{"paper_id":"68adee40-56f4-442a-a17f-6ace9737c357","body_text":"Promoter Methylation Levels of Progesterone Receptor-B (PR-B) and Expression of mRNA DNA Methyltransferase-1 (DNMT-1) In Menstrual Blood Patients 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 Short Report Promoter Methylation Levels of Progesterone Receptor-B (PR-B) and Expression of mRNA DNA Methyltransferase-1 (DNMT-1) In Menstrual Blood Patients with Endometriosis Tita Husnitawati Madjid, Mondale Saputra, Roni Rowawi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3301543/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose: This study aimed to analyze the differences and correlation of progesterone receptor-B (PR-B) promoter methylation and mRNA DNA methyltransferase-1 (DNMT-1) expression of eutopic endometrium via menstrual blood between endometriosis patients and non-endometriosis. Patients and methods: We conducted a study of PR-B promoter methylation and mRNA DNMT-1 expression in menstrual blood obtained on the first and second day of the period from forty women with endometriosis and without. Results: Methylation analysis and mRNA analysis revealed a significant difference in the level of DNA methylation of PR-B promoters in the endometriosis group compared to non-endometriosis (79.129%±4.719 vs 28.716% ± 4.732, p<0.05). DNMT-1 mRNA expression was higher, though statistically insignificant, in the endometriosis group than non-endometriosis (10.517±13.421 vs 9.532 ± 10.269, p > 0.05). Conclusion: Correlation analysis showed a positive correlation between PR-B methylation and mRNA DNMT-1 expression in endometriosis. (r=0.526, p < 0.05). Promoter methylation PR-B MRNA DNMT-1 endometriosis correlation endometrium Figures Figure 1 Author Summary The presence of progesterone resistance in some patients with endometriosis complicates the management of the disease. Progesterone resistance result in only short-term pain relief in endometriosis, and it is difficult to predict which patient present with progesterone resistance in their endometriosis. Here, we attempted to use menstrual blood to assess progesterone receptor methylation and DNA methyltransferase-1 expression in order to predict the presence of progesterone resistance in endometriosis with a non-invasive diagnostic modality. We show that the progesterone receptor B-promoter is more methylated in the menstrual slough of endometriotic patients than in patients without endometriosis. This finding can contribute to a more personalized approach to endometriosis management in the future. Introduction The pathogenesis and development of endometriosis is unclear, however research with current molecular methods can gradually deliver illustration of disease mechanisms and can provide a new approach for diagnosis and treatment. So far there is no compelling evidence linking environmental factors, such as exposure to dioxin to endometriosis. Exposure to dioxin at the right time and dose may trigger initiation or development of endometriosis through interaction with estrogen receptor (ER) 1 or suppress expression of progesterone receptor (PR). 2 In addition, the disease is thought to be caused by exposure to environmental pollution and toxins, although so far there is no adequate data, so endometriosis is suspected as an epigenetic disease. 3 – 5 Provision of progestin in endometriosis currently has not provided satisfactory results due to progesterone resistance, which occurs in 20–50% of patients. Progestin is only able to relieve pain in a short time, so long-term progestin also does not give better results. 6 , 7 This is thought to be due to a transcriptional disorder of the progesterone B receptor due to an epigenetic mechanism, that is the deoxyribonucleic acid (DNA) methylation of the PR-B promoter, resulting in a decrease in the number of such receptors. Further research in the field of immune and epigenetic function in women with endometriosis is therefore necessary. Evidence has supported the hypothesis of endometriosis as an epigenetic disease. A study by Wu et al in 2005 showed that the promoters of HOXA10 in the endometrium of women with endometriosis were hypermethylated compared to women without endometriosis. 8 A study by Wu et al in 2006 showed that PR-B promoters were hypermethylated in endometriosis compared to non-endometriotic tissue. 9 Hypermethylation of PR-B promoter results in decreased expression of PR-B, providing support for the role of epigenetic aberrations in decreased PR-B. It is well known that there is a general trend of progesterone resistance in endometriosis. 10 A study by Rowawi in 2004 discovered that the endometrial DNA level of PRG promoters in women with endometriosis was higher than in normal women, however DNMT-1 mRNA expression and endometrial PR-B promoter-level methylation in endometriosis were higher than in normal women. 11 Several studies have also provided further evidence for epigenetic changes in endometriosis. Steroidogenic factor-1 (SF-1), an important transcription factor for the activation of some steroidogenic genes for estrogen biosynthesis, is usually undetectable in normal endometrial stromal cells but subjected to irregularities expressed in endometrial stromal cells. A study by Xue et al in 2007 showed that SF-1 promoter is hypermethylated in endometrial cells, but hypomethylated in endometriotic cells. 12 Another evidence that suggests endometriosis is an epigenetic disease is the significant increase of DNA enzyme methyltransferase (DNMT), namely DNMT1, DNMT3A and DNMT3B, the three coding genes for DNMTs involved in genomic DNA methylation in endometriosis. 9 Because these genes are involved in the maintenance of methylation, their distorted expression suggests that distorted methylation may be widespread in endometriosis. Bird's research in 1986 on a mice model of endometriosis suggested that there was a significant increase in DNMT-1 methylation in PR-B promoters compared to DNMTs. 13 Increased DNA methylation in these receptor promoters inhibits progesterone work resulting in progesterone resistance. 14 , 15 Regardless, as previously mentioned, progesterone resistance is not something that happened to all endometriosis patient. Elucidating the mechanism behind this phenomenon and designing a non-invasive test may help tailor a more personalized approach to the management of endometriosis for each individual. Material and methods This research was conducted in the Endocrinology and Gynecology Clinic of Hasan Sadikin General Hospital, January to August 2016, and was approved by the Ethical Committee for Medical Research, Hasan Sadikin General Hospital. Recruitment of study object Study subjects were selected based on an inclusion and exclusion criteria. Inclusion criteria for the cases were as follows: women diagnosed with endometriosis based on clinical characteristics, ultrasound, and confirmed histopathology results following diagnostic laparoscopy. Women with confirmed endometriosis who have received hormonal therapy or GnRH analogue were excluded from the study. Controls were taken from women who underwent diagnostic laparoscopy for pelvic pain but whose histopathology results were negative for endometriosis. Menstrual blood collection and DNA isolation 1 cc of menstrual blood was collected from each subject on the first or second day of the cycle. Blood sample was placed in a Vacuette® EDTA tube and kept in -80 o C for further analysis. DNA was isolated from each blood sample using the QIAamp DNA mini kit (Qiagen, Germany) using the blood protocol as recommended by the manufacturer (Wong, 1999). DNA methylation analysis Methylation rates were analyzed by ethylation-specific high-resolution melting (MS-HRM). Following DNA isolation, the sample was taken from the fridge and thawed. In 200 µl PCR tubes, 1 ng – 0.5 µg DNA was aliquoted and mixed with 59 µl RNAse-free water; 85 µl dissolved Bisulfate mix and 35 µl DNA protect buffer. PCR tubes were briefly mixed and added to the thermal cycler with following conditions: 5 minutes of initial denaturation at 95 o C; 25 minutes of incubation at 60 o C; 5 minutes of repeated denaturing at 95 o C; 85 minutes of incubation at 60 o C; 5 minutes of denaturation at 95 o C; and final incubation for 175 minutes at 60 o C. Primers used in RT-PCR is detailed in Table 1. Table 1. Primer sequence PR-B Methylated PR-B Un-Methylated Forward 5’-AGTATGGAGTTAGTAGAAGTT-3’ 5’-AGTACGGAGCCAGCAGAAGTC-3’ Reverse 5’-TCACAAGTCCAACACTTAAATAACT-3’ 5’-TCACAAGTCCGGCACTTGAGTGGCT-3’ Abbreviations: PR-B, progesterone receptor-B DNA methylation analysis Quantitative real-time PCR (qRT-PCR) was used to determine the expression of DNMT-1 mRNA. Forty mg of samples were homogenized using liquid nitrogen and tissue mortar soaked in DEPC. Sample was then transferred to a microtube with 1 ml RNA lysis buffer. 175 µl of lysate was transferred to a new microtube and 350 ul of dilution buffer was added. Sample was incubated in 70o C for 3 minutes; and centrifuged for 10 minutes at 8000 x g. The resulting supernatant was transferred to a new microtube containing 200 ul of 95% ethanol. Sample was transferred to spin basket assembly and spun for 1 minute at 8000 x g. 600 ul of RNA was solution was added to the sample, and then the sample spun for 1 minute at 8000 x g. DNA incubation mix (40 ul yellow core buffer, 5 ul DNAse 1 and 5 ul MnCl 2 ) was added and sample incubated at room temperature for 15 minutes. DNAse stop solution was added next, and sample was centrifuged at 8000 x g. 100 ul of nuclease-free water was added and spun for 1 minute at 8000 x g. The resulting RNA was quantified by spectrophotometry at 260, 280 and 320 nm. cDNA synthesis cDNA synthesis was conducted using BioRad iScript cDNA according to manufacturer’s instructions. Reaction mix was incubated for 5 minutes at 25 o C, 30 minutes at 42 o C, and 5 minutes at 85 o C and added to thermal cycler under these conditions: 3 minutes of incubation at 95 o , followed by 39 cycles of denaturation at 95 o C for 15 seconds, annealing at 57 o C and extension at 72 o C for 25 seconds. Primers used for the reaction was as follows: DNMT-1 gene forward 5ʹ-AACCTTCACCTAGCCCCAG-3ʹ reverse 5ʹ-CTCATCCGATTTGGCTCTTCA-3ʹ; GAPDH gene forward 5ʹ-GAA GGT GAA GGT CGG AGTC-3ʹ reverse 5ʹ-GAA GAT GGT GAT GGG ATT TC-3ʹ. Melting curve is detailed in Figure 1. Results Forty volunteers (n=20 per group) were enrolled between January to August of 2016. Baseline characteristics of the subjects are presented in Table 2. Table 2. Baseline characteristics Variables Group P value Endometriosis (n=20) Non-Endometriosis (n=20) Age (year) 0.102 Mean (SD) 36.10(4.53) 33.45(5.44) Range 27.00-43.00 24.00-43.00 Parity 0.342 <2 19(95%) 16(80%) >2 1(5%) 4(20%) There was a significant difference in the level of PR-B promoter DNA methylation between endometriosis and non-endometriosis tissue (79.13 vs 28.72, p < 0.001). However, DNMT-1 expression was not significantly increased in the endometriotic group (p = 0.738) as presented in Table 3. Table 3. Levels of PR-B promoter DNA Methylation and DNMT-1 expression Variables Group P value Endometriosis (n=20) Non-Endometriosis (n=20) Levels of Methylation (%) <0.001 Mean (SD) 79.13(4.72) 28.72(4.73) Range 65.53-86.81 20.95-39.69 DNMT-1 expression 0.738 Mean (SD) 10.52(13.42) 9.53(10.27) Range 0.15-45.73 0.35-36.07 Next, we conducted a correlational analysis between the level of PR-B promoter methylation with DNMT-1 expression in endometriotic tissue. Spearman’s test showed a moderately strong, significantly positive correlation between the two variables (r = 0.526, p < 0.05) Discussion Multiple gene expression disorders have been demonstrated in endometriosis; for example, inactivation of 17-bHSD2, PR-B, HOXA10 enzymes and an increase in the CYP19A1 gene (aromatase). 16 In patients with endometriosis it has been reported that both isoforms are expressed but unregulated (PR-B), in the eutopic endometrial PR-B is low, even undetectable. Progesterone and progestin have long been used for the treatment of endometriosis for pain relief, by inducing pseudo pregnancy, suppressing the biosynthesis of estrogen by the ovaries thus suppressing growth and inflammation of endometriosis. Unfortunately, the function of progestin here is only short-lived to relieve pain, and it is estimated that 20% -50% of patients with endometriosis are unresponsive to progestin administration. Progesterone resistance in eutopic and ectopic endometrium in women with endometriosis has been noted in several studies. 9 The results of this study showed differences in levels of DNA methylation of PR-B promoters in the endometriosis group compared with controls. The level of DNA methylation of PR-B promoters in the endometriosis group was higher than that of control. This will disrupt the appearance of PR-B receptors, thus disrupting the pathway of progesterone in cells or tissues of endometriosis. A study by Wu et al in 2006 demonstrated hypermethylation at PR-B receptor promoters in endometrial and endometrial eutopic endometrial tissue, as well as significantly reduced PR-B expression in ectopic and eutectic endometrial tissue compared with controL. 8 A study by Rowawi in 2014 also discovered that the level of PR-B promoter methylation of endometriosis patients' biopsy samples was higher than those in the control group. 11 A study by Jichan in 2011 showed that there was hypermethylation in PR-B promoters in adenomyotic tissue compared to normal patients (p <0.05). 17 This study aimed to provide therapy with trichostatin A (TSA), a histone deacetylase inhibitor (HDI), and 5-aza-2-deoxycytidine (ADC), a demyelation agent in the DRR gene and protein expression. The provision of TSA and ADC in this study increased the number of PR-B genes and protein expression in endometrial ectopic tissue but not in normal endometrial tissue. TSA and ADC can also suppress the development of the cell cycle in the ectopic endometrial stromal cell. 17 PR-B receptor dysfunction has also been observed in endometrial cancer. A study by Masahiro et al In 2001 found PR-B receptor disorders in endometrial cancer, explaining that CpG island methylation deviation, which results in the hypermethylation of PR-B promoter and under-expression of PR-B in endometrial cancer tissues. 16 Administration of a 5-aza-2-deoxycytidine (ADC) demethylation agent in this study restored PR-B expression in all layers of endometrial cells. 16 Increased DNMT-1 expression is thought to be a factor in high levels of methylation. The result of this study showed the appearance of DNMT-1 mRNA in endometrial eutopic tissue in menstrual blood with high endometriosis compared with the control group but no statistically significant difference (p>0.05). A study by Wu et al in 2007 explained that the expression of DNMT-1, DNMT3A, and DNMT3B genes is higher in endometrial ectopic tissue compared to control and eutopic endometrium tissue of women with endometriosis. 9 Ectopic and eutopic samples of endometrial tissue in Wu’s study were taken during the proliferation phase of the menstrual cycle, only 1 sample each was taken during the menstrual phase of the 17 samples of the endometriosis group and 8 control group samples. 9 A study by Yamagata in 2009 describes the appearance of DNMT-1, DNMT3A, and DNMT3B mRNAs low in the secretion phase versus the proliferative phase (p <0.01). 18 By contrast, a study by Van Kam J et al in 2011 explains that DNMTs mRNA expression is higher in the secretion instead of the proliferation phase of the menstrual cycle. 19 Vincent et al in 2011 also explained that DNMT expression of mRNA changes according to menstrual cycles i.e, mRNA expression of DNMT is higher in the proliferative compared to the secretion phase. 20 A study by Liao et al in 2008 demonstrated DNMT-1 expression in epithelial nuclei and endometrial stromal cells during the proliferative phase and was not found in the secretion phase. 21 This is in accordance with the research of Ghabreau in 2004 who explained that DNA methylation is high in the proliferation phase and decreases at the end of the secretion phase. 22 Some of the above studies suggest that the appearance of DNMT-1 mRNA may be influenced by steroids, which is higher in the proliferative phase and decreased in the secretion and menstrual phase. The sample in this study was obtained from menstrual blood – which may explain the insignificant difference between DNMT-1 mRNA in endometriosis and non-endometriosis (p> 0.05), however, the rate of DNA methylation of PR-B promoters remained high in the endometriosis group compared with non-endometriosis. This study also showed a positive correlation between the level of DNA methylation of PR-B promoter with DNMT-1 expression of mRNA in eutopic cell of endometriosis, with p = 0,017 indicating a significant or statistically significant correlation with moderate correlation strength (r = 0.526). Improving the appearance of DNMT-1 mRNA increases the DNA methylation of PRB promoters that will play a role in the progression of endometriosis disease. The correlation between DNMT-1 expression and methylation rate was also found in endometrial cancer, in Liao et al. 2008 study. The DNMT-1 protein expression level was significantly correlated with promoter hypermethylation of RASSF1A and RASSF2A (p = 0.043 and 0.004) in endometrial cancer. 21 If proven, it can be the basis or consideration with other relevant studies to examine the 5-aza-2-deoxycytidine (ADC) agent as a demethylation agent for progesterone resistance therapy, such as some studies that already exist in endometrial cancer using agent 5 -aza-2- deoxycytidine (ADC) to reactivate the PR-B genes by DNA demethylation and histone deacetylation. 16, 17, 23 Molecular changes of eutopic endometriotic tissue is new field of endometriosis research. Increased endometrial promoter PR-B metastasis of eutopic endometriosis further decreases levels of PR-B mRNA. This study may also add to the literature that non-invasive collection of endometrial cells, obtained from menstrual blood, can be identified and isolated for various studies in order to reveal further etiopathogenesis, simple diagnosis and directional therapy of endometriosis. Conclusion Increased expression of DNMT-1 mRNA will increase the DNA methylation of PR-B promoters which will play a role in the proliferation of endometriosis disease, due to low PR-B levels so as not to induce retinoic acid that increases the expression of the 17-bHSD2 enzyme to alter estradiol to estrone, estradiol remains high and will stimulate endometriosis to continue to grow and develop. The PR-B promoter methylation leads to a decrease in PR-B appearance which can then cause the endometriosis tissue to no longer respond to progesterone. If the relationship between PR-B promoter hypermethylation and progesterone resistance can be aligned, then the hypermethylation rate of the PR-B promoter may be a biomarker of progesterone resistance. Abbreviations PR-B : Progesterone receptor-B DNMT-1 : DNA Methyltransferase-1 Declarations Ethics approval and consent to participate The Faculty of Medicine, Universitas Padjadjaran, Ethics Committee Review Board approved this study protocol, and all study participants gave verbal and written informed consent. All authors hereby declare that all patients have been examined in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. Consent for publication Not applicable Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author on reasonable request Competing interests The authors declare that they have no competing interests Funding This research was funded by the authors themselves. We received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors Authors’ contributions THM researched literature and conceived the study. RR was involved in protocol development, gaining ethical approval, patient recruitment, and data analysis. MS wrote the first draft of the manuscript. All authors reviewed and edited the manuscript and approved the final version of the manuscript. Acknowledgments: Not applicable References Ohtake F, Takeyama KI, Matsumoto T, et al. Modulation of estrogen receptor signaling by association with the activated dioxin receptor. Nature . 2003;456:545-550. Igarashi TM, Bruner-Tran KL, Yeaman GR, et al. Reduced expression of progesterone receptor-B in the endometrium of women with endometriosis and in cocultures of endometrial cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Fertil Steril . Jul 2005;84(1):67-74. Guo SW. The link between exposure to dioxin and endometriosis: a critical reappraisal of primate data. Gynecologic and Obstetric Investigation . 2004;57(3):157-173. Rier SE. The potential role of exposure to environmental toxicants in the pathophysiology of endometriosis. Annals of the New York Academy of Sciences . 2002;955 Rier SE, Martin DC, Bowman RE, Dmowski WP, Becker JL. Endometriosis in Rhesus Monkeys (Macaca mulatta) Following Chronic Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin. Fundam Appl Toxicol . 1993;21(4):433-441. Surrey ES. The role of progestins in treating the pain of endometriosis. Journal of Minimally Invasive Gynecology . 2006;13(6):528-534. Vercellini P, Cortesi I, Crosignani PG. Progestins for symptomatic endometriosis: a critical analysis of the evidence. Fertil Steril . 1997;68(3):393-401. Wu Y, Strawn E, Basir Z, Halverson G, Guo SW. Promoter hypermethylation of progesterone receptor isoform B (PR-B) in endometriosis. Epigenetics . 2006;1(2):106-111. Wu Y, Strawn E, Basir Z, Halverson G, Guo SW. Aberrant expression of deoxyribonucleic acid methyltransferases DNMT1, DNMT3A, and DNMT3B in women with endometriosis. Fertil Steril . 2007;87(1):24-32. Guo SW. Epigenetics of endometriosis. Molecular Human Reproduction . 2009;15(10):587-607. Rowawi R. Hubungan kadar estradiol zalir peritoneum, tampilan mRNA reseptor progesteron - B, dan mRNA DNA Metil Transferase - 1 serta tingkat metilasi pengaju reseptor progesteron - B endometrium dengan kejadian endometriosis. Universitas Padjadjaran; 2014. Xue Q, Lin Z, Yin P, et al. Transcriptional activation of steroidogenic factor-1 by hypomethylation of the 5' CpG island in endometriosis. J Clin Endocrinol Metab . 2007;92(8):3261-3267. Bird AP. CpG-rich islands and the function of DNA methylation. Nature . 1986;321:209-213. Issa JP. Aging, DNA methylation and cancer. Crit Rev Oncol Hematol . 1999;32(1):31-43. Stirzaker C, Song JZ, Davidson B, Clark SJ. Transcriptional gene silencing promotes DNA hypermethylation through a sequential change in chromatin modifications in cancer cells. Cancer Res . 2004;64(11):3871-3877. Sasaki M, Dharia A, Oh BR, Tanaka Y, Fujimoto S, Dahiya R. Progesterone receptor b gene inactivation and CpG hypermethylation in human uterine endometrial cancer. Cancer Res . 2001;61(1):97-102. Nie J, Liu X, Guo SW. Promoter hypermethylation of progesterone receptor isoform B (PR-B) in adenomyosis and its rectification by a histone deacetylase inhibitor and a demethylation agent. Reprod Sci . 2010;17(11):995-1005. Yamagata Y, Asada H, Tamura I, et al. DNA methyltransferase expression in the human endometrium: Down-regulation by progesterone and estrogen. Hum Reprod . 2009;24(5):1126-1132. Van Kaam KJAF, Delvoux B, Romano A, D’Hooghe T, Dunselman GAJ, Groothuis PG. Deoxyribonucleic acid methyltransferases and methyl-CpG-binding domain proteins in human endometrium and endometriosis. Fertil Steril . 2011;95(4):1421-1427. Vincent ZL, Farquhar CM, Mitchell MD, Ponnampalam AP. Expression and regulation of DNA methyltransferases in human endometrium. Fertil Steril . 2011;95:4. Liao X, Siu MK, Chan KY, et al. Hypermethylation of RAS effector related genes and DNA methyltransferase 1 expression in endometrial carcinogenesis. Int J Cancer . 2008;123(2):296-302. Ghabreau L, Roux JP, Niveleau A, et al. Correlation between the DNA global methylation status and progesterone receptor expression in normal endometrium, endometrioid adenocarcinoma and precursors. Virchows Arch . 2004;445(2):129-134. Xiong Y, Dowdy SC, Bosquet JG, et al. Epigenetic-mediated upregulation of progesterone receptor B gene in endometrial cancer cell lines. Gynecol Oncol . 2005;99(1):135-141. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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-3301543\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Short Report\",\"associatedPublications\":[],\"authors\":[{\"id\":231109293,\"identity\":\"8c3d5545-36f5-413e-b77c-2534c29c35d6\",\"order_by\":0,\"name\":\"Tita Husnitawati Madjid\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYDACCRBhAGEf+AAk2NgJ6OCBaAHpYWY4OAOkhZkoLQwQLcw8DGAaP7CX7jH+XFDwR97g+PmDh21+bZPnY2Zg/PAxB48tMmfMpGcYGBhuOJPMcDi377ZhG9A2yZnb8Dksx4yZx8CAcdsBkJae24xALWzMvPi1GH8GarHfdv4xw2HLntv2xGgxkAZqSdx2A2gLw4/biYS13EgrA2oxTt5/47HBwd6G28ltzIzNeP3CPiN582eeP3K2M/sTH3/48ee27fz25oMfPuLRggoY28BkA7HqQeAPKYpHwSgYBaNgpAAAWvZND+B8Ar4AAAAASUVORK5CYII=\",\"orcid\":\"\",\"institution\":\"Padjadjaran University / Dr. Hasan Sadikin General Hospital\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Tita\",\"middleName\":\"Husnitawati\",\"lastName\":\"Madjid\",\"suffix\":\"\"},{\"id\":231109294,\"identity\":\"6c2b0182-6aea-4b8f-92d1-6e4333dd22ce\",\"order_by\":1,\"name\":\"Mondale Saputra\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Padjadjaran University / Dr. Hasan Sadikin General Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Mondale\",\"middleName\":\"\",\"lastName\":\"Saputra\",\"suffix\":\"\"},{\"id\":231109295,\"identity\":\"ebf3dd44-e67d-4159-8a56-c48bb2b5b877\",\"order_by\":2,\"name\":\"Roni Rowawi\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Maranatha Christian University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Roni\",\"middleName\":\"\",\"lastName\":\"Rowawi\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2023-08-28 02:29:12\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-3301543/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-3301543/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":42857475,\"identity\":\"42651222-0d73-4523-b080-5ddd5b98f0b4\",\"added_by\":\"auto\",\"created_at\":\"2023-09-08 20:13:59\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":74472,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eqRT-PCR melting curve.\\u003c/strong\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3301543/v1/a6b6c310468b7592be5842f9.jpg\"},{\"id\":58134305,\"identity\":\"3fb1d0ce-0e53-45fb-b1ef-f72edbd6cd0a\",\"added_by\":\"auto\",\"created_at\":\"2024-06-11 15:17:48\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":579113,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3301543/v1/5ee92748-5463-4f30-a539-de11e1a8a3f3.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Promoter Methylation Levels of Progesterone Receptor-B (PR-B) and Expression of mRNA DNA Methyltransferase-1 (DNMT-1) In Menstrual Blood Patients with Endometriosis\",\"fulltext\":[{\"header\":\"Author Summary\",\"content\":\"\\u003cp\\u003eThe presence of progesterone resistance in some patients with endometriosis complicates the management of the disease. Progesterone resistance result in only short-term pain relief in endometriosis, and it is difficult to predict which patient present with progesterone resistance in their endometriosis. Here, we attempted to use menstrual blood to assess progesterone receptor methylation and DNA methyltransferase-1 expression in order to predict the presence of progesterone resistance in endometriosis with a non-invasive diagnostic modality. We show that the progesterone receptor B-promoter is more methylated in the menstrual slough of endometriotic patients than in patients without endometriosis. This finding can contribute to a more personalized approach to endometriosis management in the future. \\u0026nbsp;\\u003c/p\\u003e\"},{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eThe pathogenesis and development of endometriosis is unclear, however research with current molecular methods can gradually deliver illustration of disease mechanisms and can provide a new approach for diagnosis and treatment. So far there is no compelling evidence linking environmental factors, such as exposure to dioxin to endometriosis. Exposure to dioxin at the right time and dose may trigger initiation or development of endometriosis through interaction with estrogen receptor (ER)\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e or suppress expression of progesterone receptor (PR).\\u003csup\\u003e\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e\\u003c/sup\\u003e In addition, the disease is thought to be caused by exposure to environmental pollution and toxins, although so far there is no adequate data, so endometriosis is suspected as an epigenetic disease.\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR4\\\" citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003cp\\u003eProvision of progestin in endometriosis currently has not provided satisfactory results due to progesterone resistance, which occurs in 20\\u0026ndash;50% of patients. Progestin is only able to relieve pain in a short time, so long-term progestin also does not give better results.\\u003csup\\u003e\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e\\u003c/sup\\u003e This is thought to be due to a transcriptional disorder of the progesterone B receptor due to an epigenetic mechanism, that is the deoxyribonucleic acid (DNA) methylation of the PR-B promoter, resulting in a decrease in the number of such receptors. Further research in the field of immune and epigenetic function in women with endometriosis is therefore necessary.\\u003c/p\\u003e \\u003cp\\u003eEvidence has supported the hypothesis of endometriosis as an epigenetic disease. A study by Wu et al in 2005 showed that the promoters of HOXA10 in the endometrium of women with endometriosis were hypermethylated compared to women without endometriosis.\\u003csup\\u003e\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u003c/sup\\u003e A study by Wu et al in 2006 showed that PR-B promoters were hypermethylated in endometriosis compared to non-endometriotic tissue.\\u003csup\\u003e\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e\\u003c/sup\\u003e Hypermethylation of PR-B promoter results in decreased expression of PR-B, providing support for the role of epigenetic aberrations in decreased PR-B. It is well known that there is a general trend of progesterone resistance in endometriosis.\\u003csup\\u003e\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003cp\\u003eA study by Rowawi in 2004 discovered that the endometrial DNA level of PRG promoters in women with endometriosis was higher than in normal women, however DNMT-1 mRNA expression and endometrial PR-B promoter-level methylation in endometriosis were higher than in normal women.\\u003csup\\u003e\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u003c/sup\\u003e Several studies have also provided further evidence for epigenetic changes in endometriosis. Steroidogenic factor-1 (SF-1), an important transcription factor for the activation of some steroidogenic genes for estrogen biosynthesis, is usually undetectable in normal endometrial stromal cells but subjected to irregularities expressed in endometrial stromal cells. A study by Xue et al in 2007 showed that SF-1 promoter is hypermethylated in endometrial cells, but hypomethylated in endometriotic cells.\\u003csup\\u003e\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e\\u003c/sup\\u003e Another evidence that suggests endometriosis is an epigenetic disease is the significant increase of DNA enzyme methyltransferase (DNMT), namely DNMT1, DNMT3A and DNMT3B, the three coding genes for DNMTs involved in genomic DNA methylation in endometriosis.\\u003csup\\u003e\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e\\u003c/sup\\u003e Because these genes are involved in the maintenance of methylation, their distorted expression suggests that distorted methylation may be widespread in endometriosis. Bird's research in 1986 on a mice model of endometriosis suggested that there was a significant increase in DNMT-1 methylation in PR-B promoters compared to DNMTs.\\u003csup\\u003e\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e\\u003c/sup\\u003e Increased DNA methylation in these receptor promoters inhibits progesterone work resulting in progesterone resistance.\\u003csup\\u003e\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003cp\\u003eRegardless, as previously mentioned, progesterone resistance is not something that happened to all endometriosis patient. Elucidating the mechanism behind this phenomenon and designing a non-invasive test may help tailor a more personalized approach to the management of endometriosis for each individual.\\u003c/p\\u003e\"},{\"header\":\"Material and methods\",\"content\":\"\\u003cp\\u003e This research was conducted in the Endocrinology and Gynecology Clinic of Hasan Sadikin General Hospital, January to August 2016, and was approved by the Ethical Committee for Medical Research, Hasan Sadikin General Hospital.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eRecruitment of study object\\u003c/h2\\u003e \\u003cp\\u003eStudy subjects were selected based on an inclusion and exclusion criteria. Inclusion criteria for the cases were as follows: women diagnosed with endometriosis based on clinical characteristics, ultrasound, and confirmed histopathology results following diagnostic laparoscopy. Women with confirmed endometriosis who have received hormonal therapy or GnRH analogue were excluded from the study. Controls were taken from women who underwent diagnostic laparoscopy for pelvic pain but whose histopathology results were negative for endometriosis.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMenstrual blood collection and DNA isolation\\u003c/h2\\u003e \\u003cp\\u003e1 cc of menstrual blood was collected from each subject on the first or second day of the cycle. Blood sample was placed in a Vacuette\\u0026reg; EDTA tube and kept in -80\\u003csup\\u003eo\\u003c/sup\\u003e C for further analysis. DNA was isolated from each blood sample using the QIAamp DNA mini kit (Qiagen, Germany) using the blood protocol as recommended by the manufacturer (Wong, 1999).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eDNA methylation analysis\\u003c/h2\\u003e \\u003cp\\u003eMethylation rates were analyzed by ethylation-specific high-resolution melting (MS-HRM). Following DNA isolation, the sample was taken from the fridge and thawed. In 200 \\u0026micro;l PCR tubes, 1 ng \\u0026ndash; 0.5 \\u0026micro;g DNA was aliquoted and mixed with 59 \\u0026micro;l RNAse-free water; 85 \\u0026micro;l dissolved Bisulfate mix and 35 \\u0026micro;l DNA protect buffer. PCR tubes were briefly mixed and added to the thermal cycler with following conditions: 5 minutes of initial denaturation at 95\\u003csup\\u003eo\\u003c/sup\\u003e C; 25 minutes of incubation at 60\\u003csup\\u003eo\\u003c/sup\\u003e C; 5 minutes of repeated denaturing at 95\\u003csup\\u003eo\\u003c/sup\\u003eC; 85 minutes of incubation at 60\\u003csup\\u003eo\\u003c/sup\\u003eC; 5 minutes of denaturation at 95\\u003csup\\u003eo\\u003c/sup\\u003eC; and final incubation for 175 minutes at 60\\u003csup\\u003eo\\u003c/sup\\u003eC. Primers used in RT-PCR is detailed in Table\\u0026nbsp;1.\\u003c/p\\u003e \\u003cp\\u003e\\u003cstrong\\u003eTable 1. Primer sequence\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\" width=\\\"570\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"10.526315789473685%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"44.73684210526316%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003ePR-B Methylated\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"44.73684210526316%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003ePR-B Un-Methylated\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"10.526315789473685%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eForward\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"44.73684210526316%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e5\\u0026rsquo;-AGTATGGAGTTAGTAGAAGTT-3\\u0026rsquo;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"44.73684210526316%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e5\\u0026rsquo;-AGTACGGAGCCAGCAGAAGTC-3\\u0026rsquo;\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"10.526315789473685%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eReverse\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"44.73684210526316%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e5\\u0026rsquo;-TCACAAGTCCAACACTTAAATAACT-3\\u0026rsquo;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"44.73684210526316%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e5\\u0026rsquo;-TCACAAGTCCGGCACTTGAGTGGCT-3\\u0026rsquo;\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAbbreviations:\\u003c/strong\\u003e PR-B, progesterone receptor-B\\u003c/p\\u003e\\n\\u003ch2\\u003eDNA methylation analysis\\u003c/h2\\u003e\\n\\u003cp\\u003eQuantitative real-time PCR (qRT-PCR) was used to determine the expression of DNMT-1 mRNA. Forty mg of samples were homogenized using liquid nitrogen and tissue mortar soaked in DEPC. Sample was then transferred to a microtube with 1 ml RNA lysis buffer. 175 \\u0026micro;l of lysate was transferred to a new microtube and 350 ul of dilution buffer was added. Sample was incubated in 70o C for 3 minutes; and centrifuged for 10 minutes at 8000 x \\u003cem\\u003eg.\\u0026nbsp;\\u003c/em\\u003eThe resulting supernatant was transferred to a new microtube containing 200 ul of 95% ethanol. Sample was transferred to spin basket assembly and spun for 1 minute at 8000 x \\u003cem\\u003eg.\\u0026nbsp;\\u003c/em\\u003e600 ul of RNA was solution was added to the sample, and then the sample spun for 1 minute at 8000 x \\u003cem\\u003eg.\\u0026nbsp;\\u003c/em\\u003eDNA incubation mix (40 ul yellow core buffer, 5 ul DNAse 1 and 5 ul MnCl\\u003csub\\u003e2\\u003c/sub\\u003e) was added and sample incubated at room temperature for 15 minutes. DNAse stop solution was added next, and sample was centrifuged at 8000 x \\u003cem\\u003eg.\\u0026nbsp;\\u003c/em\\u003e100 ul of nuclease-free water was added and spun for 1 minute at 8000 x \\u003cem\\u003eg. \\u0026nbsp;\\u003c/em\\u003eThe resulting RNA was quantified by spectrophotometry at 260, 280 and 320 nm.\\u003c/p\\u003e\\n\\u003ch2\\u003ecDNA synthesis\\u003c/h2\\u003e\\n\\u003cp\\u003ecDNA synthesis was conducted using BioRad iScript cDNA according to manufacturer\\u0026rsquo;s instructions. Reaction mix was incubated for 5 minutes at 25\\u003csup\\u003eo\\u003c/sup\\u003eC, 30 minutes at 42\\u003csup\\u003eo\\u003c/sup\\u003eC, and 5 minutes at 85\\u003csup\\u003eo\\u003c/sup\\u003eC and added to thermal cycler under these conditions: 3 minutes of incubation at 95\\u003csup\\u003eo\\u003c/sup\\u003e, followed by 39 cycles of denaturation at 95\\u003csup\\u003eo\\u003c/sup\\u003eC for 15 seconds, annealing at 57\\u003csup\\u003eo\\u003c/sup\\u003eC and extension at 72\\u003csup\\u003eo\\u003c/sup\\u003eC for 25 seconds. Primers used for the reaction was as follows: DNMT-1 gene forward 5ʹ-AACCTTCACCTAGCCCCAG-3ʹ reverse 5ʹ-CTCATCCGATTTGGCTCTTCA-3ʹ; GAPDH gene forward 5ʹ-GAA GGT GAA GGT CGG AGTC-3ʹ reverse 5ʹ-GAA GAT GGT GAT GGG ATT TC-3ʹ. Melting curve is detailed in Figure 1.\\u003c/p\\u003e\\u003c/div\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003eForty volunteers (n=20 per group) were enrolled between January to August of 2016. Baseline characteristics of the subjects are presented in Table 2.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTable 2. Baseline characteristics\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"22.71186440677966%\\\" rowspan=\\\"2\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eVariables\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"52.3728813559322%\\\" colspan=\\\"2\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eGroup\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"24.915254237288135%\\\" rowspan=\\\"2\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eP value\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"50%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eEndometriosis\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e(n=20)\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"50%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eNon-Endometriosis\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e(n=20)\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"22.750424448217316%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eAge (year)\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"26.146010186757216%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"26.146010186757216%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"24.95755517826825%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0.102\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"22.750424448217316%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMean (SD)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"26.146010186757216%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e36.10(4.53)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"26.146010186757216%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e33.45(5.44)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"24.95755517826825%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"22.750424448217316%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eRange\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"26.146010186757216%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e27.00-43.00\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"26.146010186757216%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e24.00-43.00\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"24.95755517826825%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"22.750424448217316%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eParity\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"26.146010186757216%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"26.146010186757216%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"24.95755517826825%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0.342\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"22.750424448217316%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026lt;2\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"26.146010186757216%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e19(95%)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"26.146010186757216%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e16(80%)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"24.95755517826825%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"22.750424448217316%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026gt;2\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"26.146010186757216%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1(5%)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"26.146010186757216%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e4(20%)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"24.95755517826825%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003eThere was a significant difference in the level of PR-B promoter DNA methylation between endometriosis and non-endometriosis tissue (79.13 vs 28.72, p \\u0026lt; 0.001).\\u0026nbsp;However, DNMT-1 expression was not significantly increased in the endometriotic group (p = 0.738) as presented in Table 3.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTable 3. Levels of PR-B promoter DNA Methylation and DNMT-1 expression\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\" width=\\\"620\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"22.86634460547504%\\\" rowspan=\\\"2\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eVariables\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"51.368760064412236%\\\" colspan=\\\"2\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eGroup\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"25.764895330112722%\\\" rowspan=\\\"2\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eP value\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"50%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eEndometriosis\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e(n=20)\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"50%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eNon-Endometriosis\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e(n=20)\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"22.829581993569132%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eLevels of Methylation (%)\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"25.723472668810288%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"25.723472668810288%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"25.723472668810288%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026lt;0.001\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"22.829581993569132%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMean (SD)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"25.723472668810288%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e79.13(4.72)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"25.723472668810288%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e28.72(4.73)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"25.723472668810288%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"22.829581993569132%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eRange\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"25.723472668810288%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e65.53-86.81\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"25.723472668810288%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e20.95-39.69\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"25.723472668810288%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"22.829581993569132%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eDNMT-1 expression\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"25.723472668810288%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"25.723472668810288%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"25.723472668810288%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0.738\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"22.829581993569132%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMean (SD)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"25.723472668810288%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e10.52(13.42)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"25.723472668810288%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e9.53(10.27)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"25.723472668810288%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"22.829581993569132%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eRange\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"25.723472668810288%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.15-45.73\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"25.723472668810288%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.35-36.07\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"25.723472668810288%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003eNext, we conducted a correlational analysis between the level of PR-B promoter methylation with DNMT-1 expression in endometriotic tissue. Spearman\\u0026rsquo;s test showed a moderately strong, significantly positive correlation between the two variables (r = 0.526, p \\u0026lt; 0.05)\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eMultiple gene expression disorders have been demonstrated in endometriosis; for example, inactivation of 17-bHSD2, PR-B, HOXA10 enzymes and an increase in the CYP19A1 gene (aromatase).\\u003csup\\u003e16\\u003c/sup\\u003e In patients with endometriosis it has been reported that both isoforms are expressed but unregulated (PR-B), in the eutopic endometrial PR-B is low, even undetectable. Progesterone and progestin have long been used for the treatment of endometriosis for pain relief, by inducing pseudo pregnancy, suppressing the biosynthesis of estrogen by the ovaries thus suppressing growth and inflammation of endometriosis. Unfortunately, the function of progestin here is only short-lived to relieve pain, and it is estimated that 20% -50% of patients with endometriosis are unresponsive to progestin administration. Progesterone resistance in eutopic and ectopic endometrium in women with endometriosis has been noted in several studies.\\u003csup\\u003e9\\u003c/sup\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe results of this study showed differences in levels of DNA methylation of PR-B promoters in the endometriosis group compared with controls. The level of DNA methylation of PR-B promoters in the endometriosis group was higher than that of control. This will disrupt the appearance of PR-B receptors, thus disrupting the pathway of progesterone in cells or tissues of endometriosis.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eA study by Wu et al in 2006 demonstrated hypermethylation at PR-B receptor promoters in endometrial and endometrial eutopic endometrial tissue, as well as significantly reduced PR-B expression in ectopic and eutectic endometrial tissue compared with controL.\\u003csup\\u003e8\\u003c/sup\\u003e A study by Rowawi in 2014 also discovered that the level of PR-B promoter methylation of endometriosis patients\\u0026apos; biopsy samples was higher than those in the control group.\\u003csup\\u003e11\\u003c/sup\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eA study by Jichan in 2011 showed that there was hypermethylation in PR-B promoters in adenomyotic tissue compared to normal patients (p \\u0026lt;0.05).\\u003csup\\u003e17\\u003c/sup\\u003e This study aimed to provide therapy with trichostatin A (TSA), a histone deacetylase inhibitor (HDI), and 5-aza-2-deoxycytidine (ADC), a demyelation agent in the DRR gene and protein expression. The provision of TSA and ADC in this study increased the number of PR-B genes and protein expression in endometrial ectopic tissue but not in normal endometrial tissue. TSA and ADC can also suppress the development of the cell cycle in the ectopic endometrial stromal cell.\\u003csup\\u003e17\\u003c/sup\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003ePR-B receptor dysfunction has also been observed in endometrial cancer. A study by Masahiro et al In 2001 found PR-B receptor disorders in endometrial cancer, explaining that CpG island methylation deviation, which results in the hypermethylation of PR-B promoter and under-expression of PR-B in endometrial cancer tissues.\\u003csup\\u003e16\\u003c/sup\\u003e Administration of a 5-aza-2-deoxycytidine (ADC) demethylation agent in this study restored PR-B expression in all layers of endometrial cells.\\u003csup\\u003e16\\u003c/sup\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eIncreased DNMT-1 expression is thought to be a factor in high levels of methylation. The result of this study showed the appearance of DNMT-1 mRNA in endometrial eutopic tissue in menstrual blood with high endometriosis compared with the control group but no statistically significant difference (p\\u0026gt;0.05).\\u003c/p\\u003e\\n\\u003cp\\u003eA study by Wu et al in 2007 explained that the expression of DNMT-1, DNMT3A, and DNMT3B genes is higher in endometrial ectopic tissue compared to control and eutopic endometrium tissue of women with endometriosis.\\u003csup\\u003e9\\u003c/sup\\u003e Ectopic and eutopic samples of endometrial tissue in Wu\\u0026rsquo;s study were taken during the proliferation phase of the menstrual cycle, only 1 sample each was taken during the menstrual phase of the 17 samples of the endometriosis group and 8 control group samples.\\u003csup\\u003e9\\u003c/sup\\u003e A study by Yamagata in 2009 describes the appearance of DNMT-1, DNMT3A, and DNMT3B mRNAs low in the secretion phase versus the proliferative phase (p \\u0026lt;0.01).\\u003csup\\u003e18\\u003c/sup\\u003e By contrast, a study by Van Kam J et al in 2011 explains that DNMTs mRNA expression is higher in the secretion instead of the proliferation phase of the menstrual cycle.\\u003csup\\u003e19\\u003c/sup\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eVincent et al in 2011 also explained that DNMT expression of mRNA changes according to menstrual cycles i.e, mRNA expression of DNMT is higher in the proliferative compared to the secretion phase.\\u003csup\\u003e20\\u003c/sup\\u003e A study by Liao et al in 2008 demonstrated DNMT-1 expression in epithelial nuclei and endometrial stromal cells during the proliferative phase and was not found in the secretion phase.\\u003csup\\u003e21\\u003c/sup\\u003e This is in accordance with the research of Ghabreau in 2004 who explained that DNA methylation is high in the proliferation phase and decreases at the end of the secretion phase.\\u003csup\\u003e22\\u003c/sup\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eSome of the above studies suggest that the appearance of DNMT-1 mRNA may be influenced by steroids, which is higher in the proliferative phase and decreased in the secretion and menstrual phase. The sample in this study was obtained from menstrual blood \\u0026ndash; which may explain the insignificant difference between DNMT-1 mRNA in endometriosis and non-endometriosis (p\\u0026gt; 0.05), however, the rate of DNA methylation of PR-B promoters remained high in the endometriosis group compared with non-endometriosis.\\u003c/p\\u003e\\n\\u003cp\\u003eThis study also showed a positive correlation between the level of DNA methylation of PR-B promoter with DNMT-1 expression of mRNA in eutopic cell of endometriosis, with p = 0,017 indicating a significant or statistically significant correlation with moderate correlation strength (r = 0.526). Improving the appearance of DNMT-1 mRNA increases the DNA methylation of PRB promoters that will play a role in the progression of endometriosis disease. The correlation between DNMT-1 expression and methylation rate was also found in endometrial cancer, in Liao et al. 2008 study. The DNMT-1 protein expression level was significantly correlated with promoter hypermethylation of RASSF1A and RASSF2A (p = 0.043 and 0.004) in endometrial cancer.\\u003csup\\u003e21\\u003c/sup\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eIf proven, it can be the basis or consideration with other relevant studies to examine the 5-aza-2-deoxycytidine (ADC) agent as a demethylation agent for progesterone resistance therapy, such as some studies that already exist in endometrial cancer using agent 5 -aza-2- deoxycytidine (ADC) to reactivate the PR-B genes by DNA demethylation and histone deacetylation.\\u003csup\\u003e16, 17, 23\\u003c/sup\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eMolecular changes of eutopic endometriotic tissue is new field of endometriosis research. Increased endometrial promoter PR-B metastasis of eutopic endometriosis further decreases levels of PR-B mRNA. This study may also add to the literature that non-invasive collection of endometrial cells, obtained from menstrual blood, can be identified and isolated for various studies in order to reveal further etiopathogenesis, simple diagnosis and directional therapy of endometriosis.\\u003c/p\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eIncreased expression of DNMT-1 mRNA will increase the DNA methylation of PR-B promoters which will play a role in the proliferation of endometriosis disease, due to low PR-B levels so as not to induce retinoic acid that increases the expression of the 17-bHSD2 enzyme to alter estradiol to estrone, estradiol remains high and will stimulate endometriosis to continue to grow and develop. The PR-B promoter methylation leads to a decrease in PR-B appearance which can then cause the endometriosis tissue to no longer respond to progesterone. If the relationship between PR-B promoter hypermethylation and progesterone resistance can be aligned, then the hypermethylation rate of the PR-B promoter may be a biomarker of progesterone resistance.\\u0026nbsp;\\u003c/p\\u003e\"},{\"header\":\"Abbreviations\",\"content\":\"\\u003cp\\u003ePR-B \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; : Progesterone receptor-B\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eDNMT-1 \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; : DNA Methyltransferase-1\\u0026nbsp;\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eEthics approval and consent to participate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe\\u0026nbsp;Faculty of Medicine, Universitas Padjadjaran, Ethics Committee Review Board approved this study protocol, and all study participants gave verbal and written informed consent. All authors hereby declare that all patients have been examined in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAvailability of data and materials\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe datasets used and analyzed during the current study are available from the corresponding author on reasonable request\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare that they have no competing interests\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis research was funded by the authors themselves. We received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthors\\u0026rsquo; contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTHM researched literature and conceived the study. RR was involved in protocol development, gaining ethical approval, patient recruitment, and data analysis. MS wrote the first draft of the manuscript. All authors reviewed and edited the manuscript and approved the final version of the manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgments:\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eOhtake F, Takeyama KI, Matsumoto T, et al. Modulation of estrogen receptor signaling by association with the activated dioxin receptor. \\u003cem\\u003eNature\\u003c/em\\u003e. 2003;456:545-550. \\u003c/li\\u003e\\n\\u003cli\\u003eIgarashi TM, Bruner-Tran KL, Yeaman GR, et al. Reduced expression of progesterone receptor-B in the endometrium of women with endometriosis and in cocultures of endometrial cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin. \\u003cem\\u003eFertil Steril\\u003c/em\\u003e. Jul 2005;84(1):67-74. \\u003c/li\\u003e\\n\\u003cli\\u003eGuo SW. The link between exposure to dioxin and endometriosis: a critical reappraisal of primate data. \\u003cem\\u003eGynecologic and Obstetric Investigation\\u003c/em\\u003e. 2004;57(3):157-173. \\u003c/li\\u003e\\n\\u003cli\\u003eRier SE. The potential role of exposure to environmental toxicants in the pathophysiology of endometriosis. \\u003cem\\u003eAnnals of the New York Academy of Sciences\\u003c/em\\u003e. 2002;955\\u003c/li\\u003e\\n\\u003cli\\u003eRier SE, Martin DC, Bowman RE, Dmowski WP, Becker JL. Endometriosis in Rhesus Monkeys (Macaca mulatta) Following Chronic Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin. \\u003cem\\u003eFundam Appl Toxicol\\u003c/em\\u003e. 1993;21(4):433-441. \\u003c/li\\u003e\\n\\u003cli\\u003eSurrey ES. The role of progestins in treating the pain of endometriosis. \\u003cem\\u003eJournal of Minimally Invasive Gynecology\\u003c/em\\u003e. 2006;13(6):528-534. \\u003c/li\\u003e\\n\\u003cli\\u003eVercellini P, Cortesi I, Crosignani PG. Progestins for symptomatic endometriosis: a critical analysis of the evidence. \\u003cem\\u003eFertil Steril\\u003c/em\\u003e. 1997;68(3):393-401. \\u003c/li\\u003e\\n\\u003cli\\u003eWu Y, Strawn E, Basir Z, Halverson G, Guo SW. Promoter hypermethylation of progesterone receptor isoform B (PR-B) in endometriosis. \\u003cem\\u003eEpigenetics\\u003c/em\\u003e. 2006;1(2):106-111. \\u003c/li\\u003e\\n\\u003cli\\u003eWu Y, Strawn E, Basir Z, Halverson G, Guo SW. Aberrant expression of deoxyribonucleic acid methyltransferases DNMT1, DNMT3A, and DNMT3B in women with endometriosis. \\u003cem\\u003eFertil Steril\\u003c/em\\u003e. 2007;87(1):24-32. \\u003c/li\\u003e\\n\\u003cli\\u003eGuo SW. Epigenetics of endometriosis. \\u003cem\\u003eMolecular Human Reproduction\\u003c/em\\u003e. 2009;15(10):587-607. \\u003c/li\\u003e\\n\\u003cli\\u003eRowawi R. Hubungan kadar estradiol zalir peritoneum, tampilan mRNA reseptor progesteron - B, dan mRNA DNA Metil Transferase - 1 serta tingkat metilasi pengaju reseptor progesteron - B endometrium dengan kejadian endometriosis. Universitas Padjadjaran; 2014.\\u003c/li\\u003e\\n\\u003cli\\u003eXue Q, Lin Z, Yin P, et al. Transcriptional activation of steroidogenic factor-1 by hypomethylation of the 5\\u0026apos; CpG island in endometriosis. \\u003cem\\u003eJ Clin Endocrinol Metab\\u003c/em\\u003e. 2007;92(8):3261-3267. \\u003c/li\\u003e\\n\\u003cli\\u003eBird AP. CpG-rich islands and the function of DNA methylation. \\u003cem\\u003eNature\\u003c/em\\u003e. 1986;321:209-213. \\u003c/li\\u003e\\n\\u003cli\\u003eIssa JP. Aging, DNA methylation and cancer. \\u003cem\\u003eCrit Rev Oncol Hematol\\u003c/em\\u003e. 1999;32(1):31-43. \\u003c/li\\u003e\\n\\u003cli\\u003eStirzaker C, Song JZ, Davidson B, Clark SJ. Transcriptional gene silencing promotes DNA hypermethylation through a sequential change in chromatin modifications in cancer cells. \\u003cem\\u003eCancer Res\\u003c/em\\u003e. 2004;64(11):3871-3877. \\u003c/li\\u003e\\n\\u003cli\\u003eSasaki M, Dharia A, Oh BR, Tanaka Y, Fujimoto S, Dahiya R. Progesterone receptor b gene inactivation and CpG hypermethylation in human uterine endometrial cancer. \\u003cem\\u003eCancer Res\\u003c/em\\u003e. 2001;61(1):97-102. \\u003c/li\\u003e\\n\\u003cli\\u003eNie J, Liu X, Guo SW. Promoter hypermethylation of progesterone receptor isoform B (PR-B) in adenomyosis and its rectification by a histone deacetylase inhibitor and a demethylation agent. \\u003cem\\u003eReprod Sci\\u003c/em\\u003e. 2010;17(11):995-1005. \\u003c/li\\u003e\\n\\u003cli\\u003eYamagata Y, Asada H, Tamura I, et al. DNA methyltransferase expression in the human endometrium: Down-regulation by progesterone and estrogen. \\u003cem\\u003eHum Reprod\\u003c/em\\u003e. 2009;24(5):1126-1132. \\u003c/li\\u003e\\n\\u003cli\\u003eVan Kaam KJAF, Delvoux B, Romano A, D\\u0026rsquo;Hooghe T, Dunselman GAJ, Groothuis PG. Deoxyribonucleic acid methyltransferases and methyl-CpG-binding domain proteins in human endometrium and endometriosis. \\u003cem\\u003eFertil Steril\\u003c/em\\u003e. 2011;95(4):1421-1427. \\u003c/li\\u003e\\n\\u003cli\\u003eVincent ZL, Farquhar CM, Mitchell MD, Ponnampalam AP. Expression and regulation of DNA methyltransferases in human endometrium. \\u003cem\\u003eFertil Steril\\u003c/em\\u003e. 2011;95:4. \\u003c/li\\u003e\\n\\u003cli\\u003eLiao X, Siu MK, Chan KY, et al. Hypermethylation of RAS effector related genes and DNA methyltransferase 1 expression in endometrial carcinogenesis. \\u003cem\\u003eInt J Cancer\\u003c/em\\u003e. 2008;123(2):296-302. \\u003c/li\\u003e\\n\\u003cli\\u003eGhabreau L, Roux JP, Niveleau A, et al. Correlation between the DNA global methylation status and progesterone receptor expression in normal endometrium, endometrioid adenocarcinoma and precursors. \\u003cem\\u003eVirchows Arch\\u003c/em\\u003e. 2004;445(2):129-134. \\u003c/li\\u003e\\n\\u003cli\\u003eXiong Y, Dowdy SC, Bosquet JG, et al. Epigenetic-mediated upregulation of progesterone receptor B gene in endometrial cancer cell lines. \\u003cem\\u003eGynecol Oncol\\u003c/em\\u003e. 2005;99(1):135-141. \\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Promoter methylation, PR-B, MRNA DNMT-1, endometriosis, correlation, endometrium\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-3301543/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-3301543/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003e\\u003cstrong\\u003ePurpose: \\u003c/strong\\u003eThis study aimed to analyze the differences and correlation of progesterone receptor-B (PR-B) promoter methylation and mRNA DNA methyltransferase-1 (DNMT-1) expression of eutopic endometrium via menstrual blood between endometriosis patients and non-endometriosis.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ePatients and methods: \\u003c/strong\\u003eWe conducted a study of PR-B promoter methylation and mRNA DNMT-1 expression in menstrual blood obtained on the first and second day of the period from forty women with endometriosis and without.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eResults: \\u003c/strong\\u003eMethylation analysis and mRNA analysis revealed a significant difference in the level of DNA methylation of PR-B promoters in the endometriosis group compared to non-endometriosis (79.129%±4.719 vs 28.716% ± 4.732, p\\u0026lt;0.05). DNMT-1 mRNA expression was higher, though statistically insignificant, in the endometriosis group than non-endometriosis (10.517±13.421 vs 9.532 ± 10.269, p \\u0026gt; 0.05).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConclusion: \\u003c/strong\\u003eCorrelation analysis showed a positive correlation between PR-B methylation and mRNA DNMT-1 expression in endometriosis. (r=0.526, p \\u0026lt; 0.05).\\u003c/p\\u003e\",\"manuscriptTitle\":\"Promoter Methylation Levels of Progesterone Receptor-B (PR-B) and Expression of mRNA DNA Methyltransferase-1 (DNMT-1) In Menstrual Blood Patients with Endometriosis\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2023-09-08 20:13:54\",\"doi\":\"10.21203/rs.3.rs-3301543/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"ef046015-0b4f-4a08-bdfb-6da0c4ad8137\",\"owner\":[],\"postedDate\":\"September 8th, 2023\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2024-06-11T15:09:42+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2023-09-08 20:13:54\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-3301543\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-3301543\",\"identity\":\"rs-3301543\",\"version\":[\"v1\"]},\"buildId\":\"_2-kVJe1T_tPrBINL-cwx\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC0","license_restricted":false}