Uterine leiomyomas express a molecular pattern that lowers retinoic acid exposure  William H. Catherino, M.D., Ph.D., Minnie Malik, Ph.D.  Fertility and Sterility  Volume 87, Issue 6, Pages 1388-1398 (June 2007) DOI: 10.1016/j.fertnstert.2006.11.093 Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

FIGURE 1 Representative Western blots demonstrating protein data. Histograms represent the average fold difference in protein amount in leiomyoma (L) compared with myometrium (M). The data are representative of at least three patients. (A) Expression of CRBP-1 protein and (B) CRABP-I/II protein; (C) Expression of the nuclear receptors RARs: RARα, RARβ, and RARγ; and RXRs: RXRα, RXRβ, and RXRγ (D) β-actin was used as an internal standard between samples in all experiments. Catherino. Leiomyomas lower retinoic acid exposure. Fertil Steril 2007. Fertility and Sterility 2007 87, 1388-1398DOI: (10.1016/j.fertnstert.2006.11.093) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

FIGURE 2 Representative immunohistochemical stain for CRABP-1 (A) and RARβ (B). No regional differences in staining were noted for either protein. The negative controls are [1] nonimmune serum and [2] no secondary antibody. Magnification 40×. Catherino. Leiomyomas lower retinoic acid exposure. Fertil Steril 2007. Fertility and Sterility 2007 87, 1388-1398DOI: (10.1016/j.fertnstert.2006.11.093) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

FIGURE 3 Western blot representation of CYP26A1 protein amount. Histograms represent the average fold difference in leiomyoma (L) measured in six patients. β-actin was used as an internal standard between samples. Catherino. Leiomyomas lower retinoic acid exposure. Fertil Steril 2007. Fertility and Sterility 2007 87, 1388-1398DOI: (10.1016/j.fertnstert.2006.11.093) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

FIGURE 4 Representative HPLC chromatograms of commercial and endogenous ATRA and 9-cis RA. (A) HPLC profile of ATRA and 9-cis RA added at equimolar concentration. (B) Positive control sample containing ATRA and 9-cis RA (1:1 v/v) but no tissue samples. (C) and (E) Profile of endogenous ATRA and 9-cis RA in myometrium (C) and leiomyoma (E) samples. (D) and (F) Myometrium and leiomyoma sample to which commercial RAs have been added at equimolar concentration. Catherino. Leiomyomas lower retinoic acid exposure. Fertil Steril 2007. Fertility and Sterility 2007 87, 1388-1398DOI: (10.1016/j.fertnstert.2006.11.093) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

FIGURE 5 Model of retinol metabolizing pathway. Down-regulation of critical enzymes involved in the first step in retinol signaling, ALDH and ADH, as well as up-regulation of metabolizing enzyme CYP26, in a leiomyoma cell, results in a decreased amount of all-trans RA and 9-cis RA. In the absence of biologically active RAs, the expression of RA-dependent genes including those involved in apoptosis and differentiation may be affected. Catherino. Leiomyomas lower retinoic acid exposure. Fertil Steril 2007. Fertility and Sterility 2007 87, 1388-1398DOI: (10.1016/j.fertnstert.2006.11.093) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions