{"paper_id":"768e3f48-e053-4fc4-8938-1d88a01a4e4f","body_text":"565\nTumor Necrosis Factor Affects Steroidogenic Factor-1 \nExpression in Ovarian Granular Cells Via Inducing \nGene Methylation\nCopy Right@ Xiaoxi Sun\nThis work is licensed under Creative Commons Attribution 4.0 License  AJBSR.MS.ID.002154.\nAmerican Journal of\nBiomedical Science & Research\nwww.biomedgrid.com\n---------------------------------------------------------------------------------------------------------------------------------\nISSN: 2642-1747\nResearch Article\nXiang Lu, Lu Li, Junling Chen, Wenbi Zhang and Xiaoxi Sun*\nAffiliated Obstetrics and Gynecology Hospital of Fudan University, Shanghai JIAI Genetics and IVF institute, China\n*Corresponding author: Xiaoxi Sun, Affiliated Obstetrics and Gynecology Hospital of Fudan University, Shanghai JIAI Genetics and \nIVF institute, Shanghai, People’s Republic of China.\nTo Cite This Article: Xiang Lu, Lu Li, Junling Chen, Wenbi Zhang and Xiaoxi Sun. Tumor Necrosis Factor Affects Steroidogenic Factor-1 Expression \nin Ovarian Granular Cells Via Inducing Gene Methylation. Am J Biomed Sci & Res. 2022 - 15(5). AJBSR.MS.ID.002154.\nDOI: 10.34297/AJBSR.2022.15.002154\nReceived: \n  February 28, 2022;  Published: \n   March 11, 2022\nAbstract\nObjective: The expression of SF-1 in ovarian granulosa cells is critical for follicular growth and development, and the decreased \nexpression of SF-1 in ovarian granulosa cells under endometriosis (EMs) pathology is not clear. This study aims to investigate \nthe mechanism of tumor necrosis factor (TNF), a factor promoting endometriosis, in regulating the promoter methylation and \nexpression of steroidogenic factor-1 (SF-1) in ovarian granulosa cells.\nMethods: Human ovarian granulosa cell line KGN was used. Methylation inhibition was mediated by cells treated with 5-Aza-2 \n‘deoxycytidine (5-Aza). The inhibition of the p65 expression gene in KGN cells was mediated by a lentiviral CRISPR/Cas9 vector (p65-\n/-KGN cells). The methylation level in the promoter region of SF-1 was detected by BSP assay and SF-1 expression was determined \nusing quantitative real-time PCR and western blot.\nResults: In KGN cells, inhibition methylation increases the mRNA level of SF-1. TNF treatment causes an increased methylation \nlevel of SF-1 and inhibits the mRNA and protein expression of SF-1 in a concentration-dependent manner. Inhibition methylation of \nwild-type KGN (p65wt) and p65-/- KGN cells enhances SF-1 expression. TNF treatment significantly increase the methylation level of \nSF-1 and decreased the expression of SF-1 in p65wt KGN cells but does not affect that in p65-/- KGN cells.\nConclusion: TNF induces the increase in methylation of the SF-1 gene, which leads to the decrease of SF-1 expression in KGN \ncells through the ReIA/p65 pathway. It is suggested that the decrease of SF-1 expression in ovarian granulosa cells of EMs is related \nto the immune mechanism of EMs disease.\nKeywords: Endometriosis; Ovarian granulosa cells; Steroidogenic factor 1; Tumor necrosis factor\nIntroduction\nEndometriosis (EMs) is an immune-related chronic \ninflammatory disease and can lead to infertility in reproductive-\nage women through multiple factors and mechanisms [1]. In \nvitro fertilization embryo transfer (IVF-ET) is one of the effective \ntreatment methods for EMs-induced infertility, but EMs has  \n \nadverse effects on the treatment outcome of IVF-ET . The research \nhas pointed out that EM-derived egg embryos had a limited growth \nrate and clinical pregnancy rate in recipients with non-EMs, mostly \nbecause of follicular development obstacles and decline in the \nquality of oocytes leading to a decline in embryonic plant capacity \n\nAmerican Journal of Biomedical Science & Research\nAm J Biomed Sci & Res                                     Copy@ Xiaoxi Sun\n566\n[2,3]. This suggests the necessity of conducting basic research to \nexplore the mechanism of egg quality loss in EMs-induced infertility.\nSteroidogenic factor-1 (SF-1), a member of the myonuclear \nreceptor family, is abnormally expressed in intrinsic and ectopic \nendometrial tissue of EMs [4] and plays an important role in the \npathogenesis of EMs [5]. In the ectopic endometrium, SF-1 promotes \nthe high expression of its target gene P450 aromatase, which \nenables the ectopic endometrium to synthesize estrogen locally \nwithout relying on circulating estrogen, and further promotes the \ngrowth of the ectopic endometrium [5]. Our previous study found \nthat the transcription level of SF-1 in ovarian granulosa cells of \npatients with EMs was downregulated and the level of estradiol \nsynthesis in granulosa cells was decreased [6,7]. The significance \nof SF-1 expression in ovarian granulosa cells is its necessity for \nnormal follicular development and the maintenance of ovarian \nreproductive function, which is different from its promoting role in \nthe pathological growth of the endometrium. SF-1 knockout mice \noften show follicular arrest, follicular atresia, and infertility [8].\nHerein, understanding the molecular mechanism of the \ndecreased expression of SF-1 in ovarian granulosa cells in the \npathological state of EMs can help us to understand the pathological \nmechanism of infertility induced by EMs. The regulation of SF-1 \ngene expression is mainly realized through DNA methylation. \nAs one of the important epigenetic mechanisms, the methylation \nof CpG island in gene promoters is an important way to regulate \ngene expression. A study has shown that the CpG site in the \npromoter region of the SF-1 gene is hypermethylated [9]. Whether \nthe hypermethylated state of the SF-1 promoter is related to the \ndecreased expression of SF-1 and the underlying mechanisms of \nSF-1 expression in granulosa cells of EMs population has not been \nknown yet.\nExisting theories support that the inflammatory response \ninvolved by macrophages plays an important role in the occurrence \nand development of EMs [10,11]. Tumor necrosis factor (TNF), a \ncytokine secreted by macrophages, is an on-off activating factor \nin early inflammation. The level of TNF in follicular fluid of EMs \nwas significantly higher than that in normal follicular fluid [12,13] \nwhich is involved in the pathogenesis of EMs and is one of the \ninitiating factors for the occurrence, development, and invasion \nof ectopic endometrium. TNF has a function in regulating the \nexpression of its target genes by activating transcription factors \n(i.e., nuclear factor-kappa B, NF- κB) to change the methylation \nlevel of target gene promoters [14,15]. In this study, we tried to \ninvestigate whether TNF affected the methylation level of the SF-1 \ngene promoter, thereby regulating the transcription and expression \nof SF-1 in granulosa cells.\nMaterial and Methods\nReagents \nConsidering the obtained ovarian granulosa cells purified \nfrom follicular fluid in IVF puncture egg extraction is luteinated \ngranulosa cells and may have an influence on the accuracy and \nsignificance for the experiment, so the selection of ovarian granular \ncell lines (KGN) as the research object. KGN cells were donated \nby Professor Yi-ming Mu of the Chinese people’s liberation army \ngeneral hospital. Cell transfection reagent Lipofectamine2000, \nDMEM high glucose medium, fetal bovine serum, trypsin, and PBS \nbuffer was purchased from Thermo Fisher Scientific (USA). P65 \nlentivirus kit was provided by Shanghai Heyuan Biotech (China). \nECL substrate chromophase solution and Trizol kit were purchased \nfrom Sigma (USA). PCR reagent and reverse transcription kit were \npurchased from Takara (China). TIAN amp Genomic DNA Kit was \npurchased from Beijing Tiangen Biochemical Technology (China). \nEZ DNA Methylation Gold kit was purchased from Zymo Research, \nInc (USA).\nCell Culture and Drug Treatment\nAbout 104 KGN cells were resuspended in a DMEM culture \nmedium (containing 10% FBS and 1% double-antibody) and \ncultured in an incubator at 37℃ and 5% CO 2. The medium was \nchanged every 2-3 days. When the cells grew to about 90% \nconfluence, the cells were passed or frozen stored. Before the cell \nintervention experiment, the cells were starved with the DMEM \nculture medium for 12-24h.\nTo explore the effect of inhibition of methylation on KGN \ncell expression of SF-1, cells were treated with 1μM 5-Aza-2 \n‘deoxycytidine (a DNA methylation inhibitor, 5-aza). Cells treated \nwith an equal volume of DMSO served as the negative control.\nCell Transfection for SF-1 Knockdown \nA total of 5×104 KGN cells was inoculated on a 24-well plate one \nday before transfection, and 50, 100, and 200 nmol/L siRNA (siRNA \nfor SF-1 designed by Suzhou Jima Company, China) were added \nto 50μL DMEM serum-free medium. Lipofectamin2000 reagent \nwas diluted with serum-free DMEM (1μL:50μL) (2.4μg RNAi-mate \nreagent was added when DNA transfection). The diluted siRNA \nand lipofectamin2000 reagent were mixed to form the siRNA/\nlipofectamine (or DNA/ lipofectamine) complex, and the complex \nwas added to the wells containing the cells and the culture medium.\nLentiviral CRISPR/Cas9 Mediated p65 Inhibition\nLentiviral CRISPR/Cas9 vector was used to inhibit p65 in KGN \ncells (p65-/- KGN cells) with empty vector in KGN cells (p65wild type(wt.) \n\nAm J Biomed Sci & Res\nAmerican Journal of Biomedical Science & Research\nCopy@ Xiaoxi Sun\n567\nKGN cells) as control. In brief, a total of 3~5×104 KGN cells in 500μL \nmedium were inoculated in a 24-well culture plate. Lentivirus \ninfection begins when cells grew to 30-50% confluence. KGN cells \nwere transfected with p65 CRISPR/Cas9 lentivirus or control vector \naccording to the instructions of the lentivirus kit. After mixing \nwith vectors, the cells were placed in an incubator (37℃, 5% CO 2) \novernight and the culture medium was changed 12-20 hours later. \nThe infection effect was observed 24 hours later by laser confocal \nmicroscopy and GFP fluorescence.\nQuantitative Real-Time PCR Detects SF-1 Transcription \nTotal RNA was extracted by the Trizol method, and cDNA \nwas synthesized referring to the instructions of the TAKARA \nreverse transcription kit (RR047A, TaKaRa, China). SF-1 primer \nwas synthesized by Suzhou Jinweizi Company (China). SF-1 \nForward: 5’-TGGACHHAATCGGAACACG-3’; SF-1 Reverse: 5’ \nTGGCTATGGCACCTTGAAAAAC-3’ . the reaction conditions of general \nquantitative PCR reagent system were as follows: pre-denaturation \nat 95℃ for 10 minutes, denaturation at 95℃ for 25 seconds, \nannealing at 55℃ for 25 seconds, extension at 72℃ for 30 seconds, \ncirculation for 40 times, extension at 72℃ for 5 minutes to end the \nreaction, and fluorescence was collected at this step. GAPDH gene \nwas an internal reference.\nWestern Blot Detects SF-1 Protein Content\nKGN cells were transfected with SF-1 siRNA plasmid and was \nadded to with cell lysate to extract total proteins. The protein \nsample was quantified by the BCA method and a volume of 40μL \nwas loaded on PAGE gel. After electrophoresis, proteins were \ntransferred to the PVDF membrane (Merck, German). Primary \nantibodies against SF-1 and GAPDH (Proteintech, USA) were added \nto the membrane and incubated at 4℃ overnight. On the next day, \nthe PVDF membrane was incubated with a secondary antibody \n(HRP-labeled sheep anti-rabbit IgG) (Biotin, China) for 2 hours. \nPVDF membrane was washed at room temperature and added with \nECL. The blot was visualized and photographed.\nBisulfite Sequencing PCR (BSP) Detects Methylation \nLevel at 5 ‘CpG Site of SF-1 Gene\nThe centrifugal type of column genomic DNA extraction kit \nwas used for the extraction of genomic DNA, and the EZ DNA \nMethylation-Gold kit for bisulfite conversion. The genomic DNA \nafter bisulfite conversion was used for specific PCR amplification \n(SF-1 primer and the PCR reaction conditions were the same as the \nformer). PCR products were purified and connected to PMD18-T \ncarrier. The positive plaque was selected for plasmid extraction and \nsequencing (10 cloning). \nStatistical Analysis\nData were analyzed on SPSS version 11.0 and presented as \nmean standard deviation (SD). Analysis of significant differences \nbetween groups was conducted using a student’s t-test. More than \n3 independent experiments were performed for each protocol. A \np-value less than 0.05 was considered statistical significance.\nResults\nMethylation Inhibition Promotes SF-1 Expression\nWe examined the SF-1 expression levels in methylation \ninhibitor 5-aza treated cells. The results showed that compared \nwith DMSO, 5-aza induced the increase in mRNA and protein levels \nof SF-1 (Figure 1a and 1b, P<0.01), suggesting that hypomethylation \npromoted SF-1 transcription and protein expression in KGN cells.\nFigure 1: Effect of methylation inhibitor on SF-1 expression. KGN cells were treated with 1 μM of 5-aza-2’deoxycytidine and that treated with \nDMSO as a negative control (NC). Cells were collected for analysis of expression level of mRNA (A) and protein (B) using qRT-PCR and western \nblot. **P<0.01.\n\nAmerican Journal of Biomedical Science & Research\nAm J Biomed Sci & Res                                     Copy@ Xiaoxi Sun\n568\nTNF Enhances Methylation Level of the SF-1 Gene\nBSP assay detected the effect of different concentrations of \nTNF on methylation level of the proximal promoter region of SF-1 \ngene via sequencing on gene fragments covering 13 CpG loci (from \nCpG-84 to the CpG+168) near transcription start site and part of \nthe exon 1 area (122-/129/+). The result showed that 10ng/mL, \n20ng/mL, 30ng/mL, and 40ng/mL TNF caused the increase in the \nmethylation level of the SF-1 gene and the methylation level on the \nSF-1 gene was increased with the increase of TNF concentration in \nKGN cells (Figure 2).\nFigure 2: Effects of different concentrations of TNF on SF-1 methylation in KGN cells. Cells were treated with TNF as indicated doses and that \ntreated with DMSO as a negative control (NC). Cells were collected for a measure of methylation level at 5’CpG site of SF-1 gene using BSP \nassay. *P<0.05, **P<0.01.\nTNF Inhibits SF-1 Expression\nQuantitative real-time PCR and Western blot determined SF-1 \nexpression in KGN cell processing different concentrations (10 ng/\nml, 20 ng/ml, 30 ng/ml, and 40 ng/ml) of TNF. The results showed \nthat compared with negative control, cells in the TNF treatment \ngroup expressed a lower level of SF-1 mRNA and protein. The \ninhibitory effect of TNF on SF-1 mRNA and protein expression was \nin a dose-dependent manner and the inhibition effect was strongest \nat 30ng/ml (Figure 3A and B).\n\nAm J Biomed Sci & Res\nAmerican Journal of Biomedical Science & Research\nCopy@ Xiaoxi Sun\n569\nFigure 3: Effects of different concentrations of TNF on SF-1 expression in KGN cells. KGN cells were treated with TNF as indicated doses and \nthat treated with DMSO as the negative control (NC). Cells were collected for analysis of expression level of mRNA (A) and protein (B) using \nqRT-PCR and western blot. **P<0.01.\nTNF Affects Methylation Level and SF-1 Expression in p65wt And p65-/- KGN Cells\nFigure 4: TNF affects methylation level and SF-1 expression in p65wt and p65-/- KGN cells. (A)qRT-PCR detects SF-1 mRNA expression \nin cells treated with methylation inhibitor 5-aza-2’deoxycytidine. **P<0.01. (B) BSP assay examines methylation levels in TNF-treated cells. \n*P<0.05; **P<0.01. (C) qRT-PCR detects SF-1 mRNA expression in TNF-treated cells. **P<0.01.\n\nAmerican Journal of Biomedical Science & Research\nAm J Biomed Sci & Res                                     Copy@ Xiaoxi Sun\n570\nThe above results suggest that TNF may inhibit the \ntranscription and expression of SF-1 in KGN cells by regulating \nthe methylation level of the SF-1 promoter. Studies have shown \nthat NF-κB (a transcription factor with an active subunit of REIA/\np65) was cascaded by TNF and thereby promoted the expression \nof its target genes [14,15]. Our results showed that p65 -/-KGN cells \nexpressed higher levels of SF-1 than p65 wt cells when the negative \ncontrol treatment and after methylation inhibitor 5-aza treatment, \nSF-1 expression levels were significantly increased in both of p65wt \nand p65-/-KGN cells. These data suggest that p65 plays a role in the \nmethylation and expression of SF-1 and the regulatory expression \npathway by p65 is still effective even in p65-/-KGN cells (Figure 4a).\nTo confirm whether REIA/p65 is involved in the process of \nTNF induced the increase in SF-1 methylation and the decrease in \nSF-1 expression in KGN cells, we treated p65 wt and p65-/-KGN cells \nwith different concentrations of TNF (10ng/mL, 20ng/mL, 30ng/\nmL, and 40ng/mL) to detect the methylation level of SF-1. The \nresult of the BSP assay showed that in p65 wt KGN cells, 10ng/mL \nof TNF did not change the methylation level of SF-1, while 20, 30, \nand 40ng/mL of TNF significantly increased the methylation level \nof SF-1 (Figure 4b, P<0.01). However, in p65 -/- KGN cells, TNF did \nnot affect SF-1 methylation and SF-1 expression (Figure 4b and 4c). \nBesides, at any concentration of TNF, the SF-1 methylation level of \np65wtKGN cells was significantly higher than that of p65-/- KGN cells, \nwhile the SF-1 expression level of p65 wtKGN cells was lower than \nthat of p65-/- KGN cells (Figure 4b, 4c, P<0.01). It is suggested that \np65 is an important switch in TNF regulation of SF-1 methylation \nand expression in KGN cells.\nDiscussion\nInflammation and immune cell dysregulation affect infertility \nassociated with EMs. The level of cytokine TNF is significantly \nincreased in the peritoneal fluid of patients with EMs complicated \nwith infertility and is closely related to the occurrence of EMs \n[16,17]. The results in this study show that TNF decreases \nthe expression of SF-1, an essential molecular for follicular \ndevelopment, via increasing the methylation level of the SF-1 gene \nin KGN cells suggesting that TNF-induced downregulation of SF-1 \nmay be a pathological mechanism of EMs-induced infertility.\nPreviously, we detected high methylation status in the CpG \nsites of SF-1 gene promoter region in the ovarian granular cells of \nEMs patients using the BSP cloning sequencing method, especially \nfocused in CpG loci near the transcription start site +77, +121, and \n+141 [18]. In this study, the low methylation level of SF-1 promotes \nSF-1 mRNA and protein expression in KGN cells suggesting that \ngene methylation level affects SF-1 mRNA and protein expression. \nThis result is in accord with the finding by Xue et al., in which the \nSF-1 gene is demethylated in the interstitial cells of the intrinsic \nand ectopic endometrium, and this abnormal demethylated state \nis an important mechanism for the expression of SF-1 in ectopic \nendometrium (while the hypermethylated SF-1 gene in normal \nendometrium silences the expression of SF-1) [9,19].\nWe found that TNF can significantly reduce the mRNA and \nprotein expression level of SF-1 in KGN cells. In the pathogenesis \nof EMs, TNF is one of the initiating factors for the occurrence, \ndevelopment, and invasion of ectopic endometrium. In the follicular \nfluid of EMs patients, the level of TNF is significantly increased [12, \n13]. Our pre-test supported this fact (TNF 363 ±87ng/L of EMs vs. \n78±36ng/L of normal, P<0.05). As a switch-acting promoter for \nimmune response, TNF forms a cascade reaction with transcription \nfactor NF- κB via activating its active subunit REIA/p65, leading \nto abnormal expression of the target genes [14, 15]. The cascade \nreaction induced by TNF and NF- κB-mediated transcription \ninhibition of targeted genes is commonly found in tumors [15,20]. \nBy searching the bioinformatics software, we found that NF-κB has \nbinding sites in the SF-1 gene near the transcription initiation site \n(search at www.genecards.org). This leads us to further investigate \nwhether TNF associated with SF-1 methylation also occurred in \novarian granulosa cells.\nCampbell et al., for the first time put forward the definition \nof REIA/p65 to “active repressor [21], and study in tumor cells \nfound that REIA/p65 signal pathway plays an important role in cell \nproliferation, differentiation, apoptosis under the stimulation of \ncytokines (i.e., TNF and IL- 6) [14]. The molecular mechanism by \nwhich REIA/p65 acts is to suppress the transcription level of the \ngene by enhancing the methylation of the gene promoter. In KGN \ncells, we found that TNF increased the methylation level of the SF-1 \ngene in a concentration-dependent manner indicating that TNF \ninhibits the transcription and expression of SF-1 by controlling \nSF-1 gene methylation levels. REIA/p65 is a key molecule in this \nprocess. TNF did not affect the methylation level and expression of \nthe SF-1 gene in p65-/- KGN cells. The effect of TNF on enhancing the \nmethylation level of the SF-1 gene and decreasing the expression of \nSF-1 occurred only in p65wt KGN cells, although the methylation \nof p65-/- KGN cells was still effective in regulating the expression of \nSF-1. It is suggested that the REIA/p65 pathway plays a key role in \nthe increase of SF-1 methylation and the downregulation of SF-1 \nexpression in KGN cells induced by TNF, which is consistent with \nthe issue that REIA/p65 is a potential regulatory factor of DNA \nmethylation in oncologic studies.\nIn summary, the present research found that TNF can induce an \nincrease in gene methylation and thereby contributes the decrease \nin SF-1 mRNA and protein expression levels via REIA/p65 pathway \n\nAm J Biomed Sci & Res\nAmerican Journal of Biomedical Science & Research\nCopy@ Xiaoxi Sun\n571\nin KGN cells suggesting that the reduced expression of SF-1 in \novarian granulosa cells is related to the inflammation and immune \ncell dysregulation pathomechanism of EMs disease. Whether drugs \ncan be used to reduce the TNF level of follicular fluid of EMs and \nrestore the normal expression of SF-1 in granulosa cells, to improve \nthe quality of eggs and embryos is a research direction worthy of \nexploration.\nAcknowledgement\nNone\nReferences\n1. Y Wang, K Nicholes, I M Shih (2020) The Origin and Pathogenesis of \nEndometriosis. Annu Rev Pathol 15: 71-95.\n2. E E Hauzman, J A Garcia Velasco, A Pellicer (2013) Oocyte donation and \nendometriosis: What are the lessons? Semin Reprod Med 31(2): 173-\n177.\n3. S Senapati, M D Sammel, C Morse, K T Barnhart (2016) Impact of \nendometriosis on in vitro fertilization outcomes: an evaluation of the \nSociety for Assisted Reproductive Technologies Database. Fertil Steril \n106(1): 164-171 e1.\n4. Y Tian, B Kong, W Zhu, S Su, Y Kan (2009) Expression of steroidogenic \nfactor 1 (SF-1) and steroidogenic acute regulatory protein (StAR) in \nendometriosis is associated with endometriosis severity, J Int Med Res \n37(5): 1389-1395.\n5. S E Bulun, H Utsunomiya, Z Lin, P Yin, Y H Cheng, et al. (2009) \nSteroidogenic factor-1 and endometriosis. Mol Cell Endocrinol 300(1-\n2): 104-108.\n6. X Lu, Y Wu, X H Gao, Y W Wang, L Wang, et al. (2012) Effect of letrozole \non estradiol production and P450 aromatase messenger RNA expression \nof cultured luteinized granulosa cells from women with and without \nendometriosis. Fertil Steril 98(1): 131-135.\n7. X Lu, Z M Wu, Y W Wang, M Wang, W W Cheng, et al. (2015) Liver \nreceptor homologue-1 and steroidogenic factor-1 expression in cultured \ngranulosa cells from patients with endometriosis: A preliminary study. J \nObstet Gynaecol Res 41(12): 1927-1934.\n8. M C Meinsohn, O E Smith, K Bertolin, B D Murphy (2019) The Orphan \nNuclear Receptors Steroidogenic Factor-1 and Liver Receptor \nHomolog-1: Structure, Regulation, and Essential Roles in Mammalian \nReproduction. Physiol Rev 99(2): 1249-1279.\n9. Q Xue, Y Xu, H Yang, L Zhang, J Shang, et al. (2014) Methylation of a \nnovel CpG island of intron 1 is associated with steroidogenic factor 1 \nexpression in endometriotic stromal cells. Reprod Sci 21(3): 395-400.\n10. C Hogg, A W Horne, E Greaves (2020) Endometriosis-Associated \nMacrophages: Origin, Phenotype, and Function. Front Endocrinol \n(Lausanne) 11: 7.\n11. J Vallve Juanico, X Santamaria, K C Vo, S Houshdaran, L C Giudice (2019) \nMacrophages display proinflammatory phenotypes in the eutopic \nendometrium of women with endometriosis with relevance to an \ninfectious etiology of the disease. Fertil Steril 112(6): 1118-1128.\n12. H Falconer, J Sundqvist, K Gemzell Danielsson, B von Schoultz, T M D \nHooghe, et al. (2009) outcome in women with endometriosis in relation \nto tumour necrosis factor and anti-Mullerian hormone. Reprod Biomed \nOnline 18(4): 582-588.\n13. A K Singh, M Dutta, R Chattopadhyay, B Chakravarty, K Chaudhury, et al. \n(2016) Intrafollicular interleukin-8, interleukin-12, and adrenomedullin \nare the promising prognostic markers of oocyte and embryo quality in \nwomen with endometriosis. J Assist Reprod Genet 33(10): 1363-1372.\n14. L Xia, S Tan, Y Zhou, J Lin, H Wang, et al. (2018) Role of the NFkappaB-\nsignaling pathway in cancer. Onco Targets Ther 11: 2063-2073.\n15. Y Liu, M W Mayo, A S Nagji, P W Smith, C S Ramsey, et al. (2012) \nPhosphorylation of RelA/p65 promotes DNMT-1 recruitment to \nchromatin and represses transcription of the tumor metastasis \nsuppressor gene BRMS1. Oncogene 31(9): 1143-1154.\n16. X M Wang, Z Y Ma, N Song, (2018) Inflammatory cytokines IL-6, IL-\n10, IL-13, TNF-alpha and peritoneal fluid flora were associated with \ninfertility in patients with endometriosis. Eur Rev Med Pharmacol Sci \n22(9): 2513-2518.\n17. B Babaabasi, A Ahani, F Sadeghi, H Bashizade Fakhar, (2019) The \nAssociation between TNF-alpha Gene Polymorphisms and Endometriosis \nin An Iranian Population. Int J Fertil Steril 13(1): 6-11.\n18. X Lu, Wu Z M, Wang W, Cheng W W, (2016) Study on the methylation \nstatus of SF-1 gene promoter in ovarian granulosa cells in patients with \nendometriosis. Reproduction Contraception 36(6): 812.\n19. Q Xue, Z Lin, P Yin, M P Milad, Y H Cheng, et al. (2007) Transcriptional \nactivation of steroidogenic factor-1 by hypomethylation of the 5’ CpG \nisland in endometriosis, J Clin Endocrinol Metab 92(8): 3261-3267.\n20. H Wehbe, R Henson, F Meng, J Mize Berge (2006) T Patel, Interleukin-6 \ncontributes to growth in cholangiocarcinoma cells by aberrant promoter \nmethylation and gene expression. Cancer Res 66(21): 10517-10524.\n21. K J Campbell, S Rocha (2004) N D Perkins Active repression of \nantiapoptotic gene expression by RelA(p65) NF-kappa B. Mol Cell 13(6): \n853-865.","source_license":"CC0","license_restricted":false}