Quantitative analysis of cellular morphology during in vitro decidualization

Quantitative analysis of cellular morphology during in vitro decidualization | 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 Method Article Quantitative analysis of cellular morphology during in vitro decidualization Luciana Ant, François Le Dily, Miguel Beato, Patricia Saragüeta This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2565851/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Oct, 2023 Read the published version in Current Protocols → Version 1 posted You are reading this latest preprint version Abstract Decidualization is a differentiation process involving shape reorganization from a fibroblast to an epithelioid-like appearance of endometrial stromal cells. Specificities of these cells impede the use of existing automated tools to follow morphological changes during differentiation; we therefore developed a simple but accurate methodology to quantify the phenotypical changes that occur in an in vitro decidualization system. The approach consists of the analysis of the circularity of the cells directly from light microscopy images. Here, we used this methodology to follow the effects of progesterone or progestin R5020 in combination with estradiol (E2) and cAMP on inducing the decidualization of human endometrial cells. We further implemented a statistical model to detect the differences in the kinetics of decidualization of the two hormonal stimuli before all the cell population acquired the decidual phenotype. We found that 2 days afterstimulation are sufficient to detect statistical differences in morphology between decidualization induced and control cells. Here, we detail the model and scripts in order to provide a useful, practical and low cost tool to evaluate morphological aspects of endometrial stromal differentiation. Figures Figure 1 Introduction Decidualization is the transdifferentiation process of stromal endometrial cells into decidual cells, which involves changes in cytoplasm and nuclei morphology (2,3) and changes in gene expression associated to a secretory signature to support the embryo in case of implantation success (6). This process can be recapitulated in vitro by the use of hormonal treatments. Although decidualization markers can be used to analyze the differentiation by immune-fluorescence (3,8), for example at the end point of the process once the whole population of cells are committed, these approaches may not be always suitable to follow the dynamic of those changes in a time course manner. In order to better approach this problem and analyze the proportion of decidualized cells during the time course of hormonal stimulation, we aimed at quantifying the morphological changes that occur during this process. Although decidualization associated phenotypic changes are recapitulated in vitro and detectable on light microscopy images, the extremely flat phenotype of endometrial cells in 2D culture conditions impede the use of existing tools developed to follow morphological changes in other cell models (7). In this work, we therefore implemented a bioinformatic and statistical model to quantify the morphological changes accompanying differentiation in an in vitro decidualization system directly from light microscopy images. The approach presented here covers the necessity to quantify morphology changes in a simple, low cost, and accurate way allowing to follow the dynamic of differentiation before all the cell population acquires a decidual phenotype. Results In order to determine potential differences in the kinetic of decidualization induced by different hormonal combinations, we treated human endometrial T-HESC cells (4) up to 6 days with EtOH vehicle as control or with E2 and cAMP in combination with different progestins, either P4 or R5020 (see materials and methods and Fig. 1a). The main workflow of the process is described in figure 1b. The decidualized final state at 6 days was confirmed by qualitative changes in morphology of treated cells compared to cells cultured in presence of the vehicle (Fig S1a) as well as by the detection of decidualization markers, PRL and IGFBP1 by qRT-PCR (Fig S1b). To quantify the kinetic of the changes in cell morphology, light transmitted images were taken before treatment (T0) and every day during 6 days for each condition of treatment (5 plates by condition x 2 areas per plate). The circularity of the cells measured from manually curated images was used as a proxy of morphological changes. Exploratory analysis comparing the distributions of the cell circularity measures showed normal distribution of this parameter (Fig S2). Thus, mean circularity weighted by the number of cells per image was used as the response variable that did adjust to a normal distribution (Fig S3). In order to compare the effects of time and treatment, we used these data to implement simple Mixed-Model Regression. Since the T0 plates did not share time or treatment with any other conditions, we applied two different statistical models for the comparison: one to compare the circularity between T0 and Day1 and one to compare the circularity of the cells from day 1 to day 6, using the variables “time” and “treatment” (see materials and Methods). The first comparison did not show any significant differences between the T0 condition and the decidualizing treatments after one day (inset of Fig 1C), In contrast, the dynamics of morphological changes from day 1 to 6 appears to happen in a two-phase manner (Fig. 1C). From day 2, both decidualization treatments differentiate from control cells treated with EtOH, showing that our approach is sufficient to detect early morphological changes in the cell population. This difference is maintained throughout the remaining days. At day 4 the cells from all treatments show a leap in their circularity. This could be due to an increment in cell density, that causes the cells to be smaller and less elongated. From day 4, cells treated with R5020 showed increased circularity as compared to cells induced with P4. The final state at day 6 shows the same circularity for both hormonal treatments. The changes in circularity observed in decidualized cells are a result of mild changes in circularity in the majority of the cells, and not an increase in a few cells, as can be seen in the density plots (figure S2a). Discussion Here a model to quantify in vitro decidualization was implemented without defining the gene expression signature. This method was practical and reports a result of multiple processes that comprise the phenotypic transformation during decidualization. This approach allows to verify the establishment of the decidual phenotype without the need to use cell culture in RT-PCR or immunohistochemistry analysis. The quantitative analysis of the morphological changes during decidualization allowed us to gain insight that would not be possible to obtain through a qualitative analysis. The difference in circularity observed for the decidualization treatments at day 4 and 5 could be attributable to the difference in metabolization of the two progestins. R5020 is a synthetic progestin that cannot be metabolized in the cell, making the concentration levels of this drug stable during the experiment. Progestin on the other hand is metabolized as the days go by, decreasing its concentration levels. This could have slowed down the morphological changes observed in progesterone treated cells compared to R5020. Materials And Methods Implementation Circularity measures were acquired in ImageJ using custom macros, in order to render the process as efficient as possible. Data analysis was done entirely in R using basic packages such as nlme and emmeans. All the scripts and macros were developed for this analysis and can be found in the supplementary material. Cell culture and treatment Human endometrial stromal cells (t-HESC, ATCC CRL-4003) were cultured for 48 hs. in DMEM F12 (D2906, Sigma Aldrich) with 10% DCC-FBS (5). The cells were then trypsinized and re-cultured in 15 100mm plates with a density of 1x10 6 cells per plate. After 24 hours the medium was replaced with DMEM-F12 2%-DCC-FBS and either Progesterone 10 -6 M or R5020 1x10 8 , Estradiol 10nM and 8- Bromo-AMPc 0,5mM, while the control plates were given DMEM-F12 2%-DCC-FBS EtOH 0,1. RT-qPCR Cell extracts were collected in denaturing solution (4 M Guanidine thiocyanate, 25 mM Sodium citrate pH 7, 0.1 M 2-Mercaptoethanol, 0.5% Sarkosyl) and total RNA was prepared following phenol-chloroform protocol (1). Integrity-checked RNA was used to synthesize cDNA with oligodT (Biodynamics) and MMLV reverse transcriptase (Thermo Fisher Scientific). Quantification of candidate gene products was assessed by real-time PCR. Expression values were corrected by GAPDH and expressed as mRNA levels over time zero (T0). Primer sequences are: dPRL fw TGACCAGGAAAAGGGAAACGA rev GCACAGGAGCAGGTTTGACA IGFBP-1 fw TGAAAGCCCAGAGAGCACG rev CTCCTGATGTCTCCTGTGCC Image acquisition Images of two random areas of each plate were taken before treatment, and once a day from day 1 to 6. Images were obtained using a Leica DMI6000 B inverted microscope. Cell segmentation and manual image curation. As cultured tHESC cells are too flat to be recognized by an AI algorithm the images were manually curated. Circularity was measured through ImageJ. The segmenting of the cells from each image was performed by hand. Then, circularity was measured as where a circularity value of 1.0 indicates a perfect circle, and an increasingly elongated polygon as the value approaches 0.0. Statistical Model Mean circularity per photo was used as the response variable that did adjust to a normal distribution (Fig S3). The number of cells in each photo were taken into account as the “weight” for each mean. Due to the fact that the plates used for T0 images did not share time or treatment with any other plate we applied two statistical models: one to compare the circularity of the cells from day 1 to day 6, using the variables “time” and “treatment”, and another one to compare the circularity between T0 and Day1. The latter only comprised one variable, called treatment, which categories were: “T0”, “Day1 EtOH”, “Day1 R5020” and “Day1 P4”. The generalized linear mixed model used to compare plates from day 1 to 6 followed the equation below: In this equation α represents the treatment effect, β the day effect, and αβ the interaction effect between them. B denotes the random effect given by the plate. The Anova test showed a significant p-value (p-value < 0.05) for time, treatment, and the interaction between them, stating that both had an effect over the circularity of the cell, and that effect could change depending on their combination (Table S1). On the other hand, the model comparing T0 and Day 1 matched the equation below: The Anova test also showed the treatment has a significant effect over circularity (Table S2). Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and material All data generated or analyzed during this study are included in this published article and its supplementary information files. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by grants to PS from Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 2015-682) and Fondo para la Investigación Científica y Tecnológica (PICT 2015-3426), and grant to MB from H2020 European Research Council FP7/2007-2013 grant agreement 609989. Authors' contributions L.A. formal analysis, methodology, writing -review and editing- F.L. data curation, writing -review and editing- M.B. Writing -review and editing- P.S. Research hyphotesis, supervision, writing -manuscript, review and editing- Acknowledgements We are grateful to members of Saragüeta laboratories for help and suggestions and Dr. Gerardo Rubén Cueto from FCEN-UBA. References Chomczynski, P., and N. Sacchi. “Single-Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction.” Analytical Biochemistry, vol. 162, no. 1, Apr. 1987, pp. 156–59. PubMed, https://doi.org/10.1006/abio.1987.9999. Gellersen, B., and J. Brosens. “Cyclic AMP and Progesterone Receptor Cross-Talk in Human Endometrium: A Decidualizing Affair.” The Journal of Endocrinology, vol. 178, no. 3, Sept. 2003, pp. 357–72. PubMed, https://doi.org/10.1677/joe.0.1780357. Gellersen, Birgit, and Jan J. Brosens. “Cyclic Decidualization of the Human Endometrium in Reproductive Health and Failure.” Endocrine Reviews, vol. 35, no. 6, Dec. 2014, pp. 851–905. PubMed, https://doi.org/10.1210/er.2014-1045. Krikun, Graciela, et al. “A Novel Immortalized Human Endometrial Stromal Cell Line with Normal Progestational Response.” Endocrinology, vol. 145, no. 5, May 2004, pp. 2291–96. PubMed, https://doi.org/10.1210/en.2003-1606. Mestre Citrinovitz, Ana Cecilia, et al. “Decreased Autophagy Impairs Decidualization of Human Endometrial Stromal Cells: A Role for ATG Proteins in Endometrial Physiology.” International Journal of Molecular Sciences, vol. 20, no. 12, June 2019, p. E3066. PubMed, https://doi.org/10.3390/ijms20123066. Rytkönen, Kalle T., et al. “Decidualization of Human Endometrial Stromal Fibroblasts Is a Multiphasic Process Involving Distinct Transcriptional Programs.” Reproductive Sciences (Thousand Oaks, Calif.), vol. 26, no. 3, Mar. 2019, pp. 323–36. PubMed, https://doi.org/10.1177/1933719118802056. Stringer, Carsen, et al. “Cellpose: A Generalist Algorithm for Cellular Segmentation.” Nature Methods, vol. 18, no. 1, Jan. 2021, pp. 100–06. www.nature.com, https://doi.org/10.1038/s41592-020-01018-x. Telgmann, R., and B. Gellersen. “Marker Genes of Decidualization: Activation of the Decidual Prolactin Gene.” Human Reproduction Update, vol. 4, no. 5, Oct. 1998, pp. 472–79. PubMed, https://doi.org/10.1093/humupd/4.5.472. Additional Declarations No competing interests reported. Supplementary Files 021022Ant2022Supp.docx Scriptsupplementarymethods2.pdf Cite Share Download PDF Status: Published Journal Publication published 01 Oct, 2023 Read the published version in Current Protocols → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-2565851","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Method Article","associatedPublications":[],"authors":[{"id":175860501,"identity":"091a268b-970d-4189-9bde-ae38bb997ba1","order_by":0,"name":"Luciana Ant","email":"","orcid":"","institution":"Instituto de Biología y Medicina Experimental (IBYME-CONICET)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Luciana","middleName":"","lastName":"Ant","suffix":""},{"id":175860502,"identity":"b51c58ce-a28a-4d1f-b0d5-88e85285ba68","order_by":1,"name":"François Le Dily","email":"","orcid":"","institution":"Centre for Genomic Regulation","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"François","middleName":"Le","lastName":"Dily","suffix":""},{"id":175860503,"identity":"a65afd7f-add5-4782-a28b-9ca904b571f4","order_by":2,"name":"Miguel Beato","email":"","orcid":"","institution":"Centre for Genomic Regulation","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Miguel","middleName":"","lastName":"Beato","suffix":""},{"id":175860504,"identity":"d9af56dd-e8ac-44b4-bec2-f8d470c8426d","order_by":3,"name":"Patricia Saragüeta","email":"data:image/png;base64,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","orcid":"","institution":"Instituto de Biología y Medicina Experimental (IBYME-CONICET)","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Patricia","middleName":"","lastName":"Saragüeta","suffix":""}],"badges":[],"createdAt":"2023-02-08 18:14:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2565851/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2565851/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1002/cpz1.895","type":"published","date":"2023-10-01T11:44:39+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":32988998,"identity":"1d3d40a0-dd49-47e9-86fb-28790e51c4a7","added_by":"auto","created_at":"2023-02-15 15:48:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":290174,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eMorphology quantification of in vitro decidualization. A) Schematic overview of the decidualization system. Decidualizing stimuli (cAMP, E2 and P4/R5020), key transcription factors (HAND2, STATs, PGR, FOXO1) and decidualization markers (PRL and IGBP1) upregulated during this process are noted. B) Workflow used in this study. C) Estimated marginal circularity means for each treatment using the conditional model m1. Different letters indicate statistical differences between treatments at a given day. Inset denotes circularity means estimated by the simple model comparing T0 and Day 1. Each dot represents the mean circularity predicted for each treatment. Bar represents the interval of confidence. Different letters indicate statistical differences between different treatments and days.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-2565851/v1/8a9bd897c04a9d532bf2eacb.png"},{"id":53164108,"identity":"a3d1eaa0-fdb0-430d-8595-afaca43177c6","added_by":"auto","created_at":"2024-03-21 11:44:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":510946,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2565851/v1/21c365fc-6333-4afc-8e6c-747e13d19dc6.pdf"},{"id":32989000,"identity":"e5b23d81-bad2-41e7-884c-27b75d27d881","added_by":"auto","created_at":"2023-02-15 15:48:42","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1095070,"visible":true,"origin":"","legend":"","description":"","filename":"021022Ant2022Supp.docx","url":"https://assets-eu.researchsquare.com/files/rs-2565851/v1/926927470f2ec684917b196c.docx"},{"id":32988999,"identity":"a2f7a76c-1260-4288-8f55-165fa690c223","added_by":"auto","created_at":"2023-02-15 15:48:42","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":338689,"visible":true,"origin":"","legend":"","description":"","filename":"Scriptsupplementarymethods2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2565851/v1/3cc4c029d3292a824e5dde1d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Quantitative analysis of cellular morphology during in vitro decidualization","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDecidualization is the transdifferentiation process of stromal endometrial cells into decidual cells, which involves changes in cytoplasm and nuclei morphology (2,3) and\u0026nbsp;changes in gene expression associated to a secretory signature to support the embryo in case of implantation success (6). This process can be recapitulated \u003cem\u003ein vitro\u003c/em\u003e by the use of hormonal treatments. Although decidualization markers can be used to analyze the differentiation by immune-fluorescence\u0026nbsp;(3,8), for example at the end point of the process once the whole population of cells are\u0026nbsp;committed, these approaches may not be always suitable to follow the dynamic of those changes in a time course manner. In order to better approach this problem and analyze the proportion of\u0026nbsp;decidualized\u0026nbsp;cells during the time course of hormonal stimulation, we aimed at quantifying the morphological changes that occur during this process. Although decidualization associated\u0026nbsp;phenotypic\u0026nbsp;changes are recapitulated \u003cem\u003ein vitro\u003c/em\u003e and detectable on light microscopy images, the extremely flat phenotype of endometrial cells in 2D culture conditions\u0026nbsp;impede\u0026nbsp;the use of existing tools developed to follow morphological changes in other cell models (7). In this work, we therefore implemented a bioinformatic and statistical model to quantify the morphological changes accompanying differentiation in an \u003cem\u003ein vitro\u003c/em\u003e decidualization system directly from light microscopy images. The approach presented here covers the necessity to quantify morphology changes in a simple, low cost, and accurate way allowing to follow the dynamic of differentiation before all the cell population acquires a decidual phenotype.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIn order to determine potential differences in the kinetic of decidualization induced by different hormonal combinations, we treated human endometrial T-HESC cells (4) up to 6 days with EtOH vehicle as control or with E2 and cAMP in combination with different progestins, either P4 or R5020 (see materials and methods and Fig. 1a). The main workflow of the process is described in figure 1b.\u003c/p\u003e\n\u003cp\u003eThe decidualized final state at 6 days was confirmed by qualitative changes in morphology of treated cells compared to cells cultured in presence of the vehicle (Fig S1a) as well as by the detection of decidualization markers, PRL and IGFBP1 by qRT-PCR (Fig S1b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo quantify the kinetic of the changes in cell morphology, light transmitted images were taken before treatment (T0) and every day during 6 days for each condition of treatment (5 plates by condition x 2 areas per plate). The circularity of the cells measured from manually curated images was used\u0026nbsp;as a proxy\u0026nbsp;of morphological changes. Exploratory analysis comparing the distributions of the cell circularity measures showed normal distribution of this parameter (Fig S2). Thus, mean circularity weighted by the number of cells per image was used as the response variable that did adjust to a normal distribution (Fig S3). In order to compare the effects of time and treatment, we used these data to implement simple Mixed-Model Regression. Since the T0 plates did not share time or treatment with any other conditions, we applied two different statistical models for the comparison: one to compare the circularity between T0 and Day1 and one to compare the circularity of the cells from day 1 to day 6, using the variables \u0026ldquo;time\u0026rdquo; and \u0026ldquo;treatment\u0026rdquo; (see materials and Methods). The first comparison did not show any significant differences between the T0 condition and the decidualizing treatments after one day (inset of Fig 1C), In contrast, the dynamics of morphological changes from day 1 to 6 appears to happen in a two-phase manner (Fig. 1C). From day 2, both decidualization treatments differentiate from control cells treated with EtOH, showing that our approach is sufficient to detect early morphological changes in the cell population. This difference is maintained throughout the remaining days.\u003c/p\u003e\n\u003cp\u003eAt day 4 the cells from all treatments show a leap in their circularity. This could be due to an increment in cell density, that causes the cells to be smaller and less elongated. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;From day 4, cells treated with R5020 showed increased circularity as compared to cells induced with P4. The final state at day 6 shows the same circularity for both hormonal treatments.\u003c/p\u003e\n\u003cp\u003eThe changes in circularity observed in decidualized cells are a result of mild changes in circularity in the majority of the cells, and not an increase in a few cells, as can be seen in the density plots (figure S2a).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere a model to quantify in vitro decidualization was implemented without defining the gene expression signature. This method was practical and reports a result of multiple processes that comprise the phenotypic transformation during decidualization. This approach allows to verify the establishment of the decidual phenotype without the need to use cell culture in RT-PCR or immunohistochemistry\u0026nbsp;analysis.\u003c/p\u003e\n\u003cp\u003eThe quantitative analysis of the morphological changes during decidualization allowed us to gain insight that would not be possible to obtain through a qualitative analysis. The difference in circularity observed for the decidualization treatments at day 4 and 5 could be attributable to the difference in metabolization of the two progestins. R5020 is a synthetic progestin that cannot be metabolized in the cell, making the concentration levels of this drug stable during the experiment. Progestin on the other hand is metabolized as the days go by, decreasing its concentration levels. This could have slowed down the morphological changes observed in progesterone treated cells compared to R5020.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003ch2\u003eImplementation\u003c/h2\u003e\n\u003cp\u003eCircularity measures were acquired in ImageJ using custom macros, in order to render the process as efficient as possible. Data analysis was done entirely in R using basic packages such as nlme and emmeans. All the scripts and macros were developed for this analysis and can be found in the supplementary material.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eCell culture and treatment\u003c/h2\u003e\n\u003cp\u003eHuman endometrial stromal cells (t-HESC, ATCC CRL-4003) were cultured for 48 hs. in DMEM F12 (D2906, Sigma Aldrich) with 10% DCC-FBS (5). The cells were then\u0026nbsp;trypsinized\u0026nbsp;and re-cultured in 15 100mm plates with a density of 1x10\u003csup\u003e6\u003c/sup\u003e cells per plate. After 24 hours the medium was replaced with DMEM-F12 2%-DCC-FBS and either Progesterone 10\u003csup\u003e-6\u003c/sup\u003e M or R5020 1x10\u003csup\u003e8\u003c/sup\u003e, Estradiol 10nM and 8- Bromo-AMPc 0,5mM, while the control plates were given DMEM-F12 2%-DCC-FBS EtOH 0,1.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eRT-qPCR\u003c/h2\u003e\n\u003cp\u003eCell extracts were collected in denaturing solution (4 M Guanidine thiocyanate, 25 mM Sodium citrate pH 7, 0.1 M 2-Mercaptoethanol, 0.5% Sarkosyl) and total RNA was prepared following phenol-chloroform protocol (1). Integrity-checked RNA was used to synthesize cDNA with oligodT (Biodynamics) and MMLV reverse transcriptase (Thermo Fisher Scientific).\u003c/p\u003e\n\u003cp\u003eQuantification of candidate gene products was assessed by real-time PCR. Expression values were corrected by GAPDH and expressed as mRNA levels over time zero (T0). Primer sequences are:\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"322\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" width=\"22.291021671826627%\"\u003e\n \u003cp\u003edPRL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.074303405572756%\"\u003e\n \u003cp\u003efw\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"65.63467492260062%\"\u003e\n \u003cp\u003eTGACCAGGAAAAGGGAAACGA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.53784860557769%\"\u003e\n \u003cp\u003erev\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"84.4621513944223%\"\u003e\n \u003cp\u003eGCACAGGAGCAGGTTTGACA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" width=\"22.291021671826627%\"\u003e\n \u003cp\u003eIGFBP-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.074303405572756%\"\u003e\n \u003cp\u003efw\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"65.63467492260062%\"\u003e\n \u003cp\u003eTGAAAGCCCAGAGAGCACG\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.53784860557769%\"\u003e\n \u003cp\u003erev\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"84.4621513944223%\"\u003e\n \u003cp\u003eCTCCTGATGTCTCCTGTGCC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003ch2\u003eImage acquisition\u003c/h2\u003e\n\u003cp\u003eImages of two random areas of each plate were taken before treatment, and once a day from day 1 to 6. Images were obtained using a Leica DMI6000 B inverted microscope. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eCell segmentation and manual image curation.\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eAs cultured tHESC cells are too flat to be recognized by an AI algorithm the images were manually curated. Circularity was measured through ImageJ. The segmenting of the cells from each image was performed by hand. Then, circularity was measured as\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAANAAAAA3CAYAAAB95cROAAAMSElEQVR4nO2d34sbVRvHv3l5b6s9u16JeNFpQWmlIIkragsV6gQtpYtIVixFcFHSXlVkF6b2ojeFBLve2V3xUjspbW8Wk21W2AUzXaobZYKKvegE6XWmsfUPOF6sz+zJ/EhmkslmJz0fCOxOZs485+Q858dzznmeFOecQyKR9MX/Ri2ARJJkpAJJJAMgFUgytti2PfR3jKUCLS0tYWJiAs1mc9SiSEbI1NQULly4MFxF4mNEq9XiuVyOA+CFQmHU4khGTLlc5owxnk6nuWVZQ3lHivPxsMLZto1sNot6vQ5d1zEzMzNqkSS7gEajgWPHjgEA6vU69u3bF+8LhqKWI0D2PJIgarUaB8DT6TRvtVqxpj0WCrS4uMgBcFVVRy2KZJdSKBQ4AJ7L5WJNN/EKZFkWZ4xxAEMb5+4UtVrN6Unpxy4UClxRlI77yuUyT6fTHADP5/Oc8635Xz6fd571Kwtd17mqqs49qqoGtsi6rjvvYIzxfD7PNU2LvQLuJIqicAC8XC7HlmbiFYgqHFWkpKLrupMPqtSUN1GBSHlarZajDKZpOs9pmuZROLqXMcYXFxc559u9tqZpHllIEeneVqvlVL4klzOVsbt8BiHRCkRj26T3PpQP9xCUrrsrLSkYKVCvSk2KKLa8rVbLd0hDQx33XJKu67oeOX+7CWoI4spHohWIWsokt4qcbyuCuxHoVWlp6GqaZmDaQcpJPRD1MpxvKRVjzLeFDpIxaVC+4+qFEqtAlmU5vU+cY9qdxjRNx0LkhhoIPwWh/Peak1Aauq7zVqvFa7Wac839bLdhnaIonDEWMXe7D+p546o3id2J8MMPPwAAGGN4++23RyxN/9y9excA8NZbb3m+W11dBWMMhw8f9nz3888/AwBefvnlrumvrq4CAN5//30888wzOH/+PACgXC6jVCp13Lu+vg4AeP311zuuN5tNWJblK2PSmJychKqqAIDvv/9+4PT+P3AKI+Kbb74B4F/xksSjR48AAE8//XTH9VKpBMuynB/bzW+//QYAeO2117qmb1kWAICHWC//+++/AQBPPfVUx/UvvvgCQG9lTQrT09OoVqu4evUqvvrqq4HSSmQPZNs26vU6AODUqVMjliZ+bNvGlStXAABvvvmm7z3Us7z44otd02KM+V5vNBqeHsgPwzCcd/VS1qTw6quvOn9XKpWB0oqsQMViEalUCqlUaqAXDwIN34DeFWi3Qz3M2toabNuGYRi4ePEiLl26BGCrh2o0Gshmsx2bIuv1OhRFweTkZNf08/k8ADjKYts2isUijh075ulppqenAQArKyvOMysrK/j4448BAI8fP0alUkn8NilxSHznzp3BEosyYaIJL31GhaZpI5chTig/jDGuaRpvtVq81Wo5C5m5XK7D+kWWtTCLmrTAShY7RVF4oVDwXUAV15YURXEsdKZpcsZYh3xJh/LpZ7yJQqQaSD/oqCsvZV5u3ZH0S1yNcOghXLFYRL1eD5zU7iTVahUAsHfv3hFLIkkqotGm0Wj0nU4oBTIMA/Pz89A0LXBSu1OI84BxsQpJdh7RIPLPP//0nU5PBbJtGx9++CEURcGnn34aOuGlpSXs37/fMTiE/fSyDP3555+hZZBIwrCxsdH3sz3XgRYWFmBZFmq1Wk+LD3H27FlcvXq1L4GiWNUOHTrU1zskkmeffTaWdLr2QIZh4PLly9A0DW+88UaoBJeWllAqlaDrOjjn0DQNAFCr1cC3jBYd1zVN67jut+ouIrYWbjOsRBIW8WTqX3/91Xc6gT2Qbds4efJk5KFbo9HA8vKyo3C//PILAG/PQotzL730UmShJZI4GcT5TKACnTt3Du12G8vLy6GHbgA8WyOq1apnwa/ZbDo7CV555ZWoMkskuwZfBapUKrh+/Try+XyooVsmk8F7772Hubm5juuGYQDw7lejjZCKosTv5MHFKHdMSEYH3yFfOR4FajabOH36NADgxIkTjhIQ4njRMAxsbGygXq9jdnbWkzjNV9zzmh9//BEAkE6nBxRfIhktHgW6efMm2u02AOCdd97p+vCRI0ecvw8ePOj5/tdff/X9juY/5G5omOxUSyR5MvFY4ebm5jqsYu5PoVBw7hWv+w31SFHE72zbdrbYi7tiJZJRMciOlqEdZ6hUKmi3255hmrgQ2stk7Ydc+5HEQVw7WoamQHTaz21AoHlRv3vqxLWfP/74o0/pJE86ce1oGYoC2bbtbMkJWue5f/8+gC1DRCaTCW2L37Nnj/M3neaUSAbh+eef91wLe+4tkgLZto2vv/7a+T9oF+vCwoJjiHCv89AuWMuykEqlcOTIEWQymdDmbHHYJxXoyaRUKmFmZgYTExNIpVKYmJjA2bNnI0VhEEcvzz33XP/ChD33QIeqIJwHgo/LJfHQnZ/roKBDW1Ggc0nyPNCTCf47CEeHDMn9VxT3ZvQMAN8DguL3XWWJJvrugBwFjoObJUl04OOSKmqD6uf1VYQO3PU6sZpIpyK0ftRut3ckCtkwMAwDqVQq8f4FRgHnvMOVGU0lopxVo61kQV6drl+/DgD48ssvu6aTSAUS149++umnEUrSP2SN3InF5HHGtm3Mzs4inU7jo48+CvUM+bkDgKNHj3q+LxaLsCwLuq733soWus/bZZCPYz8vmpL+UVV1YEcbOwU5Xoka94eczCPAVbGqqqG9liayBwKAXC4HYLurlQyOYRioVquJcFZJEQkzmQw2NzcjnRgQ92L6WX9v374d3tttaLXdZYjWvl4Oz2u1Wtd4Ooyxrt76KW6P6BpK9FcdJV6PaN1xt/R+6ViW1WE0oZZRzJOiKF3LoJf8pml2xA0SP36WLcuyeD6fd0YB7vIzTdORWewdCoWCI4N4P8VAEq+LMvmFa6Gepx9Ihn4swG4Sq0Ccb5uzu4V1pIomVmjLsriqqtyyrI7YN37DALLGkD80tzP4fuL1+DlxD0qHfMLRe1VV5bquO+lTWkFl0Et+v/LsNhzSdZ0zxgLLz7IsR0GpsSiXyzyfz3PTNHm5XO5oUDRNc5RUzB/lJ51Oe5SYGgNRTspXL+j97uf7JdEKFDZgkl88HbHw6HqtVut4LihaQTqd7jChR43XExS2pJec9MOL8pCTRT8FCiu/mH63sgxSPlJS6tXEHoeUIqiHdOePHD/2ksHvE6ZHijsgW6IViPNoAZPoXne4ED8Folg5QS2dn1fQMPF6OO8etiQoHXqvoigd8lAldQ9HosofFMxLJKicgkKfUGUNM1QKyl+ciCFx4opzlHgFoi45TC8U1Er5VQwx5CLnWxWMxvDiKjgRNl4P591j7QSlQ/K4W2eqpO7eM6r8vYJ5kVxUzqZpcl3Xnby4398rn25I3jjmJUEMI5J74hWI820F6FYwpGh+99DQSEQ0AFDFyeVyTqAqN0EV3A1VxKBV86B0aJjkrqjUW7mJKj9VrqBeUTT9UnmpqsoXFxd9W/Ne+QyS108R44B62Lh7uMTGBxL57rvvcODAAczPz0NVVd9zRr///jsA73ki2jXu3hFAu8NrtVoovxBh4/WQP4igVfOgdMi7kSiLYRhot9u+R0Oiyk+HH4POaD148AAAUCgUPL4v/Lh37x6A8LsDNjc3ASC0+7QokHNQALh161Ykk3cvErsOJDI5OYn19XUwxvDuu+/6bu9ZW1sD0HmeyLZtfP7552CM4bPPPuu4P+iUIoUHcRM2Xg9VxCBFC0qnWq16DifSjmKqpGK+o8pPu+fF+8Td9u4AYCLFYtFT5hQ2JMwBSIr3NCwfGdls1tlZ0M8hzm6MhQIBWy3n+vo6Hj58iGw26zlqQQ7paQtNs9nEBx98gIcPH+Lbb7/1LKidOXMGAHDt2jXnWqlUwoEDB3zfHzZeD/H48WPnLFSvuD/k2CWTyQSm12g0MDU15aQVVX4KxGXbNprNJrLZrFNmAHD8+HEwxnDjxg3nHSQ/+b4QoR7zhRde6FIKW9Dhtm756wdabK3X69B1fTj7DmMbDO4STNN0zLQ01hXXUMQQLblcrqvFTFzgY4zxfD7vO96PEq9HPBbiNlMHpdPN0ibGEHLnJaz8nG+v7+A/Q4vfXERc6KV3Bs1Z6J1hCMrfoJARY5hBqMdOgQjxx6D1kDCmbsn4UC6Xhx4MbCyMCH588sknzt80nEt6OEhJNHYievvYzIG6QRNz0Z+CRBIHKc6l50GJpF/+BXalOgC1QXmmAAAAAElFTkSuQmCC\" width=\"208\" height=\"55\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003ewhere a circularity value of 1.0 indicates a perfect circle, and an increasingly elongated polygon as the value approaches 0.0.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eStatistical Model\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eMean circularity per photo was used as the response variable that did adjust to a normal distribution (Fig S3). The number of cells in each photo were\u0026nbsp;taken into account as the \u0026ldquo;weight\u0026rdquo; for each mean. Due to the fact that the plates used for T0 images did not share time or treatment with any other plate we applied two statistical models: one to compare the circularity of the cells from day 1 to day 6, using the variables \u0026ldquo;time\u0026rdquo; and \u0026ldquo;treatment\u0026rdquo;, and another one to compare the circularity between T0 and Day1. The latter only comprised one variable, called treatment, which categories\u0026nbsp;were: \u0026ldquo;T0\u0026rdquo;, \u0026ldquo;Day1 EtOH\u0026rdquo;, \u0026ldquo;Day1 R5020\u0026rdquo; and \u0026ldquo;Day1 P4\u0026rdquo;.\u003c/p\u003e\n\u003cp\u003eThe generalized linear mixed model used to compare plates from day 1 to 6 followed the equation below:\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" style=\"width: 719px; height: 143.478px;\" width=\"719\" height=\"143.478\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eIn this equation \u0026alpha; represents the treatment effect, \u0026beta; the day effect, and \u0026alpha;\u0026beta; the interaction effect between them. B denotes the random effect given by the plate. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Anova test showed a significant p-value (p-value \u0026lt; 0.05) for time, treatment, and the interaction between them, stating that both had an effect over the circularity of the cell, and that effect could change depending on their combination (Table S1). \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn the other hand, the model comparing T0 and Day 1 matched the equation below: \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"570\" height=\"156\"\u003e\u003c/p\u003e\n\u003cp\u003eThe Anova test also showed the treatment has a significant effect over circularity (Table S2).\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants to PS from Consejo Nacional de Investigaciones Cient\u0026iacute;ficas y T\u0026eacute;cnicas (PIP 2015-682) and Fondo para la Investigaci\u0026oacute;n Cient\u0026iacute;fica y Tecnol\u0026oacute;gica (PICT 2015-3426), and grant to MB from H2020 European Research Council FP7/2007-2013 grant agreement 609989.\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.A. formal analysis, methodology, writing -review and editing- F.L. data curation, writing -review and editing- M.B. Writing -review and editing- P.S. Research hyphotesis, supervision, writing -manuscript, review and editing-\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to members of Sarag\u0026uuml;eta laboratories for help and suggestions and Dr. Gerardo Rub\u0026eacute;n Cueto from FCEN-UBA.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChomczynski, P., and N. Sacchi. \u0026ldquo;Single-Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction.\u0026rdquo; Analytical Biochemistry, vol. 162, no. 1, Apr. 1987, pp. 156\u0026ndash;59. PubMed, https://doi.org/10.1006/abio.1987.9999. \u003c/li\u003e\n\u003cli\u003eGellersen, B., and J. Brosens. \u0026ldquo;Cyclic AMP and Progesterone Receptor Cross-Talk in Human Endometrium: A Decidualizing Affair.\u0026rdquo; The Journal of Endocrinology, vol. 178, no. 3, Sept. 2003, pp. 357\u0026ndash;72. PubMed, https://doi.org/10.1677/joe.0.1780357. \u003c/li\u003e\n\u003cli\u003eGellersen, Birgit, and Jan J. Brosens. \u0026ldquo;Cyclic Decidualization of the Human Endometrium in Reproductive Health and Failure.\u0026rdquo; Endocrine Reviews, vol. 35, no. 6, Dec. 2014, pp. 851\u0026ndash;905. PubMed, https://doi.org/10.1210/er.2014-1045. \u003c/li\u003e\n\u003cli\u003eKrikun, Graciela, et al. \u0026ldquo;A Novel Immortalized Human Endometrial Stromal Cell Line with Normal Progestational Response.\u0026rdquo; Endocrinology, vol. 145, no. 5, May 2004, pp. 2291\u0026ndash;96. PubMed, https://doi.org/10.1210/en.2003-1606. \u003c/li\u003e\n\u003cli\u003eMestre Citrinovitz, Ana Cecilia, et al. \u0026ldquo;Decreased Autophagy Impairs Decidualization of Human Endometrial Stromal Cells: A Role for ATG Proteins in Endometrial Physiology.\u0026rdquo; International Journal of Molecular Sciences, vol. 20, no. 12, June 2019, p. E3066. PubMed, https://doi.org/10.3390/ijms20123066. \u003c/li\u003e\n\u003cli\u003eRytk\u0026ouml;nen, Kalle T., et al. \u0026ldquo;Decidualization of Human Endometrial Stromal Fibroblasts Is a Multiphasic Process Involving Distinct Transcriptional Programs.\u0026rdquo; Reproductive Sciences (Thousand Oaks, Calif.), vol. 26, no. 3, Mar. 2019, pp. 323\u0026ndash;36. PubMed, https://doi.org/10.1177/1933719118802056. \u003c/li\u003e\n\u003cli\u003eStringer, Carsen, et al. \u0026ldquo;Cellpose: A Generalist Algorithm for Cellular Segmentation.\u0026rdquo; Nature Methods, vol. 18, no. 1, Jan. 2021, pp. 100\u0026ndash;06. www.nature.com, https://doi.org/10.1038/s41592-020-01018-x. \u003c/li\u003e\n\u003cli\u003eTelgmann, R., and B. Gellersen. \u0026ldquo;Marker Genes of Decidualization: Activation of the Decidual Prolactin Gene.\u0026rdquo; Human Reproduction Update, vol. 4, no. 5, Oct. 1998, pp. 472\u0026ndash;79. PubMed, https://doi.org/10.1093/humupd/4.5.472.\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":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":"","lastPublishedDoi":"10.21203/rs.3.rs-2565851/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2565851/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDecidualization is a differentiation process involving shape reorganization from a fibroblast to an epithelioid-like appearance of endometrial stromal cells. Specificities of these cells impede the use of existing automated tools to follow morphological changes during differentiation; we therefore developed a simple but accurate methodology to quantify the phenotypical changes that occur in an \u003cem\u003ein vitro\u003c/em\u003e decidualization system.\u003c/p\u003e\n\u003cp\u003eThe approach consists of the analysis of the circularity of the cells directly from light microscopy images. Here, we used this methodology to follow the effects of progesterone or progestin R5020 in combination with estradiol (E2) and cAMP on inducing the decidualization of human endometrial cells. We further implemented a statistical model to detect the differences in the kinetics of decidualization of the two hormonal stimuli before all the cell population acquired the decidual phenotype. We found that 2 days afterstimulation are sufficient to detect statistical differences in morphology between decidualization induced and control cells. Here, we detail the model and scripts in order to provide a useful, practical and low cost tool to evaluate morphological aspects of endometrial stromal differentiation.\u003c/p\u003e","manuscriptTitle":"Quantitative analysis of cellular morphology during in vitro decidualization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-02-15 15:48:37","doi":"10.21203/rs.3.rs-2565851/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":"5afd46b3-a990-4a24-8929-04b94128ecd9","owner":[],"postedDate":"February 15th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-03-21T11:44:39+00:00","versionOfRecord":{"articleIdentity":"rs-2565851","link":"https://doi.org/10.1002/cpz1.895","journal":{"identity":"current-protocols","isVorOnly":true,"title":"Current Protocols"},"publishedOn":"2023-10-01 11:44:39","publishedOnDateReadable":"October 1st, 2023"},"versionCreatedAt":"2023-02-15 15:48:37","video":"","vorDoi":"10.1002/cpz1.895","vorDoiUrl":"https://doi.org/10.1002/cpz1.895","workflowStages":[]},"version":"v1","identity":"rs-2565851","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2565851","identity":"rs-2565851","version":["v1"]},"buildId":"omnImTCwR2MFx8CMYfrG7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}