Free access to water was provided via an automatic watering system. All animal procedures were approved by Emory University’s IACUC. The care and handling of animals was conducted in a humane manner consistent with institutional, federal, and regulatory requirements. The facility and its programs are fully AAALAC accredited.\nRecords search.\nStudy animals were selected, and data were collected and managed using Research Electronic Data Capture (REDCap) tools hosted at ENPRC.28,29 This digital records system contains necropsy reports for animals at ENPRC from 2010 to the present day. A project report was created with specific criteria to filter the database for necropsy results relevant to this study. The keyword “endometriosis” was used as a filter term to generate a list of all the necropsy reports in this time frame that described endometriosis in animals evaluated at the institution. Exclusion criteria included primates other than rhesus macaques, incomplete necropsy reports, or a lack of definitive endometriosis diagnosis. As of June 2024, 7,157 records were queried, and 68 total reports remained that contained rhesus macaques diagnosed with endometriosis that met study criteria. These animals were searched in REDCap, as well as ENPRC’s animal records system for data from their complete medical history pertaining to the following: reproductive history, clinical presentations, and physical examinations that led to an endometriosis diagnosis and the characteristics of their endometriosis lesions. The endometriosis lesions of each animal were categorized into 4 subtypes based on location and biologic behavior as follows: (1) pelvic, (2) abdominal, (3) extra-abdominal, and (4) invasive (Figure 2): (1) pelvic: lesions associated with organs in the pelvic canal, including the uterus, ovaries, urinary bladder, and any connective tissues surrounding those organs; (2) abdominal: lesions associated with organs in the abdominal cavity, excluding those encompassed by the pelvic category; (3) extraabdominal: lesions present outside of the abdominal cavity; and (4) invasive: lesions that invade through the serosal layer and into deeper tissues of the organ, regardless of location within the body, as this is a distinct biologic behavior. Based on similar categorization described in literature for humans,17,19 these subtypes were developed to encompass the broad manner in which endometriosis lesions present. This facilitated the inclusion of less prevalent forms of the disease1,5,12–15,22 in this study.\nRandom selection.\nA subset of approximately 30 animals was randomly selected for immunohistochemistry from the initial 68 animals diagnosed with endometriosis due to budgetary constraints. Eight animals were intentionally included due to the presence of extra-abdominal endometriosis, invasive endometriosis, or other endometriosis subtypes in the absence of concurrent pelvic endometriosis. These presentations are infrequently reported5 and are therefore important to include in this study focused on discerning trends of endometriosis lesion development. Random selection of the remaining 22 animals was performed using the SURVEYSELECT procedure in SAS/STAT software, version 15.1 (SAS Institute, Cary, NC; 2018).30 A sampling frame of 60 animals was used, accounting for the 8 already intentionally included. The animals’ information selected from this procedure was then provided to the ENPRC Division of Pathology for the cutting, sectioning, and staining of slides. If archived tissues of the animals’ endometriosis lesions were not stored, the animal was removed from the study. This resulted in a total of 27 animals remaining that were affected by endometriosis and included in this portion of this study. As a negative control, a convenience sample of 6 animals was selected that were not diagnosed with endometriosis. A total of 33 animal tissues were then sent for histologic and immunohistochemical examination.\nHistopathology and IHC.\nTissue samples from rhesus macaques were fixed in 10% neutral buffered formalin, processed, and embedded in paraffin for histologic analysis. All samples were sectioned at 5 μm and stained with hematoxylin and eosin for routine histopathology.\nImmunohistochemistry for CD10 (Leica, Wetzlar, Germany; PA0131), progesterone (Invitrogen, Waltham, MA; cat. no. MA5-12581), and estrogen (Invitrogen; cat. no. MA1-27107) antigens at 1:200 dilution was performed on the Bond RX automated system with the Polymer Define Detection System (Leica) used per the manufacturer’s protocol. Sample and control tissue sections were dewaxed with Bond Dewaxing Solution (Leica) at 72 °C for 30 minutes and then subsequently rehydrated with graded alcohol washes and 1 × Leica bond wash. Heat-induced epitope retrieval (HIER) was performed using Epitope Retrieval Solution 1 (Leica), heated to 100 °C for 20 minutes. A peroxide block (Leica) was applied for 5 minutes to quench endogenous peroxidase activity before applying each of the antibodies. Controls performed appropriately, confirming the validity of each staining procedure. Sections were examined under light microscopy by an American College of Veterinary Pathologists board-certified pathologist using an Olympus BX51 microscope, and photomicrographs were taken using an Olympus DP73 camera.\nIHC scoring and evaluation.\nThe respective IHC sections were examined and scored based on a standardized scoring system (Table 1). Eutopic endometrium was divided into stroma, basalis epithelium, functionalis epithelium, and superficial epithelium. Ectopic endometrium was divided into stroma and epithelium. Each component was assigned a subjective score based on staining intensity and distribution. Staining intensity was graduated into scores ranging from negative (0), mostly negative with scattered weak positive cells (1), mostly weak positive with scattered positive cells (2), mostly positive with some strong positive cells (3), and mostly or all strongly positive (4). Distribution of positivity was graduated into scores of negative (0), up to 25% positivity (1), 25%-49% positivity (2), 50%-74% positivity (3), and 75%-100% positivity (4). This scoring system was used to score only the ERα and PR samples (Figures 3 and 4).\n| Degree of staining intensity | Corresponding score | Distribution of staining positivity | Corresponding score |\n|---|---|---|---|\n| Negative | 0 | Negative | 0 |\n| Mostly negative with scattered weak positive cells | 1 | Up to 25% positivity | 1 |\n| Mostly weak positive with scattered positive cells | 2 | 25%-49% positivity | 2 |\n| Mostly positive with some strong positive cells | 3 | 50%-74% positivity | 3 |\n| Mostly or all strong positive cells | 4 | 75%-100% positivity | 4 |\nClinical data evaluation.\nThe historical clinical data of all 68 study animals diagnosed with endometriosis were collected and assessed relating to their reproductive histories, clinical presentations that led to an endometriosis diagnosis, and necropsy findings. The data were categorized into physical exam findings, other observations and concurrent findings, and reproductive findings. The prevalence of these findings in the overall study population was calculated generally and with respect to the different endometriosis subtypes.\nStatistical analysis.\nDue to the complexity of the clinical dataset, including multiple findings per animal, and the small sample sizes within rare endometriosis subtypes, inferential statistical analysis was not feasible for all variables. Therefore, clinical data were evaluated using descriptive statistics, and findings were considered noteworthy if present in at least 5% of the study population.\nFor the analysis of endometriosis lesion staining intensity, measured on an ordinal scale (0-4), we used a linear-mixed effects model, treating the outcome as a continuous variable. The mixed-model for repeated measures analysis was implemented using the PROC MIXED procedure in SAS software, version 15.1 (SAS Institute, Cary, NC).30 The model included type of antibody (ER and PR), endometriosis subtype, and the interaction between antibody and subtype as predictors. Since cases of extra-abdominal endometriosis lesions also showed invasive behavior, it is assumed that this is how the disease spreads outside the abdominal cavity. Because of this and the low prevalence of invasive and extra-abdominal endometriosis, those categories were combined for this analysis. A random intercept for animal was incorporated to account for the nested structure of the data and the repeated measurements within each animal. To help ensure reproducibility, all statistical analyses were performed using the SAS procedure MIXED. Model assumptions, particularly linearity and normality of residuals, were assessed through visual inspection of residual plots. The primary outcome is the mean staining intensity for each antibody type and endometriosis subtype, along with its 95% CI. A P value ≤ 0.05 was considered statistically significant for the main effects (antibody and subtype) and for the interaction between antibody and endometriosis subtype. Specific statistical tests were done within the framework of the mixed effects linear model. All statistical tests were 2-sided and unadjusted for multiple comparisons. The same linear-mixed effects model was used to analyze staining positivity.\nFor the eutopic endometrium data, a similar linear-mixed effects model was used to analyze staining intensity and distribution of positivity. The model included type of antibody (ERα and PR), uterus sample status (animals with an affected uterus or animals with an unaffected uterus), and the interaction between antibody and type of uterus (unaffected or affected) as predictors. The primary outcome is the mean staining intensity and positivity for each antibody type and uterus status, along with its 95% CI.\nResults\nClinical findings.\nThe age range of endometriosis diagnosis in these rhesus macaques was 5. 7-26.9 years, with an average age of diagnosis at approximately 18 years. This range includes animals that were diagnosed with the condition ante- and/or postmortem. Twenty-one of the 68 total animals were suspected or confirmed to have endometriosis before necropsy. In addition, 79% (54/68) of all study animals were euthanized for clinical reasons, often described as “poor prognosis” for their clinical condition.\nThe most frequent physical exam finding was an abdominal mass, occurring in 24% of the study population. The next most frequent findings were vaginal bleeding not associated with menstruation and an enlarged uterus, occurring in 18% and 15% of the study population, respectively. Other findings include heavy menstruation, anemia, an irregular uterus on palpation, and abdominal distention (Table 2). When assessed by endometriosis subtype (Table 3), abdominal masses, vaginal bleeding, and an enlarged uterus were the physical exam findings with the highest prevalence across all subtypes, but with varying prevalence for each one.\n| Physical exam findings | Prevalence | Percentage of prevalence |\n|---|---|---|\n| Abdominal mass a | 16 | 23.53% |\n| Vaginal bleeding b | 12 | 17.65% |\n| Enlarged uterus | 10 | 14.71% |\n| Heavy mense | 8 | 11.76% |\n| Anemia c | 7 | 10.29% |\n| Irregular uterus d | 5 | 7.35% |\n| Abdominal distention | 4 | 5.88% |\nSome findings were grouped to account for varied verbiage used by individual veterinarians; n = 68 rhesus macaques.\n- a\n“Abdominal mass” includes any notation of an abdominal mass without an affected organ specified.\n- b\n“Vaginal bleeding” includes any notation of vaginal bleeding of varying severities that was not attributed to menstruation.\n- c\n“Anemia” includes any notation of the term as well as “pallor.”\n- d\n“Irregular uterus” includes any notation of atypical uterine palpation not directly attributed to a cyst. Specific examples include irregular margins, multilobulated consistency, crepitus, and firm palpation.\n| Endometriosis subtype | Pelvic (n = 65) | Abdominal (n = 38) | Extra-abdominal/invasive (n = 14) | |||\n|---|---|---|---|---|---|---|\n| Physical exam findings | Prevalence | Percentage of Prevalence | Prevalence | Percentage of Prevalence | Prevalence | Percentage of Prevalence |\n| Abdominal mass a | 15 | 23.1% | 12 | 31.6% | 5 | 35.7% |\n| Vaginal bleeding b | 12 | 18.5% | 8 | 21.1% | 6 | 42.9% |\n| Enlarged uterus | 9 | 13.8% | 9 | 23.6% | 3 | 21.4% |\n| Heavy mense | 8 | 12.3% | 7 | 18.4% | 2 | 14.3% |\n| Anemia c | 6 | 9.2% | 5 | 13.2% | 1 | 7.1% |\n| Irregular uterus d | 5 | 7.7% | 4 | 10.5% | 3 | 21.4% |\n| Abdominal distension | 3 | 4.6% | 3 | 7.9% | 0 | 0 |\nSome findings were grouped to account for varied verbiage used by individual veterinarians; n = 68 rhesus macaques.\n- a\n“Abdominal mass” includes any notation of an abdominal mass without an affected organ specified.\n- b\n“Vaginal bleeding” includes any notation of vaginal bleeding of varying severities that was not attributed to menstruation.\n- c\n“Anemia” includes any notation of the term as well as “pallor.”\n- d\n“Irregular uterus” includes any notation of atypical uterine palpation not directly attributed to a cyst. Specific examples include irregular margins, multilobulated consistency, crepitus, and firm palpation.\nThe most frequent observation was weight loss, observed in 53% of the study population. Inappetence was reported in 16% of the study population, and lethargy and diarrhea/dehydration were both reported in 15%. Other observations include alopecia, abnormal gait, abnormal behavior, and dental issues (Table 4). When assessed for endometriosis subtypes (Table 5), weight loss had the highest prevalence across all subtypes.\n| Observations/concurrent diagnoses | Prevalence | Percentage of prevalence |\n|---|---|---|\n| Weight loss | 36 | 52.94% |\n| Inappetence a | 11 | 16.18% |\n| Diarrhea/dehydration b | 10 | 14.71% |\n| Lethargy | 10 | 14.71% |\n| Alopecia | 6 | 8.82% |\n| Abnormal gait c | 6 | 8.82% |\n| Abnormal behavior | 4 | 5.88% |\n| Dental issues d | 4 | 5.88% |\nSome findings were grouped to account for varied verbiage used by individual veterinarians; n = 68 rhesus macaques.\n- a\n“Inappetence” includes any notation of the term, as well as anorexia.\n- b\n“Diarrhea/dehydration” includes any notation of either term, regardless of chronicity, due to the occasional interchangeable nature in which these symptoms were listed within records.\n- c\n“Abnormal gait” includes any notation of an atypical gait, including lameness.\n- d\n“Dental issues” includes any notation of pathology related to teeth. Specific examples include gingivitis, tooth root abscesses, and pulp exposure.\n| Endometriosis subtype | Pelvic (n = 65) | Abdominal (n = 38) | Extra-abdominal/invasive (n = 14) | |||\n|---|---|---|---|---|---|---|\n| Observations/concurrent findings | Prevalence | Percentage of prevalence | Prevalence | Percentage of prevalence | Prevalence | Percentage of prevalence |\n| Weight loss | 34 | 52.3% | 18 | 47.4% | 9 | 64.3% |\n| Inappetence a | 9 | 13.8% | 7 | 18.4% | 3 | 21.4% |\n| Diarrhea/dehydration b | 10 | 15.4% | 8 | 21.1% | 3 | 21.4% |\n| Lethargy | 10 | 15.4% | 4 | 10.5% | 3 | 21.4% |\n| Alopecia | 6 | 9.2% | 3 | 7.9% | 0 | 0 |\n| Abnormal gait c | 6 | 9.2% | 2 | 5.3% | 2 | 14.3% |\n| Abnormal behavior | 4 | 6.2% | 1 | 2.6% | 0 | 0 |\n| Dental issues d | 3 | 4.6% | 3 | 7.9% | 1 | 7.1% |\nSome findings were grouped to account for varied verbiage used by individual veterinarians; n = 68 rhesus macaques.\n- a\n“Inappetence” includes any notation of the term, as well as anorexia.\n- b\n“Diarrhea/dehydration” includes any notation of either term, regardless of chronicity, due to the occasional interchangeable nature in which these symptoms were listed within records.\n- c\n“Abnormal gait” includes any notation of an atypical gait, including lameness.\n- d\n“Dental issues” includes any notation of pathology related to teeth. Specific examples include gingivitis, tooth root abscesses, and pulp exposure.\nThe mean number of pregnancies each animal had during their lifetime was 4.9. The most common reproductive finding was retained placenta after parturition in 15% of the study population (10 cases). Other findings had similar prevalence, including Cesarean section surgeries, mastitis, endometritis, and abortions, each seen in 10% of the study population (7 animals), and dystocia and Depo-Provera injections each occurring in 9% (6 animals) (Table 6). When evaluated by endometriosis subtype (Table 7), this category had the smallest number of occurrences of findings throughout the study population, with the highest being 10.\n| Reproductive findings | Prevalence | Percentage of prevalence |\n|---|---|---|\n| Retained placenta | 10 | 14.71% |\n| Mastitis | 7 | 10.29% |\n| Cesarean section | 7 | 10.29% |\n| Endometritis | 7 | 10.29% |\n| Abortion | 7 | 10.29% |\n| Dystocia | 6 | 8.82% |\n| Depo Provera injections | 6 | 8.82% |\nThe reported occurrences do not account for some animals having multiple instances of these findings; n = 68 rhesus macaques.\n| Endometriosis subtype | Pelvic (n = 65) | Abdominal (n = 38) | Extra-abdominal/invasive (n = 14) | |||\n|---|---|---|---|---|---|---|\n| Reproductive findings | Prevalence | Percentage of prevalence | Prevalence | Percentage of prevalence | Prevalence | Percentage of prevalence |\n| Retained placenta | 10 | 15.4% | 4 | 10.5% | 3 | 21.4% |\n| Mastitis | 7 | 10.8% | 5 | 13.2% | 5 | 35.7% |\n| Cesarean section | 7 | 10.8% | 3 | 7.9% | 2 | 14.3% |\n| Endometritis | 6 | 9.2% | 3 | 7.9% | 2 | 14.3% |\n| Abortion | 7 | 10.8% | 5 | 13.2% | 3 | 21.4% |\n| Dystocia | 6 | 9.2% | 2 | 5.3% | 1 | 7.1% |\n| Depo Provera Injections | 6 | 9.2% | 1 | 2.6% | 0 | 0 |\nThe reported occurrences do not account for some animals having multiple instances of these findings; n = 68 rhesus macaques.\nIHC findings.\nCD10 subjectively showed uniform positivity in stromal cells across all groups (Figure 5), except for one sample with decidualized stroma, which tested negative for CD10. No additional scoring or statistical analysis of this antibody was conducted. There was no subjective variability in CD10 expression on endometriosis lesions, regardless of subtype.\nThe mean ERα staining intensity (Table 8) was significantly higher in abdominal endometriosis compared with the invasive/extra-abdominal category (mean difference = 0/60, P = 0.043). In addition, ERα staining intensity was significantly higher in abdominal endometriosis compared with the eutopic endometrium of affected animals (mean difference = 0.64, P = 0.007). There were no statistically significant differences in ERα staining positivity across endometriosis subtypes.\n| Label | Mean difference in staining intensity | SE | 95% CI | P value | |\n|---|---|---|---|---|---|\n| ERα: abdominal compared with invasive/extra-abdominal | 0.60 | 0.30 | 0.02 | 1.18 | 0.043 |\n| ERα: abdominal compared with eutopic uterus | 0.64 | 0.24 | 0.18 | 1.11 | 0.007 |\n| ERα: pelvic compared with invasive/extra-abdominal | 0.45 | 0.28 | −0.11 | 1.00 | 0.117 |\nThe mean PR staining intensity (Table 9) was significantly higher in abdominal endometriosis than in the invasive/extra-abdominal subtype (mean difference = 0.60, P = 0.040). In addition, PR staining intensity was significantly higher in abdominal endometriosis compared with the eutopic endometrium of affected animals (mean difference = 0.68, P = 0.005). There were no statistically significant differences in PR staining positivity levels across endometriosis subtypes.\n| Label | Mean difference in staining intensity | SE | 95% CI | P value | |\n|---|---|---|---|---|---|\n| PR: abdominal compared with invasive/extra-abdominal | 0.60 | 0.29 | 0.03 | 1.17 | 0.040 |\n| PR: abdominal compared with eutopic uterus | 0.68 | 0.24 | 0.21 | 1.14 | 0.005 |\n| PR: pelvic compared with invasive/extra-abdominal | 0.34 | 0.28 | −0.20 | 0.88 | 0.221 |\nFor eutopic endometrium, the mean staining intensity of ERα was 2.6 for unaffected animals, and the mean staining intensity was 2.3 for affected animals (mean difference = 0.349, P = 0.56). For PR, the mean staining intensity is 2.7 for unaffected animals and 1.8 for affected animals (mean difference = 0.886, P = 0.14). There was no statistically significant difference in ERα or PR staining intensity in eutopic endometrial tissue between animals with or without endometriosis. For ERα, the mean staining positivity is 2.6 for unaffected animals, and the mean staining positivity is 2.8 for affected animals (mean difference = −0.16, P = 0.79). For PR, the mean staining positivity is 2.7 for unaffected animals and 2.0 for affected animals (mean difference = 0.685, P = 0.26). There was no statistically significant difference in ERα or PR staining positivity in eutopic endometrial tissue between animals with or without endometriosis.\nDiscussion\nSome of the general historical clinical findings in this study were similar to those from previous studies. For instance, the average age of diagnosis in this rhesus colony was 17.9 years, which aligns with the typical age range in which the disease is reported, approximately 11-24 years, with more cases in older animals within the colony.7,10,21,23Aging at a rate of approximately 3 times that of humans,20,23,26 this is comparable to the average diagnostic age range of 15-45 years in people.2 In this study, we also observed some of the more well-established nonspecific clinical signs such as dysmenorrhea, inappetence, weight loss, and abdominal discomfort, seen in both humans and rhesus macaques.1–8 Weight loss was the most common finding across all endometriosis subtypes, with at least 25% higher prevalence than other findings in this assessment category. Other common observations across all subtypes were lethargy, inappetence, and diarrhea/dehydration. While these signs are not specific to a particular disease, they are regularly seen in animals exhibiting pain and discomfort, which is often associated with endometriosis.2–8,11,17 The prevalence of symptoms in this study suggests that these clinical presentations can exist across all studied endometriosis subtypes. The most common physical exam findings in this study were also nonspecific but varied more across endometriosis subtypes. An abdominal mass, vaginal bleeding, and an enlarged uterus were the 3 most prevalent physical exam findings, but they did not have similar patterns of prevalence within each subtype. It remains undetermined whether there is an association between the clinical findings in this study and a specific endometriosis subtype.\nMany studies have identified potential risk factors for endometriosis in rhesus macaques and humans relating to their reproductive history. These factors include familial history, nulliparity, heavy or retrograde menstruation, cesarean section, hysterectomy, and long-term estrogen administration.2–11,13,15,17–19,22,23 The prevalence of symptoms related to the reproductive system was compiled for our study population and analyzed according to endometriosis subtype. Overall, the frequency of these reproductive findings was lower and less variable than the other clinical categories studied, with the highest number of individual occurrences being 10. The recorded prevalence in this study reflects each reproductive finding as an individual data point per animal. It does not account for an animal experiencing multiple iterations of a finding over its lifetime, and therefore, it is undetermined currently if this factor impacts endometriosis and its subtypes. The literature generally accepts that reproductive history is related to endometriosis development, but in this study, there were fewer occurrences of reproductive findings compared with the other clinical categories assessed. This suggests that out of the 3 clinical categories, reproductive findings may be less widely observed in a population affected by endometriosis and across endometriosis subtypes.\nNecropsy data for study animals’ postmortem examinations indicated that most subjects (79%) were euthanized for clinical reasons rather than experimental endpoints. “Poor prognosis” is often listed in the animal’s history for clinical euthanasia. The poor prognosis was presumed to be due to complications from endometriosis lesions, or due to the diagnosis or suspicion of the condition itself considering the lack of definitive treatment and the risk of uncommon but severe presentations.7–9,13 By conveying the vast presentations of endometriosis lesions and describing some of the poor outcomes of the condition, this study helps underscore the importance of understanding endometriosis due to its large impact on research populations.\nImmunohistochemistry was used to analyze endometriosis lesions and their expression of various markers based on clinical behavior. CD10, ERα, and PR antibodies were selected because of their established use as an ancillary diagnostic for endometriosis.1,14 CD10 hormone functions as a cell surface enzyme that reduces a cell’s response to peptide hormones, and therefore, many hormone-sensitive cells and their related neoplasms express the CD10 antigen.14,31 While the other roles of CD10 in endometrial tissues are not completely understood, it is expressed in these tissues and has been shown to play an important role in cell proliferation and adhesion.32 Eutopic and ectopic endometrial tissue respond to estradiol and progesterone, and they both contain immunoreactive estrogen and progesterone receptors.1,33 Furthermore, endometriosis is known as a highly estrogen-dependent disease.4,10,12,13,16,17,33,34 While it is known that endometrial lesions have these receptors and that they are influenced by these hormones,16 the role of them on the pathogenesis of the disease has yet to be determined.\nIn this study, the CD10 antibody displayed subjectively consistent cytoplasmic reactivity in the stromal cells of all samples assessed, except for one with decidualized stroma. This is not unexpected, as previous studies35 have shown CD10 IHC to have negative staining in decidualized endometrial stroma. What has not been previously characterized is that CD10 consistently shows immunoreactivity across endometriosis subtypes. This suggests that the stroma in these endometrial lesions is active and behaves similarly to eutopic endometrium, regardless of location within the body or biologic behavior. ER and PR both showed more variability in staining intensity and the distribution of staining positivity and were scored and compared across the different endometriosis subtypes. The staining intensities of both ERα and PR were significantly higher in abdominal endometriosis compared with extra-abdominal/invasive endometriosis, as well as to eutopic endometrium in affected animals. This suggests a unique hormonal expression of estrogen and progesterone receptors in the abdominal cavity, and these may influence the development of endometriosis lesions in this area.\nSince endometriosis is a highly estrogen-dependent disease,4,10,12,13,16,17,33,34 the regular cyclical changes that take place in menstruating animals can also impact endometriosis lesions.6,13,22 Given the normal fluctuations in estrogen and progesterone during the menstrual cycle, variation in their receptor expression is expected depending on the cycle phase at the time of sample collection. As cycle phase was not assessed in this study, its potential contribution to the observed variability cannot be determined. ERα and PR staining intensity were significantly higher in abdominal endometriosis when compared with eutopic endometrium, suggesting an association between this endometriosis subtype and receptor expression. In addition, multiple studies have reported variable outcomes of ERα and PR antigen expression in endometriosis lesions. Reports1,36 show ERα expression in most endometriotic lesions corresponds to their morphologic appearance. Others1,37 found that receptor levels in endometriotic tissue were heterogenous, unlike eutopic endometrium, and did not undergo predictable changes in response to endogenous hormones. In eutopic endometrium, there is variation of IHC expression between the various compartments, defined here as the stroma, basal glands, functionalis glands, and superficial epithelium. Since there is often no such organization in ectopic lesions, only glandular epithelium and stroma were evaluated. Scores for glandular epithelium often showed variance throughout ectopic lesions, which could suggest that a particular compartment of eutopic endometrium is recapitulated. Because of the limited number of resident uterus samples included in our study, we were not able to assess for this potential reason for the observed variability. Our finding of increased ER and PR staining intensity scores in abdominal endometriosis suggests a pattern of variance inherent to this specific location not previously established. There was no statistically significant variability when assessing staining intensity of ERα and PR when comparing the remaining endometriosis subtypes. This finding suggests that the hormonal variation in abdominal endometriosis is unique and highlights a potential direction for future research.\nThere was no statistically significant variability in staining intensity of ERα and PR on eutopic endometrium of animals with or without endometriosis. This suggests that ER,α PR, and CD10 are not associated with any variable present in eutopic endometrium that could predispose the development of endometriosis. In addition, there was no statistically significant variability in the distribution of ERα and PR staining across endometriosis subtypes or in the eutopic endometrium of both affected and unaffected animals. This indicates that although hormonal receptor expression may influence how endometriosis manifests, the distribution of these hormonal receptors does not appear to be significantly associated with endometriosis subtype.\nThere were a few limitations with this study. For instance, this study only includes data available through digital records. This was the only source used because of its accessibility and reliability for retrieving each animals’ complete history and searching for specific information within it. Including physical records could have greatly increased the sample size and the study’s significance. Another limitation involves the lack of standardized terminology for medical records. Although diagnoses and results are generally consistent, variability in how individual veterinarians may describe findings and apply diagnostic approaches may introduce inconsistency.\nThe information from this study helped create opportunities for potential research in the future. For instance, including rhesus macaques from multiple institutions could reveal more findings and possible differences between institutions. Further studies could also use additional antibodies or inflammatory markers.3,12 Since endometriosis is a known inflammatory disease1,3–5,12,32,38 that affects tissues across all organ systems, additional tests could shed light on how and why endometriosis lesions develop in specific locations with a particular severity. Other modalities could also be incorporated, like genetic analysis with spatial transcriptomics. Such research could also address questions about the various subtypes of endometriosis and deepen our understanding of the disease’s overall pathogenesis.\nConclusion.\nThe prevalence patterns of historical clinical findings were established, and the characteristics of CD10, ERα, and PR expression using IHC were described across endometriosis subtypes. The staining intensities of both ERα and PR were significantly higher in abdominal endometriosis when compared with extra-abdominal/invasive subtypes and eutopic endometrium. This indicates significant variability among some endometriosis subtypes when assessing antigen expression.\nThe insights gained from this study are important to help understand how and why endometriosis lesions manifest by identifying characteristics associated with certain subtypes. This study establishes a framework for future investigations to evaluate differences between subtypes. Continued investigation of this condition could help alleviate some of its long-standing effects, ultimately offering answers and improving the quality of life for both affected animals and humans.\nContributor Notes","source_license":"public-domain-us","license_restricted":false}