Endometrial stromal cells from women with endometriosis reveal peculiar migratory behavior in response to ovarian steroids  Davide Gentilini, Ph.D., Paola Vigano, Ph.D., Edgardo Somigliana, M.D., Lucia Maria Vicentini, M.D., Michele Vignali, M.D., Mauro Busacca, M.D., Anna Maria Di Blasio, M.D.  Fertility and Sterility  Volume 93, Issue 3, Pages 706-715 (February 2010) DOI: 10.1016/j.fertnstert.2008.10.014 Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Schematic illustration of the chemotaxis assay. Cells are inoculated in the upper chamber. After 6 hours of incubation at 37°C, the non-migrated cells on the upper surface of the filter are removed by scraping. The cells that migrate to the lower side of the filter are stained and are counted at ×200 magnification using a microscope. Fertility and Sterility 2010 93, 706-715DOI: (10.1016/j.fertnstert.2008.10.014) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Effect of progesterone on migration of endometrial stromal cells (ESCs) from patient with and without endometriosis. All experiments were performed in a Boyden chamber using collagen IV to coat the filters in the chemotaxis assay. (A) Cells were treated for 24 hours with and without 10 nM progesterone before the assay. Left panel: Results expressed as mean chemotactic index ± standard deviation. The number of cells migrated in response to treatments are compared with basal values. Right panel: Percentage of stimulation of migration. (B) Treated and untreated cells were also exposed to PDGF-BB (25 ng/mL) used as chemoattractant in the Boyden's lower chamber. Left panel: Results expressed as mean chemotactic index ± SD. Right panel: Effect of progesterone on PDGF-BB-stimulated migration expressed as percentage of reduction. The outlined arrow indicates the level of basal migration. ∗P<.05 versus cells from control women. ∗∗P<.05 versus correspondent unstimulated cells. Fertility and Sterility 2010 93, 706-715DOI: (10.1016/j.fertnstert.2008.10.014) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Effect of 17β-estradiol on migration of endometrial stromal cells (ESCs) from patients with and without endometriosis. (A) Cells were treated for 24 hours with and without 10 nM 17β-estradiol. The results are expressed as chemotactic index ± standard deviation (SD). (B) The experiments were also performed with PDGF-BB (25 ng/mL) as chemoattractant in the Boyden's lower chamber on untreated or 17β-estradiol-treated cells left in the Boyden's upper chamber. The results are expressed as chemotactic index ± SD. The outlined arrow indicates the level of basal migration. ∗P<.05 versus cells from control women, ∗∗P<.05 versus correspondent unstimulated cells. Fertility and Sterility 2010 93, 706-715DOI: (10.1016/j.fertnstert.2008.10.014) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Representative chemotaxis experiment. Endometrial stromal cells (ESCs) that reached the lower side of the coated filter after a chemotaxis experiment were stained from controls (A–F) and from women with endometriosis (G–I, L–N). The image shows basal migration (A, G), migration after 24 hours of treatment with 10 nM progesterone (B, H), migration after 24 hours of treatment with 10 nM 17β-estradiol (C, I), migration induced by PDGF-BB (D, L), effect of 24 hours of treatment with 10 nM progesterone on PDGF-BB–induced migration (E, M), and effect of 24 hours of treatment with 10 nM 17β-estradiol on PDGF-BB–induced migration (F, N). The greater promigratory effect of progesterone on ESCs from women with endometriosis compared with those of controls is particularly evident. Fertility and Sterility 2010 93, 706-715DOI: (10.1016/j.fertnstert.2008.10.014) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 5 Effect of PDGF-BB, 17β-estradiol and progesterone on actin cytoskeleton of endometrial stromal cells (ESCs) from controls (A–D) and from women with endometriosis (E–H). Cells were serum-starved for 24 hours before the experiment. The image shows actin organization in untreated cells (A, E), in cells treated with 25 ng/mL of PDGF-BB (B, F), with 10 nM 17β-estradiol (C–G), and with 10 nM progesterone (D, H). Cells were stained with fluorescein isothiocyanate–labeled phalloidin to visualize F-actin. Micrographs at ×100 magnification. Fertility and Sterility 2010 93, 706-715DOI: (10.1016/j.fertnstert.2008.10.014) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 6 Steroid hormone effects on endometrial stromal cell (ESC) cytoskeleton organization. Cells were treated with 10 nM 17β-estradiol or 10 nM progesterone for 20 minutes, 1 hour, and 24 hours. Cells were stained with fluorescein isothiocyanate–labeled phalloidin to visualize F-actin and photographed at ×100 magnification. The results are expressed as percentage of cells that manifest a migratory phenotype (loss of stress fibers, progressive localization of actin toward the edge of the cell membrane, and simultaneous presence of numerous stress fiber arcs) and are shown as mean values ± standard deviation of 12 independent experiments. ∗P<.05 versus cells from control women, ∗∗P<.05 versus correspondent unstimulated cells. Fertility and Sterility 2010 93, 706-715DOI: (10.1016/j.fertnstert.2008.10.014) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions