{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T17:47:22Z","timestamp":1790876842326,"version":"4.1.0"},"reference-count":45,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2025,4,10]],"date-time":"2025-04-10T00:00:00Z","timestamp":1744243200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003500","name":"University of Padua","doi-asserted-by":"publisher","award":["ANDR_BIRD24_01"],"award-info":[{"award-number":["ANDR_BIRD24_01"]}],"id":[{"id":"10.13039\/501100003500","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Endometriosis is a complicated condition characterized by inflammation, low oocyte quality, and decreased uterus receptivity, associated with fertility issues. This study aims to better understand the reduced pregnancy outcome in endometriosis by analyzing both the granulosa cells (GCs) and the follicular fluids (FFs) obtained during the assisted reproductive technology (ART)-related oocyte pick-up. Seventy patients, approaching our ART Center with the diagnosis of infertility for Age-Idiopathic Factor (AIF) (n = 36), endometriosis (ENDO) (n = 23), or male factor (MF) (n = 11), were enrolled in this study. GCs from each group were separately analyzed for morphology, replication, and expression of Connexin-43 and Follicle-Stimulating Hormone Receptor (FSHR) by microscopy, flow cytometry, and immunocytochemistry. Results show that FF in a culture medium allowed GCs to survive and replicate. Upon culturing GCs from each group with ENDO follicular fluid, increases were observed in both population doublings and in the development of fibroblast-like and muscle-like morphologies. Despite undergoing morphological changes, GCs consistently expressed FSHR. However, exposure to ENDO follicular fluid led to an upregulation of Connexin-43 expression across all GC groups. These findings suggest that in endometriosis, FF contains unidentified factors that can induce aberrant replication, morphological differentiation, and overexpression of Connexin-43, potentially contributing to follicular dysfunction.<\/jats:p>","DOI":"10.3390\/biom15040561","type":"journal-article","created":{"date-parts":[[2025,4,10]],"date-time":"2025-04-10T08:46:20Z","timestamp":1744274780000},"page":"561","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Endometriotic Follicular Fluid Affects Granulosa Cells\u2019 Morphology and Increases Duplication Rate and Connexin-43 Expression"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4771-031X","authenticated-orcid":false,"given":"Loris","family":"Marin","sequence":"first","affiliation":[{"name":"Department of Women\u2019s and Children\u2019s Health, University of Padova, 35128 Padova, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chiara","family":"Sabbadin","sequence":"additional","affiliation":[{"name":"Endocrine Unit, Department of Medicine (DIMED), University of Padova, 35128 Padova, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5443-5638","authenticated-orcid":false,"given":"Giovanni","family":"Faggin","sequence":"additional","affiliation":[{"name":"Department of Molecular Medicine, University of Padua, 35128 Padua, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5548-9785","authenticated-orcid":false,"given":"Claudia Maria","family":"Radu","sequence":"additional","affiliation":[{"name":"Thrombotic and Haemorrhagic Diseases Unit, Department of Medicine (DIMED), University of Padua, 35128 Padua, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6433-5155","authenticated-orcid":false,"given":"Decio","family":"Armanini","sequence":"additional","affiliation":[{"name":"Endocrine Unit, Department of Medicine (DIMED), University of Padova, 35128 Padova, Italy"},{"name":"Studium Patavinum, University of Padova, 35128 Padova, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-3985-7739","authenticated-orcid":false,"given":"Michele","family":"Paccagnella","sequence":"additional","affiliation":[{"name":"Department of Molecular Medicine, University of Padua, 35128 Padua, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5136-7406","authenticated-orcid":false,"given":"Cristiano","family":"Salata","sequence":"additional","affiliation":[{"name":"Department of Molecular Medicine, University of Padua, 35128 Padua, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4238-3153","authenticated-orcid":false,"given":"Luciana","family":"Bordin","sequence":"additional","affiliation":[{"name":"Department of Women\u2019s and Children\u2019s Health, University of Padova, 35128 Padova, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0390-6823","authenticated-orcid":false,"given":"Eugenio","family":"Ragazzi","sequence":"additional","affiliation":[{"name":"Studium Patavinum, University of Padova, 35128 Padova, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guido","family":"Ambrosini","sequence":"additional","affiliation":[{"name":"Department of Women\u2019s and Children\u2019s Health, University of Padova, 35128 Padova, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9428-4363","authenticated-orcid":false,"given":"Alessandra","family":"Andrisani","sequence":"additional","affiliation":[{"name":"Department of Women\u2019s and Children\u2019s Health, University of Padova, 35128 Padova, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,4,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Bonavina, G., and Taylor, H.S. (2022). Endometriosis-Associated Infertility: From Pathophysiology to Tailored Treatment. Front. Endocrinol., 13.","DOI":"10.3389\/fendo.2022.1020827"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Xie, J., Xu, X., and Liu, S. (2023). Intercellular Communication in the Cumulus\u2013Oocyte Complex during Folliculogenesis: A Review. Front. Cell Dev. Biol., 11.","DOI":"10.3389\/fcell.2023.1087612"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Mulchandani, V., Banerjee, A., Vadlamannati, A.V., Kumar, S., and Das Sarma, J. (2023). Connexin 43 Trafficking and Regulation of Gap Junctional Intercellular Communication Alters Ovarian Cancer Cell Migration and Tumorigenesis. Biomed. Pharmacother., 159.","DOI":"10.1016\/j.biopha.2023.114296"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1016\/j.bbamem.2017.05.016","article-title":"Gap Junction Gene and Protein Families: Connexins, Innexins, and Pannexins","volume":"1860","author":"Beyer","year":"2018","journal-title":"Biochim. Biophys. Acta BBA-Biomembr."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"340","DOI":"10.1093\/humupd\/dmv007","article-title":"Gap Junction Connexins in Female Reproductive Organs: Implications for Women\u2019s Reproductive Health","volume":"21","author":"Winterhager","year":"2015","journal-title":"Hum. Reprod. Update"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Erickson, G.F. (2009). Follicle Growth and Development. Glob. Libr. Women\u2019s Med.","DOI":"10.3843\/GLOWM.10289"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"bqae023","DOI":"10.1210\/endocr\/bqae023","article-title":"Disturbed Follicular Microenvironment in Polycystic Ovary Syndrome: Relationship to Oocyte Quality and Infertility","volume":"165","author":"Dai","year":"2024","journal-title":"Endocrinology"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Qi, Q., Li, Y., Chen, Z., Luo, Z., Zhou, T., Zhou, J., Zhang, Y., Chen, S., and Wang, L. (2025). Update on the Pathogenesis of Endometriosis-Related Infertility Based on Contemporary Evidence. Front. Endocrinol., 16.","DOI":"10.3389\/fendo.2025.1558271"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Retis-Resendiz, A.M., G\u00f3mez-Su\u00e1rez, S.K., Garc\u00eda-G\u00f3mez, E., and V\u00e1zquez-Mart\u00ednez, E.R. (2025). Molecular Basis of Impaired Decidualization in the Eutopic Endometrium of Endometriosis Patients. Cells, 14.","DOI":"10.3390\/cells14050326"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"773","DOI":"10.1016\/j.rbmo.2014.01.016","article-title":"Increased Oxidation-Related Glutathionylation and Carbonic Anhydrase Activity in Endometriosis","volume":"28","author":"Andrisani","year":"2014","journal-title":"Reprod. BioMed. Online"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1616","DOI":"10.1016\/j.fertnstert.2009.10.038","article-title":"Evaluation of Erythrocyte Band 3 Phosphotyrosine Level, Glutathione Content, CA-125, and Human Epididymal Secretory Protein E4 as Combined Parameters in Endometriosis","volume":"94","author":"Bordin","year":"2010","journal-title":"Fertil. Steril."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1616","DOI":"10.1016\/j.fertnstert.2006.05.043","article-title":"Regulated upon Activation, Normal T Cell Expressed and Secreted (RANTES) and Monocyte Chemotactic Protein 1 in Follicular Fluid Accumulate Differentially in Patients with and without Endometriosis Undergoing in Vitro Fertilization","volume":"86","author":"Xu","year":"2006","journal-title":"Fertil. Steril."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1363","DOI":"10.1007\/s10815-016-0782-5","article-title":"Intrafollicular Interleukin-8, Interleukin-12, and Adrenomedullin Are the Promising Prognostic Markers of Oocyte and Embryo Quality in Women with Endometriosis","volume":"33","author":"Singh","year":"2016","journal-title":"J. Assist. Reprod. Genet."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1530\/REP-14-0438","article-title":"Alteration in the Intrafollicular Thiol\u2013Redox System in Infertile Women with Endometriosis","volume":"149","author":"Choi","year":"2015","journal-title":"Reproduction"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1016\/S0015-0282(00)00552-5","article-title":"Effect of Peritoneal Fluid from Women with Endometriosis on Implantation in the Mouse Model","volume":"74","author":"Illera","year":"2000","journal-title":"Fertil. Steril."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1304","DOI":"10.1177\/1933719116687658","article-title":"Peritoneal Fluid From Infertile Women With Minimal\/Mild Endometriosis Compromises the Meiotic Spindle of Metaphase II Bovine Oocytes: A Pilot Study","volume":"24","author":"Jianini","year":"2017","journal-title":"Reprod. Sci."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"926","DOI":"10.1016\/S0015-0282(16)53533-X","article-title":"Peritoneal Fluid from Endometriosis Patients Affects Reproductive Outcome in an in Vivo Model","volume":"53","author":"Steinleitner","year":"1990","journal-title":"Fertil. Steril."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1109","DOI":"10.1016\/0002-9378(86)90360-1","article-title":"Experimental Evidence for Failure to Implant as a Mechanism of Infertility Associated with Endometriosis","volume":"155","author":"Hahn","year":"1986","journal-title":"Am. J. Obstet. Gynecol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"709","DOI":"10.1089\/ten.2005.11.709","article-title":"Follicle-Like Model by Granulosa Cell Encapsulation in a Barium Alginate\u2013Protamine Membrane","volume":"11","author":"Vigo","year":"2005","journal-title":"Tissue Eng."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1842","DOI":"10.1093\/humrep\/deu139","article-title":"The Bologna Criteria for the Definition of Poor Ovarian Responders: Is There a Need for Revision?","volume":"29","author":"Ferraretti","year":"2014","journal-title":"Hum. Reprod."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"The ESHRE Working Group on Ultrasound in ART, D\u2019Angelo, A., Panayotidis, C., Amso, N., Marci, R., Matorras, R., Onofriescu, M., Turp, A.B., Vandekerckhove, F., and Veleva, Z. (2019). Recommendations for Good Practice in Ultrasound: Oocyte Pick Up. Hum. Reprod. Open, 2019, hoz025.","DOI":"10.1093\/hropen\/hoz025"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Stirling, D.R., Swain-Bowden, M.J., Lucas, A.M., Carpenter, A.E., Cimini, B.A., and Goodman, A. (2021). CellProfiler 4: Improvements in Speed, Utility and Usability. BMC Bioinform., 22.","DOI":"10.1186\/s12859-021-04344-9"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Shan, G., and Gerstenberger, S. (2017). Fisher\u2019s Exact Approach for Post Hoc Analysis of a Chi-Squared Test. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0188709"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Alviggi, C., Conforti, A., Esteves, S.C., Vallone, R., Venturella, R., Staiano, S., Castaldo, E., Andersen, C.Y., and De Placido, G. (2018). Understanding Ovarian Hypo-Response to Exogenous Gonadotropin in Ovarian Stimulation and Its New Proposed Marker\u2014The Follicle-To-Oocyte (FOI) Index. Front. Endocrinol., 9.","DOI":"10.3389\/fendo.2018.00589"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"300","DOI":"10.1007\/s13577-020-00439-2","article-title":"Characterization of Stem Cells from Human Ovarian Follicular Fluid; a Potential Source of Autologous Stem Cell for Cell-Based Therapy","volume":"34","author":"Rungsiwiwut","year":"2021","journal-title":"Hum. Cell"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Jozkowiak, M., Hutchings, G., Jankowski, M., Kulcenty, K., Mozdziak, P., Kempisty, B., Spaczynski, R.Z., and Piotrowska-Kempisty, H. (2020). The Stemness of Human Ovarian Granulosa Cells and the Role of Resveratrol in the Differentiation of MSCs\u2014A Review Based on Cellular and Molecular Knowledge. Cells, 9.","DOI":"10.3390\/cells9061418"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"210","DOI":"10.1634\/stemcells.2008-0233","article-title":"The Multipotency of Luteinizing Granulosa Cells Collected from Mature Ovarian Follicles","volume":"27","author":"Martin","year":"2009","journal-title":"Stem Cells"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"939","DOI":"10.1007\/s10815-011-9617-6","article-title":"Proliferative Potential and Phenotypic Analysis of Long-Term Cultivated Human Granulosa Cells Initiated by Addition of Follicular Fluid","volume":"28","author":"Bruckova","year":"2011","journal-title":"J. Assist. Reprod. Genet."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1612","DOI":"10.1007\/s13577-022-00757-7","article-title":"Establishment and Characterization of IPS-OGC-C1: A Novel Induced Pluripotent Stem Cell Line from Healthy Human Ovarian Granulosa Cells","volume":"35","author":"Wang","year":"2022","journal-title":"Hum. Cell"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"603","DOI":"10.1016\/S0015-0282(16)58162-X","article-title":"The Effect of Endometriosis on Implantation: Results from the Yale University in Vitro Fertilization and Embryo Transfer Program","volume":"65","author":"Arici","year":"1996","journal-title":"Fertil. Steril."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"593","DOI":"10.1093\/humupd\/dmz012","article-title":"Reproductive, Obstetric, and Perinatal Outcomes of Women with Adenomyosis and Endometriosis: A Systematic Review and Meta-Analysis","volume":"25","author":"Horton","year":"2019","journal-title":"Hum. Reprod. Update"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1015","DOI":"10.1177\/1933719115570909","article-title":"Driving Human Granulosa-Luteal Cells Recovered From In Vitro Fertilization Cycles Toward the Follicular Phase Phenotype","volume":"22","author":"Vireque","year":"2015","journal-title":"Reprod. Sci."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Horisberger, M.A. (2006). A method for prolonged survival of primary cell lines. Vitr. Cell. Dev. Biol. Anim., 42.","DOI":"10.1290\/0511081"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"2063","DOI":"10.1089\/ten.tea.2009.0684","article-title":"A Novel Three-Dimensional Culture System Allows Prolonged Culture of Functional Human Granulosa Cells and Mimics the Ovarian Environment","volume":"16","author":"Pelczar","year":"2010","journal-title":"Tissue Eng. Part A"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1453","DOI":"10.1007\/s43032-022-01083-x","article-title":"Endometriosis: Cellular and Molecular Mechanisms Leading to Fibrosis","volume":"30","author":"Vannuzzi","year":"2023","journal-title":"Reprod. Sci."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1053","DOI":"10.1177\/1933719115592713","article-title":"Pathogenetic Mechanisms of Deep Infiltrating Endometriosis","volume":"22","author":"Tosti","year":"2015","journal-title":"Reprod. Sci."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"160","DOI":"10.1186\/s13048-024-01480-7","article-title":"Extracellular Vesicles and Their Content in the Context of Polycystic Ovarian Syndrome and Endometriosis: A Review","volume":"17","author":"Duval","year":"2024","journal-title":"J. Ovarian Res."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Gebremedhn, S., Ali, A., Gad, A., Prochazka, R., and Tesfaye, D. (2020). Extracellular Vesicles as Mediators of Environmental and Metabolic Stress Coping Mechanisms During Mammalian Follicular Development. Front. Vet. Sci., 7.","DOI":"10.3389\/fvets.2020.602043"},{"key":"ref_39","first-page":"182","article-title":"Extracellular Vesicles: Roles in Gamete Maturation, Fertilization and Embryo Implantation","volume":"22","author":"Machtinger","year":"2016","journal-title":"Hum. Reprod. Update"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1016\/j.mce.2007.11.001","article-title":"Gap Junctions in the Ovary: Expression, Localization and Function","volume":"282","author":"Gershon","year":"2008","journal-title":"Mol. Cell. Endocrinol."},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Zhang, M., Wang, Z.-Z., and Chen, N.-H. (2023). Connexin 43 Phosphorylation: Implications in Multiple Diseases. Molecules, 28.","DOI":"10.3390\/molecules28134914"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"464","DOI":"10.1016\/j.bbapap.2012.06.018","article-title":"Redox Regulation of Protein Tyrosine Phosphatase Activity by Hydroxyl Radical","volume":"1834","author":"Meng","year":"2013","journal-title":"Biochim. Biophys. Acta BBA-Proteins Proteom."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"178","DOI":"10.1038\/nrm3758","article-title":"Molecular Mechanisms of Epithelial\u2013Mesenchymal Transition","volume":"15","author":"Lamouille","year":"2014","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1016\/B978-0-12-394305-7.00004-5","article-title":"New Insights into the Regulation of Epithelial\u2013Mesenchymal Transition and Tissue Fibrosis","volume":"Volume 294","author":"Lee","year":"2012","journal-title":"International Review of Cell and Molecular Biology"},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Igietseme, J.U., Omosun, Y., Stuchlik, O., Reed, M.S., Partin, J., He, Q., Joseph, K., Ellerson, D., Bollweg, B., and George, Z. (2015). Role of Epithelial-Mesenchyme Transition in Chlamydia Pathogenesis. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0145198"}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/15\/4\/561\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T17:12:17Z","timestamp":1760029937000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/15\/4\/561"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,4,10]]},"references-count":45,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2025,4]]}},"alternative-id":["biom15040561"],"URL":"https:\/\/doi.org\/10.3390\/biom15040561","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,4,10]]}}}