Merja Bläuer, Ph. D. , Päivi H. Rovio, M. D. , Timo Ylikomi, M. D

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Vitamin D inhibits myometrial and leiomyoma cell proliferation in vitro Merja Bläuer, Ph.D., Päivi H. Rovio, M.D., Timo Ylikomi, M.D., Pentti K. Heinonen, M.D. Fertility and Sterility Volume 91, Issue 5, Pages 1919-1925 (May 2009) DOI: 10.1016/j.fertnstert.2008.02.136 Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Immunohistochemical characterization of myometrial and leiomyoma cells in culture. The pictures are representative of leiomyoma cells, but both cell types displayed similar staining patterns. (A) The majority of the cultivated cells were smooth muscle actin (SMA) positive. Arrows point to immunonegative cells. (B) Fewer than 10% of cells were positive for desmin. (C) The intermediate filament vimentin was strongly expressed in all cells. (D) A few scattered nuclei (arrow) were immunopositive for estrogen receptor (ER). (E) No progesterone receptor (PR) labeling was detected. (F) Vitamin D receptor (VDR) immunoreactivity was localized in discrete foci within the nuclei. Bar = 50 μm. Fertility and Sterility 2009 91, 1919-1925DOI: (10.1016/j.fertnstert.2008.02.136) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Immunohistochemical localization of vitamin D receptor (VDR) in myometrial and leiomyoma tissues. The VDR immunoreactivity (arrows) was localized in the nuclei of smooth muscle cells in matched (A) myometrial and (B) leiomyoma tissues. Bars = 50 μm. Fertility and Sterility 2009 91, 1919-1925DOI: (10.1016/j.fertnstert.2008.02.136) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Growth curves of paired myometrial and leiomyoma cell cultures. Cells were seeded at 1000 to 2000 cells/well in 96-well microplates and allowed to proliferate in culture medium supplemented with 1 nM estradiol for 15 days. Growth medium was renewed every third day. At 3-day intervals, proliferation was determined by the colorimetric crystal violet assay. Absorbance values (ABS) were read at 590 nm. Each point represents the mean ± SEM of cell cultures from six patients (at day 15, n = 4). Both cell types exhibited similar growth patterns with no statistically significant differences in their mean rates of proliferation. Fertility and Sterility 2009 91, 1919-1925DOI: (10.1016/j.fertnstert.2008.02.136) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Effect of various concentrations of (A, B) 1,25(OH)2D3 or (C, D) 25(OH)D3 on myometrial and leiomyoma cells. Proliferation was determined using the colorimetric crystal violet assay after 9 days of treatment with increasing doses of the two compounds as indicated. The cell growth is presented as a percentage of ethanol-treated controls (C on the x-axis). Six individual cultures were examined in each concentration (in 500 nM 25(OH)D3, n = 4). Horizontal lines represent the median. Fertility and Sterility 2009 91, 1919-1925DOI: (10.1016/j.fertnstert.2008.02.136) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions