Eutopic and ectopic stromal cells from patients with endometriosis exhibit differential invasive, adhesive, and proliferative behavior Ali-Akbar Delbandi,

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Eutopic and ectopic stromal cells from patients with endometriosis exhibit differential invasive, adhesive, and proliferative behavior Ali-Akbar Delbandi, M.Sc., Mahmoud Mahmoudi, Ph.D., Adel Shervin, M.D., Elham Akbari, M.D., Mahmood Jeddi-Tehrani, Ph.D., Mojtaba Sankian, Ph.D., Somayeh Kazemnejad, Ph.D., Amir-Hassan Zarnani, Ph.D. Fertility and Sterility Volume 100, Issue 3, Pages 761-769 (September 2013) DOI: 10.1016/j.fertnstert.2013.04.041 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Immunophenotyping of ESCs, which were isolated from ectopic and eutopic endometrial samples of patients with endometriosis and nonendometriotic controls and characterized by immunofluorescent staining and flow cytometry analysis. (A) Representative immunofluorescent staining of ESCs showing the expression of vimentin (a) and nestin (b) and negative immunoreactivity for cytokeratin (c). ESCs from all three sources exhibited the same pattern. Panels d, e, and f show negative reagent controls for vimentin, nestin, and cytokeratin, respectively. (B) Representative flow cytometric analysis of CD9, CD10, CD29, CD34, CD38, CD44, CD45, CD73, CD105, and CD133 in ectopic ESCs (a), eutopic ESCs (b), and control ESCs (c). Data are presented as mean ± SD. Fertility and Sterility 2013 100, 761-769DOI: (10.1016/j.fertnstert.2013.04.041) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Comparative proliferation, invasion, and adhesion analysis of EESCs, EuESCs, and CESCs. Proliferation of isolated stromal cells was assessed by XTT assay in untreated (A) and fibronectin-coated (B) plates. Invasive capacity of stromal cells was evaluated by matrigel invasion assay at 200× magnification (C). Stromal cell attachment was measured at different time intervals (D). Each bar represents the median with a range of 10–24 different samples. EESCs = stromal cells from ectopic site (endometrioma); EuESCs = stromal cells from the eutopic endometrium of patients with endometriosis; CESCs = stromal cells from nonendometriotic controls; OD = optical density. *P<.05, **P<.01, ***P<.001. Fertility and Sterility 2013 100, 761-769DOI: (10.1016/j.fertnstert.2013.04.041) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 EuSCs and EESCs show different invasive capacity. EESCs (B), EuESCs (C), and CESCs (D) were isolated, and their matrigel invasive capacity was assessed. Ovarian cancer cell line (SKOV3) (A) was used in parallel as a positive control. EESCs = stromal cells from ectopic site (endometrioma); EuESCs = stromal cells from the eutopic endometrium of patients with endometriosis; CESCs = stromal cells from nonendometriotic controls. Scale bar = 50 μm. Fertility and Sterility 2013 100, 761-769DOI: (10.1016/j.fertnstert.2013.04.041) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Cytokine production by EESCs, EuESCs, and CESCs. Stromal cells from 18 patients with endometriosis and 14 nonendometriotic controls were cultured in DMEM-F12 medium, and the levels of interleukin (IL)-8 and IL-6 were measured by capture ELISA. IL-6 (A) and IL-8 (B). EESCs = stromal cells from ectopic site (endometrioma); EuESCs = stromal cells from the eutopic endometrium of patients with endometriosis; CESCs = stromal cells from nonendometriotic controls. **P<.01, ***P<.001. Fertility and Sterility 2013 100, 761-769DOI: (10.1016/j.fertnstert.2013.04.041) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions