{"paper_id":"acc7982e-c8c6-4698-bad6-eafff2438907","body_text":"Endometriosis is an inflammatory, estrogen-dependent\ndisease that is characterized by presence of ectopic\nendometrial-like tissue outside of the uterine cavity ( 1 ,\n 2 ). Endometriosis is associated with pelvic pain and\ninfertility in most patients ( 2 ). Several theories explained\nthe pathogenesis of the condition ( 3 ), but the Sampson’s\nhypothesis is the most-widely accepted one which\nsuggested a retrograde movement of endometrial cells\nvia the fallopian tubes into the peritoneal cavity during\nmenstruation ( 4 ). It seems that approximately 90%\nof women possess retrograde menstruation; however,\nrefluxed endometrial cells are usually cleared by\nmacrophages, natural killer (NK) cells, and lymphocytes\nbut in endometriosis patients, a combination of impaired\nimmunological clearance and aberrant cytokine\nexpression interferes with clearance of the ectopic lesions\nleading to establishment and development of the disease\n( 5 ). The impaired immune response is related to reduced\ncytotoxic activity of NK cells, increased number of T\ncells and accumulation of activated macrophages ( 6 ,  7 ).\nMacrophage migration inhibitory factor (MIF) is\na pleiotropic pro-inflammatory cytokine ( 8 ,  9 ) that\nis produced by T lymphocytes and accumulative\nmacrophages and activates several molecular\npathways in the ectopic endometrial tissue ( 10 ). The\nMIF-induced extracellular mitogen-activated (MAP)\nkinase pathway causes an increase in prostaglandin\nE2 (PGE2) and estrogen, also negatively regulates\np53 and promotes apoptosis; thus, its inhibition may\nenhance proliferation ( 11 ,  12 ). Several studies as well\nas our group suggested that mRNA and plasma levels\nof MIF are increased in the ectopic and eutopic tissues\nof endometriosis patients ( 12 - 15 ) but no genetic\nvariation was described.\nThe  MIF  gene is located in the chromosome\n22q11.2 region and consists of three exons ( 16 ).\nPolymorphisms with potential functional relevance\nwere also identified in the  MIF  promoter ( 17 ); a single\nnucleotide polymorphism (SNP) at position -173G/C\n(rs755622) and a short tandem repeat polymorphism\n(STRP), -794 (CATT) 5–8  (rs5844572), were shown by\nseveral meta-analyses to increase susceptibility some\nimmune and inflammatory diseases and their severity\n( 18 - 21 ).\nSince endometriosis is an inflammatory disorder and\nincreased levels of  MIF  are observed in ectopic tissues,\nwe aimed to evaluate  MIF  promoter variations that\ncould be involved in development of endometriosis and\nsusceptibility towards this disorder. Also,  MIF  mRNA\nexpression levels in ectopic tissues from patients with\nendometriosis who carried different genotypes for the\ntwo promoter variations were determined.\n\nThis case-control study was approved by Ethics Committee\nof Royan Institute (No.EC/91/1137) and each participant\nsigned an informed consent form. The stage of endometriosis\nlesions was categorized based on the revised classification\nof the American Fertility Society (rAFS). In the current\nstudy, 106 patients with diagnosed endometriosis, who\nhad undergone laparoscopy from 2015 to 2017 and had\nendometrioma cysts confirmed by histological tests, were\nenrolled. The endometrioma tissues were collected during\nlaparoscopic surgery in the secretory phases of menstrual\ncycle (days 16-19). The 110 controls were recruited from\nsubjects who were not diagnosed with endometriosis,\nunderwent diagnostic laparoscopy or fertile women with no\nsign of endometriosis in Doppler ultrasonography. None of\nthe participants had endometrial hyperplasia, neoplasia, or\ninflammatory and autoimmune disorders, and none of them\nwere receiving anti-inflammatory or hormonal medication\nfor at least 3 months before laparoscopy. The subjects’ age\nwas between 20 and 40 years old and the control individuals\nwere matched with the endometriosis patients in terms of\nbody mass index (BMI).\nDNA was extracted from whole blood anticoagulated\nwith ethylenediamine tetraacetic acid (EDTA)-2Na.\nPatients’ genomic DNA was extracted by using the\nGene All® kit (Korea), according to the manufacturer’s\ninstructions. Salting-out method was used to obtain the\ncontrols’ genomic DNA.\nThe polymerase chain reaction (PCR) was used to\namplify the studied fragments of MIF gene. The PCR\nincluded a hot start at 95˚C for 5 minutes, followed by 35\nPCR cycles, each including denaturation for 30 seconds\nat 94˚C, primer annealing (depending on the primer pairs)\nfor 30 seconds, and extension for 60 seconds at 72˚C. A\nfinal extension step was conducted at 72˚C for 10 minutes.\nRestriction fragment length polymorphism (RFLP)\nwas performed to detect -173G/C SNP. PCR was used to\namplify a 303 bp fragment.\nSense primer was:\n5´- CCT-CCT-GGC-GAC-TAA-CAT-CGG-TGA-CT-3´\nand the anti-sense primer was:\n5´- TAC-GTG-CCT-GAC-TTC-TCG-GAC-ACC-ACT -3´\nThe annealing temperature was set at 63˚C. The\nresulting fragment was digested using AluI restriction\nendonuclease (Fermentase Biolabs, MA, USA) for\n15 minutes at 37˚C, and the digested fragments were\nresolved using 1.7% agarose gel stained with ethidium\nbromide, and visualized using Molecular Imager® Gel\nDoc™ XR+ (BioRad, California, USA) under ultraviolet\n(UV) light. The GG genotype revealed a single band (303\nbp) because no cutting site for this enzyme, while two\nsmall PCR fragments containing 98 and 205 bp represent\nCC genotype. The RFLP pattern for heterozygous GC\nwas characterized using the following 3 bands: 303, 205\nand 98bp. More than 10% of the samples with different\ngenotypes, were randomly selected to be sequenced\n(Macro gen, Geumcheon-gu, Korea) to confirm the\ngenotypes obtained by PCR- RFLP method.\nOligonucleotide primers (sense primer:\n5´-TAT- GGA-TTG-CAC-CTA-TCA-GAG-ACC-3´\nand anti-sense primer:\n5´-TCT-CAT-AGA-GCC-CTT-GGT-GAAT-3´),\nwere designed to amplify a 250 bp segment of the -794(CATT) 5-8  promoter region. The annealing temperature of PCR cycle\nwas 58˚C. Purified PCR products of -794(CATT) 5-8  and\nORF region were sequenced using an ABI automated DNA\nsequencer (Macro gen, Geumcheon-gu, Korea).\nEndometriotic tissues were collected from 17\nendometrioma lesions in women who were genotyped for\nthe  MIF  promoter. The RNA was isolated using TRIzol\n(TRI<sup>®</sup> reagent, Sigma-Aldrich, St Louis, MO, USA) based\non the manufacturer’s instructions. cDNA was amplified\nby One-Step Reverse transcriptase (RT)-PCR using a\ntranscriptase kit (Fermentase Biolabs, Ipswich, MA,\nUSA) in the presence of random hexamers. The reaction\nwas incubated at 25˚C for 5 minutes, 42˚C for 60 minutes,\nand 70˚C for 5 minutes. The RT-PCR products were run\non agarose gel. Quantitative real-time PCR was performed\nin an ABI 7000 Thermal Cycler (Applied Biosystems,\nFoster City, CA, USA). Each standard PCR reaction\ncontained 2 μl cDNA templates, 1 μl of each primer (final\nconcentration, 0.1 mmol/L), and 10 μl SYBR Green PCR\nMaster Mix (Applied Biosystems, Foster City, CA, USA)\ncontaining Taq DNA polymerase buffer, deoxynucleotide\ntriphosphate mix, SYBR green I, MgCl2, and Taq DNA\npolymerase. After denaturation (for 4 minutes at 95˚C),\namplification and quantification were repeated 40 times\n(10 seconds at 95˚C, 30 seconds at 60˚C, and 30 seconds\nat 72˚C). The primer pairs (in the 5’-3’direction) used for\nhuman  MIF :\n[(sense:\n5´-AGA-ACC-GCT-CCT-ACA-GCA-AG-3´\nantisense:\n]\n5´-GAG-TTG-TTC-CAG-CCC-ACA-TT-3´\nand amplicon size: 121bp) and\n(sense:\n5´-CAA-GAT-CAT-TGC-TCC-TCC-TG-3´\nand antisense:\n5´-ATC-CAC-ATC-TGC-TGG-AAG-G-3´\nand amplicon size: 90bp)\nfor  β-Actin  were described in our previous study ( 15 ).\nThe specificity of the PCR product was estimated by\nmelting curve analysis. All experiments were carried out\nin triplicate and the relative expression was evaluated\nusing the 2 -ΔΔCt  method.\nComparison of Hardy-Weinberg equilibrium\ntest results was made and allelic and genotype\ndistributions were compared between endometriosis\npatients and controls using the Pearson’s Chi-square\nanalysis by SHEsis ( http://analysis.bio-x.cn ) ( 22 ).\nThe homozygotes for -794(CATT) 5-8  and -173G/C\nwere evaluated by SHEsis for haplotype distribution,\nodds ratios (ORs) and 95% confidence interval (CI)\n( 23 ). For haplotype analyses, scale significantly was\nconsidered global P values<0.05. Clinical features of\nendometriosis patients were studied and comparisons\nof  MIF  mRNA levels in endometrioma lesions were\nmade using one-way ANOVA and the Tukey’s test, and\nthe results were presented as mean ± standard deviation\n(SD). Differences were considered statistically\nsignificant at P<0.05. All statistical analyses were\nperformed using Statistical Package for the Social Sciences\n(SPSS Inc., version 22, Chicago, IL, USA) software.\n\nAll endometriosis participants had severe endometriosis\n(stage III and IV). The two groups matched on age with\na mean distribution of 31 ± 3.74 and 31.75 ± 3.52 years\nin endometriosis patients and controls, respectively. BMI\nhas not significant difference between both groups (25.2\n± 3.52 and 24.8 ± 3.8 Kg/m2 in endometriosis and control\ngroups, respectively). All participants in the current study\nhave a regular menstruation cycle.\nGenotype and allele frequencies were in Hardy–\nWeinberg equilibrium in both groups (P>0.05). As shown\nin Table 1, the CATT8 allele was not detected neither\nin endometriosis patients nor in controls. Homozygote\nof CATT7 was observed only in endometriosis whilst\nCATT6 and CATT7 alleles were more prevalent in\nendometriosis patients and CATT5 allele was more\nprevalent in controls but these differences were not\nsignificant (P>0.05). Therefore, at the first step, we\nstudied the -794 (CATT) 5-8  polymorphisms ( Table 1 ) and\nfound that 53 out of 106 endometriosis patients and 53\nsubjects out of 110 controls were homozygous. In order\nto assess the effect of simultaneous occurrence of two\npromoter polymorphisms (-794(CATT) 5-8  and -173G/\nC), we continued to evaluate the frequencies of -173G/\nC polymorphism in 53 endometriosis patients and 53\ncontrols with homozygous genotypes of -794(CATT) 5-8 \npolymorphism ( Table 2 ). The data obtained from -173G/\nC genotyping in these patients were used for this\nanalysis extracted from our previous related study ( 24 ).\nFinally, considering the results presented in Tables 1\nand 2, samples from 43 endometriosis patients and 46\ncontrols who were homozygotes for both -794(CATT) 5-8 \nand -173G/C polymorphisms, were investigated to\nestimate the haplotype frequencies ( Table 3 ). Since the\npurpose of this study was run a haplotype analysis, it\nwas essential to eliminate heterozygous subjects. The\nhomozygous for -794(CATT) 5-8  polymorphism was\nmore frequently accompanied by the GG genotype of\nthe -173GC in both groups. With respect to haplotypic\nfrequencies, CATT5/G haplotype was significantly\nmore frequent in controls [global test P=0.044]. We\nobserved similar distributions for CATT 6 /G haplotype\nin both groups; however, the carriage of the CATT 6 /C and\nCATT 7 /C haplotypes was associated with higher\nendometriosis susceptibility, but the difference was\nnot statistically significant (P>0.05). Additionally, the\nCATT 5 /C and CATT 7 /G haplotypes were not detected\nin any group ( Table 3 ). Therefore, strong linkage\nbetween decreased repetition of CATT and G allele was\ndetected.\nDistribution of genotype and allele frequency of MIF -794(CATT)5-8 in endometriosis patients and controls\nData are presented as n (%). *; Homozygote subjects identified for further analysis (in total 53 endometriosis patients and 53 controls were included in\nthis analysis).\nDistribution of genotype and allele frequency of MIF -173G/C between -794(CATT)5-8 homozygotes in endometriosis patients and controls\nData are presented as n (%). *; Homozygote subjects identified for further analysis.\nDistribution of MIF -794(CATT)5-8 and -173G/C haplotype in endometriosis patients and controls*\nData are presented as n (%). *; Homozygous of -794(CATT)5-8 and -173G/C were included in haplotype frequencies assessment, OR; Odds ratios, and CI;\nConfidence interval.\nTo evaluate the promoter haplotype -173G/C and\n-794(CATT) 5-8  and function with  MIF  mRNA expression,\nwe assessed the levels of mRNA using fluorescent\nquantitative polymerase chain reaction (FQ-PCR) in 17\npatients with endometrioma who were also genotyped\nfor promoter polymorphisms. The data were normalized\nagainst the mRNA level of the CATT 5 /G samples. We\nfound an interaction between the -173C and more copies\nof repetitions of CATT in ectopic endometriotic tissues.\nPromoter activity and subsequent expression of mRNA\nin ectopic tissue of patients with CATT 6,7 /CC haplotype\nwere significantly higher compared to other haplotypes\nincluding CATT 5,5 /GG (2.91 fold, P=0.007), CATT 5,5 /\nGC (2.48 fold, P=0.047) and CATT6,6/GG (2.08 fold,\nP=0.046). However, the higher transcriptional activity in\nindividuals carrying CATT 6,6 /GC (2.07 fold, P=0.113) and\nCATT 6,6 /CC (2.01 fold, P=0.130) was not significantly\ndifferent from those of subjects carrying CATT 6,7 /CC\nhaplotype ( Fig.1 ).\nAnalysis of MIF mRNA levels and evaluation of the promoter\nhaplotype with different MIF -794 CATT genotypes (5/5, 5/6, 6/6, and 6/7)\ntogether with -173 (GG/GC/CC). Comparison between groups was made\nby analysis of means ± SEM. Bars show the mean and SEM of experiment\nperformed in triplicate. *; P<0.05.\n\nConsidering the importance of the  MIF  gene in promoting\nthe inflammatory processes and establishment of ectopic\ntissues, we investigated possible associations between\ngenetic variants of  MIF  promoter and susceptibility to\nendometriosis. The results revealed that genetic variants\nof  MIF , including the 7 repetitions of the CATT STR\nin homozygote, were only detected in subjects with\nendometriosis. Exceptionally, the CATT 8  allele, which is\na rare allele, was not detected in this study.\nBased on our results, the -173C allele is more common in\npatients carrying -794(CATT)5-8 homozygotes suggesting\nit as a risk factor for endometriosis. This finding was\nconfirmed by haplotype analysis which revealed that\nCATT 6 /C and CATT7/C haplotypes can be considered\nas moderate risk factors and CATT 5 /G has an almost\nprotective effect against endometriosis. Additionally, the\nCATT 5 /C and CATT7/G haplotypes were not detected in\nany of the groups.\nGeographic variation in -794 STRP also exists\nand farther from Kenya and Zambia, the frequency\ndistribution of MIF-794 STRP with 5 repeats was lower\nthan other genotypes ( 25 ), while in white and Northeast\nAsian populations, the 6-repeat allele was predominant\n( 26 ). The western populations with short tandem repeat\nof MIF CATT 5  were less susceptible to autoimmune\ninflammation ( 27 ) and in Northeast Asian populations,\nthe 6-repeat allele was predominant ( 26 ). In our study, the\nmost frequent CATT5 allele showed protective properties\nagainst endometriosis, which is similar to the effect\nobserved in the Asian population, whereas the 8- repeat\nof CATT allele was not detected in this study.\nDonn et al. ( 28 ), for the first time, reported that -173C\n MIF  variation is related to inflammatory disorders. Also,\nBaugh et al. ( 29 ), first proposed that -794CATT has five to\neight-repeat units and found that short CATT repetitions\nhave a protective effect on rheumatoid arthritis (RA) and\nsuggested that CATT 7 /C haplotype is related to increased\nMIF level. Up to now, several meta-analyses investigated\npossible associations between -173C and CATT 7 /C,\nand inflammatory and autoimmune disorders such as\ntuberculosis (TB), juvenile rheumatoid arthritis (JRA),\ninflammatory bowel disease (IBD) and cancers ( 18 - 21 ,\n 30 ,  31 ). Another study evaluated associations between\nhaplotype promoter and MIF expression level and\ndemonstrated that the 7-repeat at the -794CATT and C\nallele at the -173 G/C position (7C haplotype) are related to\nincreased  MIF  expression in RA ( 19 ). Also, we previously\nshowed the over-expression of  MIF  in ectopic tissues\nfrom endometriosis patients ( 15 ). The presence of C in\nthe -173 promoter region introduces an AP-4 (activating\nenhancer binding protein 4) transcription factor binding\nsite ( 28 ). AP-4 plays an important role in cellular function\nby regulation of genes involved in cell growth, survival,\nimmune response and angiogenesis ( 32 ). Therefore, the\npresence of C allele in this region increases the tendency\nof DNA to bind AP-4 transcription factor.\nAt the promoter region, the CATT repetition contains\nseveral identification regions pituitary-specific factor\n1 (Pit-1) binding sites. Pit-1 is a transcription factor in\nneuroendocrine and mononuclear cells ( 33 ,  34 ). Also,\nrecent studies revealed that inverted CCAAT box binding\nprotein of 90 kDa (ICBP90) is the transcription factor\nrequired for interactions between  MIF  microsatellite in\nseveral immune system cells and synovial fibroblasts\n( 35 ). Pit-1 and ICBP90 regulate  MIF  promoter function\nthat is dependent on the length of tandem repetition\n( 34 ,  35 ). This may be the reason for the association\nbetween the  MIF  CATT 7 /C genotype (haplotype) and\nthe susceptibility towards endometriosis, because this\nhaplotype causes the simultaneous presence of AP-4,\nPit-1 and ICBP90 transcription factors and consequently\nenhances  MIF  promoter activity. The results showed\nan increased expression of  MIF  mRNA in individuals\nwith 6C compared to those with 5G and 7C haplotypes.\nThus, observed simultaneous attendance of longer CATT\nrepeats at -794 and the -173C allele in the gene was\nassociated with elevated MIF production and correlated\nwith increased risk of endometriosis. This was confirmed\nin haplotype analysis which revealed that CATT 5 /G has\nstrongly protective effect against endometriosis. Taken\ntogether, our findings indicate that increment of  MIF \nexpression is associated with genetic variants of  MIF \npromoter in ectopic endometriotic tissues.\nIncreased MIF activates a cascade of events and strongly\nstimulates cyclooxygenase 2 (COX2) and prostaglandin\nE2 (PGE2) expression. This finally leads to increased local\nsynthesis of estrogen in ectopic tissues, which is involved\nin maintenance and progression of endometriosis ( 36 ).\nThus, increased MIF is associated with facilitated growth,\nangiogenesis and development of endometriosis tissue\n( 11 ,  37 ).\n\nWe believe that the CATT 5 /G have a protective effect\non endometriosis. As well, increased repetitions of\nCATT and C allele in  MIF  promoter were associated\nwith increased susceptibility to endometriosis in our\npopulation and this was related to transcriptional activity\nof  MIF . These findings provide the first insight that  MIF \npromoter polymorphisms may have a significant effect\non susceptibility towards endometriosis; however, further\nstudies are required to determine contribution of MIF to\ndevelopment of endometriosis.","source_license":"CC0","license_restricted":false}