In vivo mechanisms of uterine myoma volume reduction with ulipristal acetate treatment  Guillaume E. Courtoy, M.Sc., Jacques Donnez, M.D., Ph.D., Etienne Marbaix, M.D., Ph.D., Marie-Madeleine Dolmans, M.D., Ph.D.  Fertility and Sterility  Volume 104, Issue 2, Pages 426-434.e1 (August 2015) DOI: 10.1016/j.fertnstert.2015.04.025 Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Proliferation and cell death after ulipristal acetate (UPA) treatment compared with untreated myomas. (A) Ki-67 immunohistochemistry (IHC) in myomas and (B) associated quantification. The proliferation rate per patient is expressed as a percentage out of total cells. (C) Terminal deoxynucleotide transferase–mediated dUTP nick-end labeling (TUNEL) assay on representative samples of tissue microarray. TUNEL-labeled cells are shown in false red. 6-Diamino-2-phenylindole (DAPI)–labeled DNA in nuclei appears grey. (D, G) Quantifications of TUNEL-positive cells as percentage of total cells. (E) Cleaved caspase-3 IHC in myomas. Diaminobenzidine-labeled cells appear brown (arrowheads), and counterstained cells appear blue-violet with hematoxylin staining. (F) Quantification of cleaved caspase-3–positive cells expressed as percentage of total cells. (G, H) Quantifications of TUNEL assays or cleaved caspase-3 IHC to compare the untreated group (UPA0) and short-term groups (UPA5+UPA10 = UPA-ST) with the long-term group (UPA10-LT). Boxes: median and interquartile range; whiskers: 10th–90th percentiles; dots: outlier values. *P<.05; **P<.01; ***P<.001. Fertility and Sterility 2015 104, 426-434.e1DOI: (10.1016/j.fertnstert.2015.04.025) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Histologic changes in myomas after UPA treatment compared with untreated myomas (UPA0). (A) Cell density analysis assessed with the use of hematoxylin-eosin. Cell density represents the number of nuclei (in dark violet) out of total area per sample. (B) Quantification of cell density (in cells/10 μm2). (C) Extracellular matrix (ECM) fraction in myomas assessed with the use of Masson green trichrome. Extracellular components including collagen appear green, whereas nuclei are dark violet and cytoplasm is pink. (D) Quantification of the ECM. (E) Sirius Red (dye that reacts specifically with collagen) with Fast Green counterstaining (noncollagen proteins) and (F) quantification of collagen-stained areas out of total tissue area. (G) Matrix metalloproteinase (MMP) 2 spatial distribution in untreated and UPA-treated myomas, assessed by IHC. Both intracellular and secreted forms are detected. (H) Quantification of labeled areas out of total tissue area. Boxes: median and interquartile range; whiskers: 10th–90th percentiles; dots: outlier values. *P<.05; **P<.01; ***P<.001. Abbreviations as in Figure 1. Fertility and Sterility 2015 104, 426-434.e1DOI: (10.1016/j.fertnstert.2015.04.025) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Negative and positive control samples for Ki-67, terminal deoxynucleotide transferase–mediated dUTP nick-end labeling (TUNEL), and cleaved caspase-3. (A, B) Control samples for Ki-67. (A) Negative control was obtained by omission of the primary antibody on human proliferative endometrium. (B) Human proliferative endometrium was used as positive control for Ki-67 antibody (M7240, clone MIB1, 1:100; Dako). (C–H) Control samples for TUNEL assay. (C) Human tonsil was incubated without the terminal deoxynucleotidyl transferase enzyme and used for negative technical control. (D) Mouse intestine served as positive tissue control. (E, F) Human tonsil and human uterine fibroid (non–ulipristal acetate [UPA]–treated) incubated with DNAse. This treatment was done with 2 U/mL bovine pancreatic DNAse (ref. 18068–015; Invitrogen) in 1 mL 10 mmol/L Tris-HCl, pH 7.4, 1 mmol/L MgCl2, and 1 mg/mL bovine serum albumin for 20 minutes at room temperature. (G) Human tonsil without DNAse incubation served as a positive tissue control. (H) Human uterine myoma (non–UPA-treated) without DNAse incubation showed few TUNEL-positive cells. (I–L) Control samples for cleaved caspase-3 immunohistochemistry. Negative control samples for cleaved caspase-3 were obtained by omission of the primary antibody on (I) human tonsil and (J) menstrual endometrium. (K) Human tonsil and (L) menstrual endometrium were also used as positive control samples. Scale bars: A, B, I–L = 50 μm; C–H = 100 μm. Fertility and Sterility 2015 104, 426-434.e1DOI: (10.1016/j.fertnstert.2015.04.025) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions