{"paper_id":"05193684-2cc6-44ca-97c5-b62b31dc4d79","body_text":"Endometriosis is a chronic debilitating inflammatory disease that affects\napproximately 10 to 20 % of women of reproductive age and roughly\n50 % of women with infertility (Giudice, 2010). The associated clinical\nsymptoms like dysmenorrhoea, dyspareunia, and chronic pelvic pain have a\nnegative impact on the quality of life of women affected with the disorder\n(Gupta et al., 2008). Histologically, the disease is defined by the presence\nof endometrial glandular and stromal tissue in organs and tissues outside of\nthe uterine endometrium.\nExperimental studies in women are hindered by the risks and complications\nassociated with repetitive biopsy or surgical procedures. As a consequence,\nthe pathogenesis of endometriosis remains incompletely understood. It is\nlikely that endometriosis is a complex and multifactorial disorder triggered\nby hormonal, immunologic, genetic, and environmental factors. One\nhypothesized process in the pathogenesis of endometriosis is metaplasia,\ninvolving the transformation of tissues in the peritoneal cavity into\nendometrial tissue driven by hormonal or immunological factors (Sourial et\nal., 2014). Hormones play multiple roles with estrogen promoting\nproliferation of endometrial lesions and progesterone limiting endometrial\nproliferation. Inflammation, immune dysregulation, and oxidative stress\nhave also been associated with endometriosis, contributing to\ncytokine-mediated endometrial growth (Forte et al., 2014). Other\nhypothesized processes include the suppression of normal apoptosis of\nendometrial glandular cells, proliferation of a population of progenitor or\nstem cells, epigenetic alterations (Forte et al., 2014), and the oldest\ntheory of retrograde menstruation (Sampson, 1927).\nHereditary studies in women with endometriosis showed increased incidence in\nrelatives of affected women compared to women without a familial history of\nendometriosis (Simpson et al., 1980). Consequently, the role of genetics in\nendometriosis has been long hypothesized (Moen et al., 1984; Simpson et al.,\n1984; Kennedy, 1999; Ishii et al., 2003; Bischoff and Simpson, 2004;\nZondervan et al., 2001, 2004), but, like many complex\ndiseases, specific causative genes or haplotypes have been elusive. In\naddition, while early studies implicated an immunologic basis for\nendometriosis (Steele et al., 1984), there was no identified association\nwith the human leukocyte antigen (HLA), which are the genes encoding the major\nhistocompatibility complex (MHC) in humans (Moen et al., 1984; Simpson et\nal., 1984). More recent studies have been increasingly suggestive of the\nrole of immune dysfunction and inflammation in endometriosis (Ahn et al.,\n2016; Yamada-Nomoto et al., 2016), but, while there have been more\nsuggestions of an association with HLA in endometriosis (Ishii et al., 2003;\nKitawaki et al., 2002), possibly in concert with specific killer\nimmunoglobulin-like receptor (KIR)\ngenotypes (Kitawaki et al., 2007; Nowak\net al., 2015), the role of the HLA/MHC remains an open question.\nControlled experiments in humans are difficult due to limitations on\nrepeated imaging and surgical biopsies for disease monitoring (Story and\nKennedy, 2004). Therefore, animal models provide an invaluable tool for\nstudying complex diseases like endometriosis. Although the use of rodent\nmodels of endometriosis has some advantages with respect to genetic\nmanipulation and affordability, these species differ greatly from humans,\nmaking comparisons difficult. Baboons and macaques have been the best\nnonhuman primate (NHP) models to study endometriosis' pathogenesis,\npathophysiology, spontaneous evolution, and new medical treatment options\n(D'Hooghe et al., 2009; Fazleabas et al., 2002; Yamanaka et al., 2012). In\nfact spontaneous endometriosis only occurs in humans and menstruating NHPs.\nRhesus macaques share many similarities with humans,\nsuch as their reproductive physiology, which is of particular relevance.\nMenarche in rhesus monkeys occurs at about 3 years of\nage, the length of the menstrual cycle is about 28 days, and\nmenstrual bleeding lasts for about 4 days (Catchpole and van Wagenen, 1975).\nAs in women, studies have implicated genetic predisposition to endometriosis\nin macaques (Zondervan et al., 2001, 2004). Humans and\nrhesus macaques have a comparable major histocompatibility complex\n(also known as the human leukocyte antigen in humans) with two main\nantigen-presenting classes of molecules. In rhesus macaques MHC  (MhcMamu)  class I\nconsists of  Mamu-A  and  Mamu-B  and class II of  Mamu-DR ,\n Mamu-DQ , and  Mamu-DP  molecules. In both\nspecies, the genes encoding both MHC I and II molecules are characterized by\nhigh allelic variation, but, while macaques show a high degree of copy number\nvariation of class I and II, specifically  Mamu-B  genes, the equivalent of the\nhuman  C  gene is absent. The aim of our study is to examine any genetic\nsusceptibility of MHC alleles to endometriosis in two colonies of rhesus macaques.\n\nThe Biomedical Primate Research Centre (BPRC) in Rijswijk, the Netherlands,\nis fully accredited by the Association for Assessment and Accreditation of\nLaboratory Animal Care (AAALAC) and maintains a breeding colony of\napproximately 1100 rhesus macaques ( Macaca mulatta ). Animals are\nconventionally housed in large social breeding groups (one alpha male with\nseveral adult females and their juvenile and adolescent offspring), mimicking\nthe natural ecology. The housing of these groups consists of interconnected\nindoor (72 m 2 )  and outdoor (208 m 2 )  enclosures with elevated\nsitting locations and enrichment devices (Vernes and Louwerse, 2010). Animals\nare fed on a diet of commercially available monkey chow, fruits, vegetables,\nand grains. Water is available ad libitum. Housing and care is in accordance\nwith the Dutch law on animal experimentation, which follows EU\nDirective 86/609/EEC.\nThe coefficient of inbreeding is calculated annually for all\nbreeding animals according to Wrigh (1922), and the parentage is defined for\nall newborns by means of STR typing with 24 microsatellites localized on 16\ndifferent chromosomes.\nUltrasound image of endometrial lesions of a 16-year-old rhesus macaque no. 9234 from the BPRC breeding colony. Image was taken with a\nflat probe, and it shows a cystic lesion with solid compartments.\nAt the time of data collection, the New England Primate Research Center\n(NEPRC) was a specific-pathogen-free colony of approximately 2000\nprimates maintained in accordance with federal and institutional guidelines\nmandated by the Institutional Animal Care and Use Committee\n(IACUC) of\nHarvard Medical School and accredited by the AAALAC. Rhesus macaques were\nhoused in harems including one male and several adult females with\npre-weaning offspring. Colony rooms were on a 12 h light–dark cycle, and\nthe animals received a diet of monkey chow (Harlan Teklad monkey diet)\nsupplemented with fresh fruit. A variety of enrichment objects were available\nat all times. All animal procedures including euthanasia were performed in\naccordance with guidelines and recommendations of the Committee on Animals of\nHarvard Medical School and the National Institutes of Health Guide for\nthe Care and Use of Laboratory Animals (publication no. 85-23, revised\n1996). Research protocols were approved by the Harvard Medical School Animal\nCare and Use Committee.\nBoth BPRC and NEPRC maintained complete medical records and familial\nrelationships on all colony animals. After death, all animals were\nnecropsied within several hours of death, often immediately following\neuthanasia, and representative sections of tissues were collected, flash\nfrozen, and stored at  - 80   ∘ C, as well as fixed in 10 % neutral buffered\nformalin (NBF) and embedded in paraffin. The records from gross and\nhistopathological examinations were held on the computerized database.\nGross pathology of uterus from rhesus macaque with endometriosis.\nThe ovaries and fallopian tubes are embedded and distorted by accumulation of\nsolid fibrous masses (scar tissue) and formation of endometrial cysts (arrow\nshows an open large cyst with yellow fibrous nodules in the center and dark\nred-brown fluid seen at the edge).\nInformation on the presence of endometriosis was obtained through necropsy\nreports and archived gross, histological, and diagnostic representative\nimages shown in Figs. 1, 2, and 3. The main criteria for the animals\nselected for the study were the clinically (bloating, pain, dysmenorrhea,\nultrasound-detected cystic lesions) and histologically proven endometriosis\n(ectopic proliferative endometrial glandular and stromal tissues,\nhemosiderin, and hemorrhage). Necropsy records from NEPRC were examined to\nidentify female rhesus macaques ( Macaca mulatta ) over 1 year of age for which\nrepresentative tissues from all organs had been collected and examined\nhistologically by routine hematoxylin and eosin staining. Cases with a\ndiagnosis of endometriosis were reviewed and selected if frozen endometrial\ntissues were archived. A similar selection of tissues was made from the tissue\nbank at BPRC (Table 1). Control or unaffected animals were defined as such\naccording their full necropsy report providing evidence of absence of\nendometriosis. All animals with endometriosis were of Indian origin, except\nanimals 8612 and BB93, which are Indian  ×  Burmese and Indian  ×  Chinese\nmixed-breed animals, respectively, and animal 4050, which is of Burmese\norigin. The control animals from the two colonies were of Indian origin.\nCohorts of rhesus macaques with endometriosis housed at NEPRC and\nBRPC shown with age, body weight, and surgical history of caesarian sections.\nParental relationships between animals were determined from veterinary\nrecords. For most matings only a single sire was present at the time of\nconception. MHC transmission between parent and offspring was used to\nconfirm relationships with further genetic tests when warranted. There were\nno ambiguous parentage calls among the animals involved in these studies.\nUrinary bladder of rhesus macaque with endometrial lesions. The\nserosa is infiltrated by endometrial glands, endometrial stroma, and\ninflammatory cells (hematoxylin and eosin staining).\n(a)   Mamu-A1  and  Mamu-DRB  genotypes of animals\ndiagnosed with endometriosis and their family relationship. A question mark\nfor  Mamu-A1  typing indicates that the animal is most probably\nhomozygous for  Mamu-A1 . A question mark in the column “remarks”\nindicates that sharing of a MHC haplotype is possible but cannot be\nconfirmed. Alleles in bold represent those which are present at a higher\nfrequency in rhesus macaques with endometriosis than in healthy animals.\n (b)  Allele frequencies for animals with endometriosis as well as\ncolony frequencies for NEPRC and BPRC are shown. Significant  p < 0.05  values\nare bold.\nP adj  (p-adjusted) is the significance value after\nBonferroni correction for multiple tests (see methods); “Endom.” represents\nanimals with endometriosis;  n  is the number of alleles.\nUterine tissue from NEPRC study animals was frozen in liquid nitrogen and\npulverized. The powdered tissue was resuspended in digestion buffer and\ndigested with proteinase K at 55  ∘ C overnight. DNA was isolated\nvia phenol/chloroform extraction followed by ethanol precipitation. DNA\npellets were resuspended in TE buffer, and sample concentration was measured\nvia UV spectrometry at 260 nm. DNA isolation of BPRC's animals was\nperformed on fresh EDTA blood or frozen peripheral blood mononuclear\ncells (PBMCs) by a standard salting-out\nmethod (Doxiadis et al., 2013) or by using the QIAamp DNA mini kit (QIAgen,\nGermantown, USA) according to the manufacturer's instructions.\nPedigree showing the familial relationships of 12 of the 17 animals\nfrom the NEPRC colony. Legend: females are represented by circles and males\nby squares. Animals diagnosed with endometriosis are shaded in gray.\nMHC typing of both class I and class II alleles was performed on DNA samples\nfrom these monkeys, namely for ( Macaca mulatta )  Mamu-A1 \n(MHC class I, locus A1) and  Mamu-DRB  (MHC class II DR, beta-chain) by\nmicrosatellite (STR) typing with STRs D6S2854 and D6S2859, being\n Mamu-A \nspecific, and D6S2878, being  Mamu-DRB  specific markers (Doxiadis et al., 2007,\n2013). For animals from the NEPRC colony, additional high-resolution\nsequencing was done using Roche 454 technologies on blood-derived lymphocyte\ncDNA (Karl et al., 2013; Wiseman et al., 2013). In the case of the animals of\nBPRC, additional high-resolution Sanger sequencing had been performed\nbeforehand and published previously (Otting et al., 2005; Doxiadis et al.,\n2013). Since the animals were members of breeding colonies, kinship\ncoefficients and/or pedigrees of the animals are known, and some MHC\nhaplotypes could be defined as well by segregation analysis. Significance was\ndetermined by comparing the number of carriers of the haplotype with\nendometriosis to the number of carriers in the colony (colony size: BPRC,  n = 1383 ; NEPRC,  n = 380 , colony frequencies shown in Table 2b) using a\nFisher's exact test with Bonferroni correction for multiple testing (each of\nnine independent MHC  A1  alleles).\n\nEight female rhesus macaques from BPRC (ranging from 12 to 21 years of age,\nmean 17.0 years) and seventeen female rhesus macaques from NEPRC (14 to 20 years of age,\nmean 15.9 years) were identified with endometriosis based on\nclinical and histologic diagnoses (Figs. 1, 2, and 3). The mean body\nweight of NEPRC macaques with endometriosis was 9.93 kg, and for BRPC\nmacaques it was 7.78 kg. Four of the 17 macaques with endometriosis from NEPRC\nhad caesarean sections, while none of the macaques at BPRC had undergone\nsurgery (Table 1).\nThe relationship status of the animals that had been identified with\nendometriosis was determined in an attempt to first identify obvious\nMendelian segregation and to identify confounds in association analysis that\nmay result from cryptic genetic substructure within endometriosis cases\ncompared to the colony as a whole. Among the eight monkeys identified at\nBPRC, there were two pairs of siblings. Among the seventeen rhesus macaques\nwith endometriosis from NEPRC, there was one mother–daughter pair, one pair\nof half-sib, and one trio of half-sibs. Additionally, there were four more\ndistantly related animals identified with endometriosis (Fig. 4, gray\nshading). Given the breeding patterns within the colony and the animals for\nwhich tissue was available and for which pathology could be ascertained, the\nrelationships among the affected females were not different from random\nsamples using bootstrapping.\nAssociation analysis for endometriosis was robust to allele sharing between\nthe animals. The rhesus macaques from BPRC ( n = 8 ) and NEPRC ( n = 17 )\nunderwent MHC typing for their  Mamu-A1  and  Mamu-DRB  alleles\n(Table 2a). The BRPC endometriosis cohort included the following MHC I ( Mamu-A1 )\nalleles:  A1*001  (33.3 %),  *002  (13.3 %),  *004  (6.7 %),\n *007 \n(6.7 %),  *008  (26.7 %), and  *011  (6.7 %) (Table 2b), while\nanimals with endometriosis from NEPRC had the following  Mamu-A1  alleles:\n A1*001 \n(9.4 %),  *002  (12.5 %),  *003  (3.1 %),  *004  (15.6 %),  *007  (21.9 %),\n *008 \n(21.9 %),  *012  (6.3 %), and  *026  (9.4 %) (Table 2b). The allele\nfrequencies in BPRC endometriosis samples compared to controls revealed\nsignificant enrichment of  Mamu-A1*001  (33.3 vs. 11.6 %\nin healthy animals,  p =  0.007) in monkeys with endometriosis (Table 2b).\nIn the NEPRC cohort, the MHC allele  Mamu-A1*007  was significantly\noverrepresented in diseased macaques compared to controls (21.9 vs.\n6.7 %,  p =  0.003). These associations are not shared between the\ncolonies. The  Mamu-A1*026  allele is marginally overrepresented in the NEPRC\ncolony (9.4 vs 1.5 %), although this does not pass the multiple testing\ncorrection. This allele is uncommon in both colonies and is only seen in the\naffected mother–daughter pair at NEPRC. Additionally, the  Mamu-DRB \nhaplotype, which is characterized by the  DRB*W3:03  allele, may be\noverrepresented in diseased animals of the NEPRC colony (Table 2a)\n(17.64 vs. 3.74 % in healthy animals of BPRC). Although\n Mamu-DRB  typing is not routinely performed at NEPRC, the comparison to\nthe colony frequencies at BPRC may be relevant, since allele frequencies\nof  Mamu-A1  in the two colonies are comparable (Table 2b).\nNevertheless, while this is putatively suggestive and warrants further study,\nit cannot be interpreted with certainty.\n\nIn this paper, we report significant higher frequency of two\n Mamu-A1  MHC class I alleles in rhesus macaques with endometriosis\nfrom two different primate centers,  Mamu-A1*001  in BPRC macaques\nand  Mamu-A1*007  in NEPRC macaques. The familial relatedness of\nseveral macaques with endometriosis from the two colonies supports a\nhereditary risk for this disease in rhesus macaques which is similar to that seen in\nwomen (Bischoff and Simpson, 2004; Ishii et al., 2003; Kennedy, 1999). The\ndifferent  Mamu-A1  alleles may reflect the different origins of the\ntwo colonies. Since diseased and control animals of both colonies are part of\nbreeding groups, the  Mamu-A1  and  Mamu-DRB  alleles can be inferred from pedigree\nanalysis to be identical by state but not by descent, and the higher\nfrequencies observed in affected individuals are not attributable simply to\nkinship. Although NEPRC and most of the BPRC animals are of Indian origin,\nthe founder animals of both colonies may be from different parts of India.\nAdditionally, two of the macaques of BPRC are a mixed breed, Indian–Chinese or\nIndian–Burmese, and one animal is from Burmese origin. Although two different\n Mamu-A1  alleles were overrepresented in macaques with endometriosis from the\ntwo facilities, the arguably more important interpretation may be that both\ncolonies share a significant disease association with class I alleles. These\nresults are comparable to humans, where higher frequencies of different\nMHC class I  B  alleles are described in endometriosis patients; a\nsignificantly higher frequency of HLA-B 54 and CW7 is observed in Japanese\npatients (Ishii et al., 2002), whereas a significantly positive association\nwith endometriosis of HLA-B7 is defined by Kitawaki and colleagues (Kitawaki\net al., 2002). In addition, some MHC II  Mamu-DRB  alleles were\noverrepresented in animals with endometriosis at BPRC; however, conclusions\nwere limited by the low number of animals analyzed. These findings are\nconsistent with the reported higher frequency of  HLA-DRB1*1403  (Ishii et\nal., 2002) and  HLA-DQB1*0301  in women with endometriosis (Ishii et\nal., 2003).\nIn our macaque study,  Mamu-B  alleles have not been analyzed. Since\nMHC alleles, in humans as in macaques (de Groot et al., 2014), are well known\nto be subjected to linkage disequilibrium, it is plausible that the observed\ndisease associations are not caused by a specific  Mamu-A1  allele\nitself but may be due to certain alleles of adjacent loci such as  Mamu-B .\nLikewise, the disease association with certain  Mamu-DRB  alleles may\nalso be caused by linkage disequilibrium. Linkage disequilibrium with\n Mamu-DRB  alleles would also explain why no disease association with\n Mamu-DRB  alleles and endometriosis has been observed in other human\npopulations (Roszkowski et al., 2005). Accordingly, Kitawaki and coworkers\nconclude that there is a certain HLA haplotype, namely\n HLA-A24-B*0702-Cw*0702-DRB*0101 , which is linked to endometriosis\nsusceptibility (Kitawaki et al., 2002). Further analysis of extended\nhaplotypes in rhesus macaques will help to clarify these findings. It is\nimportant to interpret the present findings in rhesus macaques with caution,\nas the associated alleles may simply represent markers of associated\nhaplotypes rather than causative variants themselves.\nThere may be several ways in which immune system surveillance, function, or\ndysfunction may contribute to or promote endometriosis (Ishii et al., 2002;\nForte et al., 2014). As demonstrated in previous work, women with endometriosis\nexhibit altered or reduced innate and even adaptive immunity (Dmowski et al.,\n1981; Ota and Igarashi, 1993; Chiang and Hill, 1997; Khan et al., 2009). Additional studies\nsuggest an autoimmune component to endometriosis (Eisenberg et al., 2012).\nSpecific MHC I alleles may result in altered immune\nresponses, leading to uncontrolled growth of stem cells, progenitor cells,\nand/or ectopic glandular tissue (Forte et al., 2014). Further investigation\nof spontaneous endometriosis in primates is warranted. The MHC typing results\nsuggest the likelihood of a comparable genetic predisposition to\nendometriosis in women.\n\nThe MHC I allele overexpression in our macaque cohorts suggests a role for\nimmune system on endometriosis pathogenesis. Further research is required to\nfully understand how these MHC I ( Mamu-A1 ) alleles contribute to\ndisease.","source_license":"CC0","license_restricted":false}