{"paper_id":"05193684-2cc6-44ca-97c5-b62b31dc4d79","body_text":"Primate Biol., 4, 117–125, 2017\nhttps://doi.org/10.5194/pb-4-117-2017\n© Author(s) 2017. This work is distributed under\nthe Creative Commons Attribution 3.0 License.\nSpontaneous endometriosis in rhesus macaques:\nevidence for a genetic association with specific\nMamu-A1 alleles\nIvanela Kondova1, Gerco Braskamp1,†, Peter J. Heidt 1, Wim Collignon1, Tom Haaksma1,\nNanine de Groot2, Nel Otting 2, Gaby Doxiadis 2, Susan V . Westmoreland3, Eric J. Vallender4,5, and\nRonald E. Bontrop2\n1Animal Science Department, Division of Pathology and Microbiology, Division of Veterinary care,\nBiomedical Primate Research Centre, 2288 GJ Rijswijk, the Netherlands\n2Department of Comparative Genetics, Biomedical Primate Research Centre,\n2288 GJ Rijswijk, the Netherlands\n3AbbVie Bioresearch Center, Immunology, Pharmacology, Pathology and Exploratory Toxicology,\nWorcester, MA 01605, USA\n4Department of Psychiatry and Human Behavior, University of Mississippi Medical Center,\nJackson, MS 39216, USA\n5Division of Veterinary Medicine, Tulane National Primate Research Center,\nCovington, LA 70433, USA\n†deceased\nCorrespondence to: Ivanela Kondova (kondova@bprc.nl)\nReceived: 20 December 2016 – Revised: 19 April 2017 – Accepted: 28 April 2017 – Published: 22 June 2017\nAbstract. Endometriosis is a poorly understood common debilitating women’s reproductive disorder resulting\nfrom proliferative and ectopic endometrial tissue associated with variable clinical symptoms including dysmen-\norrhea (painful menstrual periods), dyspareunia (pain on intercourse), female infertility, and an increased risk\nof malignant transformation. The rhesus macaque (Macaca mulatta) develops a spontaneous endometriosis that\nis very similar to that seen in women. We hypothesized that specific major histocompatibility complex (MHC)\nalleles may contribute to the pathogenesis of endometriosis. As part of a collaboration between the Biomedical\nPrimate Research Centre (BPRC) in the Netherlands and the New England Primate Research Center (NEPRC)\nin the United States, we analyzed DNA sequences of MHC class I ( Macaca mulatta, Mamu-A1 ) and class II\n(Mamu-DRB) alleles from rhesus macaques with endometriosis and compared the allele frequencies with those\nof age-matched healthy macaques. We demonstrate that two MHC class I alleles are overrepresented in dis-\neased macaques compared to controls: Mamu-A1*001, 33.3 % in BPRC animals with endometriosis vs. 11.6 %\nin healthy macaques (p= 0.007), and Mamu-A1*007, 21.9 % NEPRC rhesus macaques vs. 6.7 %, (p= 0.003).\nWe provide evidence that select MHC class I alleles are associated with endometriosis in rhesus macaques and\nsuggest that the disease pathogenesis contribution of MHC class I warrants further research.\nPublished by Copernicus Publications on behalf of the Deutsches Primatenzentrum GmbH (DPZ).\n\n118 I. Kondova et al.: Spontaneous endometriosis in rhesus macaques\n1 Introduction\n1.1 Endometriosis in humans\nEndometriosis is a chronic debilitating inflammatory disease\nthat affects approximately 10 to 20 % of women of reproduc-\ntive age and roughly 50 % of women with infertility (Giu-\ndice, 2010). The associated clinical symptoms like dysmen-\norrhoea, dyspareunia, and chronic pelvic pain have a nega-\ntive impact on the quality of life of women affected with the\ndisorder (Gupta et al., 2008). Histologically, the disease is\ndefined by the presence of endometrial glandular and stro-\nmal tissue in organs and tissues outside of the uterine en-\ndometrium.\nExperimental studies in women are hindered by the risks\nand complications associated with repetitive biopsy or sur-\ngical procedures. As a consequence, the pathogenesis of en-\ndometriosis remains incompletely understood. It is likely that\nendometriosis is a complex and multifactorial disorder trig-\ngered by hormonal, immunologic, genetic, and environmen-\ntal factors. One hypothesized process in the pathogenesis of\nendometriosis is metaplasia, involving the transformation of\ntissues in the peritoneal cavity into endometrial tissue driven\nby hormonal or immunological factors (Sourial et al., 2014).\nHormones play multiple roles with estrogen promoting pro-\nliferation of endometrial lesions and progesterone limiting\nendometrial proliferation. Inflammation, immune dysregula-\ntion, and oxidative stress have also been associated with en-\ndometriosis, contributing to cytokine-mediated endometrial\ngrowth (Forte et al., 2014). Other hypothesized processes\ninclude the suppression of normal apoptosis of endometrial\nglandular cells, proliferation of a population of progenitor or\nstem cells, epigenetic alterations (Forte et al., 2014), and the\noldest theory of retrograde menstruation (Sampson, 1927).\nHereditary studies in women with endometriosis showed\nincreased incidence in relatives of affected women com-\npared to women without a familial history of endometrio-\nsis (Simpson et al., 1980). Consequently, the role of genetics\nin endometriosis has been long hypothesized (Moen et al.,\n1984; Simpson et al., 1984; Kennedy, 1999; Ishii et al., 2003;\nBischoff and Simpson, 2004; Zondervan et al., 2001, 2004),\nbut, like many complex diseases, specific causative genes or\nhaplotypes have been elusive. In addition, while early studies\nimplicated an immunologic basis for endometriosis (Steele et\nal., 1984), there was no identified association with the human\nleukocyte antigen (HLA), which are the genes encoding the\nmajor histocompatibility complex (MHC) in humans (Moen\net al., 1984; Simpson et al., 1984). More recent studies have\nbeen increasingly suggestive of the role of immune dysfunc-\ntion and inflammation in endometriosis (Ahn et al., 2016;\nYamada-Nomoto et al., 2016), but, while there have been\nmore suggestions of an association with HLA in endometrio-\nsis (Ishii et al., 2003; Kitawaki et al., 2002), possibly in con-\ncert with specific killer immunoglobulin-like receptor (KIR)\ngenotypes (Kitawaki et al., 2007; Nowak et al., 2015), the\nrole of the HLA/MHC remains an open question.\n1.2 Rhesus macaque as a model for endometriosis\nControlled experiments in humans are difficult due to limita-\ntions on repeated imaging and surgical biopsies for disease\nmonitoring (Story and Kennedy, 2004). Therefore, animal\nmodels provide an invaluable tool for studying complex dis-\neases like endometriosis. Although the use of rodent models\nof endometriosis has some advantages with respect to genetic\nmanipulation and affordability, these species differ greatly\nfrom humans, making comparisons difficult. Baboons and\nmacaques have been the best nonhuman primate (NHP) mod-\nels to study endometriosis’ pathogenesis, pathophysiology,\nspontaneous evolution, and new medical treatment options\n(D’Hooghe et al., 2009; Fazleabas et al., 2002; Yamanaka\net al., 2012). In fact spontaneous endometriosis only occurs\nin humans and menstruating NHPs. Rhesus macaques share\nmany similarities with humans, such as their reproductive\nphysiology, which is of particular relevance. Menarche in\nrhesus monkeys occurs at about 3 years of age, the length of\nthe menstrual cycle is about 28 days, and menstrual bleeding\nlasts for about 4 days (Catchpole and van Wagenen, 1975).\nAs in women, studies have implicated genetic predisposition\nto endometriosis in macaques (Zondervan et al., 2001, 2004).\nHumans and rhesus macaques have a comparable major his-\ntocompatibility complex (also known as the human leukocyte\nantigen in humans) with two main antigen-presenting classes\nof molecules. In rhesus macaques MHC (MhcMamu) class I\nconsists of Mamu-A and Mamu-B and class II of Mamu-DR,\nMamu-DQ, and Mamu-DP molecules. In both species, the\ngenes encoding both MHC I and II molecules are charac-\nterized by high allelic variation, but, while macaques show a\nhigh degree of copy number variation of class I and II, specif-\nically Mamu-B genes, the equivalent of the human C gene is\nabsent. The aim of our study is to examine any genetic sus-\nceptibility of MHC alleles to endometriosis in two colonies\nof rhesus macaques.\n2 Materials and methods\n2.1 Description of the colonies\nThe Biomedical Primate Research Centre (BPRC) in Ri-\njswijk, the Netherlands, is fully accredited by the Associa-\ntion for Assessment and Accreditation of Laboratory Animal\nCare (AAALAC) and maintains a breeding colony of approx-\nimately 1100 rhesus macaques ( Macaca mulatta ). Animals\nare conventionally housed in large social breeding groups\n(one alpha male with several adult females and their juve-\nnile and adolescent offspring), mimicking the natural ecol-\nogy. The housing of these groups consists of interconnected\nindoor (72 m2) and outdoor (208 m 2) enclosures with ele-\nvated sitting locations and enrichment devices (Vernes and\nPrimate Biol., 4, 117–125, 2017 www.primate-biol.net/4/117/2017/\n\nI. Kondova et al.: Spontaneous endometriosis in rhesus macaques 119\nFigure 1. Ultrasound image of endometrial lesions of a 16-year-old\nrhesus macaque no. 9234 from the BPRC breeding colony. Image\nwas taken with a flat probe, and it shows a cystic lesion with solid\ncompartments.\nLouwerse, 2010). Animals are fed on a diet of commercially\navailable monkey chow, fruits, vegetables, and grains. Wa-\nter is available ad libitum. Housing and care is in accordance\nwith the Dutch law on animal experimentation, which fol-\nlows EU Directive 86/609/EEC. The coefficient of inbreed-\ning is calculated annually for all breeding animals according\nto Wrigh (1922), and the parentage is defined for all new-\nborns by means of STR typing with 24 microsatellites local-\nized on 16 different chromosomes.\nAt the time of data collection, the New England Pri-\nmate Research Center (NEPRC) was a specific-pathogen-\nfree colony of approximately 2000 primates maintained in\naccordance with federal and institutional guidelines man-\ndated by the Institutional Animal Care and Use Committee\n(IACUC) of Harvard Medical School and accredited by the\nAAALAC. Rhesus macaques were housed in harems includ-\ning one male and several adult females with pre-weaning off-\nspring. Colony rooms were on a 12 h light–dark cycle, and\nthe animals received a diet of monkey chow (Harlan Teklad\nmonkey diet) supplemented with fresh fruit. A variety of en-\nrichment objects were available at all times. All animal pro-\ncedures including euthanasia were performed in accordance\nwith guidelines and recommendations of the Committee on\nAnimals of Harvard Medical School and the National Insti-\ntutes of Health Guide for the Care and Use of Laboratory An-\nimals (publication no. 85-23, revised 1996). Research proto-\ncols were approved by the Harvard Medical School Animal\nCare and Use Committee.\nBoth BPRC and NEPRC maintained complete medical\nrecords and familial relationships on all colony animals. Af-\nter death, all animals were necropsied within several hours\nof death, often immediately following euthanasia, and repre-\nsentative sections of tissues were collected, flash frozen, and\nstored at−80◦C, as well as fixed in 10 % neutral buffered\nFigure 2. Gross pathology of uterus from rhesus macaque with en-\ndometriosis. The ovaries and fallopian tubes are embedded and dis-\ntorted by accumulation of solid fibrous masses (scar tissue) and for-\nmation of endometrial cysts (arrow shows an open large cyst with\nyellow fibrous nodules in the center and dark red-brown fluid seen\nat the edge).\nformalin (NBF) and embedded in paraffin. The records from\ngross and histopathological examinations were held on the\ncomputerized database.\n2.2 Identification of animals with endometriosis and\ncase selection for the study\nInformation on the presence of endometriosis was ob-\ntained through necropsy reports and archived gross, his-\ntological, and diagnostic representative images shown in\nFigs. 1, 2, and 3. The main criteria for the animals se-\nlected for the study were the clinically (bloating, pain, dys-\nmenorrhea, ultrasound-detected cystic lesions) and histolog-\nically proven endometriosis (ectopic proliferative endome-\ntrial glandular and stromal tissues, hemosiderin, and hem-\norrhage). Necropsy records from NEPRC were examined\nto identify female rhesus macaques ( Macaca mulatta ) over\n1 year of age for which representative tissues from all or-\ngans had been collected and examined histologically by rou-\ntine hematoxylin and eosin staining. Cases with a diagnosis\nof endometriosis were reviewed and selected if frozen en-\ndometrial tissues were archived. A similar selection of tis-\nsues was made from the tissue bank at BPRC (Table 1). Con-\ntrol or unaffected animals were defined as such according\ntheir full necropsy report providing evidence of absence of\nendometriosis. All animals with endometriosis were of In-\ndian origin, except animals 8612 and BB93, which are In-\ndian× Burmese and Indian× Chinese mixed-breed animals,\nrespectively, and animal 4050, which is of Burmese origin.\nwww.primate-biol.net/4/117/2017/ Primate Biol., 4, 117–125, 2017\n\n120 I. Kondova et al.: Spontaneous endometriosis in rhesus macaques\nFigure 3. Urinary bladder of rhesus macaque with endometrial le-\nsions. The serosa is infiltrated by endometrial glands, endometrial\nstroma, and inflammatory cells (hematoxylin and eosin staining).\nThe control animals from the two colonies were of Indian\norigin.\n2.3 Pedigree analysis of macaques from BRPC and\nNEPRC\nParental relationships between animals were determined\nfrom veterinary records. For most matings only a single sire\nwas present at the time of conception. MHC transmission be-\ntween parent and offspring was used to confirm relationships\nwith further genetic tests when warranted. There were no am-\nbiguous parentage calls among the animals involved in these\nstudies.\n2.4 DNA extraction\nUterine tissue from NEPRC study animals was frozen in liq-\nuid nitrogen and pulverized. The powdered tissue was resus-\npended in digestion buffer and digested with proteinase K at\n55◦C overnight. DNA was isolated via phenol/chloroform\nextraction followed by ethanol precipitation. DNA pellets\nwere resuspended in TE buffer, and sample concentration\nwas measured via UV spectrometry at 260 nm. DNA isola-\ntion of BPRC’s animals was performed on fresh EDTA blood\nor frozen peripheral blood mononuclear cells (PBMCs) by a\nstandard salting-out method (Doxiadis et al., 2013) or by us-\ning the QIAamp DNA mini kit (QIAgen, Germantown, USA)\naccording to the manufacturer’s instructions.\n2.5 MHC typing\nMHC typing of both class I and class II alleles was per-\nformed on DNA samples from these monkeys, namely for\n(Macaca mulatta ) Mamu-A1 (MHC class I, locus A1) and\nMamu-DRB (MHC class II DR, beta-chain) by microsatel-\nlite (STR) typing with STRs D6S2854 and D6S2859, being\nMamu-A specific, and D6S2878, being Mamu-DRB specific\nmarkers (Doxiadis et al., 2007, 2013). For animals from the\nNEPRC colony, additional high-resolution sequencing was\ndone using Roche 454 technologies on blood-derived lym-\nphocyte cDNA (Karl et al., 2013; Wiseman et al., 2013). In\nthe case of the animals of BPRC, additional high-resolution\nSanger sequencing had been performed beforehand and pub-\nlished previously (Otting et al., 2005; Doxiadis et al., 2013).\nSince the animals were members of breeding colonies, kin-\nship coefficients and/or pedigrees of the animals are known,\nand some MHC haplotypes could be defined as well by seg-\nregation analysis. Significance was determined by comparing\nthe number of carriers of the haplotype with endometriosis\nto the number of carriers in the colony (colony size: BPRC,\nn= 1383; NEPRC, n= 380, colony frequencies shown in\nTable 2b) using a Fisher’s exact test with Bonferroni correc-\ntion for multiple testing (each of nine independent MHC A1\nalleles).\n3 Results\n3.1 Demographics of endometriosis cases\nEight female rhesus macaques from BPRC (ranging from 12\nto 21 years of age, mean 17.0 years) and seventeen female\nrhesus macaques from NEPRC (14 to 20 years of age, mean\n15.9 years) were identified with endometriosis based on clin-\nical and histologic diagnoses (Figs. 1, 2, and 3). The mean\nbody weight of NEPRC macaques with endometriosis was\n9.93 kg, and for BRPC macaques it was 7.78 kg. Four of the\n17 macaques with endometriosis from NEPRC had caesarean\nsections, while none of the macaques at BPRC had under-\ngone surgery (Table 1).\n3.2 Pedigree analysis\nThe relationship status of the animals that had been identi-\nfied with endometriosis was determined in an attempt to first\nidentify obvious Mendelian segregation and to identify con-\nfounds in association analysis that may result from cryptic\ngenetic substructure within endometriosis cases compared to\nthe colony as a whole. Among the eight monkeys identified\nat BPRC, there were two pairs of siblings. Among the sev-\nenteen rhesus macaques with endometriosis from NEPRC,\nthere was one mother–daughter pair, one pair of half-sib, and\none trio of half-sibs. Additionally, there were four more dis-\ntantly related animals identified with endometriosis (Fig. 4,\ngray shading). Given the breeding patterns within the colony\nand the animals for which tissue was available and for which\npathology could be ascertained, the relationships among the\naffected females were not different from random samples us-\ning bootstrapping.\nPrimate Biol., 4, 117–125, 2017 www.primate-biol.net/4/117/2017/\n\nI. Kondova et al.: Spontaneous endometriosis in rhesus macaques 121\nTable 1. Cohorts of rhesus macaques with endometriosis housed at NEPRC and BRPC shown with age, body weight, and surgical history\nof caesarian sections.\nAnimal Necrop. no. Species Sex Source Age (years) Weight (kg) Sample C-section\n1 148-90 A06-313 M. mulatta F NEPRC 16 11.1 DNA no C-section\n2 183-90 A06-314 M. mulatta F NEPRC 16 10.7 DNA no C-section\n3 265-87 A06-316 M. mulatta F NEPRC 19 11.6 DNA no C-section\n4 196-89 A06-317 M. mulatta F NEPRC 17 10.2 DNA no C-section\n5 256-89 A06-318 M. mulatta F NEPRC 17 9.4 DNA no C-section\n6 236-88 A06-323 M. mulatta F NEPRC 18 8.3 DNA no C-section\n7 536-91 A06-324 M. mulatta F NEPRC 15 10.2 DNA no C-section\n8 169-92 A06-325 M. mulatta F NEPRC 14 11 DNA no C-section\n9 369-92 A06-326 M. mulatta F NEPRC 14 6.2 DNA no C-section\n10 103-87 A06-327 M. mulatta F NEPRC 19 7.9 DNA C-section\n11 259-87 A06-342 M. mulatta F NEPRC 19 8.3 DNA no C-section\n12 419-91 A06-352 M. mulatta F NEPRC 15 9.4 DNA C-section\n13 170-87 A07-2 M. mulatta F NEPRC 19 9 DNA no C-section\n14 142-92 A07-9 M. mulatta F NEPRC 14 10.8 DNA C-section\n15 229-87 A07-10 M. mulatta F NEPRC 19 8.6 DNA no C-section\n16 127-86 A07-36 M. mulatta F NEPRC 20 10.8 DNA no C-section\n17 468-87 A07-37 M. mulatta F NEPRC 19 15.3 DNA 2 C-sections\n1 8803 06-1120 M. mulatta F BPRC 18 6 DNA no C-section\n2 1WQ 06-1129 M. mulatta F BPRC 21 7.8 DNA no C-section\n3 8851 06-1183 M. mulatta F BPRC 17 7.3 DNA no C-section\n4 8612 05-1047 M. mulatta F BPRC 18 8.41 DNA no C-section\n5 9250 05-1070 M. mulatta F BPRC 12 7.66 DNA no C-section\n6 8930 05-1107 M. mulatta F BPRC 16 6.97 DNA no C-section\n7 BB93 07-1386 M. mulatta F BPRC 16 10.35 DNA no C-section\n8 4050 07-1354 M. mulatta F BPRC 18 5.6 DNA no C-section\nFigure 4. Pedigree showing the familial relationships of 12 of the 17 animals from the NEPRC colony. Legend: females are represented by\ncircles and males by squares. Animals diagnosed with endometriosis are shaded in gray.\n3.3 MHC typing\nAssociation analysis for endometriosis was robust to allele\nsharing between the animals. The rhesus macaques from\nBPRC (n= 8) and NEPRC (n= 17) underwent MHC typing\nfor their Mamu-A1 and Mamu-DRB alleles (Table 2a). The\nBRPC endometriosis cohort included the following MHC I\n(Mamu-A1) alleles: A1*001 (33.3 %), *002 (13.3 %), *004\n(6.7 %), *007 (6.7 %), *008 (26.7 %), and *011 (6.7 %) (Ta-\nble 2b), while animals with endometriosis from NEPRC\nhad the following Mamu-A1 alleles: A1*001 (9.4 %), *002\n(12.5 %), *003 (3.1 %), *004 (15.6 %), *007 (21.9 %), *008\n(21.9 %), *012 (6.3 %), and *026 (9.4 %) (Table 2b). The al-\nlele frequencies in BPRC endometriosis samples compared\nto controls revealed significant enrichment ofMamu-A1*001\n(33.3 vs. 11.6 % in healthy animals, p= 0.007) in mon-\nkeys with endometriosis (Table 2b). In the NEPRC cohort,\nthe MHC allele Mamu-A1*007 was significantly overrepre-\nsented in diseased macaques compared to controls (21.9 vs.\n6.7 %, p= 0.003). These associations are not shared between\nthe colonies. The Mamu-A1*026 allele is marginally over-\nrepresented in the NEPRC colony (9.4 vs 1.5 %), although\nthis does not pass the multiple testing correction. This allele\nis uncommon in both colonies and is only seen in the affected\nmother–daughter pair at NEPRC. Additionally, the Mamu-\nDRB haplotype, which is characterized by the DRB*W3:03\nwww.primate-biol.net/4/117/2017/ Primate Biol., 4, 117–125, 2017\n\n122 I. Kondova et al.: Spontaneous endometriosis in rhesus macaques\nTable 2. (a) Mamu-A1 and Mamu-DRB genotypes of animals diagnosed with endometriosis and their family relationship. A question mark\nfor Mamu-A1 typing indicates that the animal is most probably homozygous for Mamu-A1. A question mark in the column “remarks”\nindicates that sharing of a MHC haplotype is possible but cannot be confirmed. Alleles in bold represent those which are present at a higher\nfrequency in rhesus macaques with endometriosis than in healthy animals. (b) Allele frequencies for animals with endometriosis as well as\ncolony frequencies for NEPRC and BPRC are shown. Significant p < 0.05 values are bold.\n(a) Animal Mamu-A1* Mamu-DRB Source Remarks\n1 148-90 008/ 007 *W3:03/*W3:03 NEPRC\n2 183-90 012/002 1*03:03/3*04:10 NEPRC\n3 265-87 004/002 1*03:10/1*03:09 NEPRC same father as 229 and 536; shared MHC?\n4 196-89 007/001 1*04:06/*W6:06 NEPRC same father as 256; no MHC sharing\n5 256-89 008/002 1*03:03/1*03:09 NEPRC same father as 196; no MHC sharing\n6 236-88 008 /007 1*03:03/1*03:18 NEPRC shared 1st MHC hapl. with 142; 2nd with 259\n7 536-91 012/008 *W3:03 /1*03:09 NEPRC same father as 229 and 265; shared MHC?\n8 169-92 026/026 1*04:06/1*03:09 NEPRC shared MHC with 127\n9 369-92 007 /? 1*04:06/1*07:01 NEPRC\n10 103-87 008/ 007 1*03:03/1*03:09 NEPRC\n11 259-87 007/007 *W6:06/1*03:18 NEPRC shared 2nd MHC haplotype with 236\n12 419-91 004/008 1*03:10/1*03:06 NEPRC\n13 170-87 004/002 1*03:09 /*W3:03 NEPRC\n14 142-92 008 /001 1*03:03 /*W3:03 NEPRC shared 1st MHC hapl. with 236\n15 229-87 004/ 001 1*04:06 /*W3:03 NEPRC same father as 265 and 536; shared MHC?\n16 127-88 004/026 1*03:09/1*03:17 NEPRC shared MHC with offspring 169\n17 468-87 003/? 1*04:04/1*03:06 NEPRC\n1 8803 011/008 3*04:10/1*04:06 BPRC\n2 1WQ 001 /008 1*04:06/1*03:03 BPRC\n3 8851 001 /002 *W6:06/1*04:06 BPRC sibling of 8612; 1 shared MHC haplotype\n4 8612 001/007 *W6:06/4*01:02 BPRC sibling of 8651; 1 shared MHC haplotype\n5 9250 001 /004 1*03:09/1*03:09 BPRC sibling of 8930; 1 shared MHC haplotype\n6 8930 001/ 002 1*03:09/1*04:03 BPRC sibling of 9250; 1 shared MHC haplotype\n7 BB93 008/? 1*03:03/1*04:06 BPRC\n8 4050 008/050 1*03:21/*W26:04 BPRC\n(b) NEPRC BPRC\nMamu-A1 Endom. (n) Colony ( n) p val Padj Endom. (n) Colony ( n) p val Padj\nA1*001 9.4 % (3) 11.9 % (90) 1 1 33.3 % (5) 11.6 % (321) 0.007 0.04\nA1*002 12.5 % (4) 12.0 % (91) 1 1 13.3 %(2) 15.8 % (437) 1 1\nA1*003 3.1 % (1) 0.3 % (2) 0.117 0.93 0 %(0) 4 % (111) 1 1\nA1*004 15.6 % (5) 17.6 % (134) 1 1 6.7 %(1) 23.2 % (642) 0.251 1\nA1*007 21.9 % (7) 6.7 %(51) 0.003 0.03 6.7 % (1) 4.7 % (130) 0.492 1\nA1*008 21.9 % (7) 23.1 % (176) 1 1 26.7 %(4) 20.4 % (564) 0.475 1\nA1*011 0 % (0) 0.2 % (1) 1 1 6.7 % (1) 2.7 % (75) 0.322 1\nA1*012 6.3 % (2) 6.8 % (52) 1 1 0 % (0) 5.6 % (155) 1 1\nA1*026 9.4 % (3) 1.5 % (11) 0.013 0.14 0 %(0) 1.1 % (30) 1 1\nTotal 100.0 % (32) 100.0 % (760) 100.0 % (15) 100.0 % (2766)\nPadj (p-adjusted) is the significance value after Bonferroni correction for multiple tests (see methods); “Endom.” represents animals with\nendometriosis; n is the number of alleles.\nallele, may be overrepresented in diseased animals of the\nNEPRC colony (Table 2a) (17.64 vs. 3.74 % in healthy an-\nimals of BPRC). Although Mamu-DRB typing is not rou-\ntinely performed at NEPRC, the comparison to the colony\nfrequencies at BPRC may be relevant, since allele frequen-\ncies of Mamu-A1 in the two colonies are comparable (Ta-\nble 2b). Nevertheless, while this is putatively suggestive and\nwarrants further study, it cannot be interpreted with certainty.\n4 Discussion\nIn this paper, we report significant higher frequency of two\nMamu-A1 MHC class I alleles in rhesus macaques with\nPrimate Biol., 4, 117–125, 2017 www.primate-biol.net/4/117/2017/\n\nI. Kondova et al.: Spontaneous endometriosis in rhesus macaques 123\nendometriosis from two different primate centers, Mamu-\nA1*001 in BPRC macaques and Mamu-A1*007 in NEPRC\nmacaques. The familial relatedness of several macaques with\nendometriosis from the two colonies supports a hereditary\nrisk for this disease in rhesus macaques which is similar\nto that seen in women (Bischoff and Simpson, 2004; Ishii\net al., 2003; Kennedy, 1999). The different Mamu-A1 al-\nleles may reflect the different origins of the two colonies.\nSince diseased and control animals of both colonies are part\nof breeding groups, the Mamu-A1 and Mamu-DRB alleles\ncan be inferred from pedigree analysis to be identical by\nstate but not by descent, and the higher frequencies ob-\nserved in affected individuals are not attributable simply to\nkinship. Although NEPRC and most of the BPRC animals\nare of Indian origin, the founder animals of both colonies\nmay be from different parts of India. Additionally, two of\nthe macaques of BPRC are a mixed breed, Indian–Chinese\nor Indian–Burmese, and one animal is from Burmese ori-\ngin. Although two different Mamu-A1 alleles were over-\nrepresented in macaques with endometriosis from the two\nfacilities, the arguably more important interpretation may\nbe that both colonies share a significant disease association\nwith class I alleles. These results are comparable to humans,\nwhere higher frequencies of different MHC class I B alleles\nare described in endometriosis patients; a significantly higher\nfrequency of HLA-B 54 and CW7 is observed in Japanese\npatients (Ishii et al., 2002), whereas a significantly posi-\ntive association with endometriosis of HLA-B7 is defined by\nKitawaki and colleagues (Kitawaki et al., 2002). In addition,\nsome MHC II Mamu-DRB alleles were overrepresented in\nanimals with endometriosis at BPRC; however, conclusions\nwere limited by the low number of animals analyzed. These\nfindings are consistent with the reported higher frequency of\nHLA-DRB1*1403 (Ishii et al., 2002) and HLA-DQB1*0301\nin women with endometriosis (Ishii et al., 2003).\nIn our macaque study, Mamu-B alleles have not been an-\nalyzed. Since MHC alleles, in humans as in macaques (de\nGroot et al., 2014), are well known to be subjected to link-\nage disequilibrium, it is plausible that the observed disease\nassociations are not caused by a specific Mamu-A1 allele it-\nself but may be due to certain alleles of adjacent loci such\nas Mamu-B. Likewise, the disease association with certain\nMamu-DRB alleles may also be caused by linkage dise-\nquilibrium. Linkage disequilibrium with Mamu-DRB alleles\nwould also explain why no disease association with Mamu-\nDRB alleles and endometriosis has been observed in other\nhuman populations (Roszkowski et al., 2005). Accordingly,\nKitawaki and coworkers conclude that there is a certain HLA\nhaplotype, namely HLA-A24-B*0702-Cw*0702-DRB*0101,\nwhich is linked to endometriosis susceptibility (Kitawaki et\nal., 2002). Further analysis of extended haplotypes in rhesus\nmacaques will help to clarify these findings. It is important\nto interpret the present findings in rhesus macaques with cau-\ntion, as the associated alleles may simply represent markers\nof associated haplotypes rather than causative variants them-\nselves.\nThere may be several ways in which immune system\nsurveillance, function, or dysfunction may contribute to or\npromote endometriosis (Ishii et al., 2002; Forte et al., 2014).\nAs demonstrated in previous work, women with endometrio-\nsis exhibit altered or reduced innate and even adaptive im-\nmunity (Dmowski et al., 1981; Ota and Igarashi, 1993; Chi-\nang and Hill, 1997; Khan et al., 2009). Additional studies\nsuggest an autoimmune component to endometriosis (Eisen-\nberg et al., 2012). Specific MHC I alleles may result in al-\ntered immune responses, leading to uncontrolled growth of\nstem cells, progenitor cells, and/or ectopic glandular tissue\n(Forte et al., 2014). Further investigation of spontaneous en-\ndometriosis in primates is warranted. The MHC typing re-\nsults suggest the likelihood of a comparable genetic predis-\nposition to endometriosis in women.\n5 Conclusions\nThe MHC I allele overexpression in our macaque cohorts\nsuggests a role for immune system on endometriosis patho-\ngenesis. Further research is required to fully understand how\nthese MHC I (Mamu-A1) alleles contribute to disease.\nData availability. All Mamu-A1 and DRB sequences are publicly\navailable at the IPD-MHC NHP database http://www.ebi.ac.uk/ipd/\nmhc/.\nCompeting interests. The authors declare that they have no con-\nflict of interest.\nAcknowledgements. We thank Charles C. Bailey from De-\npartment of Molecular and Comparative Pathology at the John\nHopkins School of Medicine, Baltimore, MD, USA for his\ntechnical assistance. We also thank F. van Hassel for the artwork.\nThis study was in part supported by NIH/NIAID contract number\nHHSN266200400088C.\nEdited by: M. Bleyer\nReviewed by: two anonymous referees\nReferences\nAhn, S. 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