Killer cell immunoglobulin-like receptors (KIR) and human leukocyte antigen-C (HLA-C) allorecognition patterns in women with endometriosis

Endometriosis shares similarities with several autoimmune diseases. The human leukocyte antigen (HLA)-C genotype is associated with several human autoimmune diseases. HLA-C is a ligand of killer cell immunoglobulin receptors (KIRs) and is an essential regulator of natural killer cell activity, which is associated with endometriosis progression. Polymorphisms in HLA-C and KIR affect the activity of NK cells and susceptibility to several diseases. Therefore, we attempted to investigate an association between HLA-C genotype and KIR polymorphism and the occurrence of endometriosis. We tested the association of certain KIR and HLA-C combinations and the development of endometriosis by characterizing both KIR and HLA-C genes in 147 women with endometriosis and 117 controls. The HLA-C genotypes and KIR polymorphisms were analyzed via DNA-based method for higher-resolution genotyping. We found that the occurrence of HLA-C*03:03*01 was increased in endometriosis than in control groups. Analysis of various KIR haplotypes revealed differences between the endometriosis and control cohorts. The number of KIR centromeric A/A haplotypes was increased in the endometriosis group than controls. Moreover, the endometriosis cohort was characterized by reduced number of KIR2DS2-positive individuals in the Han Chinese population. Our current findings suggest that the KIR and HLA-C genotypes are associated with the pathogenesis of endometriosis.


Results
frequency distributions of HLA-C alleles among endometriosis and control groups. The demographic results of endometriosis and control groups are shown in Table 1. HLA-C allele frequencies in endometriosis patients (n = 147, 294 alleles) and control patients (n = 117, 234 alleles) were determined using a sequence-based typing method. The presence of HLA-C*03:03:01 significantly increased the risk of endometriosis with p = 0.0473 [Odds Ratio (OR) = 2.811, 95% confidence interval (CI) = 1.021-7.738] and the statistical power was 43.8% (Table 2). After multiple test analyses using Bonferroni correction, the association was not significant. frequency distributions of HLA-C group among endometriosis and control groups. We evaluated whether the HLA-C group C1 (HLA-C1) and HLA-C group C2 (HLA-C2) were associated with frequency distributions of KIR genotypes among endometriosis and control groups. Using sequence-specific PCR amplification, we analyzed the KIR genotypes in the endometriosis and control groups. The frequencies of the KIR genotypes in women with endometriosis and controls and their statistical associations are presented in Table 4. The presence of KIR2DS2 significantly reduced the risk of endometriosis with p = 0.0394 [(OR) = 0.5577, 95% CI = 0.3251-0.9569] and the statistical power was 68.6%. After multiple test analyses using Bonferroni correction, the association was not significant. The two groups showed no significant differences in the remaining KIR genotypes.  Table 2. Distribution of the HLA-C alleles in the endometriosis and control groups. Each HLA allele has four unique sets denoted by different numbers that are separated by a colon. The first two digits often correspond to the serological antigen; the two digits after the first colon denote the subtypes and order in the genome from the IMGT/HLA Database (www.ebi.ac.uk/imgt/hla/). The differences in HLA-C allele frequencies between the endometriosis and control groups were analyzed using the Fisher's exact test. Significance was set at a P value < 0.05 and the statistical power was 43.8% calculated by G*Power. OR indicates odds ratio. CI indicates confidence interval.  www.nature.com/scientificreports www.nature.com/scientificreports/ frequency distributions of KIR haplotypes among endometriosis and control groups. The frequencies of the KIR haplotypes in women with endometriosis and controls and their statistical associations are presented in Table 5. The χ 2 value was calculated by Hardy-Weinberg analysis (χ 2 > 3.841 showed the subgroup was deviating from the Hardy-Weinberg equilibrium). We revealed differences between the endometriosis and control cohorts. The number of KIR centromeric A/A haplotypes was increased in the endometriosis group than controls with p = 0.0394 [(OR) = 1.793, 95% CI = 1.045-3.076] and the statistical power was 68.6%.

combinations of KiR and their HLA-c ligands. The frequencies of KIR and their HLA-C ligands
were analyzed for their statistical associations with endometriosis (Table 6). HLA-C C1 groups are recognized by KIR2DL2/2DS2 and KIR2DL3, while HLA-C C2 groups are recognized by KIR2DL1/2DS1. Moreover, KIR2DL2/2DL3 also binds to some HLA-C C2 molecules, and KIR2DS4 binds to some HLA-C1 and HLA-C2 31,34-36 . The molecular interactions of KIR gene-HLA-ligands were calculated from the KIR frequency of a total number of HLA ligands. The total number of HLA ligands is shown in Table 3. We calculated the KIR frequency in the combination of different HA ligands. Analysis of various KIR-HLA-C combinations revealed no significant differences between the endometriosis and control cohorts ( Table 6). The frequency of KIR haplotypes and HLA-C combinations also showed no significant differences between the endometriosis and control groups (Table 7).

Discussion
Several factors are involved in the pathogenesis of endometriosis including genetic, neuroendocrine, and immunological factors [43][44][45] . Abnormal immune responses are recognized in endometriosis patients, including excessive inflammatory cytokine secretion, autoantibody production, and NK cell regulation 17,18 . Endometriosis shares similar characteristics with autoimmune diseases 11,12,17,18 . HLA-C affects viral infections and is implicated in several human autoimmune diseases 22 . HLA-C*06:02 is associated with severe early-onset psoriasis. HLA-C *12:02 was found to be associated with increased susceptibility to Crohn's disease 20 . HLA-C*03 restricts the cytotoxic CD8 + T-cell responses during the Epstein-Barr virus and human immunodeficiency virus infections, as well as during co-infection with the influenza virus and the Sendai virus. Herein, we analyzed the associations between HLA-C alleles and endometriosis. Consequently, the presence of HLA-C*03:03:01 increased the risk of endometriosis in Asian women (Table 2). However, after multiple test analyses using Bonferroni correction, the association was not significant.
Previous studies reported no association between HLA genotypes and endometriosis in Caucasian women with endometriosis and controls, as assessed by serological typing [46][47][48] . A serological study showed increased frequencies of the HLA-B*54 and HLA-C*07 alleles in Japanese patients with endometriosis 49 . In a recent study, PCR-restriction fragment length polymorphism analysis revealed that the HLA-DRB1*14:03 and HLA-DQB1*03:01 alleles are associated with endometriosis in Japanese women 50  www.nature.com/scientificreports www.nature.com/scientificreports/ association between the HLA-A*24, HLA-B*07:02, HLA-C*07:02, and HLA-DRB1*01:01 haplotypes and endometriosis in Japanese women 52 . A previous study showed that HLA-DRB1 alleles were not associated with endometriosis in Polish women 53 . A literature search identified similar reports from China, which showed an association between endometriosis and the HLA-B*46, HLA-DRB1*15, and HLA-DQA1*0401 alleles [54][55][56] . The reasons underlying the discrepancies observed among these studies are unclear; however, the results may have been influenced by differences in the ethnicities of the women in the study cohorts and the differences in the detection methods.
The frequency of KIR3DS1 was significantly lower in endometriosis patients compared to control patients 57 . Moreover, the protective effect of the KIR2DS5 gene was observed in endometriosis patients 58 . Moreover, Nowak et al. showed that the protective effect of KIR2DS5 was present only in the women who harbored the HLA-C C2 group 59 . Our current findings revealed that a lower proportion of endometriosis groups, which was characterized by the presence of activating KIR2DS2 compared to the control groups (Table 4). Previous studies have shown that KIR2DL2 is in the linkage disequilibrium with KIR2DS2, which caused the relative activation of KIR receptor, which is responsible for the loss of recognition of HLA-C 60-62 . The different ethnic populations showed different values of KIR polymorphisms in elucidating genetic relationships among human populations 63 . Moreover, HLA genotyping is traditionally performed using a serological method. Therefore, these discrepancies can be influenced by ethnic or assay methods as they do for discrepancies among HLA alleles.
NK cell activity has been reported to be a crucial factor in the recognition and lysis of endometrial cells. NK cell activity and quantity were found to be controversial in women with endometriosis relative to controls 15,64-68 . The observed increase in the proportion of CD158a + (KIR2DL1) NK cells in the peripheral blood and peritoneal fluid in endometriosis patients suggested reduced NK cell cytotoxicity in endometriosis 69 Table 6. Distribution of molecular interactions of KIR gene-HLA-ligands in endometriosis and control groups. The molecular interactions of KIR gene-HLA-ligands were shown in the frequency of the KIR gene of HLAligands, which was calculated from the KIR frequency of the total number of HLA ligands. The total number of HLA ligands is shown in Table 3. Two-sided Fisher's exact test was used to estimate the differences between endometriosis and control groups. n: number of cases with relevant genotypes, OR: odds ratio, CI: confidence interval. (2020) 10:4897 | https://doi.org/10.1038/s41598-020-61702-y www.nature.com/scientificreports www.nature.com/scientificreports/ in the proportion of NK cells facilitates endometrial cell invasion and persistent growth in endometriosis 70 . Maeda et al. demonstrated that the increase in KIR2DL1 levels in women with pelvic endometriosis can inhibit NK cell activity 66 . However, the activation of chronic NK cell activation can also play a role in endometriosis 71 . The upregulation of KIR2DS1 expression in peritoneal fluid has been detected in women with endometriosis 72 . Recently, bioinformatic analysis showed that KIR2DS2 expression is upregulated in the secretory phase of endometrium in women with endometriosis relative to control women 73 . Thus, the regulation of NK cell activity is a complex process and can affect the pathogenesis of endometriosis 74 . The final activation status of functional NK cells depends on the homeostasis of all NK cell activation/inhibitory receptors and the corresponding ligands. Then, NK cells are regulated in endometrium in women with endometriosis. Further studies will be worth elucidating the functional relevance of the presence of these receptors and ligand proteins in the endometrium. The limitation of this study is that only the stage III or stage IV endometriosis patients were enrolled to investigate the genetic associations. Thus, our results did not show any association between genes and severity of disease. Further clinically relevant studies on the severity of disease and genetic associations are required.
Our current findings demonstrated the association between KIR polymorphisms and HLA-C genotypes with endometriosis in women. It is the first study addressing KIR polymorphisms and HLA-C genotypes of women with stage III or IV endometriosis in Han Chinese women. The results suggested that HLA-C and KIR genotypes influence the susceptibility for endometriosis. Further studies should investigate the role of NK cells in the pathogenesis of endometriosis.

patients and controls.
Han Chinese women were categorized into the endometriosis (n = 147) and control (n = 117) groups. Endometriosis was diagnosed via laparoscopic examination and confirmed via histological assessment. A total of 147 women were classified under stage III or IV endometriosis in accordance with the Revised American Society for Reproductive Medicine Classification. Women in the control group underwent benign gynecological surgery and showed no evidence of endometriosis including myoma, teratoma, serous cystadenoma, ovarian cyst, ovarian stroma, dermoid cyst, mucinous cystadenoma, paratubal cyst, follicular cyst, simple cyst, hydrosalpinx, corpus luteum cyst, fibrous adhesion, and struma ovarii. Considering that autoimmune disorders are associated with HLA-C alleles and KIR genotypes, the exclusion criteria comprised autoimmune disorders. The protocol was approved by the Institutional Review Board of the Taipei Medical University Hospital, and all participants have informed consent. All experiments were performed in accordance with relevant guidelines and regulations.
DnA extraction and HLA-C and KIR genotype analysis. Genomic DNA was extracted using a DNA whole-blood kit following the manufacturer's instructions (Kurabo Industries, Osaka, Japan). HLA-C was genotyped using an HLAssure ™ SE sequence-based typing kit (TBG Biotechnology Corp, Queensland, Australia), which was designed to determine HLA-C alleles via polymerase chain reaction (PCR) amplification using a www.nature.com/scientificreports www.nature.com/scientificreports/ sequence-based typing method. Sequence data were processed using allele-typing software (AccuType ™ ) to identify the HLA-C alleles. The genotypes of the KIR genes were analyzed using the Lifecodes KIR-sequence-specific oligonucleotide (SSO) typing kit (Immucor Transplant Diagnostics, Inc., Stamford, USA) to identify the KIR loci amplified in the sample. The presence or absence of the 16 KIR genes was determined using 20 different oligonucleotide probes targeting known KIR genes (KIR3DL3 as positive control, KIR2DL1, KIR2DL2*001-3/5,  KIR2DL2*004, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DP1, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4*whole exon 4, KIR2DS4*whole exon 5, KIR2DS4*-deleted exon 5, KIR2DS5, KIR3DS1, KIR3DS1*049N, and KIR3DP1). The amplicons were analyzed on a Luminex instrument according to the manufacturer's instructions. The characteristics of full-length and truncated forms of KIR2DS4 were determined using the following three probes; probe 45: KIR2DS4*all full-length, probe 175: 2DS4*full-length Exon 5, and probe 234: 2DS4*deletion Exon 5. KIR genes are divided into centromeric and telomeric haplotypes 75 . In short, centromeric A/A haplotypes contained KIR2DL3 but not with KIR2DL2 and/or KIR2DS2, centromeric A/B haplotypes contained KIR2DL3 with KIR2DL2 and/or KIR2DS2, and centromeric B/B haplotypes contained KIR2DL2 and/ or KIR2DS2 but not KIR2DL3. Meanwhile, telomeric A/A haplotypes contained KIR3DL1 and KIR2DS4 but not KIR3DS1 or KIR2DS1, telomeric A/B haplotypes contained KIR3DL1 and KIR2DS4 with KIR3DS1 and/or KIR2DS1, and telomeric B/B haplotypes lacked KIR3DL1 and/or KIR2DS4 76 .
Statistical analyses. HLA-C allele frequencies, the genotypes of the KIR genes and KIR-HLA-C pair frequency in endometriosis patients and control women were compared using the Fisher's exact test. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using the GraphPad Prism software (California, USA). P value <0.05 was considered statistically significant. Multiple tests were analyzed by the Bonferroni correction using the GraphPad Prism software. The normality was analyzed by the Kolmogorov-Smirnov test using IBM SPSS statistics version 22 (New York, USA). The continuous variables of patient demographic results were analyzed by the Mann-Whitney test using the GraphPad Prism software. The discontinuous variable of dysmenorrhea was analyzed by χ 2 test using the GraphPad Prism software. The statistical power was analyzed by the G*Power version 3.1.9.4 77 . The χ 2 value was calculated by Hardy-Weinberg analysis (χ 2 > 3.841 showed the subgroup was deviating from the Hardy-Weinberg equilibrium).