{"paper_id":"1743823b-df00-4b13-bfdb-14d62186ab62","body_text":"1Scientific  RepoRtS  |         (2020) 10:4897  | https://doi.org/10.1038/s41598-020-61702-y\nwww.nature.com/scientificreports\nKiller cell immunoglobulin-\nlike receptors (KiR) and human \nleukocyte antigen-c (HLA-c) \nallorecognition patterns in women \nwith endometriosis\nYa-ching chou 1,2,3,4, Chi-Huang chen 1,2, Ming-Jer chen 5,6, Ching-Wen chang 1, Pi-Hua chen 7,8, \nMu-Hsien Yu9, Yi-Jen chen 10,11, Eing-Mei t sai12,13, Peng-Sheng Yang1, Shyr-Yeu Lin1,2 &  \nchii-Ruey t zeng1,2*\nendometriosis shares similarities with several autoimmune diseases. the human leukocyte antigen \n(HLA)-C genotype is associated with several human autoimmune diseases. HLA-c is a ligand of killer \ncell immunoglobulin receptors (KIRs) and is an essential regulator of natural killer cell activity, which \nis associated with endometriosis progression. polymorphisms in HLA-C and KIR affect the activity of \nNK cells and susceptibility to several diseases. Therefore, we attempted to investigate an association \nbetween HLA-C genotype and KIR polymorphism and the occurrence of endometriosis. We tested \nthe association of certain KiR and HLA-c combinations and the development of endometriosis by \ncharacterizing both KIR and HLA-C genes in 147 women with endometriosis and 117 controls. The \nHLA-C genotypes and KIR polymorphisms were analyzed via DnA-based method for higher-resolution \ngenotyping. We found that the occurrence of HLA-C*03:03*01 was increased in endometriosis than in \ncontrol groups. Analysis of various KIR haplotypes revealed differences between the endometriosis and \ncontrol cohorts. the number of KIR centromeric A/A haplotypes was increased in the endometriosis \ngroup than controls. Moreover, the endometriosis cohort was characterized by reduced number of \nKIR2DS2-positive individuals in the Han Chinese population. Our current findings suggest that the KIR \nand HLA-C genotypes are associated with the pathogenesis of endometriosis.\nEndometriosis is a chronic gynecological disease with unknown etiology and is characterized by extra-uterine \ngrowth of endometrial tissue\n1. Endometriosis affects 6% to 10% of fertile women at the reproductive age and \ncauses severe pelvic pain and infertility2–4. Familial and twin studies have reported that genetic factors are asso-\nciated with the pathogenesis of endometriosis5–9. Cell-mediated and humoral immune responses are essential in \nthe pathogenesis of endometriosis, since it is associated with various immunological abnormalities, particularly \n1Center for Reproductive Medicine & Sciences, Department of Obstetrics and Gynecology, Taipei Medical University \nHospital, Taipei, Taiwan. 2Department of Obstetrics and Gynecology, School of Medicine, College of Medicine, \nTaipei Medical University, Taipei, Taiwan. 3Department of Biological Science and Technology, College of Biological \nScience and Technology, National Chiao Tung University, Hsinchu, Taiwan. 4Center for Intelligent Drug Systems \nand Smart Bio-devices (IDS 2B), National Chiao Tung University, Hsinchu, Taiwan. 5Department of Obstetrics and \nGynecology and Women’s Health, Taichung Veterans General Hospital, Taichung, Taiwan. 6School of Medicine, \nNational Yang-Ming University, Taipei, Taiwan. 7Graduate Institute of Clinical Medicine, College of Medicine, Taipei \nMedical University, Taipei, Taiwan. 8Department of Obstetrics and Gynecology, Shuang Ho Hospital, Taipei Medical \nUniversity, Taipei, Taiwan. 9Department of Obstetrics & Gynecology, Tri-Service General Hospital, National Defense \nMedical Center, Taipei, Taiwan. 10Department of Obstetrics and Gynecology, Taipei Veterans General Hospital, Taipei, \nTaiwan. 11School of Medicine, Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan. 12General \nResearch Centers of R&D office, Kaohsiung Medical University, Kaohsiung, Taiwan. 13Division of Reproductive \nMedicine, Department of Obstetrics and Gynecology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan. \n*email: tzengcr@tmu.edu.tw\nopen\n\n2Scientific  RepoRtS  |         (2020) 10:4897  | https://doi.org/10.1038/s41598-020-61702-y\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\ncell-mediated immunity10–12. The activities of cytotoxic T-cells and natural killer (NK) cells are dysregulated in \nwomen with endometriosis13–16. Increased serum levels of immunoglobulins and autoantibodies, decreased endo-\nmetrial cell apoptosis, and the production of pro-inflammatory cytokines are observed in endometriosis patients, \nindicating that endometriosis shares many similarities with autoimmune diseases11,12,17,18.\nMajor histocompatibility complex (MHC) genes, also known as human leukocyte antigen (HLA) genes, are \nlocated in chromosome 6p. The genes encoding the human MHC class I (HLA-A, HLA-B, and HLA-C) and class \nII (HLA-DR, HLA-DQ, and HLA-DP) molecules are the most polymorphic loci in the human genome. HLA \ngenes are polymorphic in binding and function in presenting antigen peptides to T-cells. HLA molecules are key \nfactors involved in regulating the specificity of T-cell-mediated immune response in autoimmune and infectious \ndiseases19–21.\nHLA Class I genes encode cell-surface proteins, whose primary functions are to present antigens to cytotoxic \nCD8+ T-cells during the early immune responses 19–21. Among these, HLA-C plays a minor role in regulating \nantigen-specific T-cell responses because of low cell surface expression 22. HLA-C acts as a ligand for killer cell \nimmunoglobulin-like receptors (KIRs), which regulate natural killer (NK) cell-mediated cytotoxicity. The human \nimmunodeficiency virus Nef protein selectively downregulates the production of HLA-A and HLA-B molecules \nto suppress cytotoxic CD8\n+ T lymphocyte responses23. However, Nef maintains stable HLA-C expression levels to \ninhibit NK cell activation and renders HLA-C as a T-cell restriction element during HIV infection24. Importantly, \nthe HLA-C genotype has been implicated in several autoimmune diseases, including Graves’ disease, psoriasis, \nand Crohn’s disease20,22,23,25,26.\nNK cells are lymphocytes that serve as vital components of the immune system by regulating early responses \nagainst infected or transformed cells via cytokine production and direct cytotoxicity27. KIRs are a family of mem-\nbrane glycoproteins expressed by NK cells. KIRs contain two or three extracellular immunoglobulin-like domain \nmolecules (D) with a long (L) or short (S) cytoplasmic tail\n28. The KIR gene is located on chromosome 19q13.4 \non the leukocyte receptor complex. KIR exhibits activating and inhibitory effects with extensive haplotypic and \nallelic polymorphisms\n29–31. The 16 KIR genes comprise the following: six genes encoding activating KIR (2DS1-5 \nand 3DS1), seven genes encoding inhibitory KIR (2DL1-3, 5 and 3DL1-2), KIR2DL4, which can exert both \ninhibitory and activating activity, and two pseudogenes (2DP1 and 3DP1). Furthermore, KIR3DL3, KIR3DP1, \nKIR2DL4, and KIR3DL2 are framework genes and are always present in the genome\n32.\nThe primary ligands of KIR are HLA-C molecules, which are divided into two groups, namely C1 and C2, \nbased on the amino acid at position 80 [HLA-C C1 groups (HLA-C1), asparagine (N) at position 80: C*01, 03, 07 \n(01–06), 08, 12 (02, 03, 06), 13, 14, 15:07, 16 (01, 03, 04); HLA-C C2 groups (HLA-C2), lysine (K) at position 80: \nC*02, 04, 05, 06, 07 (07), 12 (04, 05, 42), 15, 16 (02), 17, 18]\n32,33. The inhibitory receptors KIR2DL2 and KIR2DL3 \nand activating receptor KIR2DS2 share the same ligand HLA-C1. Activating KIR2DS2 has been reported to be in \nstrong linkage disequilibrium and highly homologous to KIR2DL2. KIR2DL1 and KIR2DS1 bind to HLA-C2\n3134–36.  \nCombinations of HLA-C with KIR2DS1 and KIR2DS2 have been reported to correlate with the occurrence of \nautoimmune diseases, leukemia, and inflammatory diseases\n37–42. Polymorphisms in the genes encoding HLA-C \nand KIR affect NK cells activity and susceptibility to several diseases 31. HLA genotyping is traditionally per -\nformed using a serological method. However, detection of the HLA-C genotype via serological typing is difficult \nbecause of the low HLA-C expression levels at the cell surface, the lack of suitable antisera, and difficulties in pro-\ntein isolation\n22. Therefore, we employed a DNA-based method for higher-resolution genotyping and investigated \nthe association between the HLA-C genotype and endometriosis. Moreover, to analyze the association between \ncertain KIR-HLA-C combinations and the development of endometriosis, we characterized both KIR and HLA-C \ngene polymorphisms in 147 women with endometriosis and 117 controls.\nResults\nfrequency distributions of HLA-C alleles among endometriosis and control groups.  The \ndemographic results of endometriosis and control groups are shown in Table  1. HLA-C allele frequencies in \nendometriosis patients (n = 147, 294 alleles) and control patients (n = 117, 234 alleles) were determined using a \nsequence-based typing method. The presence of HLA-C*03:03:01 significantly increased the risk of endometri-\nosis with p = 0.0473 [Odds Ratio (OR) = 2.811, 95% confidence interval (CI)  = 1.021–7.738] and the statistical \npower was 43.8% (Table  2). After multiple test analyses using Bonferroni correction, the association was not \nsignificant.\nfrequency distributions of HLA-C  group among endometriosis and control groups. We \nevaluated whether the HLA-C group C1 (HLA-C1) and HLA-C group C2 (HLA-C2) were associated with \nCharacteristics\nControl \nn = 117 (%)\nEndometriosis \nn = 147 (%) p value\nAge\na 38.44 (7.47) 36.08 (6.55) 0.012\nBMIa, kg/m2 23.01 (4.47) 21.58 (3.47) 0.0032\nAge of menarchea 12.53 (1.21) 12.80 (1.52) 0.5968\nDuration of Menstrual cyclea 27.95 (4.95) 28.45 (2.97) 0.1392\nDysmenorrheab, n (%) 73 (62.39) 112 (76.19) 0.015\nTable 1. Patient demographic results. Abbreviations: BMI, body mass index; SD, standard deviation Mean (SD) \nfor continuous variables. n (%) for discontinuous variables. aMann-Whitney test. bχ 2 test.\n\n3Scientific  RepoRtS  |         (2020) 10:4897  | https://doi.org/10.1038/s41598-020-61702-y\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nendometriosis. Analysis revealed no significant differences in HLA-C1 and HLA-C2 frequencies in the endome-\ntriosis and control groups (Table 3).\nfrequency distributions of KIR genotypes among endometriosis and control groups.  Using \nsequence-specific PCR amplification, we analyzed the KIR genotypes in the endometriosis and control groups. \nThe frequencies of the KIR genotypes in women with endometriosis and controls and their statistical associations \nare presented in Table 4. The presence of KIR2DS2 significantly reduced the risk of endometriosis with p = 0.0394 \n[(OR) = 0.5577, 95% CI = 0.3251–0.9569] and the statistical power was 68.6%. After multiple test analyses using \nBonferroni correction, the association was not significant. The two groups showed no significant differences in \nthe remaining KIR genotypes.\nHLA-C\nControl (n = 117,\n234 alleles)\nEndometriosis (n = 147,\n294 alleles)\nOR 95% CI P valuen % n %\nC*01:02:01 56 23.9 59 20.1 0.798 0.5273 to 1.208 0.2907\nC*01:08 0 0.0 1 0.3 2.397 0.09712 to 59.16 1\nC*02:02:02:01 0 0.0 2 0.7 4.009 0.1914 to 83.97 0.5055\nC*03:02:01 24 10.3 33 11.2 1.106 0.6342 to 1.930 0.7786\nC*03:03:01 5 2.1 17 5.8 2.811 1.021 to 7.738 0.0473*\nC*03:04:01:01 30 12.8 34 11.6 0.8892 0.5265 to 1.502 0.6886\nC*03:04:04 0 0.0 4 1.4 7.265 0.3889 to 135.7 0.1333\nC*04:01:01:01 10 4.3 12 4.1 0.9532 0.4043 to 2.247 1\nC*04:03 4 1.7 4 1.4 0.7931 0.1962 to 3.207 0.7375\nC*06:02:01:01 7 3.0 5 1.7 0.561 0.1757 to 1.792 0.3849\nC*07:01:01:01 1 0.4 0 0.0 0.2643 0.01071 to 6.523 0.4432\nC*07:02:01:01 39 16.7 61 20.7 1.309 0.8391 to 2.042 0.264\nC*07:04:01 1 0.4 1 0.3 0.7952 0.04944 to 12.79 1\nC*07:359 1 0.4 0 0.0 0.2643 0.01071 to 6.523 0.4432\nC*08:01:01 21 9.0 20 6.8 0.7404 0.3911 to 1.401 0.4138\nC*08:03:01 0 0.0 1 0.3 2.397 0.09712 to 59.16 1\nC*12:02:02 15 6.4 14 4.8 0.73 0.3449 to 1.545 0.4456\nC*12:03:01:01 3 1.3 1 0.3 0.2628 0.02714 to 2.544 0.3269\nC*14:02:01 6 2.6 9 3.1 1.2 0.4208 to 3.422 0.7975\nC*14:02:03 0 0.0 1 0.3 2.397 0.09712 to 59.16 1\nC*15:02:01 8 3.4 14 4.8 1.413 0.5822 to 3.427 0.5151\nC*15:05:01 1 0.4 0 0.0 0.2643 0.01071 to 6.523 0.4432\nC*16:02:01 1 0.4 0 0.0 0.2643 0.01071 to 6.523 0.4432\nC*16:04:01 1 0.4 1 0.3 0.7952 0.04944 to 12.79 1\nTable 2. Distribution of the HLA-C alleles in the endometriosis and control groups. Each HLA allele has four \nunique sets denoted by different numbers that are separated by a colon. The first two digits often correspond \nto the serological antigen; the two digits after the first colon denote the subtypes and order in the genome from \nthe IMGT/HLA Database (www.ebi.ac.uk/imgt/hla/). The differences in HLA-C allele frequencies between \nthe endometriosis and control groups were analyzed using the Fisher’s exact test. Significance was set at a P \nvalue < 0.05 and the statistical power was 43.8% calculated by G*Power. OR indicates odds ratio. CI indicates \nconfidence interval.\nHLA-C\nControl \n(n = 117)\nEndometriosis\n(n = 147)\nOR 95% CI p valuen % n %\nC1 115 98.3 146 99.3 2.539 0.2273 to 28.37 0.5859\nC2 29 24.8 36 24.5 0.9842 0.5602 to 1.729 1\nC1C1 88 75.2 111 75.5 1.016 0.5783 to 1.785 1\nC2C2 2 1.7 1 0.7 0.3938 0.03525 to 4.400 0.5859\nC1C2 27 23.1 35 23.8 1.042 0.5869 to 1.849 1\nTable 3. Distribution of HLA-C ligand in endometriosis and control groups. Two-sided Fisher’s exact test was \nused to estimate the differences between endometriosis and control groups. n: number of cases with relevant \ngenotypes, OR: odds ratio, CI: confidence interval, Significance was set at a P value < 0.05.\n\n4Scientific  RepoRtS  |         (2020) 10:4897  | https://doi.org/10.1038/s41598-020-61702-y\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nfrequency distributions of KIR haplotypes among endometriosis and control groups.  The fre-\nquencies of the KIR haplotypes in women with endometriosis and controls and their statistical associations are \npresented in Table 5. The χ2 value was calculated by Hardy-Weinberg analysis (χ2 > 3.841 showed the subgroup \nwas deviating from the Hardy–Weinberg equilibrium). We revealed differences between the endometriosis and \ncontrol cohorts. The number of KIR centromeric A/A haplotypes was increased in the endometriosis group than \ncontrols with p = 0.0394 [(OR) = 1.793, 95% CI = 1.045–3.076] and the statistical power was 68.6%.\ncombinations of K iR and their HLA- c ligands. The frequencies of KIR and their HLA-C ligands \nwere analyzed for their statistical associations with endometriosis (Table  6). HLA-C C1 groups are recognized \nby KIR2DL2/2DS2 and KIR2DL3, while HLA-C C2 groups are recognized by KIR2DL1/2DS1. Moreover, \nKIR2DL2/2DL3 also binds to some HLA-C C2 molecules, and KIR2DS4 binds to some HLA-C1 and HLA-C2\n31,34–36.  \nThe molecular interactions of KIR gene-HLA-ligands were calculated from the KIR frequency of a total number \nof HLA ligands. The total number of HLA ligands is shown in Table 3. We calculated the KIR frequency in the \ncombination of different HA ligands. Analysis of various KIR-HLA-C combinations revealed no significant dif-\nferences between the endometriosis and control cohorts (Table 6). The frequency of KIR haplotypes and HLA-C \ncombinations also showed no significant differences between the endometriosis and control groups (Table 7).\nDiscussion\nSeveral factors are involved in the pathogenesis of endometriosis including genetic, neuroendocrine, and immu-\nnological factors\n43–45. Abnormal immune responses are recognized in endometriosis patients, including excessive \ninflammatory cytokine secretion, autoantibody production, and NK cell regulation 17,18. Endometriosis shares \nsimilar characteristics with autoimmune diseases11,12,17,18. HLA-C affects viral infections and is implicated in sev-\neral human autoimmune diseases22. HLA-C*06:02 is associated with severe early-onset psoriasis. HLA-C *12:02 \nwas found to be associated with increased susceptibility to Crohn’s disease20. HLA-C*03 restricts the cytotoxic \nCD8+ T-cell responses during the Epstein-Barr virus and human immunodeficiency virus infections, as well as \nduring co-infection with the influenza virus and the Sendai virus. Herein, we analyzed the associations between \nHLA-C alleles and endometriosis. Consequently, the presence of HLA-C*03:03:01 increased the risk of endome-\ntriosis in Asian women (Table 2). However, after multiple test analyses using Bonferroni correction, the associa-\ntion was not significant.\nPrevious studies reported no association between HLA genotypes and endometriosis in Caucasian women \nwith endometriosis and controls, as assessed by serological typing\n46–48. A serological study showed increased \nfrequencies of the HLA-B*54 and HLA-C*07 alleles in Japanese patients with endometriosis 49. In a recent \nstudy, PCR-restriction fragment length polymorphism analysis revealed that the HLA-DRB1* 14:03 and \nHLA-DQB1*03:01 alleles are associated with endometriosis in Japanese women50,51. Another study reported an \nInhibitory KIR\nControl (n = 117)\nEndometriosis\n(n = 147)\nOR 95% CI p valuen % n %\nKIR2DL1 114 97.4 147 100.0 9.017 0.4608 to 176.5 0.0858\nKIR2DL2 37 31.6 31 21.1 0.5778 0.3314 to 1.007 0.0653\nKIR2DL3 114 97.4 146 99.3 3.842 0.3942 to 37.45 0.3252\nKIR2DL4 117 100.0 147 100.0 — — —\nKIR2DL5 52 44.4 49 33.3 0.625 0.3788 to 1.031 0.0747\nKIR3DL1 115 98.3 145 98.6 1.261 0.1748 to 9.093 1\nKIR3DL2 117 100.0 147 100.0 — — —\nKIR3DL3 117 100.0 147 100.0 — — —\nActivating KIR\nKIR2DS1 39 33.3 46 31.3 0.9109 0.5421 to 1.531 0.7911\nKIR2DS2 41 35.0 34 23.1 0.5577 0.3251 to 0.9569 0.0394*\nKIR2DS3 29 24.8 25 17.0 0.6218 0.3409 to 1.134 0.1273\nKIR2DS4# 113 96.6 143 97.3 1.265 0.3096 to 5.173 0.7358\nKIR2DS4f 89 76.1 117 79.6 1.227 0.6840 to 2.201 0.5503\nKIR2DS4d 60 51.3 76 51.7 1.017 0.6255 to 1.653 1\nKIR2DS5 24 20.5 25 17.0 0.7941 0.4264 to 1.479 0.5249\nKIR3DS1 44 37.6 48 32.7 0.8044 0.4836 to 1.338 0.4365\nPseudogene\nKIR2DP1 114 97.4 147 100.0 9.017 0.4608 to 176.5 0.0858\nKIR3DP1 117 100.0 147 100.0 — — —\nTable 4. Genetic association between KIR gene in endometriosis and control groups. #The gene was considered \npositive if either of the two forms were present. KIR2DS4f - full-length KIR2DS4 allele variant. KIR2DS4d \n- deleted KIR2DS4 allele variant. Two-sided Fisher’s exact test was used to estimate the differences between \nendometriosis and control groups. n: number of cases with relevant genotypes, OR: odds ratio, CI: confidence \ninterval, *versus controls, p < 0.05 and the statistical power was 68.6% calculated by G*Power.\n\n5Scientific  RepoRtS  |         (2020) 10:4897  | https://doi.org/10.1038/s41598-020-61702-y\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nassociation between the HLA-A*24, HLA-B*07:02, HLA-C*07:02, and HLA-DRB1*01:01 haplotypes and endome-\ntriosis in Japanese women52. A previous study showed that HLA-DRB1 alleles were not associated with endome-\ntriosis in Polish women53. A literature search identified similar reports from China, which showed an association \nbetween endometriosis and the HLA-B*46, HLA-DRB1*15, and HLA-DQA1*0401 alleles54–56. The reasons under-\nlying the discrepancies observed among these studies are unclear; however, the results may have been influenced \nby differences in the ethnicities of the women in the study cohorts and the differences in the detection methods.\nThe frequency of KIR3DS1 was significantly lower in endometriosis patients compared to control patients57. \nMoreover, the protective effect of the KIR2DS5 gene was observed in endometriosis patients58. Moreover, Nowak \net al. showed that the protective effect of KIR2DS5 was present only in the women who harbored the HLA-C C2 \ngroup59. Our current findings revealed that a lower proportion of endometriosis groups, which was characterized \nby the presence of activating KIR2DS2 compared to the control groups (Table 4). Previous studies have shown \nthat KIR2DL2 is in the linkage disequilibrium with KIR2DS2, which caused the relative activation of KIR recep-\ntor, which is responsible for the loss of recognition of HLA-C\n60–62. The different ethnic populations showed dif-\nferent values of KIR polymorphisms in elucidating genetic relationships among human populations63. Moreover, \nHLA genotyping is traditionally performed using a serological method. Therefore, these discrepancies can be \ninfluenced by ethnic or assay methods as they do for discrepancies among HLA alleles.\nNK cell activity has been reported to be a crucial factor in the recognition and lysis of endometrial cells. NK \ncell activity and quantity were found to be controversial in women with endometriosis relative to controls\n15,64–68. \nThe observed increase in the proportion of CD158a+ (KIR2DL1) NK cells in the peripheral blood and peritoneal \nfluid in endometriosis patients suggested reduced NK cell cytotoxicity in endometriosis69. Moreover, the decrease \nCentromeric\nControl (n = 117)\nEndometriosis \n(n = 147)\nOR 95% CI p valuen % n %\nCen-A/A 76 65.0 113 76.9 1.793 1.045 to 3.076 0.0394*\nCen-A/B 38 32.5 33 22.4 0.6018 0.3480 to 1.041 0.0713\nCen-B/B 3 2.6 1 0.7 0.2603 0.02670 to 2.537 0.3252\nχ 2 0.47 0.73\nTelomeric\nTel-A/A 71 60.7 96 65.3 1.22 0.7374 to 2.017 0.4442\nTel-A/B 42 35.9 47 32.0 0.8393 0.5026 to 1.402 0.5151\nTel-B/B 4 3.4 4 2.7 0.7902 0.1933 to 3.230 0.7358\nχ 2 0.55 0.39\nTable 5. Frequency of centromeric and telomeric KIR haplotypes in endometriosis and control groups. Two-\nsided Fisher’s exact test was used to estimate the differences between endometriosis and control groups. n: \nnumber of cases with relevant genotypes, OR: odds ratio, CI: confidence interval, χ2 value was calculated \nby Hardy-Weinberg analysis (χ2 > 3.841 showed the subgroup was deviating from the Hardy–Weinberg \nequilibrium). *versus controls, P < 0.05 and the statistical power was 68.6% calculated by G*Power.\nInhibitory KIR-ligand \nassociation\nControl Endometriosis\nOR 95% CI p valuen % n %\nKIR2DL1-HLA-C1/C2 27 100.0 35 100.0 — — —\nKIR2DL1-HLA-C2/C2 1 50.0 1 100.0 3 0.05947 to \n151.3 1\nKIR2DL2-HLA-C1/C1 25 28.4 20 18.0 0.5538 0.2834 to 1.083 0.0902\nKIR2DL2-HLA-C1/C2 10 37.0 11 31.4 0.7792 0.2704 to 2.245 0.7876\nKIR2DL3-HLA-C1/C1 86 97.7 110 99.1 2.558 0.2280 to 28.70 0.5847\nKIR2DL3-HLA-C1/C2 26 96.3 35 100.0 4.019 0.1573 to 102.7 0.4355\nActivating KIR-ligand association\nKIR2DS1-HLA-C1/C2 5 18.5 13 37.1 2.6 0.7918 to 8.538 0.1593\nKIR2DS1-HLA-C2/C2 2 100.0 0 0.0 0.0667 0.0008081 \nto 5.5 0.3333\nKIR2DS2-HLA-C1/C1 28 31.8 23 20.7 0.5601 0.2947 to 1.064 0.1016\nKIR2DS2-HLA-C1/C2 11 40.7 11 31.4 0.6667 0.2337 to 1.902 0.5932\nTable 6. Distribution of molecular interactions of KIR gene-HLA-ligands in endometriosis and control groups. \nThe molecular interactions of KIR gene-HLA-ligands were shown in the frequency of the KIR gene of HLA-\nligands, which was calculated from the KIR frequency of the total number of HLA ligands. The total number \nof HLA ligands is shown in Table 3. Two-sided Fisher’s exact test was used to estimate the differences between \nendometriosis and control groups. n: number of cases with relevant genotypes, OR: odds ratio, CI: confidence \ninterval.\n\n6Scientific  RepoRtS  |         (2020) 10:4897  | https://doi.org/10.1038/s41598-020-61702-y\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nin the proportion of NK cells facilitates endometrial cell invasion and persistent growth in endometriosis 70. \nMaeda et al. demonstrated that the increase in KIR2DL1 levels in women with pelvic endometriosis can inhibit \nNK cell activity66. However, the activation of chronic NK cell activation can also play a role in endometriosis71. \nThe upregulation of KIR2DS1 expression in peritoneal fluid has been detected in women with endometriosis72. \nRecently, bioinformatic analysis showed that KIR2DS2 expression is upregulated in the secretory phase of endo-\nmetrium in women with endometriosis relative to control women73. Thus, the regulation of NK cell activity is a \ncomplex process and can affect the pathogenesis of endometriosis74. The final activation status of functional NK \ncells depends on the homeostasis of all NK cell activation/inhibitory receptors and the corresponding ligands. \nThen, NK cells are regulated in endometrium in women with endometriosis. Further studies will be worth elu-\ncidating the functional relevance of the presence of these receptors and ligand proteins in the endometrium. The \nlimitation of this study is that only the stage III or stage IV endometriosis patients were enrolled to investigate the \ngenetic associations. Thus, our results did not show any association between genes and severity of disease. Further \nclinically relevant studies on the severity of disease and genetic associations are required.\nOur current findings demonstrated the association between KIR polymorphisms and HLA-C genotypes with \nendometriosis in women. It is the first study addressing KIR polymorphisms and HLA-C genotypes of women \nwith stage III or IV endometriosis in Han Chinese women. The results suggested that HLA-C and KIR genotypes \ninfluence the susceptibility for endometriosis. Further studies should investigate the role of NK cells in the patho-\ngenesis of endometriosis.\nMethods\npatients and controls. Han Chinese women were categorized into the endometriosis (n = 147) and control \n(n = 117) groups. Endometriosis was diagnosed via laparoscopic examination and confirmed via histological \nassessment. A total of 147 women were classified under stage III or IV endometriosis in accordance with the \nRevised American Society for Reproductive Medicine Classification. Women in the control group underwent \nbenign gynecological surgery and showed no evidence of endometriosis including myoma, teratoma, serous cys-\ntadenoma, ovarian cyst, ovarian stroma, dermoid cyst, mucinous cystadenoma, paratubal cyst, follicular cyst, \nsimple cyst, hydrosalpinx, corpus luteum cyst, fibrous adhesion, and struma ovarii. Considering that autoimmune \ndisorders are associated with HLA-C alleles and KIR genotypes, the exclusion criteria comprised autoimmune \ndisorders. The protocol was approved by the Institutional Review Board of the Taipei Medical University Hospital, \nand all participants have informed consent. All experiments were performed in accordance with relevant guide-\nlines and regulations.\nDnA extraction and HLA-C and KIR genotype analysis.  Genomic DNA was extracted using a DNA \nwhole-blood kit following the manufacturer’s instructions (Kurabo Industries, Osaka, Japan). HLA-C was gen-\notyped using an HLAssure™ SE sequence-based typing kit (TBG Biotechnology Corp, Queensland, Australia), \nwhich was designed to determine HLA-C alleles via polymerase chain reaction (PCR) amplification using a \nKIR haplotypes\nHLA-C \ngenotypes\nControl Endometriosis\nOR 95% CI P valuen % n %\nCen-A/A C1/C1 60 68.2 88 79.3 1.786 0.9397 to 3.393 0.1016\nCen-A/A C1/C2 16 59.3 24 68.6 1.5 0.5257 to 4.280 0.5932\nCen-A/A C2/C2 0 0.0 1 100.0 15 0.1818 to 1238 0.3333\nCen-A/B C1/C1 26 29.5 22 19.8 0.5895 0.3065 to 1.134 0.1338\nCen-A/B C1/C2 10 37.0 11 31.4 0.7792 0.2704 to 2.245 0.7876\nCen-A/B C2/C2 2 100.0 0 0.0 0.0667 0.0008081 to 5.5 0.3333\nCen-B/B C1/C1 2 2.3 1 0.9 0.3909 0.03484 to 4.385 0.5847\nCen-B/B C1/C2 1 3.7 0 0.0 0.2488 0.009738 to 6.358 0.4355\nCen-B/B C2/C2 0 0.0 0 0.0 — — —\nTel-A/A C1/C1 50 56.8 74 66.7 1.52 0.8529 to 2.709 0.1853\nTel-A/A C1/C2 21 77.8 21 60.0 0.4286 0.1382 to 1.329 0.1758\nTel-A/A C2/C2 0 0.0 1 100.0 15 0.1818 to 1238 0.3333\nTel-A/B C1/C1 35 39.8 33 29.7 0.6407 0.3551 to 1.156 0.1755\nTel-A/B C1/C2 6 22.2 14 40.0 2.333 0.7523 to 7.237 0.1758\nTel-A/B C2/C2 1 50.0 0 0.0 0.3333 0.0066 to 16.82 1\nTel-B/B C1/C1 3 3.4 4 3.6 1.059 0.2307 to 4.863 1\nTel-B/B C1/C2 0 0.0 0 0.0 — — —\nTel-B/B C2/C2 1 50.0 0 0.0 0.3333 0.0066 to 16.82 1\nTable 7. Distribution of molecular interactions of KIR haplotypes-HLA-ligands in endometriosis and control \ngroups. The molecular interactions of KIR haplotypes-HLA-ligands were shown in the frequency of KIR \nhaplotypes of HLA-ligands, which was calculated from the KIR haplotypes frequency of the total number of \nHLA ligands. The total number of HLA ligands is shown in Table 3. Two-sided Fisher’s exact test was used to \nestimate the differences between endometriosis and control groups. n: number of cases with relevant genotypes, \nOR: odds ratio, CI: confidence interval.\n\n7Scientific  RepoRtS  |         (2020) 10:4897  | https://doi.org/10.1038/s41598-020-61702-y\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nsequence-based typing method. Sequence data were processed using allele-typing software (AccuType™) to iden-\ntify the HLA-C alleles. The genotypes of the KIR genes were analyzed using the Lifecodes KIR-sequence-specific \noligonucleotide (SSO) typing kit (Immucor Transplant Diagnostics, Inc., Stamford, USA) to identify the KIR  \nloci amplified in the sample. The presence or absence of the 16 KIR genes was determined using 20 different \noligonucleotide probes targeting known KIR genes (KIR3DL3 as positive control, KIR2DL1, KIR2DL2*001-3/5, \nKIR2DL2* 004, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DP1, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2, \nKIR2DS3, KIR2DS4*whole exon 4, KIR2DS4* whole exon 5, KIR2DS4* -deleted exon 5, KIR2DS5, KIR3DS1, \nKIR3DS1*049N, and KIR3DP1). The amplicons were analyzed on a Luminex instrument according to the manu-\nfacturer’s instructions. The characteristics of full-length and truncated forms of KIR2DS4 were determined using \nthe following three probes; probe 45: KIR2DS4* all full-length, probe 175: 2DS4* full-length Exon 5, and probe \n234: 2DS4*deletion Exon 5. KIR genes are divided into centromeric and telomeric haplotypes\n75. In short, centro-\nmeric A/A haplotypes contained KIR2DL3 but not with KIR2DL2 and/or KIR2DS2, centromeric A/B haplotypes \ncontained KIR2DL3 with KIR2DL2 and/or KIR2DS2, and centromeric B/B haplotypes contained KIR2DL2 and/\nor KIR2DS2 but not KIR2DL3. Meanwhile, telomeric A/A haplotypes contained KIR3DL1 and KIR2DS4 but \nnot KIR3DS1 or KIR2DS1, telomeric A/B haplotypes contained KIR3DL1 and KIR2DS4 with KIR3DS1 and/or \nKIR2DS1, and telomeric B/B haplotypes lacked KIR3DL1 and/or KIR2DS4\n76.\nStatistical analyses.  HLA-C allele frequencies, the genotypes of the KIR genes and KIR-HLA-C pair fre-\nquency in endometriosis patients and control women were compared using the Fisher’s exact test. Odds ratios \n(ORs) and 95% confidence intervals (CIs) were calculated using the GraphPad Prism software (California, USA). \nP value <0.05 was considered statistically significant. Multiple tests were analyzed by the Bonferroni correc-\ntion using the GraphPad Prism software. The normality was analyzed by the Kolmogorov–Smirnov test using \nIBM SPSS statistics version 22 (New Y ork, USA). The continuous variables of patient demographic results were \nanalyzed by the Mann–Whitney test using the GraphPad Prism software. The discontinuous variable of dys-\nmenorrhea was analyzed by χ\n2 test using the GraphPad Prism software. The statistical power was analyzed by \nthe G*Power version 3.1.9.477. The χ2 value was calculated by Hardy–Weinberg analysis (χ 2 > 3.841 showed the \nsubgroup was deviating from the Hardy–Weinberg equilibrium).\nReceived: 26 September 2019; Accepted: 24 February 2020;\nPublished: xx xx xxxx\nReferences\n 1. Rogers, P . A. et al. Priorities for endometriosis research: recommendations from an international consensus workshop. Reprod. Sci. \n16, 335–346, https://doi.org/10.1177/1933719108330568 (2009).\n 2. Giudice, L. C. Clinical practice. 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Methods 41, 1149–1160, https://doi.org/10.3758/BRM.41.4.1149 (2009).\nAcknowledgements\nThis work was supported by the Ministry of Science and Technology (grant number 104-2314-B-038-\n063-MY2, grant number 106-2314-B-038-072, grant number 107-2314-B-038-006, grant number 108-2314-\nB-038-003), Academia Sinica (grant number BM10501010036, grant number BM10601010024, grant number \nBM10701010027), National Health Research Institute (grant number MG-105-SP-07, grant number MG-\n106-SP-07, grant number MG-107-SP-07) (CRT), and Ministry of Science and Technology (grant number 107-\n2314-B-009-006) (YCC). This work was financially supported by the Center for Intelligent Drug Systems and \nSmart Bio-devices (IDS\n2B) from The Featured Areas Research Center Program within the framework of the \nHigher Education Sprout Project by the Ministry of Education (MOE) in Taiwan.\nAuthor contributions\nY .-C.C. designed the study, performed experiments, analysed the data and wrote the manuscripts, C.-H.C., \nM.-J.C., C.-W .C., P .-H.C, M.-H.Y ., Y .-J.C., E.-M.T., P .-S.Y . and S.-Y .L. enrolled patients, and C.-R.T. guided the \nexperimental design, enrolled patients and wrote the manuscripts.\ncompeting interests\nThe authors declare no competing interests.\nAdditional information\nCorrespondence and requests for materials should be addressed to C.-R.T.\nReprints and permissions information is available at www.nature.com/reprints.\nPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and \ninstitutional affiliations.\nOpen Access This article is licensed under a Creative Commons Attribution 4.0 International \nLicense, which permits use, sharing, adaptation, distribution and reproduction in any medium or \nformat, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre-\native Commons license, and indicate if changes were made. The images or other third party material in this \narticle are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the \nmaterial. If material is not included in the article’s Creative Commons license and your intended use is not per-\nmitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the \ncopyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.\n \n© The Author(s) 2020","source_license":"CC0","license_restricted":false}