Patrizia Carrarelli, Ph. D. , Chih-Fen Yen, M. D. , Felice Arcuri, Ph

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Myostatin, follistatin and activin type II receptors are highly expressed in adenomyosis Patrizia Carrarelli, Ph.D., Chih-Fen Yen, M.D., Felice Arcuri, Ph.D., Lucia Funghi, Ph.D., Claudia Tosti, M.D., Tzu-Hao Wang, M.D., Joseph S. Huang, M.D., Ph.D., Felice Petraglia, M.D. Fertility and Sterility Volume 104, Issue 3, Pages 744-752.e1 (September 2015) DOI: 10.1016/j.fertnstert.2015.05.032 Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Growth factors expression in adenomyosis. The messenger RNA (mRNA) expression of activin A, activin B, inhibin-α, follistatin, crypto, and myostatin in adenomyotic nodules (adenomyosis, n = 8), in endometrium of the same patients with adenomyosis (eutopic, n = 8), and in control endometrium (control, n = 12). Fold change (y axis) represents messenger RNA expression normalized to GAPDH. ∗P<.05; ∗∗P<.01; ∗∗∗P<.001. Fertility and Sterility 2015 104, 744-752.e1DOI: (10.1016/j.fertnstert.2015.05.032) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Activin receptors messenger RNA (mRNA) expression in adenomyosis. The messenger RNA expression of ActRIIa, ActRIIb in adenomyotic nodules (adenomyosis, n = 8), in endometrium of the same patients with adenomyosis (eutopic, n = 8), and in control endometrium (control, n = 12). Fold change (y axis) represents messenger RNA expression normalized to GAPDH. ∗P<.05; ∗∗P<.01; ∗∗∗P<.001. Fertility and Sterility 2015 104, 744-752.e1DOI: (10.1016/j.fertnstert.2015.05.032) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Myostatin immunoreactivity in adenomyosis and control. Representative micrographs of immunofluorescent staining in which positive signals are fluorescein isothiocyanate conjugate (FITC)-labeled and shown in green. The blue color shows cell nuclei stained with 6-diamino-2-phenylindole (DAPI). Specimens of the control group are shown in (A) the functional layer and (B) the basal layer. Specimens from patients with adenomyosis are shown: (C) functional layer and (D) basal layer of the eutopic endometrium, (E) adenomyotic nodules. In both cases, no basal membrane separates endometrial cells from myometrial cells. Bottom panel shows HSCORE of myostatin immunoreactivity in glandular cells (light gray columns) and stromal cells (gray columns). Bar = 20 μm, ×400. For the HSCORE, bar chart (mean ± SEM) was calculated from the measurement of 12 patients in each group. ∗P<.001; †P<.01. Fertility and Sterility 2015 104, 744-752.e1DOI: (10.1016/j.fertnstert.2015.05.032) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Follistatin immunoreactivity in adenomyosis and control. Representative micrographs showing immunofluorescent staining (Cy5-labeled) were shown in red. Cell nuclei were stained with 6-diamino-2-phenylindole (DAPI) and shown in blue. Specimens of the control group are shown in (A) the functional layer and (B) the basal layer. Specimens from patients with adenomyosis are shown: (C) functional layer and (D) basal layer of the eutopic endometrium, (E) adenomyotic nodules. In both cases, no basal membrane separates endometrial cells from myometrial cells. Bottom panel shows HSCORE of follistatin immunoreactivity in glandular cells (light gray columns) and stromal cells (gray columns). Bar = 20 μm, ×400. For the HSCORE, bar chart (mean ± SEM) was calculated from the measurement of 12 patients in each group. ∗P<.001; †P<.01. Fertility and Sterility 2015 104, 744-752.e1DOI: (10.1016/j.fertnstert.2015.05.032) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions