MSX1-expression during the different phases in healthy human endometrium

MSX1-expression during the different phases in healthy human endometrium | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article MSX1-expression during the different phases in healthy human endometrium Simon Eppich, Christina Kuhn, Elisa Schmoeckel, Doris Mayr, Sven Mahner, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2458340/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Apr, 2023 Read the published version in Archives of Gynecology and Obstetrics → Version 1 posted 4 You are reading this latest preprint version Abstract Purpose: The human endometrium consists of different layers (basalis and functionalis) and undergoes different phases throughout the menstrual cycle. In a former paper our research group was able to describe MSX1 as a positive prognosticator in endometrial carcinomas. The aim of this study was to examine the MSX1 expression in healthy endometrial tissue throughout the different phases to gain more insight on the mechanics of MSX-regulation in the female reproductive system. Materials and Methods: In this retrospective study we investigated a total of 19 normal endometrial tissues (7 during proliferative phase and each 6 during early and late secretory phase). We used immunohistochemical staining and an immunoreactive score (IRS) to evaluate MSX1 expression. We also investigated correlations with other proteins, that have already been examined in our research group using the same patient collective. Results: MSX1 is highly expressed during the proliferative phase and downregulated at early and late secretory phase (p=0.018). Also, a positive correlation between MSX1 and the progesterone-receptor A (PR-A) (correlation coefficient (cc)=0.0671; p=0.024), the progesterone receptor B (PR-B) (cc=0.0691; p=0.018) was found. A trend towards negative correlation was recognized between MSX1 and Inhibin Beta-C-expression in glandular cells (cc=-0.583; p-value=0.060). Conclusion: MSX1 is known as a member of the muscle segment homeobox gene family. MSX1 is a p53-interacting protein and overexpression of homeobox MSX1 induced apoptosis of cancer cells. Here we show that MSX1 is highly expressed especially in the proliferative phase of glandular epithelial tissue of the normal endometrium. Because MSX1 is known to be downregulated by progesterone, the found correlation of MSX1 and both PR-A and -B may represent a direct regulation of the MSX1 gene by a PR-response element. MSX-1 Progesterone Receptor endometrium proliferative phase glandular cells Figures Figure 1 Figure 2 What Does This Study Add To The Clinical Work In this study we showed that MSX1 is highly expressed especially in the proliferative phase of glandular epithelial tissues of normal endometrium. By the found correlations of MSX1 and progesterone receptor A and B a direct regulation of the MSX1 gene by a PR response element (PRR) can be assumed. Introduction The human endometrium consists of different layers, which are differently affected by hormonal changes [ 1 ]. There are two regions that define the subsurface endometrium: The functionalis (stratum spongiosum) and the basalis (stratum basale), with the functionalis being the tissue with the greatest degree of hormonal responsiveness [ 1 , 2 ]. Due to the hormonal changes throughout the menstrual cycle the human, premenopausal endometrium undergoes different phases; there is the proliferative phase and the secretory phase [ 1 ]. The proliferative phase is mainly estrogen-dependent and starts after menses and terminates at ovulation [ 3 , 4 ]. In this phase a rapid cellular proliferation of all cell types happens and new extracellular matrix is being build up [ 3 ]. The secretory phase follows ovulation; now the progesterone-dependent differentiation of the endometrium takes place [ 4 ]. Characteristics are tortuous glands, sub-nuclear vacuoles in glandular epithelial cells, stromal oedema and maturation of spiral arterioles [ 4 , 5 ]. For better understanding of this cycle and the anatomy of the female reproductive tract it is important to examine, which proteins play a potential role. In a former analysis by our research group a possible protective effect of MSX1 in endometrial carcinomas was described [ 6 ]. MSX1 is a protein, that as a transcriptional repressor plays a role during embryogenesis (especially during the formation of limb-patterns, craniofacial development, odontogenesis), but also is thought to function in tumor growth inhibition [ 7 – 10 ]. In a study by Bolnick et al a reduced MSX1-level was linked to infertility [ 11 ]. So, to better understand this protein’s function in the female reproductive system, the idea for this analysis was to examine the MSX1-expression in healthy endometrium throughout the different phases and to look for correlations with other proteins, that have already been examined in the same patient collective by our research group, and by this to gain more insight on the complex biochemical mechanics of the female reproductive system. Material And Methods Patients and Tissue Collection For this study, samples of healthy endometrium were used. We retrospectively analyzed a total of 19 patients. All samples were collected at the Department of Obstetrics and Gynecology of the Ludwig-Maximilians-University. Informed consent from all patients was obtained before surgery. The material was obtained from women undergoing dilatation and curettage or hysterectomy for benign diseases. No hormone therapy was applied for 3 months prior to surgery. By using hematoxylin and eosin staining the menstrual cycle was determined. The samples were all formalin-fixed and paraffin-embedded. Endometrium tissue of the different phases was used (proliferative phase, early secretory phase, late secretory phase). Table 1 shows patients’ characteristics. Surgery took place between 1 January 1990 and 31 December 2001 in the Department of Gynecology, Ludwig-Maximilians-University Munich, Germany. Table 1 Patients’ characteristics (healthy endometrium), n = 19 Proliferative phase 7 Early secretory phase 6 Late secretory phase 6 Immunohistochemistry For the immunohistochemical staining of the endometrial tissue new samples of the original slides were taken and representative areas were selected. The paraffinized slides were put into Roticlear (a substitute-medium for xylol) for 20 min to deparaffinize the tissue. Afterwards they were put into 100% ethanol and then for another 20 min into 3% hydrogen peroxide diluted in methanol. By this the endogenous peroxidase activity was inhibited. Using a series of graded alcohols (100%, 70%, 50%) and eventually distilled water the samples were rehydrated. By heating them up to 100°C and cooking them for 5 minutes in a pressure cooker filled with an already boiling trisodium citrate buffer solution with pH = 6.0 the slides were demasked. A blocking solution (Reagent 1 of polymer detection kit (ZytoChem Plus HRP Polymer System, Mouse Rabbit, Zytomed, Berlin, Germany)) was used to saturate the electrostatic charges in the tissue. The primary antibody was placed on the samples and incubated for 16 h at 4°C. The antibody used was Anti-MSX1, Rabbit IgG polyclonal, concentration 0.2 mg/mL (Sigma; order number HPA073604. Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), diluted at a ratio of 1:200 with PBS. The following day the antibody-surplus was washed off and a post-block-reagent applied for 20 minutes. Secondary antibodies were conjugated with horseradish peroxidase (HRP) for another 30 minutes. Finally, the antibody was stained by applying DAB (chromogen substrate kit, Dako North America Inc., Carpinteria, CA, USA) for 5 min. The staining reaction was then stopped using distilled water and the material was counterstained with hematoxylin. Then the samples were dehydrated with an ascending series of graded alcohols (50%, 70%, 96%, and 100%) and Roticlear. As final step, the samples were covered with a mounting medium and cover glasses. This method has already been described by our research group [ 6 ]. For evaluation the IRS (immunoreactive score) was applied, which is the result of the percentage of stained cells (0 = 0%, 1 = 1–10%, 2 = 11–50%, 3 = 51–80%, 4 ≤ 81%) multiplied with the coded staining intensity [ 12 ]. In statistical analysis, all three results were looked at separately (staining intensity, percentage of stained cells, and IRS). Statistical Analysis The statistical analysis software SPSS (IBM SPSS Statistics, Version 25; IBM Deutschland GmbH, Ehningen, Germany) was used. For evaluation of the clinical-pathological variables, the Kruskal–Wallis test was applied. For all analyses a p-value < 0.05 was considered statistically significant. Results Our results show a significant difference in MSX1 expression throughout the different phases. The specimens of endometrium during the proliferative phase show a staining with a mean IRS of 1 (p = 0,018), while the early and the late secretory phase show no staining for MSX1 at all. Furthermore, correlations were found between MSX1 and other proteins examined in this collective by our research group (Table 2 ) [ 13 , 14 ]. Table 2 Correlations between MSX1 and formerly investigated markers (cc: correlation coefficient) PR-A PR-B Inhibin Beta-C MSX1 IRS-Score IRS-Score IRS-Score cc 0.0671 0.691 -0.583 p 0.024 0.018 0.060 n 11 11 11 With a p-value of 0.024 and a correlation-coefficient (cc) of 0.0671 a positive correlation between MSX1 and the progesterone-receptor A (PR-A) was found. Although there is no significant correlation between progesterone-receptor B (PR-B) and the menstrual cycle or Inhibin Beta-C and menstrual cycle, the following correlations with MSX1 were found: A significant positive correlation was found between MSX1 and PR-B-expression in stroma-cells (p-value = 0.018; correlation-coefficient cc = 0.691). Also a trend towards negative correlation was recognized between MSX1 and Inhibin Beta-C expression in glandular cells (p-value = 0.060; cc = -0.583). Therefore, we were able to confirm the results of our former paper on cancer tissue, where a significant positive correlation between MSX1 and the progesterone-receptors A and B were also found. [ 6 ] Discussion In our study we found that MSX1 is highly expressed during the proliferative phase and significantly downregulated at early and late secretory phase (p = 0.018). In addition, a positive correlation between MSX1 and the progesterone-receptor A (PR-A) (correlation coefficient (cc) = 0.0671; p = 0.024) and the progesterone receptor B (PR-B) (cc = 0.0691; p = 0.018) was found. Progesterone regulation of the endometrial MSX1 gene was first described in 2008 in the ovine uterus [ 15 ]. In that study, Satterfield et al. [ 15 ] described that MSX1 mRNA was decreased by P4 treatment. In addition, another animal based study found that progesterone inhibits uterine gland development in the neonatal mouse uterus and downregulated MSX1 [ 16 ]. A very recent study on the role of MSX1 in reproduction was carried out on mice embryonic diapause [ 17 ]. Embryonic diapause in mice is a reproductive strategy in which embryo development and growth are temporarily halted in utero to ensure neonatal and maternal survival in adverse external conditions [ 17 ]. In that study, the authors have shown that dormant blastocysts are recovered from these mice on day 8 of pregnancy with persistent expression of uterine MSX1, a gene critical to maintaining the uterine quiescent state [ 17 ]. Interestingly, progesterone and anti-estrogen can prolong uterine quiescence [ 17 ]. In cancer biology, on the other side MSX1 was identified as a key candidate for progestin resistance in endometrial cancer [ 18 ]. MSX1 showed significant tissue specificity and better prognostic value and its knockdown enhanced progesterone efficacy [ 18 ]. Our own investigation on MSX1 in endometrial cancer showed that a better survival was identified for patients with an MSX1 expression in more than 10% of the tumor cells [ 6 ]. We further have given the hypothesis that MSX1 could be a potential marker for a potential uterus-preserving therapy of endometrial carcinomas [ 6 ]. In agreement to our results, a recent study on development of potential prognostic biomarkers based on DNA methylation-driven genes for patients with endometrial cancer showed that high methylation and low expression of MSX1 were significantly associated with reduced endometrial cancer survival rates [ 19 ]. A similar role of MSX1 was identified for other gynecologic cancer subtypes. An early study showed that MSX1 may be involved in the regulation of cell proliferation and cell cycle using MSX1 overexpressing human ovarian cancer cells [ 20 ]. Overexpression of MSX1 inhibited cell proliferation by markedly increasing the length of the G1 phase of the cell cycle over control cells and a suppression of cyclins D1, D3, E, cyclin-dependent kinase 4, c-Jun, and Rb was observed [ 20 ]. Furthermore, and consistent with these findings, MSX1 triggers G0/G1 arrest and apoptosis by suppressing Notch signaling and is frequently methylated in cervical cancer [ 21 ]. In breast cancer, MSX1 inhibits breast cancer cell growth and metastasis and is often silenced by promoter methylation [ 22 ]. In addition to our progesterone regulation of MSX1, a trend towards negative correlation was recognized between MSX1 and Inhibin Beta-C-expression in glandular cells (cc = -0.583; p-value = 0.060). Although no direct involvement of inhibin on MSX1 is known, MSX1 represses the αGSU and GnRH receptor genes during gonadotropic development [ 23 ]. Suppression of the mouse GnRHR promoter by MSX1 is mediated by a consensus binding motif in the downstream activin regulatory element (DARE) [ 23 ]. Activin and inhibin share the same beta subunit [ 24 , 25 ]. Taken together, MSX1 is known as a member of the muscle segment homeobox gene family. MSX1 is a p53 interacting protein and overexpression of MSX1 homeobox induces apoptosis of cancer cells. Here, we show that MSX1 is highly expressed especially in the proliferative phase of glandular epithelial tissues of normal endometrium. Since MSX1 is known to be downregulated by progesterone, the found correlation of MSX1 and PR-A and -B may represent a direct regulation of the MSX1 gene by a PR response element (PRR). Declarations Funding: This study was funded by the medical faculty of the Ludwig-Maximilians-University Munich 2018-2020. Acknowledgments: We thank Kerstin Hermelink for the excellent technical support. Conflicts of interest: Sv.M: Research support, advisory board, honoraria and travel expenses from AbbVie, AstraZeneca, Clovis, Eisai, GlaxoSmithKline, Medac, MSD, Novartis, Olympus, PharmaMar, Roche, Sensor Kinesis, Teva, Tesaro; J.G. received honoraria for lectures from MSD and Roche. All other authors declare no conflict of interest. The authors declare that no funds, grants or other support were received during the preparation of this manuscript. The authors have no relevant financial or non-financial interests to disclose. Author Contributions: Conceptualization: Udo Jeschke. and Simon Eppich; methodology: Christina Kuhn and Simon Eppich; data curation: Helene Hildegard Heidegger and Udo Jeschke; article writing: Simon Eppich and Helene Hildegard Heidegger; supervision: Udo Jeschke, Sven Mahner, Julia Gallwas, Elisa Schmoeckel and Doris Mayr; All authors analyzed and interpreted the data, and read and agreed to the published version of the manuscript. Ethics Approval: This study was conducted conforming to the Declaration of Helsinki 1975 and it was approved by the Ethics Committee of the Ludwig-Maximilians-University, Munich, Germany (approval number 063-13). All patients’ data were fully anonymized, and during experimental analysis, the authors were blinded for clinical information. All tissue used was leftover material and all diagnostic procedures had already been completed, when the samples were received for the study. Informed consent from all patients was obtained before surgery. Conflicts of interest: Sv.M: Research support, advisory board, honoraria and travel expenses from AbbVie, AstraZeneca, Clovis, Eisai, GlaxoSmithKline, Medac, MSD, Novartis, Olympus, PharmaMar, Roche, Sensor Kinesis, Teva, Tesaro; J.G. received honoraria for lectures from MSD and Roche. All other authors declare no conflict of interest. References Mazur MT (2005) R.J.K., Normal Endometrium and Infertility Evaluation , in Diagnosis of Endometrial Biopsies and Curettings, A Practical Approach . Springer, New York, USA, pp 7–33 Evans J et al (2016) Fertile ground: human endometrial programming and lessons in health and disease. Nat Rev Endocrinol 12(11):654–667 Rai P et al (2010) Proteome of human endometrium: Identification of differentially expressed proteins in proliferative and secretory phase endometrium. Proteom Clin Appl 4(1):48–59 Maybin JA, Critchley HO (2012) Steroid regulation of menstrual bleeding and endometrial repair. Rev Endocr Metab Disord 13(4):253–263 Noyes RW, Hertig AT, Rock J (2019) Reprint of: Dating the Endometrial Biopsy. Fertil Steril 112(4 Suppl1):e93–e115 Eppich S et al (2020) MSX1-A Potential Marker for Uterus-Preserving Therapy of Endometrial Carcinomas .Int J Mol Sci, 21 (12) Catron KM et al (1996) Comparison of MSX-1 and MSX-2 suggests a molecular basis for functional redundancy. Mech Dev 55(2):185–199 Becic T et al (2018) Growth factors FGF8 and FGF2 and their receptor FGFR1, transcriptional factors Msx-1 and MSX-2, and apoptotic factors p19 and RIP5 participate in the early human limb development. Acta Histochem 120(3):205–214 Dai J et al (2014) Bioinformatic analysis of Msx1 and Msx2 involved in craniofacial development. J Craniofac Surg 25(1):129–134 Bonczek O et al (2018) Next generation sequencing reveals a novel nonsense mutation in MSX1 gene related to oligodontia. PLoS ONE 13(9):e0202989 Bolnick AD et al (2016) Reduced homeobox protein MSX1 in human endometrial tissue is linked to infertility. Hum Reprod 31(9):2042–2050 Remmele W, Stegner HE (1987) [Recommendation for uniform definition of an immunoreactive score (IRS) for immunohistochemical estrogen receptor detection (ER-ICA) in breast cancer tissue]. Pathologe 8(3):138–140 Mylonas I et al (2009) Immunohistochemical labelling of steroid receptors in normal and malignant human endometrium. Acta Histochem 111(4):349–359 Mylonas I et al (2010) Evidence of inhibin/activin subunit betaC and betaE synthesis in normal human endometrial tissue. Reprod Biol Endocrinol 8:143 Satterfield MC et al (2008) Progesterone regulation of the endometrial WNT system in the ovine uterus. Reprod Fertil Dev 20(8):935–946 Filant J, Zhou H, Spencer TE (2012) Progesterone inhibits uterine gland development in the neonatal mouse uterus. Biol Reprod 86(5):1–9 Matsuo M et al (2022) Targeted depletion of uterine glandular Foxa2 induces embryonic diapause in mice .Elife,11 Yang L et al (2020) Identification and Validation of MSX1 as a Key Candidate for Progestin Resistance in Endometrial Cancer. Onco Targets Ther 13:11669–11688 Lu Y et al (2021) Development of Potential Prognostic Biomarkers Based on DNA Methylation-Driven Genes for Patients with Endometrial Cancer. Int J Gen Med 14:10541–10555 Park J et al (2001) Msx1 gene overexpression induces G1 phase cell arrest in human ovarian cancer cell line OVCAR3. Biochem Biophys Res Commun 281(5):1234–1240 Yue Y et al (2018) MSX1 induces G0/G1 arrest and apoptosis by suppressing Notch signaling and is frequently methylated in cervical cancer. Onco Targets Ther 11:4769–4780 Yue Y et al (2018) Homeobox protein MSX1 inhibits the growth and metastasis of breast cancer cells and is frequently silenced by promoter methylation. Int J Mol Med 41(5):2986–2996 Xie H et al (2013) Msx1 homeodomain protein represses the alphaGSU and GnRH receptor genes during gonadotrope development. Mol Endocrinol 27(3):422–436 Kaufl SD et al (2010) Inhibin/activin-betaC subunit in human endometrial adenocarcinomas and HEC-1a adenocarcinoma cell line. Vivo 24(5):695–698 Kimmich T et al (2010) Inhibin/activin-betaC and -betaE subunits in the Ishikawa human endometrial adenocarcinoma cell line. Arch Gynecol Obstet 282(2):185–191 Cite Share Download PDF Status: Published Journal Publication published 27 Apr, 2023 Read the published version in Archives of Gynecology and Obstetrics → Version 1 posted Reviewers agreed at journal 18 Jan, 2023 Reviewers invited by journal 09 Jan, 2023 Editor assigned by journal 09 Jan, 2023 First submitted to journal 09 Jan, 2023 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 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-2458340","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":166247088,"identity":"a3400f9b-0f0d-4150-b453-0f16b7adfb54","order_by":0,"name":"Simon Eppich","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Simon","middleName":"","lastName":"Eppich","suffix":""},{"id":166247089,"identity":"bcc16764-97af-49b5-bfa6-0adc1240eb10","order_by":1,"name":"Christina Kuhn","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Christina","middleName":"","lastName":"Kuhn","suffix":""},{"id":166247090,"identity":"7e1de7ac-75bc-42af-8e9c-fec5d0bdb6cd","order_by":2,"name":"Elisa Schmoeckel","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Elisa","middleName":"","lastName":"Schmoeckel","suffix":""},{"id":166247091,"identity":"a1fd9b86-e60e-4cad-8ea6-a0bc3d33c45d","order_by":3,"name":"Doris Mayr","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Doris","middleName":"","lastName":"Mayr","suffix":""},{"id":166247092,"identity":"e2b81168-039f-491a-a3b0-5bf2598ce030","order_by":4,"name":"Sven Mahner","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sven","middleName":"","lastName":"Mahner","suffix":""},{"id":166247093,"identity":"41f73710-c64f-43c0-8004-b714a8772602","order_by":5,"name":"udo jeschke","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-2623-3235","institution":"Ludwig Maximilians Universität München","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"udo","middleName":"","lastName":"jeschke","suffix":""},{"id":166247094,"identity":"1d523cc4-41da-4f0e-9757-3050b807ffdb","order_by":6,"name":"Julia Gallwas","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Julia","middleName":"","lastName":"Gallwas","suffix":""},{"id":166247095,"identity":"673c2919-a951-4db7-b100-3172939dfc73","order_by":7,"name":"Helene Hildegard Heidegger","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Helene","middleName":"Hildegard","lastName":"Heidegger","suffix":""}],"badges":[],"createdAt":"2023-01-09 10:01:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2458340/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2458340/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00404-023-07033-5","type":"published","date":"2023-04-27T20:38:34+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":31449594,"identity":"ee6c3562-ade1-46f6-bd99-038ea8254314","added_by":"auto","created_at":"2023-01-11 22:43:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2294559,"visible":true,"origin":"","legend":"\u003cp\u003eHealthy endometrium stained for MSX1. A/a and B/b: Proliferative phase with an immunoreactive score (IRS) of 1; C/c: Early secretory phase with an IRS of 0; D/d: Late secretory phase with an IRS of 0. E: Boxplots with a median IRS of 1 for proliferative phase and with a median IRS of 0 for early and late secretory phase (p=0.018).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2458340/v1/57d31b9d14920eac2965d123.png"},{"id":31450296,"identity":"ff4e738b-94f7-44e0-a94a-2da058a7e148","added_by":"auto","created_at":"2023-01-11 22:51:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":68711,"visible":true,"origin":"","legend":"\u003cp\u003eTop left: Boxplots showing a median IRS of 4 for proliferative phase, 2.2 for early secretory and 3 for late secretory phase for PR-B (p=0.003). Top right: Boxplots showing a median IRS of 8 for all phases for Inhibin Beta-C (p=0.157). Below: Boxplots showing a median IRS of 6 for proliferative phase and 2 for early and late secretory phase for PR-A (p=0.015).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2458340/v1/30cf82868d6cc72a6e28e7c6.png"},{"id":44727052,"identity":"14be0c6e-c0b7-4c12-b788-97832cfa3c43","added_by":"auto","created_at":"2023-10-16 20:51:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1762943,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2458340/v1/11dcb314-87fc-4f67-bc84-a9c661818915.pdf"}],"financialInterests":"","formattedTitle":"MSX1-expression during the different phases in healthy human endometrium","fulltext":[{"header":"What Does This Study Add To The Clinical Work","content":"\u003cp\u003eIn this study we showed that MSX1 is highly expressed especially in the proliferative phase of glandular epithelial tissues of normal endometrium. By the found correlations of MSX1 and progesterone receptor A and B a direct regulation of the MSX1 gene by a PR response element (PRR) can be assumed.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe human endometrium consists of different layers, which are differently affected by hormonal changes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. There are two regions that define the subsurface endometrium: The functionalis (stratum spongiosum) and the basalis (stratum basale), with the functionalis being the tissue with the greatest degree of hormonal responsiveness [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDue to the hormonal changes throughout the menstrual cycle the human, premenopausal endometrium undergoes different phases; there is the proliferative phase and the secretory phase [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe proliferative phase is mainly estrogen-dependent and starts after menses and terminates at ovulation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In this phase a rapid cellular proliferation of all cell types happens and new extracellular matrix is being build up [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe secretory phase follows ovulation; now the progesterone-dependent differentiation of the endometrium takes place [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Characteristics are tortuous glands, sub-nuclear vacuoles in glandular epithelial cells, stromal oedema and maturation of spiral arterioles [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor better understanding of this cycle and the anatomy of the female reproductive tract it is important to examine, which proteins play a potential role.\u003c/p\u003e \u003cp\u003eIn a former analysis by our research group a possible protective effect of MSX1 in endometrial carcinomas was described [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. MSX1 is a protein, that as a transcriptional repressor plays a role during embryogenesis (especially during the formation of limb-patterns, craniofacial development, odontogenesis), but also is thought to function in tumor growth inhibition [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In a study by Bolnick et al a reduced MSX1-level was linked to infertility [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSo, to better understand this protein\u0026rsquo;s function in the female reproductive system, the idea for this analysis was to examine the MSX1-expression in healthy endometrium throughout the different phases and to look for correlations with other proteins, that have already been examined in the same patient collective by our research group, and by this to gain more insight on the complex biochemical mechanics of the female reproductive system.\u003c/p\u003e"},{"header":"Material And Methods","content":"\u003cp\u003ePatients and Tissue Collection\u003c/p\u003e \u003cp\u003eFor this study, samples of healthy endometrium were used. We retrospectively analyzed a total of 19 patients. All samples were collected at the Department of Obstetrics and Gynecology of the Ludwig-Maximilians-University. Informed consent from all patients was obtained before surgery.\u003c/p\u003e \u003cp\u003eThe material was obtained from women undergoing dilatation and curettage or hysterectomy for benign diseases. No hormone therapy was applied for 3 months prior to surgery. By using hematoxylin and eosin staining the menstrual cycle was determined.\u003c/p\u003e \u003cp\u003eThe samples were all formalin-fixed and paraffin-embedded. Endometrium tissue of the different phases was used (proliferative phase, early secretory phase, late secretory phase). Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows patients\u0026rsquo; characteristics. Surgery took place between 1 January 1990 and 31 December 2001 in the Department of Gynecology, Ludwig-Maximilians-University Munich, Germany.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePatients\u0026rsquo; characteristics (healthy endometrium), n\u0026thinsp;=\u0026thinsp;19\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProliferative phase\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEarly secretory phase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLate secretory phase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eImmunohistochemistry\u003c/p\u003e \u003cp\u003eFor the immunohistochemical staining of the endometrial tissue new samples of the original slides were taken and representative areas were selected. The paraffinized slides were put into Roticlear (a substitute-medium for xylol) for 20 min to deparaffinize the tissue. Afterwards they were put into 100% ethanol and then for another 20 min into 3% hydrogen peroxide diluted in methanol. By this the endogenous peroxidase activity was inhibited. Using a series of graded alcohols (100%, 70%, 50%) and eventually distilled water the samples were rehydrated. By heating them up to 100\u0026deg;C and cooking them for 5 minutes in a pressure cooker filled with an already boiling trisodium citrate buffer solution with pH\u0026thinsp;=\u0026thinsp;6.0 the slides were demasked.\u003c/p\u003e \u003cp\u003eA blocking solution (Reagent 1 of polymer detection kit (ZytoChem Plus HRP Polymer System, Mouse Rabbit, Zytomed, Berlin, Germany)) was used to saturate the electrostatic charges in the tissue. The primary antibody was placed on the samples and incubated for 16 h at 4\u0026deg;C. The antibody used was Anti-MSX1, Rabbit IgG polyclonal, concentration 0.2 mg/mL (Sigma; order number HPA073604. Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), diluted at a ratio of 1:200 with PBS. The following day the antibody-surplus was washed off and a post-block-reagent applied for 20 minutes. Secondary antibodies were conjugated with horseradish peroxidase (HRP) for another 30 minutes. Finally, the antibody was stained by applying DAB (chromogen substrate kit, Dako North America Inc., Carpinteria, CA, USA) for 5 min. The staining reaction was then stopped using distilled water and the material was counterstained with hematoxylin. Then the samples were dehydrated with an ascending series of graded alcohols (50%, 70%, 96%, and 100%) and Roticlear. As final step, the samples were covered with a mounting medium and cover glasses. This method has already been described by our research group [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor evaluation the IRS (immunoreactive score) was applied, which is the result of the percentage of stained cells (0\u0026thinsp;=\u0026thinsp;0%, 1\u0026thinsp;=\u0026thinsp;1\u0026ndash;10%, 2\u0026thinsp;=\u0026thinsp;11\u0026ndash;50%, 3\u0026thinsp;=\u0026thinsp;51\u0026ndash;80%, 4\u0026thinsp;\u0026le;\u0026thinsp;81%) multiplied with the coded staining intensity [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In statistical analysis, all three results were looked at separately (staining intensity, percentage of stained cells, and IRS).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe statistical analysis software SPSS (IBM SPSS Statistics, Version 25; IBM Deutschland GmbH, Ehningen, Germany) was used. For evaluation of the clinical-pathological variables, the Kruskal\u0026ndash;Wallis test was applied. For all analyses a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eOur results show a significant difference in MSX1 expression throughout the different phases. The specimens of endometrium during the proliferative phase show a staining with a mean IRS of 1 (p\u0026thinsp;=\u0026thinsp;0,018), while the early and the late secretory phase show no staining for MSX1 at all.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, correlations were found between MSX1 and other proteins examined in this collective by our research group (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelations between MSX1 and formerly investigated markers (cc: correlation coefficient)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePR-A\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePR-B\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInhibin Beta-C\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMSX1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIRS-Score\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIRS-Score\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIRS-Score\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ecc\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0671\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.691\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.583\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ep\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.060\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003en\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWith a p-value of 0.024 and a correlation-coefficient (cc) of 0.0671 a positive correlation between MSX1 and the progesterone-receptor A (PR-A) was found. Although there is no significant correlation between progesterone-receptor B (PR-B) and the menstrual cycle or Inhibin Beta-C and menstrual cycle, the following correlations with MSX1 were found: A significant positive correlation was found between MSX1 and PR-B-expression in stroma-cells (p-value\u0026thinsp;=\u0026thinsp;0.018; correlation-coefficient cc\u0026thinsp;=\u0026thinsp;0.691). Also a trend towards negative correlation was recognized between MSX1 and Inhibin Beta-C expression in glandular cells (p-value\u0026thinsp;=\u0026thinsp;0.060; cc = -0.583).\u003c/p\u003e \u003cp\u003eTherefore, we were able to confirm the results of our former paper on cancer tissue, where a significant positive correlation between MSX1 and the progesterone-receptors A and B were also found. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn our study we found that MSX1 is highly expressed during the proliferative phase and significantly downregulated at early and late secretory phase (p\u0026thinsp;=\u0026thinsp;0.018). In addition, a positive correlation between MSX1 and the progesterone-receptor A (PR-A) (correlation coefficient (cc)\u0026thinsp;=\u0026thinsp;0.0671; p\u0026thinsp;=\u0026thinsp;0.024) and the progesterone receptor B (PR-B) (cc\u0026thinsp;=\u0026thinsp;0.0691; p\u0026thinsp;=\u0026thinsp;0.018) was found.\u003c/p\u003e \u003cp\u003eProgesterone regulation of the endometrial MSX1 gene was first described in 2008 in the ovine uterus [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In that study, Satterfield et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] described that MSX1 mRNA was decreased by P4 treatment. In addition, another animal based study found that progesterone inhibits uterine gland development in the neonatal mouse uterus and downregulated MSX1 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. A very recent study on the role of MSX1 in reproduction was carried out on mice embryonic diapause [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Embryonic diapause in mice is a reproductive strategy in which embryo development and growth are temporarily halted in utero to ensure neonatal and maternal survival in adverse external conditions [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In that study, the authors have shown that dormant blastocysts are recovered from these mice on day 8 of pregnancy with persistent expression of uterine MSX1, a gene critical to maintaining the uterine quiescent state [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Interestingly, progesterone and anti-estrogen can prolong uterine quiescence [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn cancer biology, on the other side MSX1 was identified as a key candidate for progestin resistance in endometrial cancer [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. MSX1 showed significant tissue specificity and better prognostic value and its knockdown enhanced progesterone efficacy [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Our own investigation on MSX1 in endometrial cancer showed that a better survival was identified for patients with an MSX1 expression in more than 10% of the tumor cells [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. We further have given the hypothesis that MSX1 could be a potential marker for a potential uterus-preserving therapy of endometrial carcinomas [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In agreement to our results, a recent study on development of potential prognostic biomarkers based on DNA methylation-driven genes for patients with endometrial cancer showed that high methylation and low expression of MSX1 were significantly associated with reduced endometrial cancer survival rates [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. A similar role of MSX1 was identified for other gynecologic cancer subtypes. An early study showed that MSX1 may be involved in the regulation of cell proliferation and cell cycle using MSX1 overexpressing human ovarian cancer cells [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Overexpression of MSX1 inhibited cell proliferation by markedly increasing the length of the G1 phase of the cell cycle over control cells and a suppression of cyclins D1, D3, E, cyclin-dependent kinase 4, c-Jun, and Rb was observed [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Furthermore, and consistent with these findings, MSX1 triggers G0/G1 arrest and apoptosis by suppressing Notch signaling and is frequently methylated in cervical cancer [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In breast cancer, MSX1 inhibits breast cancer cell growth and metastasis and is often silenced by promoter methylation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition to our progesterone regulation of MSX1, a trend towards negative correlation was recognized between MSX1 and Inhibin Beta-C-expression in glandular cells (cc = -0.583; p-value\u0026thinsp;=\u0026thinsp;0.060). Although no direct involvement of inhibin on MSX1 is known, MSX1 represses the αGSU and GnRH receptor genes during gonadotropic development [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Suppression of the mouse GnRHR promoter by MSX1 is mediated by a consensus binding motif in the downstream activin regulatory element (DARE) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Activin and inhibin share the same beta subunit [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTaken together, MSX1 is known as a member of the muscle segment homeobox gene family. MSX1 is a p53 interacting protein and overexpression of MSX1 homeobox induces apoptosis of cancer cells. Here, we show that MSX1 is highly expressed especially in the proliferative phase of glandular epithelial tissues of normal endometrium. Since MSX1 is known to be downregulated by progesterone, the found correlation of MSX1 and PR-A and -B may represent a direct regulation of the MSX1 gene by a PR response element (PRR).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eFunding:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was funded by the medical faculty of the Ludwig-Maximilians-University Munich 2018-2020.\u003c/p\u003e\n\u003cp\u003eAcknowledgments:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe thank Kerstin Hermelink for the excellent technical support.\u003c/p\u003e\n\u003cp\u003eConflicts of interest:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSv.M: Research support, advisory board, honoraria and travel expenses from AbbVie, AstraZeneca, Clovis, Eisai, GlaxoSmithKline, Medac, MSD, Novartis, Olympus, PharmaMar, Roche, Sensor Kinesis, Teva, Tesaro; J.G. received honoraria for lectures from MSD and Roche. All other authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funds, grants or other support were received during the preparation of this manuscript. The authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003eAuthor Contributions:\u003c/p\u003e\n\u003cp\u003eConceptualization: Udo Jeschke. and Simon Eppich; methodology: Christina Kuhn and Simon Eppich; data curation: Helene Hildegard Heidegger and Udo Jeschke; article writing: Simon Eppich and Helene Hildegard Heidegger; supervision: Udo Jeschke, Sven Mahner, Julia Gallwas, Elisa Schmoeckel and Doris Mayr; All authors analyzed and interpreted the data, and read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003eEthics Approval:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was conducted conforming to the Declaration of Helsinki 1975 and it was approved by the Ethics Committee of the Ludwig-Maximilians-University, Munich, Germany (approval number 063-13). All patients\u0026rsquo; data were fully anonymized, and during experimental analysis, the authors were blinded for clinical information. All tissue used was leftover material and all diagnostic procedures had already been completed, when the samples were received for the study. Informed consent from all patients was obtained before surgery.\u003c/p\u003e\n\u003cp\u003e\u003cspan style=\"text-align: inherit;\"\u003eConflicts of interest:\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eSv.M: Research support, advisory board, honoraria and travel expenses from AbbVie, AstraZeneca, Clovis, Eisai, GlaxoSmithKline, Medac, MSD, Novartis, Olympus, PharmaMar, Roche, Sensor Kinesis, Teva, Tesaro; J.G. received honoraria for lectures from MSD and Roche. All other authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eMazur MT (2005) R.J.K., \u003cem\u003eNormal Endometrium and Infertility Evaluation\u003c/em\u003e, in \u003cem\u003eDiagnosis of Endometrial Biopsies and Curettings, A Practical Approach\u003c/em\u003e. Springer, New York, USA, pp 7\u0026ndash;33\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eEvans J et al (2016) Fertile ground: human endometrial programming and lessons in health and disease. Nat Rev Endocrinol 12(11):654\u0026ndash;667\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eRai P et al (2010) Proteome of human endometrium: Identification of differentially expressed proteins in proliferative and secretory phase endometrium. Proteom Clin Appl 4(1):48\u0026ndash;59\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMaybin JA, Critchley HO (2012) Steroid regulation of menstrual bleeding and endometrial repair. Rev Endocr Metab Disord 13(4):253\u0026ndash;263\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eNoyes RW, Hertig AT, Rock J (2019) Reprint of: Dating the Endometrial Biopsy. Fertil Steril 112(4 Suppl1):e93\u0026ndash;e115\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eEppich S et al (2020) \u003cem\u003eMSX1-A Potential Marker for Uterus-Preserving Therapy of Endometrial Carcinomas\u003c/em\u003e.Int J Mol Sci, \u003cstrong\u003e21\u003c/strong\u003e(12)\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eCatron KM et al (1996) Comparison of MSX-1 and MSX-2 suggests a molecular basis for functional redundancy. Mech Dev 55(2):185\u0026ndash;199\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBecic T et al (2018) Growth factors FGF8 and FGF2 and their receptor FGFR1, transcriptional factors Msx-1 and MSX-2, and apoptotic factors p19 and RIP5 participate in the early human limb development. Acta Histochem 120(3):205\u0026ndash;214\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eDai J et al (2014) Bioinformatic analysis of Msx1 and Msx2 involved in craniofacial development. J Craniofac Surg 25(1):129\u0026ndash;134\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBonczek O et al (2018) Next generation sequencing reveals a novel nonsense mutation in MSX1 gene related to oligodontia. PLoS ONE 13(9):e0202989\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBolnick AD et al (2016) Reduced homeobox protein MSX1 in human endometrial tissue is linked to infertility. Hum Reprod 31(9):2042\u0026ndash;2050\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eRemmele W, Stegner HE (1987) [Recommendation for uniform definition of an immunoreactive score (IRS) for immunohistochemical estrogen receptor detection (ER-ICA) in breast cancer tissue]. Pathologe 8(3):138\u0026ndash;140\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMylonas I et al (2009) Immunohistochemical labelling of steroid receptors in normal and malignant human endometrium. Acta Histochem 111(4):349\u0026ndash;359\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMylonas I et al (2010) Evidence of inhibin/activin subunit betaC and betaE synthesis in normal human endometrial tissue. Reprod Biol Endocrinol 8:143\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSatterfield MC et al (2008) Progesterone regulation of the endometrial WNT system in the ovine uterus. Reprod Fertil Dev 20(8):935\u0026ndash;946\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eFilant J, Zhou H, Spencer TE (2012) Progesterone inhibits uterine gland development in the neonatal mouse uterus. Biol Reprod 86(5):1\u0026ndash;9\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMatsuo M et al (2022) \u003cem\u003eTargeted depletion of uterine glandular Foxa2 induces embryonic diapause in mice\u003c/em\u003e.Elife,11\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eYang L et al (2020) Identification and Validation of MSX1 as a Key Candidate for Progestin Resistance in Endometrial Cancer. Onco Targets Ther 13:11669\u0026ndash;11688\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLu Y et al (2021) Development of Potential Prognostic Biomarkers Based on DNA Methylation-Driven Genes for Patients with Endometrial Cancer. Int J Gen Med 14:10541\u0026ndash;10555\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003ePark J et al (2001) Msx1 gene overexpression induces G1 phase cell arrest in human ovarian cancer cell line OVCAR3. 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Vivo 24(5):695\u0026ndash;698\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKimmich T et al (2010) Inhibin/activin-betaC and -betaE subunits in the Ishikawa human endometrial adenocarcinoma cell line. Arch Gynecol Obstet 282(2):185\u0026ndash;191\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-gynecology-and-obstetrics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arch","sideBox":"Learn more about [Archives of Gynecology and Obstetrics](https://www.springer.com/journal/404)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/arch/default.aspx","title":"Archives of Gynecology and Obstetrics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"MSX-1, Progesterone Receptor, endometrium, proliferative phase, glandular cells","lastPublishedDoi":"10.21203/rs.3.rs-2458340/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2458340/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human endometrium consists of different layers (basalis and functionalis) and undergoes different phases throughout the menstrual cycle. In a former paper our research group was able to describe MSX1 as a positive prognosticator in endometrial carcinomas. The aim of this study was to examine the MSX1 expression in healthy endometrial tissue throughout the different phases to gain more insight on the mechanics of MSX-regulation in the female reproductive system.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and Methods:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this retrospective study we investigated a total of 19 normal endometrial tissues (7 during proliferative phase and each 6 during early and late secretory phase). We used immunohistochemical staining and an immunoreactive score (IRS) to evaluate MSX1 expression. We also investigated correlations with other proteins, that have already been examined in our research group using the same patient collective.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMSX1 is highly expressed during the proliferative phase and downregulated at early and late secretory phase (p=0.018). Also, a positive correlation between MSX1 and the progesterone-receptor A (PR-A) (correlation coefficient (cc)=0.0671; p=0.024), the progesterone receptor B (PR-B) (cc=0.0691; p=0.018) was found. A trend towards negative correlation was recognized between MSX1 and Inhibin Beta-C-expression in glandular cells (cc=-0.583; p-value=0.060).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMSX1 is known as a member of the muscle segment homeobox gene family. MSX1 is a p53-interacting protein and overexpression of homeobox MSX1 induced apoptosis of cancer cells. Here we show that MSX1 is highly expressed especially in the proliferative phase of glandular epithelial tissue of the normal endometrium. Because MSX1 is known to be downregulated by progesterone, the found correlation of MSX1 and both PR-A and -B may represent a direct regulation of the MSX1 gene by a PR-response element.\u003c/p\u003e","manuscriptTitle":"MSX1-expression during the different phases in healthy human endometrium","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-01-11 22:43:24","doi":"10.21203/rs.3.rs-2458340/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2023-01-18T20:40:36+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-01-09T14:47:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-01-09T11:42:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Gynecology and Obstetrics","date":"2023-01-09T05:00:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-gynecology-and-obstetrics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arch","sideBox":"Learn more about [Archives of Gynecology and Obstetrics](https://www.springer.com/journal/404)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/arch/default.aspx","title":"Archives of Gynecology and Obstetrics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5aefb4e2-a1a7-458e-8acf-216b992a5ec3","owner":[],"postedDate":"January 11th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T20:43:44+00:00","versionOfRecord":{"articleIdentity":"rs-2458340","link":"https://doi.org/10.1007/s00404-023-07033-5","journal":{"identity":"archives-of-gynecology-and-obstetrics","isVorOnly":false,"title":"Archives of Gynecology and Obstetrics"},"publishedOn":"2023-04-27 20:38:34","publishedOnDateReadable":"April 27th, 2023"},"versionCreatedAt":"2023-01-11 22:43:24","video":"","vorDoi":"10.1007/s00404-023-07033-5","vorDoiUrl":"https://doi.org/10.1007/s00404-023-07033-5","workflowStages":[]},"version":"v1","identity":"rs-2458340","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2458340","identity":"rs-2458340","version":["v1"]},"buildId":"wLkW0s4AflPzk-lpfg-fK","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}