Increased expression of macrophage colony–stimulating factor and its receptor in patients with endometriosis Nicole M. Budrys, M.D., M.P.H., Hareesh.

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Increased expression of macrophage colony–stimulating factor and its receptor in patients with endometriosis Nicole M. Budrys, M.D., M.P.H., Hareesh B. Nair, Ph.D., Ya-Guang Liu, Ph.D., Nameer B. Kirma, Ph.D., Peter A. Binkley, B.S., Shantha Kumar, Ph.D., Robert S. Schenken, M.D., Rajeshwar Rao Tekmal, Ph.D. Fertility and Sterility Volume 97, Issue 5, Pages 1129-1135.e1 (May 2012) DOI: 10.1016/j.fertnstert.2012.02.007 Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Purity of endometrial stromal (ESCs) and epithelial (EECs) cells. ESCs and EECs were separated with 98.3% and 98.6% purity by flow cytometry with EECs stained for cytokeratin-18 and ESCs stained for vimentin. Fertility and Sterility 2012 97, 1129-1135.e1DOI: (10.1016/j.fertnstert.2012.02.007) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Peritoneal fluid concentration (ng/mL) of colony-stimulating factor 1 (CSF-1) in patients with endometriosis compared with patients without endometriosis (control). Peritoneal fluid concentration of CSF-1 is higher in patients with endometriosis. Values are expressed as mean ± SD. ∗P≤.002. Fertility and Sterility 2012 97, 1129-1135.e1DOI: (10.1016/j.fertnstert.2012.02.007) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Colony-stimulating factor 1 (CSF-1) and its receptor, C-FMS, expression in ectopic tissue compared with paired eutopic tissue from women with endometriosis. Representative Western blot analysis (top) shows increased CSF-1 and C-FMS levels in ectopic lesions compared with paired eutopic endometrium. A relative increase in CSF-1 (∼3-fold increase) and C-FMS (∼1.7-fold increase) expression was seen in ectopic endometrial tissue compared with eutopic endometrial tissue samples. ∗P<.01; ∗∗P<.05. Fertility and Sterility 2012 97, 1129-1135.e1DOI: (10.1016/j.fertnstert.2012.02.007) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 4 (A) Colony-stimulating factor 1 (CSF-1) and its receptor, C-FMS, expression in EECs from women with endometriosis compared with women without endometriosis (control) after coculture with peritoneal mesothelial cells (PMCs). Reverse-transcription polymerase chain reaction (RT-PCR) analysis was used to determine RNA expression of CSF-1 and C-FMS. Endometrial epithelial cells (EECs) that were not exposed to PMCs were used as baseline control. Individual data points are represented by dashes with the mean for each group indicated by an X. (B) CSF-1 and C-FMS expression in endometrial stroma cells (ESCs) from women with endometriosis compared to women without endometriosis (control) after coculture with PMCs. RT-PCR analysis was used to determine RNA expression of CSF-1 and C-FMS. ESCs that were not exposed to PMCs were used as baseline control. Individual data points are represented by dashes with the mean for each group indicated by an X. Fertility and Sterility 2012 97, 1129-1135.e1DOI: (10.1016/j.fertnstert.2012.02.007) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Colony-stimulating factor 1 (CSF-1) expression of endometrial cells is dependent on physical contact with mesothelial cells, not with time in culture. Endometrial epithelial cells (EECs) and endometrial stroma cells (ESCs) were separated from tissue culture. CSF-1 levels (A) and the CSF-1 receptor, C-FMS (B), were measured with the use of reverse-transcription polymerase chain reaction (RT-PCR) before coculture, after culture for 16 hours, and after coculture for 16 hours. CSF-1 and C-FMS levels were similar between the cultured samples and the pre-coculture samples. Fertility and Sterility 2012 97, 1129-1135.e1DOI: (10.1016/j.fertnstert.2012.02.007) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions