{"paper_id":"afa03a43-8a92-45ed-aeb4-accc39bc9fd1","body_text":"Endometriosis is a complex, heterogeneous, chronic inflammatory gynaecologic disease characterised by the presence of endometrial tissue that contains glands and stroma\noutside the uterine cavity, and mainly located in the pelvic\ncavity and pelvic organ walls (1). Several genetic and environmental factors play key roles in its development (2, 3).\nA possible genetic component of endometriosis was first\nproposed in the 1940s, and this theory was later supported\nin the 1980s (4). A twin study estimates that about 50%\nof the risk for endometriosis is inherited (2). Identifying\ndisease-causing gene variants can be done in several ways,\nand the major challenge is to select a few candidates for\nfurther investigation (5). Numerous studies have recently\ninvestigated the association between endometriosis and\nsingle nucleotide polymorphisms (SNPs), particularly\ngenes involved in inflammation and detoxification, as well\nas cell adhesion and endocrine signaling pathway gene polymorphisms (6, 7). SNPs are the most common cause of\ngenomic dissimilarity. They denote variations at a specific site in the genome with a frequency of 1-50% in the entire population. SNPs can alter protein structure and function, though they may not cause disease, and thereby affect\ndisease susceptibility, including endometriosis. Therefore,\nSNPs can be useful for describing at-risk populations (8).\nWe based our analysis on the key role played by the phosphatidylinositol 3-kinase/protein\nkinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway in endometriosis\npathogenesis. This pathway participates in various cellular functions that include cell\ngrowth, differentiation, transformation, and survival. Activation of the PI3K/AKT/ mTOR\npathway in an oestrogen-dependent manner in the human endometrium has been reported. Results\nof in vitro and in vivo studies suggest that PI3K/AKT/mTOR hyperactivation causes\nconsiderable dysregulation of various cellular functions that may lead to endometriosis (9,\n10). Overexpression of the PI3K/AKT/mTOR pathway in eutopic endometrial stromal cells of\nwomen with endometriosis was reported (11, 12).\nThe  PIK3CA  gene is mapped on chromosome 3q26.3 and encodes the p110alpha\ncatalytic subunit of phosphoinositide 3-kinases (PI3Ks) (13). The SNP rs2230461 is a genetic\nvariation that leads to the substitution of isoleucine with methionine at position 391 of\nthe enzyme. The downstream target of PI3K is AKT1. Its gene is located on chromosome 14q32\nand plays a crucial role in cancer cell survival, cell cycle progression, and glucose\nmetabolism (13).  AKT1  rs1130233 is a synonymous coding variation associated\nwith differential expression of the AKT protein (14, 15). The  mTOR  gene,\nlocated on chromosome 1p36.22, is another important target within the PI3K/AKT/mTOR pathway.\nIts protein product is a serine/threonine kinase that functions as a downstream effector in\nthe signalling pathway (16). The SNP rs2295080 in the promoter region of the\n mTOR  gene is a protective factor of some cancers through downregulation\nof endogenous protein expression (17, 18).\nAlthough it is suggested that the PI3K/AKT/mTOR pathway might influence progression and\ndifferent stages of endometriosis (19, 20), no study has investigated relations of the\n PIK3CA ,  AKT1 , and  mTOR  gene\npolymorphisms with endometriosis risk and prognosis. We intend to elucidate whether these\npolymorphisms can serve as risk and/ or prognostic markers for endometriosis by conducting a\ncase-control study to investigate the  PIK3CA  rs2230461,  \nAKT1  rs1130233, and  mTOR  rs2295080 SNPs on endometriosis risk\nand clinical stage frequency.\n\nWe used an online sample size calculator (https://clincalc.\ncom/stats/SampleSize.aspx) to determine the sample size with\na P1 of 47% that represented the frequency of the mutant allele\nin the controls and a P2 of 22% that indicated the frequency\nof the mutant allele in the cases. P1 and P2 were determined\naccording to previously reported percentages (21). The study\npower was set at 80% and a 95% confidence interval (CI) with\nan obtained critical value of <2.0. The minimum sample size\nwas adjusted for a total of 220 subjects.\nThe study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of\nthe Zahedan University of Medical Sciences (IR.ZAUMS.\nREC.1401.080), Zahedan, Iran. All participants provided written informed consent for study participation.\nPeripheral blood samples were collected from 127 endometriosis patients (age: 26-45 years) and 125 healthy women\n(control group) matched for age and body mass idex (BMI)\nwho referred to the Department of Obstetrics and Gynaecology at Ali-ibn-Abi Taleb Educational Hospital, Zahedan, Iran.\nThe control group participants had no medical history of endometriosis. Individuals were excluded if they were not Iranian, withdrew consent, or had previous medical histories of\nchronic pelvic pain, dysmenorrhoea, or dyspareunia.\nEndometriosis was confirmed by clinicians and pathologists according to the results of laparoscopy or laparotomy and other complementary diagnostic tests. Following the diagnosis, histological examination confirmed the\npresence of an endometrial gland and/or stroma in the lesions. The endometriosis was staged according to the revised classification of the American Society of Reproductive Medicine (rASRM) (22). For subgroup analysis, the\n127 endometriosis patients were divided into two groups\n– stages I and II (n=74, minimal/mild) and stages III and\nIV (n=53, moderate/severe).\nGenomic DNA was isolated from peripheral blood leukocytes using a commercial kit (DynaBio, Takapouzist,\nIran) according to the manufacturer's instructions. The\nDNA was diluted in 100 µL of deionised water and stored\nat -20˚C until needed.\nInitially, the allelic discrimination of the  PIK3CA \nrs2230461,  AKT1  rs1130233, and  mTOR  rs2295080 polymorphisms were analysed by allele-specific polymerase\nchain reaction (AS-PCR) and polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP)\naccording to a modified protocol (20, 22–24). The primer\nsequences, genotyping method, and fragment sizes are\nlisted in Table 1. The genotypes were determined by\nseparating the PCR products (digested product for  mTOR \nSNP) with 3% agarose gel electrophoresis, and the results\nwere visualised by safe stain (Sinaclon, Iran). In order to\nensure the reliability of the genotyping results, we randomly chose 20 samples (15 homozygotes and five heterozygotes) and re-genotyped them using the same method.\nStatistical analyses were conducted using the statistical\nsoftware package SPSS 21.0 (SPSS, Inc., Chicago, IL,\nUSA). The student’s t test and chi-square test were conducted to compare demographic characteristics between\npatients and controls. The allele or genotype frequencies\nin each group were counted as the number of incidences\nof an individual allele divided by the total number of alleles. In order to determine significant differences in genetic distributions between cases and controls, both allele and genotype frequencies were compared using Pearson’s\nchi-square test. Odds ratio (OR) and 95% CIs were calculated to determine the risk of endometriosis associated\nwith the given genotypes. Statistical significance was set\nat P<0.05.\n\nTable 2 lists the clinical characteristics of all participants. A study of endometriosis-associated symptoms\nand the number of deliveries revealed significant differences between the endometriosis and control groups.\nSymptoms of chronic pelvic pain, dysmenorrhoea, and\ndyspareunia were significantly common among endometriosis patients (P<0.001). However, BMI, menstrual\ncycle length and/or menstrual bleeding length, and spontaneous abortions did not significantly differ between\ncases and controls. There were 58.26% of endometriosis\npatients with stages I and II disease and 41.73% with\nstages III and IV disease .\nThe SNP primer and genotyping methods\nSNP; Single nucleotide polymorphism, AS-PCR; Allele-specific polymerase chain reaction, and PCR-RFLP; Polymerase chain reaction-restriction fragment length polymorphism.\nCharacteristic of endometriosis cases and controls\nData are presented as mean ± SD or n (%). Pearson’s chi-square (χ2) test, (95% CI) with an unconditional logistic regression model. The bold values are significance <0.05. BMI; Body\nmass index, CI; Confidence interval, *; A patient can have more than one symptom, and **; According to the revised classification of the American Society of Reproductive Medicine\n(rASRM).\nWe genotyped three potentially functional  PIK3CA, AKT1 , and mTOR SNPs in\nthe endometriosis and control samples to assess potential associations between the SNPs\nand endometriosis risk. The genotype distributions for all SNPs in healthy women were in\naccordance with the Hardy-Weinberg equilibrium (HWE). Table 3 shows the association\nbetween variants of  PIK3CA rs2230461 A>G and endometriosis risk. The GG\nhomozygous mutant genotype had a 2.58-fold increased risk (95% CI: 1.119-5.985, P=0.026).\nSignificant associations were observed for the  PIK3CA dominant variant (AA\nvs. AG+GG), which was correlated with an approximately two-fold higher prevalence of this\nvariant between endometriosis women (OR: 1.881; 95% CI: 1.065-3.322, P=0.029). We observed\na significant difference for the PIK3CA gene polymorphism in the recessive model (GG vs.\nAA+AG), with an approximately 2.5-fold increase for endometriosis (OR: 2.4, P=0.037). In\naddition, there was a high frequency of mutant allele ‘G’ in endometriosis patients (OR:\n1.938; 95% CI: 1.255-3.135, P=0.003, Table 3). The AKT1 rs1130233 mutant AA variant was\nsignificantly more frequent in endometriosis patients (OR: 2.870; 95% CI: 1.093-7.535,\nP=0.032). In the dominant GA and AA combined vs. GG model, there was a significantly\nhigher risk of endometriosis in cases compared to controls (OR: 1.835, 95% CI:1.113-3.025,\nP=0.017). Women with endometriosis were significantly more likely to have the \nAKT1  mutant allele (A) than those with the normal allele (OR: 1.671, 95% CI:\n1.135- 2.460, P=0.009, Table 3). The  mTOR  rs2295080 mutant TT genotype\nwas related to a decreased risk for endometriosis (OR: 0.317, 95% CI: 0.108-0.927,\nP=0.036). However, a lower risk for endometriosis was associated with the\n mTOR  SNP in cases under the dominant genetic model (GT/GG) (OR: 0.539,\n95% CI: 0.317-0.951, P=0.032). The results also suggested that the  mTOR \nrs2295080 T allele was more frequent in healthy controls; therefore, it has a probable\nprotective role in the development of endometriosis (OR: 0.529, 95% CI: 0.333-0.839,\nP=0.006, Table 3).\nGenotype and allele frequencies of  PIK3CA  rs2230461,  AKT1  rs1130233, and  mTOR  rs2295080 gene polymorphisms in the control and endometriosis groups\nData are presented as n (%). Pearson’s chi-square (χ2) test, (95% CI) with an unconditional logistic regression model. The bold values are significance <0.05. OR; Odds ratio, CI; Confidence interval, and HWE; Hardy-Weinberg equilibrium.\nTable 4 shows the combined effects of these SNPs and\ntheir association with endometriosis. The frequencies\nof the combined gene polymorphisms of the  PIK3CA \nrs2230461AA,  AKT1  rs1130233 GA and  mTOR  rs2295080\nGG and the  PIK3CA  rs2230461 AG,  AKT1  rs1130233\nGG and  mTOR  rs2295080 GG were 2.6- and 2.8-fold significantly higher in endometriosis group compared to the\ncontrol group (P=0.010 and P=0.047, respectively, Table\n4). The combined frequencies of the  PIK3CA  rs2230461\nGG,  AKT1  rs1130233 GA and  mTOR  rs2295080 GG as\nwell as the  PIK3CA  rs2230461 GG,  AKT1  rs1130233\nGG and  mTOR  rs2295080 GG showed an approximately\n8- and 9- fold risk for endometriosis compared with the\ncontrol group. These increases were statistically significant (P=0.014 and P=0.009, respectively). There was an\napproximately 12-fold increase of the  PIK3CA  rs2230461\nAA,  AKT1  rs1130233 AA and  mTOR  rs2295080 GG combined gene polymorphisms in the endometriosis women\ncompared to the healthy controls, which was significant\n(P=0.028, Table 4). No statistically significant association\nwas observed between other genotypic combinations in the\nendometriosis women compared with the control group.\nThe combined effects of  PIK3CA  rs2230461,  AKT1  rs1130233 and  mTOR  rs2295080 gene polymorphisms on endometriosis risk\nPearson’s chi-square (χ2) test, (95% CI) with an unconditional logistic regression model. The bold values are significance <0.05. OR; Odds ratio and CI; Confidence interval.\nPatients with stages I and II (minimal/mild) and stages\nIII and IV (moderate/severe) endometriosis were studied\nseparately. We noted a high prevalence of mutant alleles\nof  PIK3CA  rs2230461 and  AKT1  rs1130233 SNPs in patients with stages III and IV disease. According to Table 5,\nmoderate/severe endometriosis risk was about five times\ngreater in patients with  PIK3CA  rs2230461 (OR: 4.800,\n95% CI: 2.171-10.611, P<0.001) and more than two times\ngreater in patients with  AKT1  rs1130233 (OR: 2.674, 95%\nCI: 1.261-5.670, P=0.010, Table 5). These two mutant alleles may be high-risk alleles that could have a special effect on endometriosis severity. However, the  mTOR  variant\nrs2295080 mutant allele was not predominant in moderate/\nsevere cases (OR: 0.337 95% CI: 0.132-0.859, P=0.022).\nAssociation between  PIK3CA  rs2230461,  AKT1  rs1130233, and\n mTOR  rs2295080 gene polymorphisms and clinical stage frequency in endometriosis patients\nData are presented as n (%). Pearson’s chi-square (χ2\n) test, (95% CI) with an unconditional\nlogistic regression model. The bold values are significance < 0.05. SNP; Single nucleotide\npolymorphism, OR; Odds ratio, and CI; Confidence interval.\n\nThis is the first case-control study to investigate the impact of PI3K/AKT/mTOR SNPs on the\ndevelopment of endometriosis in an Iranian population. Genotyping analysis of these three\nSNPs revealed significant risk associations between carriers of the mutant alleles in the\nendometriosis group compared with healthy controls. In particular, the SNPs\n PIK3CA  rs2230461 and  AKT1  rs1130233 lead to missense\nmutations and to a more than approximately two-fold increase in disease risk in homozygous\nmutation. The rs2230461 SNP results in a missense mutation, which likely alters the function\nof the PI3K enzyme. Changes in PI3K activity can lead to abnormal cellular proliferation and\nsurvival, which are central to endometriosis development (19). The rs1130233 SNP results in\na missense mutation in the AKT1 protein, potentially altering its kinase activity. This can\naffect numerous downstream processes, including cell growth and apoptosis, and contribute to\nthe pathological environment observed in endometriosis (19). However, the results were\ndifferent for mTOR variants, and the risk of developing the disease was reduced by half or\nless in carriers of the mutant alleles. This specific SNP in the  mTOR  gene\nis a genetic variation that can potentially influence the expression or function of the mTOR\nprotein and could influence cellular processes in a way that protects against disease\ndevelopment. In addition, given mTOR's role in autophagy, a reduction in mTOR activity due\nto the polymorphism could enhance autophagy, which is a cellular process known to be\nprotective against various diseases by removing damaged organelles and proteins. It is\npossible that changes in mTOR signaling can alter cellular metabolism. A polymorphism that\nreduces mTOR activity might improve metabolic homeostasis and reduce disease risk (23). In\naddition, analysis of the association between the  PIK3CA  rs2230461, \nAKT1  rs1130233, and  mTOR  rs2295080 gene polymorphisms with\nclinical stage of endometriosis confirmed the previous results. There was a statistical\nassociation between  PIK3CA  rs2230461 and  AKT1  rs1130233\npolymorphisms and moderate/severe (stages III and IV) endometriosis. Dysregulation of the\nPI3K/AKT pathway might lead to larger and more invasive lesions characteristic of stages III\nand IV (19). The pathway also intersects with inflammatory signaling. Enhanced or aberrant\nPI3K/AKT signaling due to these polymorphisms may exacerbate inflammation, a key feature of\nsevere endometriosis (11, 12). In addition,  mTOR , downstream of\n AKT , regulates angiogenesis. Increased angiogenesis can support the\ngrowth and maintenance of endometriotic lesions, particularly in severe cases (19). In the\ncase of the  mTOR  rs2295080 SNP, the mutant allele was more frequent in\nstages I and II of the disease. Its protective effects might prevent disease progression to\nmore severe stages.\nOur recent study has shown that certain variations in important genes may make individuals\nmore susceptible to endometriosis (25). Endometriosis is an inflammatory disease where\nendometrial-like tissue colonises outside the uterine cavity after an increase in the\nproliferative, invasive, and migratory potential of phenotypically normal endometrial cells\nsuch that it has cancer-like features from the beginning of its development. Among the\nvarious signaling pathways that control cell proliferation, the PI3K/AKT/mTOR pathway is\nunder investigation as a possible component of the pathogenic mechanism that leads to\nendometriosis (26). Migration of normal endometrial cells to the peritoneal surface is\nregulated by activating the PI3K/AKT/mTOR pathway in endometriotic cells by oestrogens and\ngrowth factors (27). Its abnormal activation is a common event in endometriosis, and this\npathway may play a role in the development of endometriosis. The antiapoptotic and\nproliferative effects of the PI3K/AKT/mTOR pathway in endometriosis have been reported (28).\nTherefore, this pathway has become an attractive therapeutic target in endometriosis, and\nrecent studies have attempted to evaluate the effects of the PI3K/ AKT/mTOR signaling\npathway on disease progression.\nThe role of SNPs in the PI3K/AKT/mTOR pathway and susceptibility to various diseases has\nbeen studied (16- 18). Some of these reported data supports our findings (21).\n PIK3CA  rs2230461 is localized in the C2 domain and does not affect the\nbinding site of the protein product. Although 7% of  PIK3CA  mutations are\nlocated in the C2 domain, it is possible that all mutations far from the binding site could\naffect the function of the protein and alter its dynamic behaviour (29). Qiu et al. (30)\nshowed that the oncogenic properties of PIK3CA played a role in the development of human\nhead and neck cancers, especially those that affect the pharynx. A study conducted in Iran\nexamined the frequency of the  PIK3CA  rs2230461 SNP in stage III breast\ncancer patients and a control group. The results showed a significantly higher occurrence of\nthe mutant genotype among stage III cases compared to the control group (31). Several\nstudies reported the association of  AKT  rs1130233 polymorphisms located on\nchromosome 14 in exon 8 with increased disease risk, including bladder cancer (20),\nschizophrenia (32), gastric cancer (33), weight loss (34), nasopharyngeal carcinoma (35),\nand pancreatic cancer (36). A recent study examined the impact of  mTOR \n(rs2295080) and  AKT1  (rs1130233) gene variations on the likelihood of\ndeveloping papillary thyroid cancer. Similar to our findings, their results indicate that\nrs2295080 and rs1130233 are protective and risk factors for disease development,\nrespectively (37). Various studies have shown that  mTOR  2295080 (T > G)\nvariants have a positive or negative association with diseases. A meta-analysis found that a\nsignificant association of  mTOR  rs2295080 with reduced oesophageal squamous\ncell carcinoma risk under the homogenous (GG vs.TT) model (38). Piao et al. (33) reported an\nassociation between rs2295080 and gastric cancer risk in the Chinese population. Unlike, the\nresults of recent meta-analysis that have revealed the  mTOR  2295080 gene\npolymorphism as a genetic risk factor for cancer susceptibility and their carcinogenesis and\nclinical outcomes (39), Zhao et al. (40) had observed the protective effect of this SNP on\nbreast cancer development.\nA meta-analysis reported a significant association between the  mTOR  rs2295080 G allele in the dominant model\nand decreased risk of genitourinary cancers, along with an\nincreased risk of acute leukaemia in the recessive model\n(17). Findings from these studies contribute to the identification of potential markers for a genetic screening test that\ncan be used to detect diseases like endometriosis. A genetic\ntest that examines biomarkers in the endometrium and a\nthorough review of the family's medical history might\nidentify women who have a higher likelihood of developing endometriosis. The use of these gene polymorphisms\nin diagnostic modalities in primary care for symptomatic\npatients might reduce the time to diagnose endometriosis.\nA limitation of this study is that it focuses on an Iranian\ncohort. The findings may not be generalisable to other\npopulations because of potential differences in genetic\nbackgrounds, environmental factors, and disease prevalence. Further studies that enrol diverse populations are\nneeded to confirm the role of these gene polymorphisms\nin endometriosis susceptibility.\n\nFor the first time, we reported that rs2230461 ( PIK3CA ) and rs1130233\n( AKT1 ) variations are risk factors for endometriosis. We observed that\nthese polymorphisms had a significant relation with the III/IV stages of the disease;\ntherefore, we propose that the selected SNPs may be involved in endometriosis development.\nOn the other hand, the rs2295080 ( mTOR ) SNPs could be a protective factor\nagainst endometriosis in a subset of the Iranian population. Validation of our results in a\nlarger population of diverse ethnicities would be necessary to study the role of\n PI3K/ AKT/mTOR  variants in disease development. Knowledge of the genetics\nof endometriosis can help healthcare professionals determine the underlying biological\npathways that contribute to endometriosis, understand aetiology of this disease, define\ngenetically heterogeneous subtypes, identify those at higher risk, and determine different\ntreatments. These findings provide insights into the PI3K/ AKT/mTOR pathway's role in\ndisease progression and help guide future research on early diagnosis, risk stratification,\nand potential therapeutic targets.","source_license":"CC0","license_restricted":false}