Cell cycle gene expression is restored in eutopic primary stromal cells after endometriosis surgery

Cell cycle gene expression is restored in eutopic primary stromal cells after endometriosis surgery | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cell cycle gene expression is restored in eutopic primary stromal cells after endometriosis surgery Adriana L Invitti, Fernando Y Asanuma, Cristina V Carvalho, Gil Kamergorodsky, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3010156/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 The role of the endometrium in the pathogenesis of endometriosis has assumed prominence. Abnormality of gene and protein expression, apoptosis and changes in cell cycle have been extensively studied in endometriosis. We evaluated the cell cycle genes expression on primary stromal endometrial cells (EC) isolated from eutopic endometrium in two different moments: before and after deep infiltrating endometriosis (DIE) surgical treatment. We analysed five ECs from healthy patients (group control) and 9 from women with diagnosis of DIE. Were identified 7 cell cycle genes (p53, TFDP1, TFDP2, KPNA2, RB1, RBL2, SERTAD1) differentially expressed between pre, post-operative and controls. The p53 and KPNA2 genes were downregulated 3.34 fold (p = 0.006) and 2.62, (p = 0.042), respectively, in the endometrium of DIE compared to control. Both were upregulated (p53 - Fold 2.22, p = 0.157; KPNA2 - Fold 4.36, p = 0.017) in the post-operative DIE group in comparison to pre-operative one. Also, the RB1 gene was downregulated 9.36 fold (p = 0.029) in the DIE-post group in comparison to DIE-pre group, having no difference between DIE-pre and control group (p = 0.311). The proteins coded by these genes have association between each other, indicating that the surgical treatment could change the cell cycle regulation in the endometrium of women with endometriosis and that the changes remain after the cell isolation from the tissue. Endometriosis cell cycle genes p53 endometrium surgery Figures Figure 1 Figure 2 Figure 3 INTRODUCTION The endometriosis pathogenesis studies have revealed that the eutopic endometrium is innately aberrant and plays an important role in the pathophysiology of the disease [ 1 ].The eutopic endometrium of women with endometriosis have an increased cell proliferation and decreased programmed cell death [ 2 ]. Analysing the expression of cell cycle molecules in endometrial biopsies, our group demonstrated that endometriotic tissues have lower levels of p27 protein compared with those of healthy control [ 3 – 5 ]. The eutopic endometrium shares changes with ectopic tissue, which are not found in the eutopic endometrium of women without the disease, corroborating the thesis that the main defect in endometriosis must be primarily in the eutopic endometrium[ 1 ]. However, it is still unclear whether abnormal findings in the eutopic endometrium of patients with the disease is related to the ectopic inflammation due to the ectopic tissue or primary of the uterine mucosa [ 6 ]. Researchers suggest that foci of endometriosis can activate transduction signals that alter the gene expression of the eutopic endometrium [ 7 ] and may be an explanation for presenting better rates of embryo implantation in infertile patients when submitted to surgical treatment [ 8 ]. According to this, we have evaluated the cell cycle-related genes expression on primary eutopic endometrial cells obtained before and after endometriosis surgical treatment to understand if the alterations of endometriosis are primary or secondary to the endometrial tissue, that is, if the change of the peritoneal environment through the surgical treatment would provide reestablishment of the normal conditions of the endometrial environment. MATERIALS AND METHODS Patients’samples The study was approved by the Research Ethics Committee of the Federal University of São Paulo (Ref. 226536/2013), and free informed consent was obtained from all of the participants. ECs were isolated from pipelle biopsies of the eutopic endometrium from 14 women, aged between 20 and 35 years, undergoing surgical treatment for the diagnosis of benign ovarian cyst (control) or endometriosis. None of the participants had received hormonal preparations or used GnRH analogues in the three months before the endometrial collection. Were excluded the patients that had childbirth or breastfed in the six months before enrollment in the study, those with menstrual irregularities, teratoma, myoma, endometrial polyp or adenomyosis. Cell isolation and RNA extraction ECs were isolated according to Pelvic Pain Unit standard protocol and cultured until the second passage and then cryopreserved [ 9 ]. mRNA was extracted with TRIzol (Invitrogen – LifeTechnologies - Carlsbad, CA, USA) according to manufacture’s protocol. Quantification and quality control (260/280 ratio) were evaluated on Nanodrop (Nanodrop Technologies Inc., Rockland, DE). Real-time PCR analysis The cDNA was synthesized with RT2 First Strand Kit (Qiagen, Hilden, Germany). The gene expression was evaluated by the SYBR Green RT-PCR method with QuantiTect Primer Assay (Qiagen, Hilden, Germany) using the PAHS-020A (Qiagen, Hilden, Germany) plate following the manufacturer’s instructions. The groups studied were divided into: Control (n = 5, patients without endometriosis), Pre-operative DIE - DIE-Pre (n = 6) and Post-operative Endometriosis - DIE-Post (n = 3). The analysis included two housekeeping genes: GAPDH and β-Actin. Triplicates were performed for all samples, and reactions carried-out on Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Statistical analysis The results were analyzed in the Data Analysis V3.5 PCR software (Qiagen) and p-values calculated using Student’s t-test. A value greater than one indicated an upregulation, and a value less than one indicated a downregulation. A comparison was considered as statistically significant if the p values were less than 0.05. RESULTS AND DISCUSSION We have observed seven (p53, TFDP1, TFDP2, KPNA2, RB1, RBL2, SERTAD1) differentially expressed genes in the ECs comparing all three groups (Fig. 1 ). These genes are strongly related to the homeostasis and control of the cell cycle indicating that the surgery can implicate in the gene expression of the endometrial cells, confirmed by the interaction between the protein coded by these genes and the clusterization observed for their expression (Fig. 2 ). The differentially expressed genes found in this work codes for proteins which have an association between each other (Fig. 3 ). The cell cycle is strictly regulated by extracellular signals and the check-points (cell cycle arrest points) provides cells with the opportunity to repair damages before division, preventing the transmission of genetic errors to daughter cells. Also, these check-points are responsible for the recovery of the cells from damages preventing premature cell death[ 10 ]. The disrupted cell cycle and aberrant inflammatory molecules expression have been already described in the endometrium of women with DIE [ 11 ]. These differentially expressed genes found codes for proteins which have an association between each other and also with some endometriosis cell cycle key protein (p27 and p21) [ 4 , 5 ], according to the following diagram (Fig. 3 ). Although these studies have considered only p27 protein levels and not the mRNA levels. In this study,we did not observe differences in the expression of p27 between DIE samples and controls (FC -1.58; p = 0.3768). Indicating that possible a post-translational mechanism regulates the p27 protein content in the endometriosis samples. This can be explained by the Karyopherin alpha 2 (KPNA2) expression, which is 2.62 times downregulated in the endometriosis groups compared to control and 4.36 times upregulated after surgery in comparison to pre-surgery ECs. KPNA2 is known as a selective nucleocytoplasmic carrier of transcription factors and proteins related to DNA repair and cell cycle regulation [ 12 ]. It is directly related to the translocation of p27 in and out of the nucleous [ 13 ]. Exacerbated expression of KPNA2 has been reported in malignant tumours and its knockdown causes the accumulation of p53 protein in the cytoplasm[ 14 ]. We demonstrate an up-regulation of KPNA2 in the ECs from patients submitted to surgical treatment. p53 protein is known as a general stress sensor, not only important to inhibit cancer progression, but also to respond during viral infection, starvation or oxidative stress, reducing cell proliferation, altering cellular metabolism and inhibiting survival[ 15 ]. The gene that codes to p53 (TP53) is 3.34 less expressed in the endometriosis group than in the control group and no statiscally significant difference was found between post-operative and control groups (fold change − 1.51, p = 0.3590). p53 is downregulated in the ECs of DIE patients compared to control and its expression is restored after surgical removal of ectopic endometriosis foci. Post-operative ECs shows other sign of a tentative regulation of the G1/S transition. The gene RBL2, which encodes for a retinoblastoma (Rb) protein, was found to be 2.23 times more more expressed in endometriosis post-operative than in control group. RBL2 proteins intereact with CDKs complexes to promote a proper G1/S transition[ 16 ]. Another gene involved in controlling early cell cycle events, from G1 to S phases, is the transcription factor DP-1 (TFDP1). TFDP1 is downregulated 2.05 fold in the DIE-pre than in Control ECs. TFDP1 or TFDP2 forms a heterodimeric complex with the tumour suppressor RB1 (downregulated 9.36 and 14.28 fold in the DIE-post in comparison to DIE-pre (p = 0.0295) and Control (p = 0.0211), respectively) binded to E2F transcription factors. This interaction results in the prevention of G1/S transition [ 16 ]. In previous work, we observed the downregulation of TFDP1 in the endometrium of women with DIE, but no changes were observed on TFDP1 expression between pre and postoperative ECs (FC 1.55, p = 0.2475). By the results presented, we infer that surgical removal of ectopic endometriosis foci leads the eutopic endometrial cells to a cell cycle genes pattern more similar to the control group. Acting directly on the cell cycle arrest, blocking or at least by, reducing the proliferation and development of endometriosis cells. To the best of our knowledge this is the first report of endometrial cells expression changes after endometriosis surgery. In spite of the limited number of samples, this study can highlight the importance of the study of post-operative endometrium cells for the understanding of endometriosis pathogenesis. Declarations Ethical Approval and consent to participate The study was approved by the Research Ethics Committee of the Federal University of São Paulo (Ref. 226536/2013), and free informed consent was obtained from all of the participants. Competing interests The authors declare that they have no affiliations that would constitute a financial conflict of interest regarding the subject matter presented in this study. Authors' contributions ALI, FYA and CVC: study design, acquisition of data, analysis and interpretation of the data.GK and AK: acquisition of data and interpretation of the data. ES: study design and interpretation of the data. All authors contributed with the drafting of the article, critical revision and have approved the final version of the manuscript. Funding This work received support of the National Council for Scientific and Technological Development (CNPQ) - 134111/2013-13 as a master scholarship to FYA. Availability of data The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. References Sharpe-Timms KL (2001) Endometrial anomalies in women with endometriosis. Ann N Y Acad Sci 943:131–147. https://doi.org/10.1111/j.1749-6632.2001.tb03797.x Dmowski WP, Ding J, Shen J et al (2001) Apoptosis in endometrial glandular and stromal cells in women with and without endometriosis. Hum Reprod 16:1802–1808. https://doi.org/10.1093/humrep/16.9.1802 Gonçalves GA, Invitti AL, Parreira RM et al (2017) p27kip1 overexpression regulates IL-1β in the microenvironment of stem cells and eutopic endometriosis co-cultures. Cytokine 89:229–234. https://doi.org/10.1016/j.cyto.2015.12.015 Gonçalves GA, Camargo-Kosugi CM, Bonetti TCS et al (2015) p27kip1 overexpression regulates VEGF expression, cell proliferation and apoptosis in cell culture from eutopic endometrium of women with endometriosis. Apoptosis 20:327–335. https://doi.org/10.1007/s10495-014-1079-8 Schor E, da Silva IDCG, Sato H et al (2009) P27Kip1 is down-regulated in the endometrium of women with endometriosis. Fertil Steril 91:682–686. https://doi.org/10.1016/j.fertnstert.2007.12.070 Taniguchi F, Kaponis A, Izawa M et al (2011) Apoptosis and endometriosis. Front Biosci (Elite Ed) 3:648–62. https://doi.org/277 [pii] Ulukus M, Cakmak H, Arici A (2006) The Role of Endometrium in Endometriosis. J Soc Gynecol Investig 13:467–476. https://doi.org/10.1016/j.jsgi.2006.07.005 Jacobson TZ, Duffy JMN, Barlow DH et al (2014) Laparoscopic surgery for subfertility associated with endometriosis. Cochrane Database Syst. Rev. 2014 D’Amora P, Maciel TT, Tambellini R et al (2009) Disrupted cell cycle control in cultured endometrial cells from patients with endometriosis harboring the progesterone receptor polymorphism PROGINS. Am J Pathol 175:215–224. https://doi.org/10.2353/ajpath.2009.080966 Matsuzaki S (2001) Expression of the cyclin-dependent kinase inhibitor p27Kip1 in eutopic endometrium and peritoneal endometriosis. Fertil Steril 75:956–960. https://doi.org/10.1016/S0015-0282(01)01752-6 Uegaki T, Taniguchi F, Nakamura K et al (2015) Inhibitor of apoptosis proteins (IAPs) may be effective therapeutic targets for treating endometriosis. Hum Reprod 30:149–158. https://doi.org/10.1093/humrep/deu288 Christiansen A, Dyrskjøt L (2013) The functional role of the novel biomarker karyopherin α 2 (KPNA2) in cancer. Cancer Lett 331:18–23. https://doi.org/10.1016/j.canlet.2012.12.013 Shin I, Rotty J, Wu FY, Arteaga CL (2005) Phosphorylation of p27 Kip1 at Thr-157 Interferes with Its Association with Importin α during G 1 and Prevents Nuclear Re-entry. J Biol Chem 280:6055–6063. https://doi.org/10.1074/jbc.M412367200 Lin F, Gao L, Su Z et al (2018) Knockdown of KPNA2 inhibits autophagy in oral squamous cell carcinoma cell lines by blocking p53 nuclear translocation. Oncol Rep. https://doi.org/10.3892/or.2018.6451 Humpton TJ, Vousden KH (2016) Regulation of Cellular Metabolism and Hypoxia by p53. Cold Spring Harb Perspect Med 6:a026146. https://doi.org/10.1101/cshperspect.a026146 Wu L, Timmers C, Maiti B et al (2001) The E2F1–3 transcription factors are essential for cellular proliferation. Nature 414:457–462. https://doi.org/10.1038/35106593 Additional Declarations No competing interests reported. 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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-3010156","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":206433127,"identity":"9dcccafe-3a88-4338-9891-6fecb722769b","order_by":0,"name":"Adriana L Invitti","email":"","orcid":"","institution":"Federal University of São Paulo (UNIFESP-EPM)","correspondingAuthor":false,"prefix":"","firstName":"Adriana","middleName":"L","lastName":"Invitti","suffix":""},{"id":206433128,"identity":"01fdea4f-dbd1-40d8-a169-2709add969f7","order_by":1,"name":"Fernando Y Asanuma","email":"","orcid":"","institution":"Federal University of São Paulo (UNIFESP-EPM)","correspondingAuthor":false,"prefix":"","firstName":"Fernando","middleName":"Y","lastName":"Asanuma","suffix":""},{"id":206433129,"identity":"40cb9e97-b9d9-4c30-9c03-4f005a8ca44c","order_by":2,"name":"Cristina V Carvalho","email":"","orcid":"","institution":"Federal University of São Paulo (UNIFESP-EPM)","correspondingAuthor":false,"prefix":"","firstName":"Cristina","middleName":"V","lastName":"Carvalho","suffix":""},{"id":206433130,"identity":"4a016a04-b887-40be-94a8-64fdbaeb414c","order_by":3,"name":"Gil Kamergorodsky","email":"","orcid":"","institution":"Federal University of São Paulo (UNIFESP-EPM)","correspondingAuthor":false,"prefix":"","firstName":"Gil","middleName":"","lastName":"Kamergorodsky","suffix":""},{"id":206433131,"identity":"b3fc9075-94e2-4d71-9f1e-01b5949e3ab4","order_by":4,"name":"Alexander Kopelman","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYDACHhBRAMTsDQwMCTZwIUJaDECMA0AtaSRpkUgAEsRo4e85fOzBB4Nt8uaSb8wePEiwsTdn4D32AZ8WibNt6YYzDG4b7pydY26QkJCWuLOBL3kGXmvO85hJ8xjcZtxwO8dMIvHH4QSDAzzGeHXIg7T8Mbhtv+HmGTOJhIT/9gS1GJztMZNmMLiduOEGD0jLAcYNhLQYnjmWbthjcDt5w5m0MqCW5MSdzXzJeLXInUk+9uBHxW3bDccPb5P8kWBnb87eexivFiBgQ3MqMyENmFoI6xgFo2AUjIIRBgA+XEmsxnpbkAAAAABJRU5ErkJggg==","orcid":"","institution":"Federal University of São Paulo (UNIFESP-EPM)","correspondingAuthor":true,"prefix":"","firstName":"Alexander","middleName":"","lastName":"Kopelman","suffix":""},{"id":206433132,"identity":"08cc1bdf-bb26-400c-84d8-71dd00eabc6c","order_by":5,"name":"Eduardo Schor","email":"","orcid":"","institution":"Federal University of São Paulo (UNIFESP-EPM)","correspondingAuthor":false,"prefix":"","firstName":"Eduardo","middleName":"","lastName":"Schor","suffix":""}],"badges":[],"createdAt":"2023-06-01 13:14:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3010156/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3010156/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":38098853,"identity":"50738aa4-7020-4e64-a33a-afdfc5c842c0","added_by":"auto","created_at":"2023-06-06 14:20:42","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":550969,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBoxplot of the Differentially Expressed Cell Cycle Genes.\u003c/strong\u003e A - TP53, B - KPNA2, C - TFDP1, D - TFDP2, E - RB1, F - RBL2, G - SERTAD1 expression in primary endometrial stromal cells from patients with endometriosis pre-operative (Endometriosis - yellow), endometriosis post-operative (Post-operative - green) and controls (healthy endometrium – Control – red). The results are expressed as Normalized Expression (2\u003csup\u003e-ΔCT\u003c/sup\u003e). Control group: n = 5, Endometriosis Group = 6 ; Post-operative Group: n = 3. Brackets indicate differences statistically significant between two groups with 95% of confidence. Black dots represents the expression for each sample.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3010156/v1/cfd3a5200f6094eb78279113.jpeg"},{"id":38097790,"identity":"b59618ef-be20-4882-91dc-f7ad3dd0ee03","added_by":"auto","created_at":"2023-06-06 14:12:42","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":279218,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHierarchical heat map for the Cell Cycle genes differentially expressed. \u003c/strong\u003eThe gene expression were plotted using ΔCT values, range shown in the vertical bar from orange to blue. Columns represents sample of primary eutopic endometrial cells. Hierarchical clustering applying Pearson distance measure arranged by genes expression. The upper line represents the groups: Blue – Control (primary cells from healthy eutopic endometrium), Red – Endometriosis (primary cells from eutopic endometrium from women with DIE) and Green – Post-operative (primary cells from eutopic endometrium from women with DIE after surgical removal of endometriosis ectopic foci).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3010156/v1/bb7d09915de1ec22182e7e67.jpeg"},{"id":38097791,"identity":"06a7f873-95fe-4c06-8abd-de10daa08f8b","added_by":"auto","created_at":"2023-06-06 14:12:42","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":158343,"visible":true,"origin":"","legend":"\u003cp\u003eProtein-Protein Interactions between the proteins coded by the differentially expressed cell cycle genes TP53, TFDP1, KPNA2, RB1, SERTAD1, RBL2 and TFDP2(DP2)) and key endometriosis cell cycle proteins (p27(CDKN1B) and p21(CDKN1A)). Meaning of network edges: molecular action, medium confidence interaction score = 0.400, number of nodes = 9, number of edges = 19, expected number of edges = 3, PPI enrichment p-value = 1.42e\u003csup\u003e-10\u003c/sup\u003e. Edges represents protein to protein association, not necessarily physical interactions. Lines ended in arrows indicate positive association, ended with vertical line indicate negative association and ended in circles non-specific association. Line colors: green: activation, blue: binding, purple: catalysis, red: inhibition, black: reaction, yellow: transcriptional regulation, grey: unknown. (String version 11.0 - https://string-db.org))\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3010156/v1/a54a9ded70f0ffe5f131258c.jpeg"},{"id":39387666,"identity":"4465eb49-21a5-4ef9-ac01-42906e5e0423","added_by":"auto","created_at":"2023-06-30 18:14:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":385938,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3010156/v1/9363128b-15dc-4c6d-8031-f000642905ad.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cell cycle gene expression is restored in eutopic primary stromal cells after endometriosis surgery","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe endometriosis pathogenesis studies have revealed that the eutopic endometrium is innately aberrant and plays an important role in the pathophysiology of the disease [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].The eutopic endometrium of women with endometriosis have an increased cell proliferation and decreased programmed cell death [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Analysing the expression of cell cycle molecules in endometrial biopsies, our group demonstrated that endometriotic tissues have lower levels of p27 protein compared with those of healthy control [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe eutopic endometrium shares changes with ectopic tissue, which are not found in the eutopic endometrium of women without the disease, corroborating the thesis that the main defect in endometriosis must be primarily in the eutopic endometrium[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, it is still unclear whether abnormal findings in the eutopic endometrium of patients with the disease is related to the ectopic inflammation due to the ectopic tissue or primary of the uterine mucosa [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eResearchers suggest that foci of endometriosis can activate transduction signals that alter the gene expression of the eutopic endometrium [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and may be an explanation for presenting better rates of embryo implantation in infertile patients when submitted to surgical treatment [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. According to this, we have evaluated the cell cycle-related genes expression on primary eutopic endometrial cells obtained before and after endometriosis surgical treatment to understand if the alterations of endometriosis are primary or secondary to the endometrial tissue, that is, if the change of the peritoneal environment through the surgical treatment would provide reestablishment of the normal conditions of the endometrial environment.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients\u0026rsquo;samples\u003c/h2\u003e \u003cp\u003e The study was approved by the Research Ethics Committee of the Federal University of S\u0026atilde;o Paulo (Ref. 226536/2013), and free informed consent was obtained from all of the participants. ECs were isolated from pipelle biopsies of the eutopic endometrium from 14 women, aged between 20 and 35 years, undergoing surgical treatment for the diagnosis of benign ovarian cyst (control) or endometriosis. None of the participants had received hormonal preparations or used GnRH analogues in the three months before the endometrial collection. Were excluded the patients that had childbirth or breastfed in the six months before enrollment in the study, those with menstrual irregularities, teratoma, myoma, endometrial polyp or adenomyosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell isolation and RNA extraction\u003c/h2\u003e \u003cp\u003eECs were isolated according to Pelvic Pain Unit standard protocol and cultured until the second passage and then cryopreserved [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. mRNA was extracted with TRIzol (Invitrogen \u0026ndash; LifeTechnologies - Carlsbad, CA, USA) according to manufacture\u0026rsquo;s protocol. Quantification and quality control (260/280 ratio) were evaluated on Nanodrop (Nanodrop Technologies Inc., Rockland, DE).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eReal-time PCR analysis\u003c/h2\u003e \u003cp\u003eThe cDNA was synthesized with RT2 First Strand Kit (Qiagen, Hilden, Germany). The gene expression was evaluated by the SYBR Green RT-PCR method with QuantiTect Primer Assay (Qiagen, Hilden, Germany) using the PAHS-020A (Qiagen, Hilden, Germany) plate following the manufacturer\u0026rsquo;s instructions. The groups studied were divided into: Control (n\u0026thinsp;=\u0026thinsp;5, patients without endometriosis), Pre-operative DIE - DIE-Pre (n\u0026thinsp;=\u0026thinsp;6) and Post-operative Endometriosis - DIE-Post (n\u0026thinsp;=\u0026thinsp;3). The analysis included two housekeeping genes: GAPDH and β-Actin. Triplicates were performed for all samples, and reactions carried-out on Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe results were analyzed in the Data Analysis V3.5 PCR software (Qiagen) and p-values calculated using Student\u0026rsquo;s t-test. A value greater than one indicated an upregulation, and a value less than one indicated a downregulation. A comparison was considered as statistically significant if the p values were less than 0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003eWe have observed seven (p53, TFDP1, TFDP2, KPNA2, RB1, RBL2, SERTAD1) differentially expressed genes in the ECs comparing all three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These genes are strongly related to the homeostasis and control of the cell cycle indicating that the surgery can implicate in the gene expression of the endometrial cells, confirmed by the interaction between the protein coded by these genes and the clusterization observed for their expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe differentially expressed genes found in this work codes for proteins which have an association between each other (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The cell cycle is strictly regulated by extracellular signals and the check-points (cell cycle arrest points) provides cells with the opportunity to repair damages before division, preventing the transmission of genetic errors to daughter cells. Also, these check-points are responsible for the recovery of the cells from damages preventing premature cell death[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The disrupted cell cycle and aberrant inflammatory molecules expression have been already described in the endometrium of women with DIE [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These differentially expressed genes found codes for proteins which have an association between each other and also with some endometriosis cell cycle key protein (p27 and p21) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], according to the following diagram (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Although these studies have considered only p27 protein levels and not the mRNA levels. In this study,we did not observe differences in the expression of p27 between DIE samples and controls (FC -1.58; p\u0026thinsp;=\u0026thinsp;0.3768). Indicating that possible a post-translational mechanism regulates the p27 protein content in the endometriosis samples.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThis can be explained by the Karyopherin alpha 2 (KPNA2) expression, which is 2.62 times downregulated in the endometriosis groups compared to control and 4.36 times upregulated after surgery in comparison to pre-surgery ECs. KPNA2 is known as a selective nucleocytoplasmic carrier of transcription factors and proteins related to DNA repair and cell cycle regulation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It is directly related to the translocation of p27 in and out of the nucleous [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Exacerbated expression of KPNA2 has been reported in malignant tumours and its knockdown causes the accumulation of p53 protein in the cytoplasm[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. We demonstrate an up-regulation of KPNA2 in the ECs from patients submitted to surgical treatment.\u003c/p\u003e \u003cp\u003ep53 protein is known as a general stress sensor, not only important to inhibit cancer progression, but also to respond during viral infection, starvation or oxidative stress, reducing cell proliferation, altering cellular metabolism and inhibiting survival[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The gene that codes to p53 (TP53) is 3.34 less expressed in the endometriosis group than in the control group and no statiscally significant difference was found between post-operative and control groups (fold change \u0026minus;\u0026thinsp;1.51, p\u0026thinsp;=\u0026thinsp;0.3590). p53 is downregulated in the ECs of DIE patients compared to control and its expression is restored after surgical removal of ectopic endometriosis foci.\u003c/p\u003e \u003cp\u003ePost-operative ECs shows other sign of a tentative regulation of the G1/S transition. The gene RBL2, which encodes for a retinoblastoma (Rb) protein, was found to be 2.23 times more more expressed in endometriosis post-operative than in control group. RBL2 proteins intereact with CDKs complexes to promote a proper G1/S transition[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Another gene involved in controlling early cell cycle events, from G1 to S phases, is the transcription factor DP-1 (TFDP1). TFDP1 is downregulated 2.05 fold in the DIE-pre than in Control ECs. TFDP1 or TFDP2 forms a heterodimeric complex with the tumour suppressor RB1 (downregulated 9.36 and 14.28 fold in the DIE-post in comparison to DIE-pre (p\u0026thinsp;=\u0026thinsp;0.0295) and Control (p\u0026thinsp;=\u0026thinsp;0.0211), respectively) binded to E2F transcription factors. This interaction results in the prevention of G1/S transition [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In previous work, we observed the downregulation of TFDP1 in the endometrium of women with DIE, but no changes were observed on TFDP1 expression between pre and postoperative ECs (FC 1.55, p\u0026thinsp;=\u0026thinsp;0.2475).\u003c/p\u003e \u003cp\u003eBy the results presented, we infer that surgical removal of ectopic endometriosis foci leads the eutopic endometrial cells to a cell cycle genes pattern more similar to the control group. Acting directly on the cell cycle arrest, blocking or at least by, reducing the proliferation and development of endometriosis cells. To the best of our knowledge this is the first report of endometrial cells expression changes after endometriosis surgery. In spite of the limited number of samples, this study can highlight the importance of the study of post-operative endometrium cells for the understanding of endometriosis pathogenesis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe study was approved by the Research Ethics Committee of the Federal University of S\u0026atilde;o Paulo (Ref. 226536/2013), and free informed consent was obtained from all of the participants.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe authors declare that they have no affiliations that would constitute a financial conflict of interest regarding the subject matter presented in this study.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eALI, FYA and CVC: study design, acquisition of data, analysis and interpretation of the data.GK and AK: acquisition of data and interpretation of the data. ES: study design and interpretation of the data. All authors contributed with the drafting of the article, critical revision and have approved the final version of the manuscript.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThis work received support of the National Council for Scientific and Technological Development (CNPQ) - 134111/2013-13 as a master scholarship to FYA.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAvailability of data\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSharpe-Timms KL (2001) Endometrial anomalies in women with endometriosis. Ann N Y Acad Sci 943:131\u0026ndash;147. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1749-6632.2001.tb03797.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1749-6632.2001.tb03797.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDmowski WP, Ding J, Shen J et al (2001) Apoptosis in endometrial glandular and stromal cells in women with and without endometriosis. Hum Reprod 16:1802\u0026ndash;1808. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/humrep/16.9.1802\u003c/span\u003e\u003cspan address=\"10.1093/humrep/16.9.1802\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGon\u0026ccedil;alves GA, Invitti AL, Parreira RM et al (2017) p27kip1 overexpression regulates IL-1β in the microenvironment of stem cells and eutopic endometriosis co-cultures. Cytokine 89:229\u0026ndash;234. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cyto.2015.12.015\u003c/span\u003e\u003cspan address=\"10.1016/j.cyto.2015.12.015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGon\u0026ccedil;alves GA, Camargo-Kosugi CM, Bonetti TCS et al (2015) p27kip1 overexpression regulates VEGF expression, cell proliferation and apoptosis in cell culture from eutopic endometrium of women with endometriosis. Apoptosis 20:327\u0026ndash;335. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10495-014-1079-8\u003c/span\u003e\u003cspan address=\"10.1007/s10495-014-1079-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchor E, da Silva IDCG, Sato H et al (2009) P27Kip1 is down-regulated in the endometrium of women with endometriosis. Fertil Steril 91:682\u0026ndash;686. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.fertnstert.2007.12.070\u003c/span\u003e\u003cspan address=\"10.1016/j.fertnstert.2007.12.070\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaniguchi F, Kaponis A, Izawa M et al (2011) Apoptosis and endometriosis. Front Biosci (Elite Ed) 3:648\u0026ndash;62. https://doi.org/277 [pii]\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUlukus M, Cakmak H, Arici A (2006) The Role of Endometrium in Endometriosis. J Soc Gynecol Investig 13:467\u0026ndash;476. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jsgi.2006.07.005\u003c/span\u003e\u003cspan address=\"10.1016/j.jsgi.2006.07.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJacobson TZ, Duffy JMN, Barlow DH et al (2014) Laparoscopic surgery for subfertility associated with endometriosis. Cochrane Database Syst. Rev. 2014\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD\u0026rsquo;Amora P, Maciel TT, Tambellini R et al (2009) Disrupted cell cycle control in cultured endometrial cells from patients with endometriosis harboring the progesterone receptor polymorphism PROGINS. Am J Pathol 175:215\u0026ndash;224. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2353/ajpath.2009.080966\u003c/span\u003e\u003cspan address=\"10.2353/ajpath.2009.080966\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatsuzaki S (2001) Expression of the cyclin-dependent kinase inhibitor p27Kip1 in eutopic endometrium and peritoneal endometriosis. Fertil Steril 75:956\u0026ndash;960. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0015-0282(01)01752-6\u003c/span\u003e\u003cspan address=\"10.1016/S0015-0282(01)01752-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUegaki T, Taniguchi F, Nakamura K et al (2015) Inhibitor of apoptosis proteins (IAPs) may be effective therapeutic targets for treating endometriosis. Hum Reprod 30:149\u0026ndash;158. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/humrep/deu288\u003c/span\u003e\u003cspan address=\"10.1093/humrep/deu288\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChristiansen A, Dyrskj\u0026oslash;t L (2013) The functional role of the novel biomarker karyopherin α 2 (KPNA2) in cancer. Cancer Lett 331:18\u0026ndash;23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.canlet.2012.12.013\u003c/span\u003e\u003cspan address=\"10.1016/j.canlet.2012.12.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShin I, Rotty J, Wu FY, Arteaga CL (2005) Phosphorylation of p27 Kip1 at Thr-157 Interferes with Its Association with Importin α during G 1 and Prevents Nuclear Re-entry. J Biol Chem 280:6055\u0026ndash;6063. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1074/jbc.M412367200\u003c/span\u003e\u003cspan address=\"10.1074/jbc.M412367200\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin F, Gao L, Su Z et al (2018) Knockdown of KPNA2 inhibits autophagy in oral squamous cell carcinoma cell lines by blocking p53 nuclear translocation. Oncol Rep. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3892/or.2018.6451\u003c/span\u003e\u003cspan address=\"10.3892/or.2018.6451\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHumpton TJ, Vousden KH (2016) Regulation of Cellular Metabolism and Hypoxia by p53. Cold Spring Harb Perspect Med 6:a026146. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1101/cshperspect.a026146\u003c/span\u003e\u003cspan address=\"10.1101/cshperspect.a026146\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu L, Timmers C, Maiti B et al (2001) The E2F1\u0026ndash;3 transcription factors are essential for cellular proliferation. Nature 414:457\u0026ndash;462. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/35106593\u003c/span\u003e\u003cspan address=\"10.1038/35106593\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","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":"Endometriosis, cell cycle genes, p53, endometrium, surgery","lastPublishedDoi":"10.21203/rs.3.rs-3010156/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3010156/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe role of the endometrium in the pathogenesis of endometriosis has assumed prominence. Abnormality of gene and protein expression, apoptosis and changes in cell cycle have been extensively studied in endometriosis. We evaluated the cell cycle genes expression on primary stromal endometrial cells (EC) isolated from eutopic endometrium in two different moments: before and after deep infiltrating endometriosis (DIE) surgical treatment. We analysed five ECs from healthy patients (group control) and 9 from women with diagnosis of DIE. Were identified 7 cell cycle genes (p53, TFDP1, TFDP2, KPNA2, RB1, RBL2, SERTAD1) differentially expressed between pre, post-operative and controls. The p53 and KPNA2 genes were downregulated 3.34 fold (p\u0026thinsp;=\u0026thinsp;0.006) and 2.62, (p\u0026thinsp;=\u0026thinsp;0.042), respectively, in the endometrium of DIE compared to control. Both were upregulated (p53 - Fold 2.22, p\u0026thinsp;=\u0026thinsp;0.157; KPNA2 - Fold 4.36, p\u0026thinsp;=\u0026thinsp;0.017) in the post-operative DIE group in comparison to pre-operative one. Also, the RB1 gene was downregulated 9.36 fold (p\u0026thinsp;=\u0026thinsp;0.029) in the DIE-post group in comparison to DIE-pre group, having no difference between DIE-pre and control group (p\u0026thinsp;=\u0026thinsp;0.311). The proteins coded by these genes have association between each other, indicating that the surgical treatment could change the cell cycle regulation in the endometrium of women with endometriosis and that the changes remain after the cell isolation from the tissue.\u003c/p\u003e","manuscriptTitle":"Cell cycle gene expression is restored in eutopic primary stromal cells after endometriosis surgery","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-06-06 14:12:37","doi":"10.21203/rs.3.rs-3010156/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","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":"3de17fe0-4f17-4708-8b5f-776e55902742","owner":[],"postedDate":"June 6th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-06-30T18:14:19+00:00","versionOfRecord":[],"versionCreatedAt":"2023-06-06 14:12:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3010156","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3010156","identity":"rs-3010156","version":["v1"]},"buildId":"2u56kwukJI3zHK-uzyFNs","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}