Spontaneous endometriosis in rhesus macaques: evidence for a genetic association with specific Mamu-A1 alleles

Primate Biol., 4, 117–125, 2017 https://doi.org/10.5194/pb-4-117-2017 © Author(s) 2017. This work is distributed under the Creative Commons Attribution 3.0 License. Spontaneous endometriosis in rhesus macaques: evidence for a genetic association with specific Mamu-A1 alleles Ivanela Kondova1, Gerco Braskamp1,†, Peter J. Heidt 1, Wim Collignon1, Tom Haaksma1, Nanine de Groot2, Nel Otting 2, Gaby Doxiadis 2, Susan V . Westmoreland3, Eric J. Vallender4,5, and Ronald E. Bontrop2 1Animal Science Department, Division of Pathology and Microbiology, Division of Veterinary care, Biomedical Primate Research Centre, 2288 GJ Rijswijk, the Netherlands 2Department of Comparative Genetics, Biomedical Primate Research Centre, 2288 GJ Rijswijk, the Netherlands 3AbbVie Bioresearch Center, Immunology, Pharmacology, Pathology and Exploratory Toxicology, Worcester, MA 01605, USA 4Department of Psychiatry and Human Behavior, University of Mississippi Medical Center, Jackson, MS 39216, USA 5Division of Veterinary Medicine, Tulane National Primate Research Center, Covington, LA 70433, USA †deceased Correspondence to: Ivanela Kondova ([email protected]) Received: 20 December 2016 – Revised: 19 April 2017 – Accepted: 28 April 2017 – Published: 22 June 2017 Abstract. Endometriosis is a poorly understood common debilitating women’s reproductive disorder resulting from proliferative and ectopic endometrial tissue associated with variable clinical symptoms including dysmen- orrhea (painful menstrual periods), dyspareunia (pain on intercourse), female infertility, and an increased risk of malignant transformation. The rhesus macaque (Macaca mulatta) develops a spontaneous endometriosis that is very similar to that seen in women. We hypothesized that specific major histocompatibility complex (MHC) alleles may contribute to the pathogenesis of endometriosis. As part of a collaboration between the Biomedical Primate Research Centre (BPRC) in the Netherlands and the New England Primate Research Center (NEPRC) in the United States, we analyzed DNA sequences of MHC class I ( Macaca mulatta, Mamu-A1 ) and class II (Mamu-DRB) alleles from rhesus macaques with endometriosis and compared the allele frequencies with those of age-matched healthy macaques. We demonstrate that two MHC class I alleles are overrepresented in dis- eased macaques compared to controls: Mamu-A1*001, 33.3 % in BPRC animals with endometriosis vs. 11.6 % in healthy macaques (p= 0.007), and Mamu-A1*007, 21.9 % NEPRC rhesus macaques vs. 6.7 %, (p= 0.003). We provide evidence that select MHC class I alleles are associated with endometriosis in rhesus macaques and suggest that the disease pathogenesis contribution of MHC class I warrants further research. Published by Copernicus Publications on behalf of the Deutsches Primatenzentrum GmbH (DPZ). 118 I. Kondova et al.: Spontaneous endometriosis in rhesus macaques 1 Introduction 1.1 Endometriosis in humans Endometriosis is a chronic debilitating inflammatory disease that affects approximately 10 to 20 % of women of reproduc- tive age and roughly 50 % of women with infertility (Giu- dice, 2010). The associated clinical symptoms like dysmen- orrhoea, dyspareunia, and chronic pelvic pain have a nega- tive impact on the quality of life of women affected with the disorder (Gupta et al., 2008). Histologically, the disease is defined by the presence of endometrial glandular and stro- mal tissue in organs and tissues outside of the uterine en- dometrium. Experimental studies in women are hindered by the risks and complications associated with repetitive biopsy or sur- gical procedures. As a consequence, the pathogenesis of en- dometriosis remains incompletely understood. It is likely that endometriosis is a complex and multifactorial disorder trig- gered by hormonal, immunologic, genetic, and environmen- tal factors. One hypothesized process in the pathogenesis of endometriosis is metaplasia, involving the transformation of tissues in the peritoneal cavity into endometrial tissue driven by hormonal or immunological factors (Sourial et al., 2014). Hormones play multiple roles with estrogen promoting pro- liferation of endometrial lesions and progesterone limiting endometrial proliferation. Inflammation, immune dysregula- tion, and oxidative stress have also been associated with en- dometriosis, contributing to cytokine-mediated endometrial growth (Forte et al., 2014). Other hypothesized processes include the suppression of normal apoptosis of endometrial glandular cells, proliferation of a population of progenitor or stem cells, epigenetic alterations (Forte et al., 2014), and the oldest theory of retrograde menstruation (Sampson, 1927). Hereditary studies in women with endometriosis showed increased incidence in relatives of affected women com- pared to women without a familial history of endometrio- sis (Simpson et al., 1980). Consequently, the role of genetics in endometriosis has been long hypothesized (Moen et al., 1984; Simpson et al., 1984; Kennedy, 1999; Ishii et al., 2003; Bischoff and Simpson, 2004; Zondervan et al., 2001, 2004), but, like many complex diseases, specific causative genes or haplotypes have been elusive. In addition, while early studies implicated an immunologic basis for endometriosis (Steele et al., 1984), there was no identified association with the human leukocyte antigen (HLA), which are the genes encoding the major histocompatibility complex (MHC) in humans (Moen et al., 1984; Simpson et al., 1984). More recent studies have been increasingly suggestive of the role of immune dysfunc- tion and inflammation in endometriosis (Ahn et al., 2016; Yamada-Nomoto et al., 2016), but, while there have been more suggestions of an association with HLA in endometrio- sis (Ishii et al., 2003; Kitawaki et al., 2002), possibly in con- cert with specific killer immunoglobulin-like receptor (KIR) genotypes (Kitawaki et al., 2007; Nowak et al., 2015), the role of the HLA/MHC remains an open question. 1.2 Rhesus macaque as a model for endometriosis Controlled experiments in humans are difficult due to limita- tions on repeated imaging and surgical biopsies for disease monitoring (Story and Kennedy, 2004). Therefore, animal models provide an invaluable tool for studying complex dis- eases like endometriosis. Although the use of rodent models of endometriosis has some advantages with respect to genetic manipulation and affordability, these species differ greatly from humans, making comparisons difficult. Baboons and macaques have been the best nonhuman primate (NHP) mod- els to study endometriosis’ pathogenesis, pathophysiology, spontaneous evolution, and new medical treatment options (D’Hooghe et al., 2009; Fazleabas et al., 2002; Yamanaka et al., 2012). In fact spontaneous endometriosis only occurs in humans and menstruating NHPs. Rhesus macaques share many similarities with humans, such as their reproductive physiology, which is of particular relevance. Menarche in rhesus monkeys occurs at about 3 years of age, the length of the menstrual cycle is about 28 days, and menstrual bleeding lasts for about 4 days (Catchpole and van Wagenen, 1975). As in women, studies have implicated genetic predisposition to endometriosis in macaques (Zondervan et al., 2001, 2004). Humans and rhesus macaques have a comparable major his- tocompatibility complex (also known as the human leukocyte antigen in humans) with two main antigen-presenting classes of molecules. In rhesus macaques MHC (MhcMamu) class I consists of Mamu-A and Mamu-B and class II of Mamu-DR, Mamu-DQ, and Mamu-DP molecules. In both species, the genes encoding both MHC I and II molecules are charac- terized by high allelic variation, but, while macaques show a high degree of copy number variation of class I and II, specif- ically Mamu-B genes, the equivalent of the human C gene is absent. The aim of our study is to examine any genetic sus- ceptibility of MHC alleles to endometriosis in two colonies of rhesus macaques. 2 Materials and methods 2.1 Description of the colonies The Biomedical Primate Research Centre (BPRC) in Ri- jswijk, the Netherlands, is fully accredited by the Associa- tion for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and maintains a breeding colony of approx- imately 1100 rhesus macaques ( Macaca mulatta ). Animals are conventionally housed in large social breeding groups (one alpha male with several adult females and their juve- nile and adolescent offspring), mimicking the natural ecol- ogy. The housing of these groups consists of interconnected indoor (72 m2) and outdoor (208 m 2) enclosures with ele- vated sitting locations and enrichment devices (Vernes and Primate Biol., 4, 117–125, 2017 www.primate-biol.net/4/117/2017/ I. Kondova et al.: Spontaneous endometriosis in rhesus macaques 119 Figure 1. Ultrasound image of endometrial lesions of a 16-year-old rhesus macaque no. 9234 from the BPRC breeding colony. Image was taken with a flat probe, and it shows a cystic lesion with solid compartments. Louwerse, 2010). Animals are fed on a diet of commercially available monkey chow, fruits, vegetables, and grains. Wa- ter is available ad libitum. Housing and care is in accordance with the Dutch law on animal experimentation, which fol- lows EU Directive 86/609/EEC. The coefficient of inbreed- ing is calculated annually for all breeding animals according to Wrigh (1922), and the parentage is defined for all new- borns by means of STR typing with 24 microsatellites local- ized on 16 different chromosomes. At the time of data collection, the New England Pri- mate Research Center (NEPRC) was a specific-pathogen- free colony of approximately 2000 primates maintained in accordance with federal and institutional guidelines man- dated by the Institutional Animal Care and Use Committee (IACUC) of Harvard Medical School and accredited by the AAALAC. Rhesus macaques were housed in harems includ- ing one male and several adult females with pre-weaning off- spring. Colony rooms were on a 12 h light–dark cycle, and the animals received a diet of monkey chow (Harlan Teklad monkey diet) supplemented with fresh fruit. A variety of en- richment objects were available at all times. All animal pro- cedures including euthanasia were performed in accordance with guidelines and recommendations of the Committee on Animals of Harvard Medical School and the National Insti- tutes of Health Guide for the Care and Use of Laboratory An- imals (publication no. 85-23, revised 1996). Research proto- cols were approved by the Harvard Medical School Animal Care and Use Committee. Both BPRC and NEPRC maintained complete medical records and familial relationships on all colony animals. Af- ter death, all animals were necropsied within several hours of death, often immediately following euthanasia, and repre- sentative sections of tissues were collected, flash frozen, and stored at−80◦C, as well as fixed in 10 % neutral buffered Figure 2. Gross pathology of uterus from rhesus macaque with en- dometriosis. The ovaries and fallopian tubes are embedded and dis- torted by accumulation of solid fibrous masses (scar tissue) and for- mation of endometrial cysts (arrow shows an open large cyst with yellow fibrous nodules in the center and dark red-brown fluid seen at the edge). formalin (NBF) and embedded in paraffin. The records from gross and histopathological examinations were held on the computerized database. 2.2 Identification of animals with endometriosis and case selection for the study Information on the presence of endometriosis was ob- tained through necropsy reports and archived gross, his- tological, and diagnostic representative images shown in Figs. 1, 2, and 3. The main criteria for the animals se- lected for the study were the clinically (bloating, pain, dys- menorrhea, ultrasound-detected cystic lesions) and histolog- ically proven endometriosis (ectopic proliferative endome- trial glandular and stromal tissues, hemosiderin, and hem- orrhage). Necropsy records from NEPRC were examined to identify female rhesus macaques ( Macaca mulatta ) over 1 year of age for which representative tissues from all or- gans had been collected and examined histologically by rou- tine hematoxylin and eosin staining. Cases with a diagnosis of endometriosis were reviewed and selected if frozen en- dometrial tissues were archived. A similar selection of tis- sues was made from the tissue bank at BPRC (Table 1). Con- trol or unaffected animals were defined as such according their full necropsy report providing evidence of absence of endometriosis. All animals with endometriosis were of In- dian origin, except animals 8612 and BB93, which are In- dian× Burmese and Indian× Chinese mixed-breed animals, respectively, and animal 4050, which is of Burmese origin. www.primate-biol.net/4/117/2017/ Primate Biol., 4, 117–125, 2017 120 I. Kondova et al.: Spontaneous endometriosis in rhesus macaques Figure 3. Urinary bladder of rhesus macaque with endometrial le- sions. The serosa is infiltrated by endometrial glands, endometrial stroma, and inflammatory cells (hematoxylin and eosin staining). The control animals from the two colonies were of Indian origin. 2.3 Pedigree analysis of macaques from BRPC and NEPRC Parental relationships between animals were determined from veterinary records. For most matings only a single sire was present at the time of conception. MHC transmission be- tween parent and offspring was used to confirm relationships with further genetic tests when warranted. There were no am- biguous parentage calls among the animals involved in these studies. 2.4 DNA extraction Uterine tissue from NEPRC study animals was frozen in liq- uid nitrogen and pulverized. The powdered tissue was resus- pended in digestion buffer and digested with proteinase K at 55◦C overnight. DNA was isolated via phenol/chloroform extraction followed by ethanol precipitation. DNA pellets were resuspended in TE buffer, and sample concentration was measured via UV spectrometry at 260 nm. DNA isola- tion of BPRC’s animals was performed on fresh EDTA blood or frozen peripheral blood mononuclear cells (PBMCs) by a standard salting-out method (Doxiadis et al., 2013) or by us- ing the QIAamp DNA mini kit (QIAgen, Germantown, USA) according to the manufacturer’s instructions. 2.5 MHC typing MHC typing of both class I and class II alleles was per- formed on DNA samples from these monkeys, namely for (Macaca mulatta ) Mamu-A1 (MHC class I, locus A1) and Mamu-DRB (MHC class II DR, beta-chain) by microsatel- lite (STR) typing with STRs D6S2854 and D6S2859, being Mamu-A specific, and D6S2878, being Mamu-DRB specific markers (Doxiadis et al., 2007, 2013). For animals from the NEPRC colony, additional high-resolution sequencing was done using Roche 454 technologies on blood-derived lym- phocyte cDNA (Karl et al., 2013; Wiseman et al., 2013). In the case of the animals of BPRC, additional high-resolution Sanger sequencing had been performed beforehand and pub- lished previously (Otting et al., 2005; Doxiadis et al., 2013). Since the animals were members of breeding colonies, kin- ship coefficients and/or pedigrees of the animals are known, and some MHC haplotypes could be defined as well by seg- regation analysis. Significance was determined by comparing the number of carriers of the haplotype with endometriosis to the number of carriers in the colony (colony size: BPRC, n= 1383; NEPRC, n= 380, colony frequencies shown in Table 2b) using a Fisher’s exact test with Bonferroni correc- tion for multiple testing (each of nine independent MHC A1 alleles). 3 Results 3.1 Demographics of endometriosis cases Eight female rhesus macaques from BPRC (ranging from 12 to 21 years of age, mean 17.0 years) and seventeen female rhesus macaques from NEPRC (14 to 20 years of age, mean 15.9 years) were identified with endometriosis based on clin- ical and histologic diagnoses (Figs. 1, 2, and 3). The mean body weight of NEPRC macaques with endometriosis was 9.93 kg, and for BRPC macaques it was 7.78 kg. Four of the 17 macaques with endometriosis from NEPRC had caesarean sections, while none of the macaques at BPRC had under- gone surgery (Table 1). 3.2 Pedigree analysis The relationship status of the animals that had been identi- fied with endometriosis was determined in an attempt to first identify obvious Mendelian segregation and to identify con- founds in association analysis that may result from cryptic genetic substructure within endometriosis cases compared to the colony as a whole. Among the eight monkeys identified at BPRC, there were two pairs of siblings. Among the sev- enteen rhesus macaques with endometriosis from NEPRC, there was one mother–daughter pair, one pair of half-sib, and one trio of half-sibs. Additionally, there were four more dis- tantly related animals identified with endometriosis (Fig. 4, gray shading). Given the breeding patterns within the colony and the animals for which tissue was available and for which pathology could be ascertained, the relationships among the affected females were not different from random samples us- ing bootstrapping. Primate Biol., 4, 117–125, 2017 www.primate-biol.net/4/117/2017/ I. Kondova et al.: Spontaneous endometriosis in rhesus macaques 121 Table 1. Cohorts of rhesus macaques with endometriosis housed at NEPRC and BRPC shown with age, body weight, and surgical history of caesarian sections. Animal Necrop. no. Species Sex Source Age (years) Weight (kg) Sample C-section 1 148-90 A06-313 M. mulatta F NEPRC 16 11.1 DNA no C-section 2 183-90 A06-314 M. mulatta F NEPRC 16 10.7 DNA no C-section 3 265-87 A06-316 M. mulatta F NEPRC 19 11.6 DNA no C-section 4 196-89 A06-317 M. mulatta F NEPRC 17 10.2 DNA no C-section 5 256-89 A06-318 M. mulatta F NEPRC 17 9.4 DNA no C-section 6 236-88 A06-323 M. mulatta F NEPRC 18 8.3 DNA no C-section 7 536-91 A06-324 M. mulatta F NEPRC 15 10.2 DNA no C-section 8 169-92 A06-325 M. mulatta F NEPRC 14 11 DNA no C-section 9 369-92 A06-326 M. mulatta F NEPRC 14 6.2 DNA no C-section 10 103-87 A06-327 M. mulatta F NEPRC 19 7.9 DNA C-section 11 259-87 A06-342 M. mulatta F NEPRC 19 8.3 DNA no C-section 12 419-91 A06-352 M. mulatta F NEPRC 15 9.4 DNA C-section 13 170-87 A07-2 M. mulatta F NEPRC 19 9 DNA no C-section 14 142-92 A07-9 M. mulatta F NEPRC 14 10.8 DNA C-section 15 229-87 A07-10 M. mulatta F NEPRC 19 8.6 DNA no C-section 16 127-86 A07-36 M. mulatta F NEPRC 20 10.8 DNA no C-section 17 468-87 A07-37 M. mulatta F NEPRC 19 15.3 DNA 2 C-sections 1 8803 06-1120 M. mulatta F BPRC 18 6 DNA no C-section 2 1WQ 06-1129 M. mulatta F BPRC 21 7.8 DNA no C-section 3 8851 06-1183 M. mulatta F BPRC 17 7.3 DNA no C-section 4 8612 05-1047 M. mulatta F BPRC 18 8.41 DNA no C-section 5 9250 05-1070 M. mulatta F BPRC 12 7.66 DNA no C-section 6 8930 05-1107 M. mulatta F BPRC 16 6.97 DNA no C-section 7 BB93 07-1386 M. mulatta F BPRC 16 10.35 DNA no C-section 8 4050 07-1354 M. mulatta F BPRC 18 5.6 DNA no C-section Figure 4. Pedigree showing the familial relationships of 12 of the 17 animals from the NEPRC colony. Legend: females are represented by circles and males by squares. Animals diagnosed with endometriosis are shaded in gray. 3.3 MHC typing Association analysis for endometriosis was robust to allele sharing between the animals. The rhesus macaques from BPRC (n= 8) and NEPRC (n= 17) underwent MHC typing for their Mamu-A1 and Mamu-DRB alleles (Table 2a). The BRPC endometriosis cohort included the following MHC I (Mamu-A1) alleles: A1*001 (33.3 %), *002 (13.3 %), *004 (6.7 %), *007 (6.7 %), *008 (26.7 %), and *011 (6.7 %) (Ta- ble 2b), while animals with endometriosis from NEPRC had the following Mamu-A1 alleles: A1*001 (9.4 %), *002 (12.5 %), *003 (3.1 %), *004 (15.6 %), *007 (21.9 %), *008 (21.9 %), *012 (6.3 %), and *026 (9.4 %) (Table 2b). The al- lele frequencies in BPRC endometriosis samples compared to controls revealed significant enrichment ofMamu-A1*001 (33.3 vs. 11.6 % in healthy animals, p= 0.007) in mon- keys with endometriosis (Table 2b). In the NEPRC cohort, the MHC allele Mamu-A1*007 was significantly overrepre- sented in diseased macaques compared to controls (21.9 vs. 6.7 %, p= 0.003). These associations are not shared between the colonies. The Mamu-A1*026 allele is marginally over- represented in the NEPRC colony (9.4 vs 1.5 %), although this does not pass the multiple testing correction. This allele is uncommon in both colonies and is only seen in the affected mother–daughter pair at NEPRC. Additionally, the Mamu- DRB haplotype, which is characterized by the DRB*W3:03 www.primate-biol.net/4/117/2017/ Primate Biol., 4, 117–125, 2017 122 I. Kondova et al.: Spontaneous endometriosis in rhesus macaques Table 2. (a) Mamu-A1 and Mamu-DRB genotypes of animals diagnosed with endometriosis and their family relationship. A question mark for Mamu-A1 typing indicates that the animal is most probably homozygous for Mamu-A1. A question mark in the column “remarks” indicates that sharing of a MHC haplotype is possible but cannot be confirmed. Alleles in bold represent those which are present at a higher frequency in rhesus macaques with endometriosis than in healthy animals. (b) Allele frequencies for animals with endometriosis as well as colony frequencies for NEPRC and BPRC are shown. Significant p < 0.05 values are bold. (a) Animal Mamu-A1* Mamu-DRB Source Remarks 1 148-90 008/ 007 *W3:03/*W3:03 NEPRC 2 183-90 012/002 1*03:03/3*04:10 NEPRC 3 265-87 004/002 1*03:10/1*03:09 NEPRC same father as 229 and 536; shared MHC? 4 196-89 007/001 1*04:06/*W6:06 NEPRC same father as 256; no MHC sharing 5 256-89 008/002 1*03:03/1*03:09 NEPRC same father as 196; no MHC sharing 6 236-88 008 /007 1*03:03/1*03:18 NEPRC shared 1st MHC hapl. with 142; 2nd with 259 7 536-91 012/008 *W3:03 /1*03:09 NEPRC same father as 229 and 265; shared MHC? 8 169-92 026/026 1*04:06/1*03:09 NEPRC shared MHC with 127 9 369-92 007 /? 1*04:06/1*07:01 NEPRC 10 103-87 008/ 007 1*03:03/1*03:09 NEPRC 11 259-87 007/007 *W6:06/1*03:18 NEPRC shared 2nd MHC haplotype with 236 12 419-91 004/008 1*03:10/1*03:06 NEPRC 13 170-87 004/002 1*03:09 /*W3:03 NEPRC 14 142-92 008 /001 1*03:03 /*W3:03 NEPRC shared 1st MHC hapl. with 236 15 229-87 004/ 001 1*04:06 /*W3:03 NEPRC same father as 265 and 536; shared MHC? 16 127-88 004/026 1*03:09/1*03:17 NEPRC shared MHC with offspring 169 17 468-87 003/? 1*04:04/1*03:06 NEPRC 1 8803 011/008 3*04:10/1*04:06 BPRC 2 1WQ 001 /008 1*04:06/1*03:03 BPRC 3 8851 001 /002 *W6:06/1*04:06 BPRC sibling of 8612; 1 shared MHC haplotype 4 8612 001/007 *W6:06/4*01:02 BPRC sibling of 8651; 1 shared MHC haplotype 5 9250 001 /004 1*03:09/1*03:09 BPRC sibling of 8930; 1 shared MHC haplotype 6 8930 001/ 002 1*03:09/1*04:03 BPRC sibling of 9250; 1 shared MHC haplotype 7 BB93 008/? 1*03:03/1*04:06 BPRC 8 4050 008/050 1*03:21/*W26:04 BPRC (b) NEPRC BPRC Mamu-A1 Endom. (n) Colony ( n) p val Padj Endom. (n) Colony ( n) p val Padj A1*001 9.4 % (3) 11.9 % (90) 1 1 33.3 % (5) 11.6 % (321) 0.007 0.04 A1*002 12.5 % (4) 12.0 % (91) 1 1 13.3 %(2) 15.8 % (437) 1 1 A1*003 3.1 % (1) 0.3 % (2) 0.117 0.93 0 %(0) 4 % (111) 1 1 A1*004 15.6 % (5) 17.6 % (134) 1 1 6.7 %(1) 23.2 % (642) 0.251 1 A1*007 21.9 % (7) 6.7 %(51) 0.003 0.03 6.7 % (1) 4.7 % (130) 0.492 1 A1*008 21.9 % (7) 23.1 % (176) 1 1 26.7 %(4) 20.4 % (564) 0.475 1 A1*011 0 % (0) 0.2 % (1) 1 1 6.7 % (1) 2.7 % (75) 0.322 1 A1*012 6.3 % (2) 6.8 % (52) 1 1 0 % (0) 5.6 % (155) 1 1 A1*026 9.4 % (3) 1.5 % (11) 0.013 0.14 0 %(0) 1.1 % (30) 1 1 Total 100.0 % (32) 100.0 % (760) 100.0 % (15) 100.0 % (2766) Padj (p-adjusted) is the significance value after Bonferroni correction for multiple tests (see methods); “Endom.” represents animals with endometriosis; n is the number of alleles. allele, may be overrepresented in diseased animals of the NEPRC colony (Table 2a) (17.64 vs. 3.74 % in healthy an- imals of BPRC). Although Mamu-DRB typing is not rou- tinely performed at NEPRC, the comparison to the colony frequencies at BPRC may be relevant, since allele frequen- cies of Mamu-A1 in the two colonies are comparable (Ta- ble 2b). Nevertheless, while this is putatively suggestive and warrants further study, it cannot be interpreted with certainty. 4 Discussion In this paper, we report significant higher frequency of two Mamu-A1 MHC class I alleles in rhesus macaques with Primate Biol., 4, 117–125, 2017 www.primate-biol.net/4/117/2017/ I. Kondova et al.: Spontaneous endometriosis in rhesus macaques 123 endometriosis from two different primate centers, Mamu- A1*001 in BPRC macaques and Mamu-A1*007 in NEPRC macaques. The familial relatedness of several macaques with endometriosis from the two colonies supports a hereditary risk for this disease in rhesus macaques which is similar to that seen in women (Bischoff and Simpson, 2004; Ishii et al., 2003; Kennedy, 1999). The different Mamu-A1 al- leles may reflect the different origins of the two colonies. Since diseased and control animals of both colonies are part of breeding groups, the Mamu-A1 and Mamu-DRB alleles can be inferred from pedigree analysis to be identical by state but not by descent, and the higher frequencies ob- served in affected individuals are not attributable simply to kinship. Although NEPRC and most of the BPRC animals are of Indian origin, the founder animals of both colonies may be from different parts of India. Additionally, two of the macaques of BPRC are a mixed breed, Indian–Chinese or Indian–Burmese, and one animal is from Burmese ori- gin. Although two different Mamu-A1 alleles were over- represented in macaques with endometriosis from the two facilities, the arguably more important interpretation may be that both colonies share a significant disease association with class I alleles. These results are comparable to humans, where higher frequencies of different MHC class I B alleles are described in endometriosis patients; a significantly higher frequency of HLA-B 54 and CW7 is observed in Japanese patients (Ishii et al., 2002), whereas a significantly posi- tive association with endometriosis of HLA-B7 is defined by Kitawaki and colleagues (Kitawaki et al., 2002). In addition, some MHC II Mamu-DRB alleles were overrepresented in animals with endometriosis at BPRC; however, conclusions were limited by the low number of animals analyzed. These findings are consistent with the reported higher frequency of HLA-DRB1*1403 (Ishii et al., 2002) and HLA-DQB1*0301 in women with endometriosis (Ishii et al., 2003). In our macaque study, Mamu-B alleles have not been an- alyzed. Since MHC alleles, in humans as in macaques (de Groot et al., 2014), are well known to be subjected to link- age disequilibrium, it is plausible that the observed disease associations are not caused by a specific Mamu-A1 allele it- self but may be due to certain alleles of adjacent loci such as Mamu-B. Likewise, the disease association with certain Mamu-DRB alleles may also be caused by linkage dise- quilibrium. Linkage disequilibrium with Mamu-DRB alleles would also explain why no disease association with Mamu- DRB alleles and endometriosis has been observed in other human populations (Roszkowski et al., 2005). Accordingly, Kitawaki and coworkers conclude that there is a certain HLA haplotype, namely HLA-A24-B*0702-Cw*0702-DRB*0101, which is linked to endometriosis susceptibility (Kitawaki et al., 2002). Further analysis of extended haplotypes in rhesus macaques will help to clarify these findings. It is important to interpret the present findings in rhesus macaques with cau- tion, as the associated alleles may simply represent markers of associated haplotypes rather than causative variants them- selves. There may be several ways in which immune system surveillance, function, or dysfunction may contribute to or promote endometriosis (Ishii et al., 2002; Forte et al., 2014). As demonstrated in previous work, women with endometrio- sis exhibit altered or reduced innate and even adaptive im- munity (Dmowski et al., 1981; Ota and Igarashi, 1993; Chi- ang and Hill, 1997; Khan et al., 2009). Additional studies suggest an autoimmune component to endometriosis (Eisen- berg et al., 2012). Specific MHC I alleles may result in al- tered immune responses, leading to uncontrolled growth of stem cells, progenitor cells, and/or ectopic glandular tissue (Forte et al., 2014). Further investigation of spontaneous en- dometriosis in primates is warranted. The MHC typing re- sults suggest the likelihood of a comparable genetic predis- position to endometriosis in women. 5 Conclusions The MHC I allele overexpression in our macaque cohorts suggests a role for immune system on endometriosis patho- genesis. Further research is required to fully understand how these MHC I (Mamu-A1) alleles contribute to disease. Data availability. All Mamu-A1 and DRB sequences are publicly available at the IPD-MHC NHP database http://www.ebi.ac.uk/ipd/ mhc/. Competing interests. The authors declare that they have no con- flict of interest. Acknowledgements. We thank Charles C. Bailey from De- partment of Molecular and Comparative Pathology at the John Hopkins School of Medicine, Baltimore, MD, USA for his technical assistance. We also thank F. van Hassel for the artwork. This study was in part supported by NIH/NIAID contract number HHSN266200400088C. Edited by: M. Bleyer Reviewed by: two anonymous referees References Ahn, S. H., Khalaj, K., Young, S. 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