Investigation of TGF-β1 gene variant and expression in a group of Iranian women with endometriosis

Investigation of TGF-β1 gene variant and expression in a group of Iranian women with endometriosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Investigation of TGF-β1 gene variant and expression in a group of Iranian women with endometriosis Mahdokht Babaei, Negar Sarhangi, Maryam Shahrabi-Farahani, Farshad Sharifi, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-219449/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Purpose Endometriosis is defined as a common gynecologic and inflammatory disease. Transforming growth factor-beta 1 (TGF-β1) gene and its protein level might play a role in the pathogenesis of endometriosis. The present study aimed for the first time to assess the associations between endometriosis risk and − 509 C/T (rs1800469) variant of the TGF-β1 gene as well as TGF-β1 mRNA expression in eutopic endometrium tissue of patients with and without endometriosis among a group of Iranian women. Methods Genotyping was carried out in 100 endometriosis patients (cases) with confirmed histological diagnosis of endometriosis and 197 non-endometriosis subjects (controls). The expression level of TGF-β1 mRNA was determined using Real-Time PCR assay in 15 eutopic endometrium tissue of women with endometriosis and 15 healthy controls. Results There was a significant association for allele and genotype frequencies of rs1800469 variant and endometriosis i. No significant difference for TGF-β1 expression was observed between eutopic endometrium of patients and healthy group. Also, evaluation of TGF-β1expression across the menstrual cycle showed the same level of TGF-β1 among cases and controls subjects. Conclusion Our investigations indicated enough evidence for the effect of TGF-β1 gene polymorphism on endometriosis risk in an Iranian population. Furthermore, we could not find any relations between TGF-β1 mRNA expression and susceptibility to endometriosis. Obstetrics & Gynecology TGF-β1 gene expression variant Endometriosis Figures Figure 1 Introduction Endometriosis is defined as a common gynecologic, estrogen-dependent, chronic, and inflammatory disease that is affecting around 6–10% of women in their reproductive period and is a major contributor to chronic pelvic pain, dysmenorrhoea, dyspareunia, and subfertility [ 1 – 3 ]. It is described by the existence of endometrial-like tissue outside the uterine cavity, mainly on the pelvic peritoneum, ovaries and rectovaginal area [ 4 , 5 ]. The prevalence of endometriosis disease seems to be ~ 5% among women between 25 years and 35 years of age [ 6 ]. It is associated with primary or secondary infertility in 30% of the women [ 7 ]. Although, the pathogenesis of endometriosis is unknown, the development of this polygenic and multifactorial disease is influenced by a combination of genetic, immunologic, hormonal, and environmental factors [ 8 , 9 ]. Up until now, the exact etiology of endometriosis has been not known while there is increasing evidence that transforming growth factor-beta 1 (TGF-β1) gene and its protein level might play a role in the pathogenesis of inflammatory disease, including endometriosis [ 10 ]. Because TGF-β1 is a multifunctional cytokine that is involved in endometrial stromal cells proliferation and differentiation [ 11 ], angiogenesis [ 12 ], formation of adhesion [ 13 ] and immune response [ 10 , 14 ]. Several lines of evidence represent that susceptibility to the development of endometriosis is somewhat influenced by genetic factors. Accordingly, with respect to the role of TGF-β1 as a candidate gene in susceptibility to endometriosis, investigation of -509 C/T variant (rs1800469) of the TGF-β1 gene, which is the main determinant of plasma TGF-β1 concentration, will be beneficial for determining the role of this genetic factor in development of endometriosis [ 9 , 15 ]. Nonetheless, the role of the aforementioned polymorphism in risk of endometriosis has not been investigated in Iranian population so far. Therefore, the present study aimed to assess the associations between endometriosis and the − 509 C/T polymorphism of the TGF-β1 gene as well as TGF-B1 mRNA expression among a group of Iranian women for the first time. Method The present study aimed at two points; first, analysis of the associations between the − 509 C/T (rs1800469) variant of the TGF-β1 gene and endometriosis risk. Second, assessing the TGF-β1 mRNA expression in eutopic endometrium tissue of patients with and without endometriosis. Blood Sample Blood samples were taken from a totally 297 subjects who had undergone laparoscopy or laparotomy for diagnosis or treatment of endometriosis to assess the association between TGF-β1 -509 C/T genetic variant and endometriosis susceptibility. The samples were enrolled from two hospitals (Firoozgar and Valiasr) between April 2013 and September 2015 and the diagnosis of endometriosis was based on surgical and histologic criteria. The study group (case) was involved 100 Iranian patients with endometriosis with the histologically confirmed diagnosis of endometriosis after their operative findings, according to the presence of endometrial glands or stroma in the lesions. Since endometriosis is estrogen-dependent, the control group was selected from women at reproductive age and before menopause with a definite diagnosis of the absence of endometriosis by a surgeon. We included 197 healthy volunteers who underwent laparoscopy, and laparotomy due to tubal ligation or treatment of benign diseases such as ovarian cysts, myoma, hydrosalpinx, or other reasons. Individuals with a history of malignancy, those at menopausal age, and patients who had received hormonal treatment for at least 6 months since sampling were excluded from the study. Three peripheral blood samples (in EDTA containing vial) were assembled from all subjects (females with endometriosis and controls females) for genotyping at the time of enrollment and stored at -20 °c until DNA extraction. Tissue Sample For evaluating the TGF-β1 mRNA expression, samples of eutopic endometrium tissue were obtained by sharp curettage of the corpus of the uterine cavity of both 15 healthy controls and 15 women with endometriosis who had undergone a surgical resection between April 2013 and September 2015 at two Hospitals (Firoozgar and Valiasr) and was snap-frozen in liquid nitrogen and stored at -80°C until used. Written informed consent and answer a questionnaire about demographics and clinical characteristics such as gravidity, parity, abortion, dysmenorrhea, menes, and bleeding were received from all the participants who were volunteers for blood and tissue sampling before they enrolled. Dysmenorrhea and bleeding were classified based on the amount of the pain (Normal, Severe, Not) and bleeding (Normal, Severe, Low) in the menstrual time. Regarding menes, subjects also were categorized as regular and irregular. SNP genotyping Genomic DNA from whole blood was isolated from all subjects using phenol protocol. Extracted DNA was labeled and stored at -20°C until use. In the isolated DNA, we identified − 509 C/T polymorphic site in the promoter region of the TGF-ß1 gene with the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method. PCR primers were designed based on the Genbank reference sequence. The primers sequences that used for amplifying DNA were listed in the Table 1 . PCR amplification was carried out in a final reaction volume of 20 µl final reaction containing 1µl template DNA, 10µl Red Master Mix (Amplicon), 0.5µl each primer and 8µl H 2 O by a Biorad MJ mini (Singapore) device. PCR cycle was done under the following conditions: an initial denaturation step at 95°c for 5 minutes followed by 35 cycles of 30 seconds at 95°c, 30 seconds at 59°c; 35 seconds at 72°c, and a final elongation at 72°c for 10 minutes. The allelic variant − 509 C (a 2.5 µl PCR product C) can be digested with 0.25 µl restriction enzyme Bsu36 I (New England Biolab) at 37°C overnight in a 10 µl reaction that produced one fragment of 419 bp for the CC genotype; three fragments of 419, 229, and 190 bp for the CT genotype; and two fragments of 229, and 190 bp for the TT genotype. Table 1 Primers that used in PCR and RT-PCR assay PCR Primers TGF-ß1 -509 C/T (rs1800469) 5′-CAGACTCTAGAGACTGTCAG-3′ 5′-GTCACCAGAGAAAGAGGAC-3′ RT-PCR Primers TGF-ß1 5-CCATGAACTTTCTGCTGTCTT-3 5-TCGATCGTTCTGTATCAGTCT-3 HPRT 5'-CCTGGCGTCGTGATTAGTGAT-3' 5'- TTATGGACAGGACTGAACGTCT-3' PCR, Polymerase chain reaction; RT-PCR, Reverse transcription polymerase chain reaction ; TGF-ß1, Transforming growth factor-beta 1; HPRT, Hypoxanthine-guanine phosphoribosyltransferase 5 µl of digestion product and 7µl of Ready-Load 100-bp DNA Ladder (Invitrogen, Spain) were loaded into 3% agarose gel (Invitrogen, Spain) containing 1.4µl of sybregreen. The gel underwent electrophoresis at 120V, 100 mA for 30 min. The gel was visualized using a UV light (Wealtec, South Africa), and photographs of gels were taken after staining. We confirmed the genotypes identified with enzyme digestion by Sanger sequencing. mRNA VEGF expression Isolation of RNA and complementary DNA Synthesis In brief, total RNA from frozen tissue was extracted using the fermentas kit (Qiagen Inc., Valencia, CA, USA), in accordance with the manufacturer’s instructions. RNA concentration and purity were measured using NanoDrop 2000 (Thermo Scientific, USA) at a wavelength of 260 nm, and both mRNA and protein concentrations were measured at a wavelength of 280 nm. Two micrograms of total RNA were subjected to reverse transcription into the first-strand cDNA with a commercially available kit (Thermo Scientific, USA) according to the manufacturer’s protocol. In the first step, 0.5µl primer (Random Hex) (100µm) and 3.5µl dH 2 O was added to 2µl of total RNA and annealed at 65°c for 5 minutes. Then, 2µl primescript buffer (5x), 1µl dNTP, 0.5µl Revertaid and 0.5µl Ribolock was added in the last tube at 25°c for 5 minutes, 42°c for 1 hour and 70°c for 5 minutes. The cDNA was stored at -80°C until used. RT-PCR Transcribed products were subjected to PCR for TGF-ß1 and hypoxanthine-guanine phosphoribosyltransferase (HPRT) in a 15µl final reaction volume. For a negative control, the cDNA template was omitted from the reaction. Amplification for TGF-ß1 cDNA was started with 5 minutes denaturation at 95°c, followed by cycles of 30 seconds of denaturation at 94°c, 30 seconds of annealing at 53°c, and 5 minutes of extension at 72°c. The PCR profile for HPRT began with 95°c denaturation for 5 minutes, followed by cycles of 39 seconds of denaturation at 95°c, 40 seconds of annealing at 60°c, and 5 minutes of extension at 72°c. The primers used for TGF-ß1 and HPRT amplification were shown in the Table 1 . Final PCR products were exposed to electrophoresis through at 2% agarose gel and stained with sybregreen. The size of reverse transcription polymerase chain reaction (RT-PCR) products for TGF-ß1 and HPRT were 83 and 131 bp, respectively. Real-Time PCR Concisely, the real-time PCR amplification was accomplished in a total volume of 20µl, containing 6µl of cDNA sample, 9.8µl sybre, 0.2µl Rox, 0.6µl of each primer and 2.8 dH 2 O.The reactions were done in duplicate by ABI System (Applied Biosystems, Foster City, CA). The real-time PCR program consisted of 10 minutes at 95°C, followed 5 seconds at 95°C, 34 seconds at 53, 15 seconds at 95°c, 1hour at 53 °c and 15 seconds at 95. Statistical analysis All statistical analysis was donning using the Stata software version 14.2. Case and controls were compared using the Kruskal-Wallis test for the quantitative variables such as age, BMI, parity, gravidity, and abortion. Data were expressed as the mean ± standard deviation (SD). Chi-squared test was used for comparison of qualitative values (categorical) including dysmenorrhea, menes, and bleeding, presented by number and percentage. Differences of TGF-ß1 genotype and allele frequencies between endometriosis patients and controls were compared using Logistic regression and an odds ratio (OR) with 95% confidence interval (CI) were used as a measure of the strength of association between genotypes, allele frequencies. The Mann–Whitney U test was used to evaluate the association of the mRNA TGF-B1 expression with endometriosis risk. Deviations from Hardy-Weinberg equilibrium (HWE) in control group were assessed using Chi-squared test ( X 2 ). Probability values less than or equal to 0.05 were considered statistically significant. Result Characteristics of the patients and controls Table 2 shows characteristics of the endometriosis patients and controls including age, BMI, gravidity, parity, abortion, dysmenorrhea, menes, and bleeding. Table 2 Characteristics of endometriosis patients and controls women in a group of Iranian population Endometriosis N = 100 Normal N = 197 P-value Age (years) Number Total = 296 N = 99 N = 197 0.90 35.39 ± 7.56 35.50 ± 11.00 BMI (kg/m 2 ) < 0.01٭ Number Total = 285 90 192 25.47 ± 4.66 28.45 ± 4.15 30 14 (15.5) 68 (35.4) Gravidity < 0.01٭ Number Total = 293 99 194 1.25 ± 1.72 2.50 ± 1.61 Parity < 0.01٭ Number Total = 293 99 194 0.88 ± 1.30 2.03 ± 1.43 Abortion 0.06 Number Total = 293 99 194 0.27 ± 0.69 0.43 ± 0.73 Dysmenorrhea Number Total = 244 98 146 < 0.01* Normal 36 (36.73) 47 (24.23) Severe 32 (32.65) 11 (5.67) Not 30 (30.61) 88 (45.36) Menes 0.97 Number Total = 244 98 146 Regular 70 (71.43) 104 (53.61) Irregular 28 (28.57) 42 (21.65) Bleeding 0.41 Number Total = 244 98 146 Normal 58 (59.18) 82 (42.27) Severe 29 (29.59) 53 (27.32) Low 11 (11.22) 11 (5.67) BMI, body mass index ٭ P-value < 0.05 is significant There was no significant difference in the mean age between the endometriosis patients and the controls group ( P = 0.9) while cases had significant lower BMI compared to control group ( P < 0.01). Both groups were showed significant differences in gravidity and parity ( P < 0.01). Conversely, no significantly different was observed in abortion between the two groups ( P = 0.06). Patients with endometriosis showed some symptom of endometriosis such as dysmenorrhea higher than the control group ( P < 0.01). A significant difference was not found in the bleeding ( P = 0.41) and menes between the two groups ( P = 0.97 ). TGF-ß1 variant (-509 C/T) in Patients with Endometriosis and Controls Totally, 100 cases and 196 controls from 297 enrolled subjects were successfully genotyped for − 509 C/T variant in the promoter of TGF-ß1 gene. The genotype distribution of -509 C/T variant did not show any deviation from the HWE in control subjects ( P = 0.498). The genotype and allele frequencies for the TGF-ß1 -509 C/T SNP are summarized in Table 3 . Table 3 Comparison of un-adjusted and adjusted odds ratio for five genetic models for candidate gene variant Gene (rsID) Genotype/allele Case N = 100 Control N = 196 P-value OR (95 % CI) TT vs. CC (Homozygote) OR (95 % CI) CT vs. CC (Heterozygote) OR (95 % CI) CT + TT vs. CC (Dominant) TGF-β1 ( rs1800469) CC 21 (21) 32 (16.33) 0.452 Un-Adjusted Adjusted Un-Adjusted Adjusted Un-Adjusted Adjusted CT 44 (44) 100 (51.02) 0.833 (0.418–1.65) P = 0.603 0.303 b (0.117–0.781) P = 0.014 0.670 (0.348–1.29) P = 0.231 0.427 a (0.186–0.981) P = 0.045 0.734 (0.397–1.35) P = 0.322 0.366 b (0.168–0.796) P = 0.011 TT 35 (35) 64 (32.65) OR (95 % CI) T vs. C (Allelic) OR (95 % CI) TT vs. CT + CC (Recessive) C 86 (43) 164 (41.84) 0.786 Un-Adjusted Adjusted Un-Adjusted Adjusted T 114 (57) 228 (58.16) 0.953 (0.675–1.34) P = 0.786 0.545 a (0.352–0.844) P = 0.007 0.900 (0.541–1.469) P = 0.686 1.935 b (1.00-3.720) P = 0.048 OR, odds ratio; CI, confidence interval a Adjusted for Age, BMI, parity, abortion b Adjusted for age, BMI, parity A significant difference was not observed in the CT and TT genotype distributions of TGF-ß1 -509 C/T between the cases and controls (P = 0.4). No significant result was also found between genotype frequency of these groups after merging heterozygous and homozygous mutant genotypes (CT + TT). The frequency of the CT, TT genotypes in the patients was significantly higher than the control group after adjusted for some quantitative variables (Adjusted CT: OR = 0.427, 95% CI = 0.186–0.981, P = 0.045; Adjusted TT: OR = 0.303, 95% CI = 0.117–0.781, P = 0.014). The combined CT + TT genotype was associated with a significantly decreased risk of endometriosis compared with the CC genotype (Adjusted: OR = 0.366, 95% CI = 0.168–0.796, and P = 0.011). There were no significant differences in the allele frequencies of TGF-ß1 -509 C/T between the cases and controls ( P = 0.786). The presence of T allele appeared to be a protective allele for the development of endometriosis after multivariate analysis for age, BMI, parity, and abortion using regression test (Adjusted: OR = 0.545, CI = 0.352–0.844, P = 0.007). Analysis of mRNA TGF-ß1 Expression As shown in Fig. 1 a, no significant differences were observed between endometriosis patients and controls in TGF-ß1 mRNA expression ( P = 0.32). Evaluating the expression of TGF-ß1 level through the menstrual cycle also showed that in the proliferative phase, TGF-ß1 mRNA level in eutopic endometrium from women with endometriosis was not significantly different compared to the controls ( P = 0.10) (Fig. 1 b). In the secretory phase, endometrium tissue from women with the disease also showed similar mRNA expression compared to the controls ( P = 0.96) (Fig. 1 c). On the other hands, we found that endometrial tissue from controls showed higher TGF-ß1 mRNA levels in the proliferative phase than in the secretory phase but the results was not significant ( P = 0.20) (Fig. 1 d). Besides that, no significant difference was observed in TGF-ß1 level when comparing secretory versus proliferative phase in endometrium from women with endometriosis ( P = 0.18) (Fig. 1 e). Discussion In the present study, we undertook genotype analysis of the − 509 C/T variant and mRNA expression of the TGF-β1 gene in relation to endometriosis and found that presence of TGF-β1 509T allele was protective against development of endometriosis (decreasing 46% risk of endometriosis), while patients with C allele were prone to have endometriosis after adjustment for cofounding variables. According to the first investigation for the role of -509 C/T variant in susceptibility to endometriosis in a study by Hsieh and colleagues it has been demonstrated that TT genotype and T allele of TGF-β1 gene are related to the higher risk of advanced endometriosis in the Taiwanese women. But, no association of this variant and late-stage endometriosis was reported in their study. Based on their results, TGF-β1 may be a candidate gene for endometriosis risk. So that, genotype and allele frequencies of TGF-β1 variant can be as convenient markers for the prediction of endometriosis susceptibility [ 10 ]. Further, the contraversial result has been identified by Van Kaam et al. They did not observe any relationship between this variant and advanced-stage endometriosis in Dutch women. They also studied the role of TGF-β1 gene variant in increased risk of deep infiltrating endometriosis and no evidence of significant association was found [ 16 ]. Also, Kim and colleagues reported that the TGF-β1 -509 C/T variant may not be associated with an increased risk for advanced-stage endometriosis in Korean population. They did not observe significant differences in the genotype distributions or allele frequencies of the TGF-β1 gene − 509 C/T variant between endometriosis patients and controls [ 15 ]. Considering the findings of the study by Lee et al the TC haplotype allele of the − 509 C⁄T variants in the TGF-β1 gene may be associated with an increased risk of early-stage endometriosis and is thus one genetic factor that may determine susceptibility for endometriosis [ 17 ]. These findings corresponded to the result of one Meta-analysis of six case-control studies that showed no positive correlation of -509 C/T variant with risk of endometriosis [ 9 ]. Similarly, no significant association was identified concerning the − 509 C/T variant with endometriosis development in a recent meta-analysis [ 18 ]. In disagreement with only previous report by Johnson and colleagues and in accordance with the statistical results, our data indicate that there were no significant differences in mRNA TGF-β1 between endometriosis patients and controls. Finding of Johnson study demonstrates lower TGF-β1 mRNA and protein levels in endometrium with endometriosis than controls during the mid-secretory phase [ 19 ]. Conclusion Taken together, our study findings provided some evidence that the TGF-β1 gene variant shows a significant protection in endometriosis susceptibility in an Iranian population. Clinical data and adjustment for co-founding factors seems to be a very important issue and must be considered in the interpretation of results. However, no association between TGF-β1 expressions in endometrial tissue and endometriosis risk was observed. Since the lack of association might come from the small sample size, which is the main limitation in our study therefore further investigations with a larger sample size including larger groups of patients with the same stage of endometriosis should be performed so that peritoneal fluid (PF) and peripheral blood could be obtained during the same stage of the menstrual cycle. Declarations Funding No funding was obtained. Compliance with ethical standards Conflict of interest There was no conflict of interest to report. Ethical approval The study was approved by the ethics committee of Tehran University of medical sciences. Informed consent Informed consents were obtained from the study participants. Acknowledgement The authors would like to express their gratitude to all participants enrolled in this project. References Young VJ, Ahmad S, Duncan WC, Horne AW (2017) The role of TGF-β in the pathophysiology of peritoneal endometriosis. Hum Reprod Update 23(5):548–559. https://doi.org/10.1093/humupd/dmx016 Bulletti C, Coccia ME, Battistoni S, Borini A (2010) Endometriosis and infertility. J Assist Reprod Genet 27(8):441–447. https://doi.org/10.1007/s10815-010-9436-1 Rashidi BH, Sarhangi N (2019) Association of vascular endothelial growth factor (VEGF) Gene polymorphisms and expression with the risk of endometriosis: a case-control study. 46(3):3445–50. https://doi.org/ 10.1007/s11033-019-04807-6 Giudice LC (2010) Clinical practice. Endometriosis. N Engl J Med 362(25):2389–2398. https://doi.org/ 10.1056/NEJMcp1000274 Wee-Stekly W-W, Kew CCY, Chern BSM (2015) Endometriosis: a review of the diagnosis and pain management. Gynecol Minim Invasive Ther 4(4):106–109. https://doi.org/10.1016/j.gmit.2015.06.005 Vercellini P, Vigano P, Somigliana E, Fedele L (2014) Endometriosis: pathogenesis and treatment. Nat Rev Endocrinol 10(5):261–275. https://doi.org/10.1038/nrendo.2013.255 Acien P, Velasco I (2013) Endometriosis: a disease that remains enigmatic. ISRN Obstet Gynecol 2013:242149. https://doi.org/10.1155/2013/242149 Giudice LC, Kao LC (2004) Endometriosis. Lancet (London, England) 364(9447):1789-99. https://doi.org/10.1016/S0140-6736(04)17403-5 Zhang F, Yang Y, Wang Y (2012) Association between TGF-beta1-509C/T polymorphism and endometriosis: a systematic review and meta-analysis. Eur J Obstet Gynecol Reprod Biol 164(2):121–126. https://doi.org/10.1016/j.ejogrb.2012.05.004 Hsieh YY, Chang CC, Tsai FJ et al (2005) Polymorphism for transforming growth factor beta 1-509 (TGF-B1-509): association with endometriosis. Biochem Genet 43(5–6):203–210. https://doi.org/ 10.1007/s10528-005-5211-x Hammond MG, Oh S-T, Anners J, Surrey ES, Halme J (1993) The effect of growth factors on the proliferation of human endometrial stromal cells in culture. Am J Obstet Gynecol 168(4):1131–1136. https://doi.org/10.1016/0002-9378(93)90356-n Bertolino P, Deckers M, Lebrin F, ten Dijke P (2005) Transforming growth factor-β signal transduction in angiogenesis and vascular disorders. Chest 128(6):585S–585S90S. https://doi.org/10.1378/chest.128.6_suppl.585S Chegini N, editor TGF-β system: the principal profibrotic mediator of peritoneal adhesion formation. Seminars in reproductive medicine; 2008: © Thieme Medical Publishers Omwandho CO, Konrad L, Halis G, Oehmke F, Tinneberg H-R (2009) Role of TGF-βs in normal human endometrium and endometriosis. Hum Reprod 25(1):101–109. https://doi.org/10.1093/humrep/dep382 Kim JJ, Choi YM, Choung SH et al (2010) Analysis of the transforming growth factor β1 gene – 509 C/T polymorphism in patients with advanced-stage endometriosis. Fertil Steril 93(7):2121–2124. https://doi.org/10.1016/j.fertnstert.2009.01.082 van Kaam K, Romano A, Dunselman G, Groothuis P (2007) Transforming Growth Factor β1 Gene Polymorphism—509C/T in Deep Infiltrating Endometriosis. Reprod Sci 14(4):367–373. https://doi.org/10.1177/1933719107303436 Lee HJ, Kim H, Ku SY, Kim SH, Kim JG (2011) Transforming Growth Factor-β1 Gene Polymorphisms in Korean Women With Endometriosis. Am J Reprod Immunol 66(5):428–434. https://doi.org/10.1111/j.1600-0897.2011.01009.x Mear L, Herr M, Fauconnier A, Pineau C, Vialard F (2020) Polymorphisms and endometriosis: a systematic review and meta-analyses. Hum Reprod Update 26(1):73–102. https://doi.org/10.1093/humupd/dmz034 Johnson MC, Torres M, Alves A et al (2005) Augmented cell survival in eutopic endometrium from women with endometriosis: expression of c-myc, TGF-beta1 and bax genes. Reprod Biol Endocrinol 3(1):45. https://doi.org/10.1186/1477-7827-3-45 Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 07 Aug, 2021 Reviewers invited by journal 07 Aug, 2021 Editor invited by journal 06 Aug, 2021 First submitted to journal 04 Apr, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-219449","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":44457282,"identity":"81f30119-08e6-438a-9d57-5b40304d6f5c","order_by":0,"name":"Mahdokht Babaei","email":"","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mahdokht","middleName":"","lastName":"Babaei","suffix":""},{"id":44457283,"identity":"22de1172-5fc9-41a6-833a-7a645c2a17c9","order_by":1,"name":"Negar Sarhangi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYJACZhDBxsx88MEHEIOdaC3sbMmGM8B6idXCwM9jJs2D4OIG5vyHn30uqLlj18fMY2xs82ubPB8zA+OHjzm4tVjOSDOePePYs+Q2ZrbCx7l9tw3bmBmYJWduw63F4AaDMTMP2+FkoPc3G+f23GYEamFj5sWn5fzxz8w8/0BaGMykLXtu2xPWciDHmJm37bAdGzOLmTTDj9uJhLXcyClm5u07nMDGDAzk3obbQE8xNuP3y/njm5l5vh22l+8/fPDBjz+3bee3Nx/88BGPFhhIbACRjG1gsoGweiCwh1B/iFI8CkbBKBgFIwwAACtFSz0o0ePNAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-2385-7333","institution":"Tehran University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Negar","middleName":"","lastName":"Sarhangi","suffix":""},{"id":44457284,"identity":"94b82ffa-8790-447e-8a14-21f62d352f06","order_by":2,"name":"Maryam Shahrabi-Farahani","email":"","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Maryam","middleName":"","lastName":"Shahrabi-Farahani","suffix":""},{"id":44457285,"identity":"0b8d38ec-29c2-47b0-b88a-fab575f34396","order_by":3,"name":"Farshad Sharifi","email":"","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Farshad","middleName":"","lastName":"Sharifi","suffix":""},{"id":44457286,"identity":"75cbc878-4ca5-44c5-b740-77acb776d489","order_by":4,"name":"Majid Alipor","email":"","orcid":"https://orcid.org/0000-0003-0889-7476","institution":"Islamic Azad University","correspondingAuthor":false,"prefix":"","firstName":"Majid","middleName":"","lastName":"Alipor","suffix":""},{"id":44457287,"identity":"725576d2-9390-405e-9f6e-6577c5a8a8ea","order_by":5,"name":"Soheila Aminimoghaddam","email":"","orcid":"","institution":"Iran University of Medical Sciences, Tehran","correspondingAuthor":false,"prefix":"","firstName":"Soheila","middleName":"","lastName":"Aminimoghaddam","suffix":""},{"id":44457288,"identity":"e74b95b1-0f3f-4cb5-9105-e606dd1b86c0","order_by":6,"name":"Mahsa M.Amoli","email":"","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mahsa","middleName":"","lastName":"M.Amoli","suffix":""}],"badges":[],"createdAt":"2021-02-07 13:43:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-219449/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-219449/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":12256931,"identity":"6e21ac68-4a87-4a18-90c2-6e4f17e9bd1e","added_by":"auto","created_at":"2021-08-09 17:49:57","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":55624,"visible":true,"origin":"","legend":"The mRNA expression differences of TGF-β1gene in eutopic endometrium tissue of endometriosis patients and normal subjects an also proliferative and secretory phases of the menstrual cycle. a) The mRNA expression differences between endometriosis and normal subjects, b) The mRNA expression differences between endometriosis and normal subjects in proliferative phase, c) The mRNA expression differences between endometriosis and normal subjects in secretory phase, d) The mRNA expression differences between proliferative and secretory phases in normal subjects, and e) The mRNA expression differences between proliferative and secretory phases in endometriosis patients.","description":"","filename":"F1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-219449/v1/c27f55544117707e8a1b31b9.jpg"},{"id":13707933,"identity":"806c1b58-a334-46e6-a057-4b41399f00cb","added_by":"auto","created_at":"2021-09-17 14:05:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":413920,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-219449/v1/01068ea7-abb8-4b41-a88d-6556946e1f3b.pdf"}],"financialInterests":"","formattedTitle":"Investigation of TGF-β1 gene variant and expression in a group of Iranian women with endometriosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEndometriosis is defined as a common gynecologic, estrogen-dependent, chronic, and inflammatory disease that is affecting around 6\u0026ndash;10% of women in their reproductive period and is a major contributor to chronic pelvic pain, dysmenorrhoea, dyspareunia, and subfertility [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. It is described by the existence of endometrial-like tissue outside the uterine cavity, mainly on the pelvic peritoneum, ovaries and rectovaginal area [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The prevalence of endometriosis disease seems to be ~\u0026thinsp;5% among women between 25 years and 35 years of age [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. It is associated with primary or secondary infertility in 30% of the women [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Although, the pathogenesis of endometriosis is unknown, the development of this polygenic and multifactorial disease is influenced by a combination of genetic, immunologic, hormonal, and environmental factors [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Up until now, the exact etiology of endometriosis has been not known while there is increasing evidence that \u003cem\u003etransforming growth factor-beta 1 (TGF-β1)\u003c/em\u003e gene and its protein level might play a role in the pathogenesis of inflammatory disease, including endometriosis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Because \u003cem\u003eTGF-β1\u003c/em\u003e is a multifunctional cytokine that is involved in endometrial stromal cells proliferation and differentiation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], angiogenesis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], formation of adhesion [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and immune response [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral lines of evidence represent that susceptibility to the development of endometriosis is somewhat influenced by genetic factors. Accordingly, with respect to the role of \u003cem\u003eTGF-β1\u003c/em\u003e as a candidate gene in susceptibility to endometriosis, investigation of -509 C/T variant (rs1800469) of the \u003cem\u003eTGF-β1\u003c/em\u003e gene, which is the main determinant of plasma TGF-β1 concentration, will be beneficial for determining the role of this genetic factor in development of endometriosis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Nonetheless, the role of the aforementioned polymorphism in risk of endometriosis has not been investigated in Iranian population so far. Therefore, the present study aimed to assess the associations between endometriosis and the \u0026minus;\u0026thinsp;509 C/T polymorphism of the \u003cem\u003eTGF-β1\u003c/em\u003e gene as well as TGF-B1 mRNA expression among a group of Iranian women for the first time.\u003c/p\u003e"},{"header":"Method","content":"\u003cp\u003eThe present study aimed at two points; first, analysis of the associations between the \u0026minus;\u0026thinsp;509 C/T (rs1800469) variant of the \u003cem\u003eTGF-β1\u003c/em\u003e gene and endometriosis risk. Second, assessing the TGF-β1 mRNA expression in eutopic endometrium tissue of patients with and without endometriosis.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBlood Sample\u003c/h2\u003e \u003cp\u003eBlood samples were taken from a totally 297 subjects who had undergone laparoscopy or laparotomy for diagnosis or treatment of endometriosis to assess the association between \u003cem\u003eTGF-β1\u003c/em\u003e -509 C/T genetic variant and endometriosis susceptibility.\u003c/p\u003e \u003cp\u003eThe samples were enrolled from two hospitals (Firoozgar and Valiasr) between April 2013 and September 2015 and the diagnosis of endometriosis was based on surgical and histologic criteria.\u003c/p\u003e \u003cp\u003eThe study group (case) was involved 100 Iranian patients with endometriosis with the histologically confirmed diagnosis of endometriosis after their operative findings, according to the presence of endometrial glands or stroma in the lesions.\u003c/p\u003e \u003cp\u003eSince endometriosis is estrogen-dependent, the control group was selected from women at reproductive age and before menopause with a definite diagnosis of the absence of endometriosis by a surgeon.\u003c/p\u003e \u003cp\u003eWe included 197 healthy volunteers who underwent laparoscopy, and laparotomy due to tubal ligation or treatment of benign diseases such as ovarian cysts, myoma, hydrosalpinx, or other reasons.\u003c/p\u003e \u003cp\u003eIndividuals with a history of malignancy, those at menopausal age, and patients who had received hormonal treatment for at least 6 months since sampling were excluded from the study.\u003c/p\u003e \u003cp\u003eThree peripheral blood samples (in EDTA containing vial) were assembled from all subjects (females with endometriosis and controls females) for genotyping at the time of enrollment and stored at -20 \u0026deg;c until DNA extraction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eTissue Sample\u003c/h2\u003e \u003cp\u003eFor evaluating the TGF-β1 mRNA expression, samples of eutopic endometrium tissue were obtained by sharp curettage of the corpus of the uterine cavity of both 15 healthy controls and 15 women with endometriosis who had undergone a surgical resection between April 2013 and September 2015 at two Hospitals (Firoozgar and Valiasr) and was snap-frozen in liquid nitrogen and stored at -80\u0026deg;C until used.\u003c/p\u003e \u003cp\u003e Written informed consent and answer a questionnaire about demographics and clinical characteristics such as gravidity, parity, abortion, dysmenorrhea, menes, and bleeding were received from all the participants who were volunteers for blood and tissue sampling before they enrolled. Dysmenorrhea and bleeding were classified based on the amount of the pain (Normal, Severe, Not) and bleeding (Normal, Severe, Low) in the menstrual time. Regarding menes, subjects also were categorized as regular and irregular.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSNP genotyping\u003c/h2\u003e \u003cp\u003eGenomic DNA from whole blood was isolated from all subjects using phenol protocol. Extracted DNA was labeled and stored at -20\u0026deg;C until use. In the isolated DNA, we identified \u0026minus;\u0026thinsp;509 C/T polymorphic site in the promoter region of the \u003cem\u003eTGF-\u0026szlig;1\u003c/em\u003e gene with the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method. PCR primers were designed based on the Genbank reference sequence. The primers sequences that used for amplifying DNA were listed in the Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. PCR amplification was carried out in a final reaction volume of 20 \u0026micro;l final reaction containing 1\u0026micro;l template DNA, 10\u0026micro;l Red Master Mix (Amplicon), 0.5\u0026micro;l each primer and 8\u0026micro;l H\u003csub\u003e2\u003c/sub\u003eO by a Biorad MJ mini (Singapore) device. PCR cycle was done under the following conditions: an initial denaturation step at 95\u0026deg;c for 5 minutes followed by 35 cycles of 30 seconds at 95\u0026deg;c, 30 seconds at 59\u0026deg;c; 35 seconds at 72\u0026deg;c, and a final elongation at 72\u0026deg;c for 10 minutes. The allelic variant \u0026minus;\u0026thinsp;509 C (a 2.5 \u0026micro;l PCR product C) can be digested with 0.25 \u0026micro;l restriction enzyme \u003cem\u003eBsu36\u003c/em\u003eI (New England Biolab) at 37\u0026deg;C overnight in a 10 \u0026micro;l reaction that produced one fragment of 419 bp for the CC genotype; three fragments of 419, 229, and 190 bp for the CT genotype; and two fragments of 229, and 190 bp for the TT genotype.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e Primers that used in PCR and RT-PCR assay\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePCR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimers\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTGF-\u0026szlig;1\u003c/p\u003e \u003cp\u003e-509 C/T\u003c/p\u003e \u003cp\u003e(rs1800469)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-CAGACTCTAGAGACTGTCAG-3\u0026prime;\u003c/p\u003e \u003cp\u003e5\u0026prime;-GTCACCAGAGAAAGAGGAC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRT-PCR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003ePrimers\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTGF-\u0026szlig;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5-CCATGAACTTTCTGCTGTCTT-3\u003c/p\u003e \u003cp\u003e5-TCGATCGTTCTGTATCAGTCT-3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHPRT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-CCTGGCGTCGTGATTAGTGAT-3'\u003c/p\u003e \u003cp\u003e5'- TTATGGACAGGACTGAACGTCT-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003ePCR, Polymerase chain reaction; RT-PCR, Reverse transcription polymerase chain reaction\u0026nbsp;; TGF-\u0026szlig;1, Transforming growth factor-beta 1; HPRT, Hypoxanthine-guanine phosphoribosyltransferase\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e5 \u0026micro;l of digestion product and 7\u0026micro;l of Ready-Load 100-bp DNA Ladder (Invitrogen, Spain) were loaded into 3% agarose gel (Invitrogen, Spain) containing 1.4\u0026micro;l of sybregreen. The gel underwent electrophoresis at 120V, 100 mA for 30 min. The gel was visualized using a UV light (Wealtec, South Africa), and photographs of gels were taken after staining. We confirmed the genotypes identified with enzyme digestion by Sanger sequencing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003emRNA VEGF expression\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003eIsolation of RNA and complementary DNA Synthesis\u003c/h2\u003e \u003cp\u003eIn brief, total RNA from frozen tissue was extracted using the fermentas kit (Qiagen Inc., Valencia, CA, USA), in accordance with the manufacturer\u0026rsquo;s instructions. RNA concentration and purity were measured using NanoDrop 2000 (Thermo Scientific, USA) at a wavelength of 260 nm, and both mRNA and protein concentrations were measured at a wavelength of 280 nm. Two micrograms of total RNA were subjected to reverse transcription into the first-strand cDNA with a commercially available kit (Thermo Scientific, USA) according to the manufacturer\u0026rsquo;s protocol. In the first step, 0.5\u0026micro;l primer (Random Hex) (100\u0026micro;m) and 3.5\u0026micro;l dH\u003csub\u003e2\u003c/sub\u003eO was added to 2\u0026micro;l of total RNA and annealed at 65\u0026deg;c for 5 minutes. Then, 2\u0026micro;l primescript buffer (5x), 1\u0026micro;l dNTP, 0.5\u0026micro;l Revertaid and 0.5\u0026micro;l Ribolock was added in the last tube at 25\u0026deg;c for 5 minutes, 42\u0026deg;c for 1 hour and 70\u0026deg;c for 5 minutes. The cDNA was stored at -80\u0026deg;C until used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003eRT-PCR\u003c/h2\u003e \u003cp\u003eTranscribed products were subjected to PCR for \u003cem\u003eTGF-\u0026szlig;1\u003c/em\u003e and hypoxanthine-guanine phosphoribosyltransferase \u003cem\u003e(HPRT)\u003c/em\u003e in a 15\u0026micro;l final reaction volume. For a negative control, the cDNA template was omitted from the reaction. Amplification for \u003cem\u003eTGF-\u0026szlig;1\u003c/em\u003e cDNA was started with 5 minutes denaturation at 95\u0026deg;c, followed by cycles of 30 seconds of denaturation at 94\u0026deg;c, 30 seconds of annealing at 53\u0026deg;c, and 5 minutes of extension at 72\u0026deg;c. The PCR profile for \u003cem\u003eHPRT\u003c/em\u003e began with 95\u0026deg;c denaturation for 5 minutes, followed by cycles of 39 seconds of denaturation at 95\u0026deg;c, 40 seconds of annealing at 60\u0026deg;c, and 5 minutes of extension at 72\u0026deg;c.\u003c/p\u003e \u003cp\u003eThe primers used for \u003cem\u003eTGF-\u0026szlig;1\u003c/em\u003e and HPRT amplification were shown in the Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Final PCR products were exposed to electrophoresis through at 2% agarose gel and stained with sybregreen. The size of reverse transcription polymerase chain reaction (RT-PCR) products for \u003cem\u003eTGF-\u0026szlig;1\u003c/em\u003e and HPRT were 83 and 131 bp, respectively.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eReal-Time PCR\u003c/h2\u003e \u003cp\u003eConcisely, the real-time PCR amplification was accomplished in a total volume of 20\u0026micro;l, containing 6\u0026micro;l of cDNA sample, 9.8\u0026micro;l sybre, 0.2\u0026micro;l Rox, 0.6\u0026micro;l of each primer and 2.8 dH\u003csub\u003e2\u003c/sub\u003eO.The reactions were done in duplicate by ABI System (Applied Biosystems, Foster City, CA). The real-time PCR program consisted of 10 minutes at 95\u0026deg;C, followed 5 seconds at 95\u0026deg;C, 34 seconds at 53, 15 seconds at 95\u0026deg;c, 1hour at 53 \u0026deg;c and 15 seconds at 95.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analysis was donning using the Stata software version 14.2.\u003c/p\u003e \u003cp\u003eCase and controls were compared using the Kruskal-Wallis test for the quantitative variables such as age, BMI, parity, gravidity, and abortion. Data were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD).\u003c/p\u003e \u003cp\u003eChi-squared test was used for comparison of qualitative values (categorical) including dysmenorrhea, menes, and bleeding, presented by number and percentage.\u003c/p\u003e \u003cp\u003eDifferences of \u003cem\u003eTGF-\u0026szlig;1\u003c/em\u003egenotype and allele frequencies between endometriosis patients and controls were compared using Logistic regression and an odds ratio (OR) with 95% confidence interval (CI) were used as a measure of the strength of association between genotypes, allele frequencies. The Mann\u0026ndash;Whitney U test was used to evaluate the association of the mRNA TGF-B1 expression with endometriosis risk.\u003c/p\u003e \u003cp\u003eDeviations from Hardy-Weinberg equilibrium (HWE) in control group were assessed using Chi-squared test (\u003cem\u003eX\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e).\u003c/p\u003e \u003cp\u003eProbability values less than or equal to 0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Result","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCharacteristics of the patients and controls\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows characteristics of the endometriosis patients and controls including age, BMI, gravidity, parity, abortion, dysmenorrhea, menes, and bleeding.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of endometriosis patients and controls women in a group of Iranian population\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEndometriosis\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;100\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNormal\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;197\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber Total\u0026thinsp;=\u0026thinsp;296\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;197\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35.39\u0026thinsp;\u0026plusmn;\u0026thinsp;7.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35.50\u0026thinsp;\u0026plusmn;\u0026thinsp;11.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI (kg/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"6\" rowspan=\"7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01٭\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber Total\u0026thinsp;=\u0026thinsp;285\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e192\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25.47\u0026thinsp;\u0026plusmn;\u0026thinsp;4.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.45\u0026thinsp;\u0026plusmn;\u0026thinsp;4.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;18.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (5.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18.5\u0026ndash;24.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44 (48.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42 (52)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25-29.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27 (30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e82 (42.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14 (15.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e68 (35.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGravidity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01٭\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber Total\u0026thinsp;=\u0026thinsp;293\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e194\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01٭\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber Total\u0026thinsp;=\u0026thinsp;293\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e194\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.88\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.03\u0026thinsp;\u0026plusmn;\u0026thinsp;1.43\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbortion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber Total\u0026thinsp;=\u0026thinsp;293\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e194\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eDysmenorrhea\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber Total\u0026thinsp;=\u0026thinsp;244\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e146\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36 (36.73)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47 (24.23)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32 (32.65)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11 (5.67)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNot\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30 (30.61)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e88 (45.36)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eMenes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e0.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber Total\u0026thinsp;=\u0026thinsp;244\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e146\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRegular\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70 (71.43)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e104 (53.61)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIrregular\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28 (28.57)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42 (21.65)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBleeding\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e0.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber Total\u0026thinsp;=\u0026thinsp;244\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e146\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e58 (59.18)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e82 (42.27)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29 (29.59)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e53 (27.32)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11 (11.22)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11 (5.67)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eBMI, body mass index\u003c/p\u003e \u003cp\u003e٭ P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 is significant\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThere was no significant difference in the mean age between the endometriosis patients and the controls group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.9) while cases had significant lower BMI compared to control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003eBoth groups were showed significant differences in gravidity and parity (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Conversely, no significantly different was observed in abortion between the two groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.06).\u003c/p\u003e \u003cp\u003ePatients with endometriosis showed some symptom of endometriosis such as dysmenorrhea higher than the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). A significant difference was not found in the bleeding (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.41) and menes between the two groups (\u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.97\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTGF-\u0026szlig;1 variant (-509 C/T) in Patients with Endometriosis and Controls\u003c/h2\u003e \u003cp\u003eTotally, 100 cases and 196 controls from 297 enrolled subjects were successfully genotyped for \u0026minus;\u0026thinsp;509 C/T variant in the promoter of \u003cem\u003eTGF-\u0026szlig;1\u003c/em\u003e gene.\u003c/p\u003e \u003cp\u003eThe genotype distribution of -509 C/T variant did not show any deviation from the HWE in control subjects (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.498).\u003c/p\u003e \u003cp\u003eThe genotype and allele frequencies for the \u003cem\u003eTGF-\u0026szlig;1\u003c/em\u003e -509 C/T SNP are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of un-adjusted and adjusted odds ratio for five genetic models for candidate gene variant\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003cp\u003e(rsID)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenotype/allele\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCase\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;100\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;196\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eOR (95 % CI)\u003c/p\u003e \u003cp\u003eTT vs. CC (Homozygote)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eOR (95 % CI)\u003c/p\u003e \u003cp\u003eCT vs. CC (Heterozygote)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003eOR (95 % CI)\u003c/p\u003e \u003cp\u003eCT\u0026thinsp;+\u0026thinsp;TT vs. CC (Dominant)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e\u003cem\u003eTGF-β1\u003c/em\u003e\u003c/p\u003e \u003cp\u003e( rs1800469)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eCC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21 (21)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32 (16.33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.452\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUn-Adjusted\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAdjusted\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUn-Adjusted\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAdjusted\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eUn-Adjusted\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eAdjusted\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eCT\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44 (44)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100 (51.02)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.833\u003c/p\u003e \u003cp\u003e(0.418\u0026ndash;1.65)\u003c/p\u003e \u003cp\u003eP\u0026thinsp;=\u0026thinsp;0.603\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.303 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e(0.117\u0026ndash;0.781)\u003c/p\u003e \u003cp\u003eP\u0026thinsp;=\u0026thinsp;0.014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.670\u003c/p\u003e \u003cp\u003e(0.348\u0026ndash;1.29)\u003c/p\u003e \u003cp\u003eP\u0026thinsp;=\u0026thinsp;0.231\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.427 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e(0.186\u0026ndash;0.981)\u003c/p\u003e \u003cp\u003eP\u0026thinsp;=\u0026thinsp;0.045\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.734\u003c/p\u003e \u003cp\u003e(0.397\u0026ndash;1.35)\u003c/p\u003e \u003cp\u003eP\u0026thinsp;=\u0026thinsp;0.322\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.366 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e(0.168\u0026ndash;0.796)\u003c/p\u003e \u003cp\u003eP\u0026thinsp;=\u0026thinsp;0.011\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eTT\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35 (35)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64 (32.65)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003cb\u003eOR (95 % CI)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eT vs. C (Allelic)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e\u003cb\u003eOR (95 % CI)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eTT vs. CT\u0026thinsp;+\u0026thinsp;CC (Recessive)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e86 (43)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e164 (41.84)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.786\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUn-Adjusted\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAdjusted\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUn-Adjusted\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAdjusted\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eT\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e114 (57)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e228 (58.16)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.953\u003c/p\u003e \u003cp\u003e(0.675\u0026ndash;1.34)\u003c/p\u003e \u003cp\u003eP\u0026thinsp;=\u0026thinsp;0.786\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.545 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e(0.352\u0026ndash;0.844)\u003c/p\u003e \u003cp\u003eP\u0026thinsp;=\u0026thinsp;0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.900\u003c/p\u003e \u003cp\u003e(0.541\u0026ndash;1.469)\u003c/p\u003e \u003cp\u003eP\u0026thinsp;=\u0026thinsp;0.686\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.935 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e(1.00-3.720)\u003c/p\u003e \u003cp\u003eP\u0026thinsp;=\u0026thinsp;0.048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"11\" nameend=\"c11\" namest=\"c1\"\u003e \u003cp\u003eOR, odds ratio; CI, confidence interval\u003c/p\u003e \u003cp\u003e\u003csup\u003ea\u003c/sup\u003e Adjusted for Age, BMI, parity, abortion\u003c/p\u003e \u003cp\u003e\u003csup\u003eb\u003c/sup\u003e Adjusted for age, BMI, parity\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eA significant difference was not observed in the CT and TT genotype distributions of \u003cem\u003eTGF-\u0026szlig;1\u003c/em\u003e -509 C/T between the cases and controls (P\u0026thinsp;=\u0026thinsp;0.4). No significant result was also found between genotype frequency of these groups after merging heterozygous and homozygous mutant genotypes (CT\u0026thinsp;+\u0026thinsp;TT). The frequency of the CT, TT genotypes in the patients was significantly higher than the control group after adjusted for some quantitative variables (Adjusted CT: OR\u0026thinsp;=\u0026thinsp;0.427, 95% CI\u0026thinsp;=\u0026thinsp;0.186\u0026ndash;0.981, P\u0026thinsp;=\u0026thinsp;0.045; Adjusted TT: OR\u0026thinsp;=\u0026thinsp;0.303, 95% CI\u0026thinsp;=\u0026thinsp;0.117\u0026ndash;0.781, P\u0026thinsp;=\u0026thinsp;0.014).\u003c/p\u003e \u003cp\u003eThe combined CT\u0026thinsp;+\u0026thinsp;TT genotype was associated with a significantly decreased risk of endometriosis compared with the CC genotype (Adjusted: OR\u0026thinsp;=\u0026thinsp;0.366, 95% CI\u0026thinsp;=\u0026thinsp;0.168\u0026ndash;0.796, and P\u0026thinsp;=\u0026thinsp;0.011).\u003c/p\u003e \u003cp\u003eThere were no significant differences in the allele frequencies of \u003cem\u003eTGF-\u0026szlig;1\u003c/em\u003e -509 C/T between the cases and controls (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.786). The presence of T allele appeared to be a protective allele for the development of endometriosis after multivariate analysis for age, BMI, parity, and abortion using regression test (Adjusted: OR\u0026thinsp;=\u0026thinsp;0.545, CI\u0026thinsp;=\u0026thinsp;0.352\u0026ndash;0.844, P\u0026thinsp;=\u0026thinsp;0.007).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of mRNA TGF-\u0026szlig;1 Expression\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, no significant differences were observed between endometriosis patients and controls in TGF-\u0026szlig;1 mRNA expression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.32).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEvaluating the expression of TGF-\u0026szlig;1 level through the menstrual cycle also showed that in the proliferative phase, TGF-\u0026szlig;1 mRNA level in eutopic endometrium from women with endometriosis was not significantly different compared to the controls (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.10) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). In the secretory phase, endometrium tissue from women with the disease also showed similar mRNA expression compared to the controls (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.96) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). On the other hands, we found that endometrial tissue from controls showed higher TGF-\u0026szlig;1 mRNA levels in the proliferative phase than in the secretory phase but the results was not significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.20) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Besides that, no significant difference was observed in TGF-\u0026szlig;1 level when comparing secretory versus proliferative phase in endometrium from women with endometriosis (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.18) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we undertook genotype analysis of the \u0026minus;\u0026thinsp;509 C/T variant and mRNA expression of the \u003cem\u003eTGF-β1\u003c/em\u003e gene in relation to endometriosis and found that presence of \u003cem\u003eTGF-β1\u003c/em\u003e 509T allele was protective against development of endometriosis (decreasing 46% risk of endometriosis), while patients with C allele were prone to have endometriosis after adjustment for cofounding variables.\u003c/p\u003e \u003cp\u003eAccording to the first investigation for the role of -509 C/T variant in susceptibility to endometriosis in a study by Hsieh and colleagues it has been demonstrated that TT genotype and T allele of \u003cem\u003eTGF-β1\u003c/em\u003e gene are related to the higher risk of advanced endometriosis in the Taiwanese women. But, no association of this variant and late-stage endometriosis was reported in their study. Based on their results, \u003cem\u003eTGF-β1\u003c/em\u003e may be a candidate gene for endometriosis risk. So that, genotype and allele frequencies of \u003cem\u003eTGF-β1\u003c/em\u003e variant can be as convenient markers for the prediction of endometriosis susceptibility [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Further, the contraversial result has been identified by Van Kaam et al. They did not observe any relationship between this variant and advanced-stage endometriosis in Dutch women. They also studied the role of \u003cem\u003eTGF-β1\u003c/em\u003e gene variant in increased risk of deep infiltrating endometriosis and no evidence of significant association was found [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlso, Kim and colleagues reported that the \u003cem\u003eTGF-β1\u003c/em\u003e-509 C/T variant may not be associated with an increased risk for advanced-stage endometriosis in Korean population. They did not observe significant differences in the genotype distributions or allele frequencies of the \u003cem\u003eTGF-β1\u003c/em\u003e gene \u0026minus;\u0026thinsp;509 C/T variant between endometriosis patients and controls [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConsidering the findings of the study by Lee et al the TC haplotype allele of the \u0026minus;\u0026thinsp;509 C\u0026frasl;T variants in the \u003cem\u003eTGF-β1\u003c/em\u003e gene may be associated with an increased risk of early-stage endometriosis and is thus one genetic factor that may determine susceptibility for endometriosis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThese findings corresponded to the result of one Meta-analysis of six case-control studies that showed no positive correlation of -509 C/T variant with risk of endometriosis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Similarly, no significant association was identified concerning the \u0026minus;\u0026thinsp;509 C/T variant with endometriosis development in a recent meta-analysis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn disagreement with only previous report by Johnson and colleagues and in accordance with the statistical results, our data indicate that there were no significant differences in mRNA TGF-β1 between endometriosis patients and controls. Finding of Johnson study demonstrates lower TGF-β1 mRNA and protein levels in endometrium with endometriosis than controls during the mid-secretory phase [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTaken together, our study findings provided some evidence that the \u003cem\u003eTGF-β1\u003c/em\u003e gene variant shows a significant protection in endometriosis susceptibility in an Iranian population. Clinical data and adjustment for co-founding factors seems to be a very important issue and must be considered in the interpretation of results.\u003c/p\u003e \u003cp\u003eHowever, no association between TGF-β1 expressions in endometrial tissue and endometriosis risk was observed.\u003c/p\u003e \u003cp\u003eSince the lack of association might come from the small sample size, which is the main limitation in our study therefore further investigations with a larger sample size including larger groups of patients with the same stage of endometriosis should be performed so that peritoneal fluid (PF) and peripheral blood could be obtained during the same stage of the menstrual cycle.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNo funding was obtained.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with ethical standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was no conflict of interest to report.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the ethics committee of Tehran University of medical sciences.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consents were obtained from the study participants.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to express their gratitude to all participants enrolled in this project.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYoung VJ, Ahmad S, Duncan WC, Horne AW (2017) The role of TGF-β in the pathophysiology of peritoneal endometriosis. Hum Reprod Update 23(5):548\u0026ndash;559. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/humupd/dmx016\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBulletti C, Coccia ME, Battistoni S, Borini A (2010) Endometriosis and infertility. J Assist Reprod Genet 27(8):441\u0026ndash;447. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10815-010-9436-1\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRashidi BH, Sarhangi N (2019) Association of vascular endothelial growth factor (VEGF) Gene polymorphisms and expression with the risk of endometriosis: a case-control study. 46(3):3445\u0026ndash;50. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/ 10.1007/s11033-019-04807-6\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGiudice LC (2010) Clinical practice. Endometriosis. N Engl J Med 362(25):2389\u0026ndash;2398. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/ 10.1056/NEJMcp1000274\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWee-Stekly W-W, Kew CCY, Chern BSM (2015) Endometriosis: a review of the diagnosis and pain management. Gynecol Minim Invasive Ther 4(4):106\u0026ndash;109. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.gmit.2015.06.005\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVercellini P, Vigano P, Somigliana E, Fedele L (2014) Endometriosis: pathogenesis and treatment. Nat Rev Endocrinol 10(5):261\u0026ndash;275. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrendo.2013.255\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAcien P, Velasco I (2013) Endometriosis: a disease that remains enigmatic. ISRN Obstet Gynecol 2013:242149. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2013/242149\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGiudice LC, Kao LC (2004) Endometriosis. Lancet (London, England) 364(9447):1789-99. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0140-6736(04)17403-5\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang F, Yang Y, Wang Y (2012) Association between TGF-beta1-509C/T polymorphism and endometriosis: a systematic review and meta-analysis. Eur J Obstet Gynecol Reprod Biol 164(2):121\u0026ndash;126. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ejogrb.2012.05.004\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHsieh YY, Chang CC, Tsai FJ et al (2005) Polymorphism for transforming growth factor beta 1-509 (TGF-B1-509): association with endometriosis. Biochem Genet 43(5\u0026ndash;6):203\u0026ndash;210. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/ 10.1007/s10528-005-5211-x\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHammond MG, Oh S-T, Anners J, Surrey ES, Halme J (1993) The effect of growth factors on the proliferation of human endometrial stromal cells in culture. Am J Obstet Gynecol 168(4):1131\u0026ndash;1136. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0002-9378(93)90356-n\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBertolino P, Deckers M, Lebrin F, ten Dijke P (2005) Transforming growth factor-β signal transduction in angiogenesis and vascular disorders. Chest 128(6):585S\u0026ndash;585S90S. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1378/chest.128.6_suppl.585S\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChegini N, editor TGF-β system: the principal profibrotic mediator of peritoneal adhesion formation. Seminars in reproductive medicine; 2008: \u0026copy; Thieme Medical Publishers\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOmwandho CO, Konrad L, Halis G, Oehmke F, Tinneberg H-R (2009) Role of TGF-βs in normal human endometrium and endometriosis. Hum Reprod 25(1):101\u0026ndash;109. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/humrep/dep382\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim JJ, Choi YM, Choung SH et al (2010) Analysis of the transforming growth factor β1 gene \u0026ndash; 509 C/T polymorphism in patients with advanced-stage endometriosis. Fertil Steril 93(7):2121\u0026ndash;2124. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.fertnstert.2009.01.082\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan Kaam K, Romano A, Dunselman G, Groothuis P (2007) Transforming Growth Factor β1 Gene Polymorphism\u0026mdash;509C/T in Deep Infiltrating Endometriosis. Reprod Sci 14(4):367\u0026ndash;373. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1177/1933719107303436\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee HJ, Kim H, Ku SY, Kim SH, Kim JG (2011) Transforming Growth Factor-β1 Gene Polymorphisms in Korean Women With Endometriosis. Am J Reprod Immunol 66(5):428\u0026ndash;434. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1600-0897.2011.01009.x\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMear L, Herr M, Fauconnier A, Pineau C, Vialard F (2020) Polymorphisms and endometriosis: a systematic review and meta-analyses. Hum Reprod Update 26(1):73\u0026ndash;102. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/humupd/dmz034\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJohnson MC, Torres M, Alves A et al (2005) Augmented cell survival in eutopic endometrium from women with endometriosis: expression of c-myc, TGF-beta1 and bax genes. Reprod Biol Endocrinol 3(1):45. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/1477-7827-3-45\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-gynecology-and-obstetrics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arch","sideBox":"Learn more about [Archives of Gynecology and Obstetrics](https://www.springer.com/journal/404)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/arch/default.aspx","title":"Archives of Gynecology and Obstetrics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"TGF-β1, gene expression, variant, Endometriosis","lastPublishedDoi":"10.21203/rs.3.rs-219449/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-219449/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eEndometriosis is defined as a common gynecologic and inflammatory disease. Transforming growth factor-beta 1 \u003cem\u003e(TGF-β1)\u003c/em\u003e gene and its protein level might play a role in the pathogenesis of endometriosis. The present study aimed for the first time to assess the associations between endometriosis risk and \u0026minus;\u0026thinsp;509 C/T (rs1800469) variant of the \u003cem\u003eTGF-β1\u003c/em\u003e gene as well as TGF-β1 mRNA expression in eutopic endometrium tissue of patients with and without endometriosis among a group of Iranian women.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eGenotyping was carried out in 100 endometriosis patients (cases) with confirmed histological diagnosis of endometriosis and 197 non-endometriosis subjects (controls). The expression level of TGF-β1 mRNA was determined using Real-Time PCR assay in 15 eutopic endometrium tissue of women with endometriosis and 15 healthy controls.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThere was a significant association for allele and genotype frequencies of rs1800469 variant and endometriosis i. No significant difference for TGF-β1 expression was observed between eutopic endometrium of patients and healthy group. Also, evaluation of TGF-β1expression across the menstrual cycle showed the same level of TGF-β1 among cases and controls subjects.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur investigations indicated enough evidence for the effect of \u003cem\u003eTGF-β1\u003c/em\u003e gene polymorphism on endometriosis risk in an Iranian population. Furthermore, we could not find any relations between TGF-β1 mRNA expression and susceptibility to endometriosis.\u003c/p\u003e","manuscriptTitle":"Investigation of TGF-β1 gene variant and expression in a group of Iranian women with endometriosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-08-09 17:49:55","doi":"10.21203/rs.3.rs-219449/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2021-08-07T17:14:50+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-08-07T13:35:29+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Archives of Gynecology and Obstetrics","date":"2021-08-07T02:01:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Gynecology and Obstetrics","date":"2021-04-04T23:43:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-gynecology-and-obstetrics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arch","sideBox":"Learn more about [Archives of Gynecology and Obstetrics](https://www.springer.com/journal/404)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/arch/default.aspx","title":"Archives of Gynecology and Obstetrics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"fbd66c7a-a9bd-4e87-8056-63a749c14276","owner":[],"postedDate":"August 9th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":6328229,"name":"Obstetrics \u0026 Gynecology"}],"tags":[],"updatedAt":"2021-08-18T08:51:21+00:00","versionOfRecord":[],"versionCreatedAt":"2021-08-09 17:49:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-219449","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-219449","identity":"rs-219449","version":["v1"]},"buildId":"WvIrzKhiLBfengagbw6Ux","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}