Introduction

Endometriosis is a chronic gynecological disease with unknown etiology and is characterized by extra-uterine growth of endometrial tissue1. Endometriosis affects 6% to 10% of fertile women at the reproductive age and causes severe pelvic pain and infertility2,3,4. Familial and twin studies have reported that genetic factors are associated with the pathogenesis of endometriosis5,6,7,8,9. Cell-mediated and humoral immune responses are essential in the pathogenesis of endometriosis, since it is associated with various immunological abnormalities, particularly cell-mediated immunity10,11,12. The activities of cytotoxic T-cells and natural killer (NK) cells are dysregulated in women with endometriosis13,14,15,16. Increased serum levels of immunoglobulins and autoantibodies, decreased endometrial cell apoptosis, and the production of pro-inflammatory cytokines are observed in endometriosis patients, indicating that endometriosis shares many similarities with autoimmune diseases11,12,17,18.

Major histocompatibility complex (MHC) genes, also known as human leukocyte antigen (HLA) genes, are located in chromosome 6p. The genes encoding the human MHC class I (HLA-A, HLA-B, and HLA-C) and class II (HLA-DR, HLA-DQ, and HLA-DP) molecules are the most polymorphic loci in the human genome. HLA genes are polymorphic in binding and function in presenting antigen peptides to T-cells. HLA molecules are key factors involved in regulating the specificity of T-cell-mediated immune response in autoimmune and infectious diseases19,20,21.

HLA Class I genes encode cell-surface proteins, whose primary functions are to present antigens to cytotoxic CD8+ T-cells during the early immune responses19,20,21. Among these, HLA-C plays a minor role in regulating antigen-specific T-cell responses because of low cell surface expression22. HLA-C acts as a ligand for killer cell immunoglobulin-like receptors (KIRs), which regulate natural killer (NK) cell-mediated cytotoxicity. The human immunodeficiency virus Nef protein selectively downregulates the production of HLA-A and HLA-B molecules to suppress cytotoxic CD8+ T lymphocyte responses23. However, Nef maintains stable HLA-C expression levels to inhibit NK cell activation and renders HLA-C as a T-cell restriction element during HIV infection24. Importantly, the HLA-C genotype has been implicated in several autoimmune diseases, including Graves’ disease, psoriasis, and Crohn’s disease20,22,23,25,26.

NK cells are lymphocytes that serve as vital components of the immune system by regulating early responses against infected or transformed cells via cytokine production and direct cytotoxicity27. KIRs are a family of membrane glycoproteins expressed by NK cells. KIRs contain two or three extracellular immunoglobulin-like domain molecules (D) with a long (L) or short (S) cytoplasmic tail28. The KIR gene is located on chromosome 19q13.4 on the leukocyte receptor complex. KIR exhibits activating and inhibitory effects with extensive haplotypic and allelic polymorphisms29,30,31. The 16 KIR genes comprise the following: six genes encoding activating KIR (2DS1-5 and 3DS1), seven genes encoding inhibitory KIR (2DL1-3, 5 and 3DL1-2), KIR2DL4, which can exert both inhibitory and activating activity, and two pseudogenes (2DP1 and 3DP1). Furthermore, KIR3DL3, KIR3DP1, KIR2DL4, and KIR3DL2 are framework genes and are always present in the genome32.

The primary ligands of KIR are HLA-C molecules, which are divided into two groups, namely C1 and C2, based on the amino acid at position 80 [HLA-C C1 groups (HLA-C1), asparagine (N) at position 80: C*01, 03, 07 (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: C*02, 04, 05, 06, 07 (07), 12 (04, 05, 42), 15, 16 (02), 17, 18]32,33. The inhibitory receptors KIR2DL2 and KIR2DL3 and activating receptor KIR2DS2 share the same ligand HLA-C1. Activating KIR2DS2 has been reported to be in strong linkage disequilibrium and highly homologous to KIR2DL2. KIR2DL1 and KIR2DS1 bind to HLA-C23134,35,36. Combinations of HLA-C with KIR2DS1 and KIR2DS2 have been reported to correlate with the occurrence of autoimmune diseases, leukemia, and inflammatory diseases37,38,39,40,41,42. Polymorphisms in the genes encoding HLA-C and KIR affect NK cells activity and susceptibility to several diseases31. HLA genotyping is traditionally performed using a serological method. However, detection of the HLA-C genotype via serological typing is difficult because of the low HLA-C expression levels at the cell surface, the lack of suitable antisera, and difficulties in protein isolation22. Therefore, we employed a DNA-based method for higher-resolution genotyping and investigated the association between the HLA-C genotype and endometriosis. Moreover, to analyze the association between certain KIR-HLA-C combinations and the development of endometriosis, we characterized both KIR and HLA-C gene polymorphisms in 147 women with endometriosis and 117 controls.

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.

Table 1 Patient demographic results.
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Table 2 Distribution of the HLA-C alleles in the endometriosis and control groups.
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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 endometriosis. Analysis revealed no significant differences in HLA-C1 and HLA-C2 frequencies in the endometriosis and control groups (Table 3).

Table 3 Distribution of HLA-C ligand in endometriosis and control groups.
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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 4 Genetic association between KIR gene in endometriosis and control groups.
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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 χ2 value was calculated by Hardy-Weinberg analysis (χ2 > 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%.

Table 5 Frequency of centromeric and telomeric KIR haplotypes in endometriosis and control groups.
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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-C231,34,35,36. 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).

Table 6 Distribution of molecular interactions of KIR gene-HLA-ligands in endometriosis and control groups.
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Table 7 Distribution of molecular interactions of KIR haplotypes-HLA-ligands in endometriosis and control groups.
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Discussion

Several factors are involved in the pathogenesis of endometriosis including genetic, neuroendocrine, and immunological factors43,44,45. Abnormal immune responses are recognized in endometriosis patients, including excessive inflammatory cytokine secretion, autoantibody production, and NK cell regulation17,18. Endometriosis shares similar characteristics with autoimmune diseases11,12,17,18. HLA-C affects viral infections and is implicated in several human autoimmune diseases22. 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 disease20. 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 typing46,47,48. A serological study showed increased frequencies of the HLA-B*54 and HLA-C*07 alleles in Japanese patients with endometriosis49. 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 women50,51. Another study reported an association between the HLA-A*24, HLA-B*07:02, HLA-C*07:02, and HLA-DRB1*01:01 haplotypes and endometriosis in Japanese women52. A previous study showed that HLA-DRB1 alleles were not associated with endometriosis in Polish women53. 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 alleles54,55,56. 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 patients57. Moreover, the protective effect of the KIR2DS5 gene was observed in endometriosis patients58. Moreover, Nowak et al. showed that the protective effect of KIR2DS5 was present only in the women who harbored the HLA-C C2 group59. 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-C60,61,62. The different ethnic populations showed different values of KIR polymorphisms in elucidating genetic relationships among human populations63. 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 controls15,64,65,66,67,68. 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 endometriosis69. Moreover, the decrease in the proportion of NK cells facilitates endometrial cell invasion and persistent growth in endometriosis70. Maeda et al. demonstrated that the increase in KIR2DL1 levels in women with pelvic endometriosis can inhibit NK cell activity66. However, the activation of chronic NK cell activation can also play a role in endometriosis71. The upregulation of KIR2DS1 expression in peritoneal fluid has been detected in women with endometriosis72. Recently, bioinformatic analysis showed that KIR2DS2 expression is upregulated in the secretory phase of endometrium in women with endometriosis relative to control women73. Thus, the regulation of NK cell activity is a complex process and can affect the pathogenesis of endometriosis74. 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.

Methods

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 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 haplotypes75. 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 KIR2DS476.

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 χ2 test using the GraphPad Prism software. The statistical power was analyzed by the G*Power version 3.1.9.477. The χ2 value was calculated by Hardy–Weinberg analysis (χ2 > 3.841 showed the subgroup was deviating from the Hardy–Weinberg equilibrium).