Expression of erythropoietin messenger ribonucleic acid in wild-type MED12 uterine leiomyomas under estrogenic influence: new insights into related growth.

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Expression of erythropoietin messenger ribonucleic acid in wild-type MED12 uterine leiomyomas under estrogenic influence: new insights into related growth disparities Ryoko Asano, M.D., Ph.D., Mikiko Asai-Sato, M.D., Ph.D, Shoichi Matsukuma, Ph.D., Taichi Mizushima, M.D., Ph.D., Masataka Taguri, Ph.D., Mitsuyo Yoshihara, B.S., Mae Inada, Atsuko Fukui, B.S., Yukio Suzuki, M.D., Yohei Miyagi, M.D., Ph.D., Etsuko Miyagi, M.D., Ph.D. Fertility and Sterility Volume 111, Issue 1, Pages 178-185 (January 2019) DOI: 10.1016/j.fertnstert.2018.09.014 Copyright © 2018 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Relation between MED12 exon 2 mutation status and EPO mRNA expression level. (A) Comparison of EPO mRNA expression levels in MED12 WT (n=56) and MED12 MUT (n=52) leiomyomas (P-value calculated via Mann-Whitney U test). *P<.001. (B) Waterfall plot of EPO mRNA expression by real-time RT-PCR in leiomyoma tissue samples. The samples were categorized into five groups by EPO mRNA expression level. Group 1: −100∼−89%, Group 2: −89∼−62%, Group 3: −62∼−19%, Group 4: −19∼+51%, Group 5: over +51% of the mean EPO mRNA level, respectively. The correlation between higher EPO mRNA level and wild-type MED12 was calculated via χ2 test for trend. *P<.001. Fertility and Sterility 2019 111, 178-185DOI: (10.1016/j.fertnstert.2018.09.014) Copyright © 2018 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Relation between HMGA and EPO mRNA expression levels. Spearman correlation coefficients (ρ) reflecting correlations between expression levels of HMGA1 or HMGA2 mRNA and EPO mRNA. (A, B) HMGA1 and EPO mRNA expression levels; and (C, D) HMGA2 and EPO mRNA expression levels (A, C: all samples; B, D: MED12 WT samples). Fertility and Sterility 2019 111, 178-185DOI: (10.1016/j.fertnstert.2018.09.014) Copyright © 2018 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Expression levels of EPO mRNA in primary cultures of normal myometrium and leiomyomas. (A) Comparison of EPO mRNA expression levels in non-estrogen treated leiomyoma (LM) and myometrial (MM) paired cellular samples (n=8). In all samples, EPO mRNA expression levels of LM exceeded those of MM; (B) Fold changes in EPO mRNA expression after E2 treatment in cultures of MM (n=8) and LM (n=10) samples (P-value calculated by Wilcoxon signed-rank test); (C) Changes in EPO mRNA expression levels after E2 treatment in MED12 WT and MED12 MUT leiomyomas; (D) Effect of E2 on EPO mRNA expression with or without ICI blocking (LM samples used for experiment clearly showing induction of EPO by E2). *P<.05 (Dunnett's test). Error bar, SD. Fertility and Sterility 2019 111, 178-185DOI: (10.1016/j.fertnstert.2018.09.014) Copyright © 2018 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Relation between diameter of leiomyoma and MED12 mutation status. P-value calculated via Mann-Whitney U test. *P<.05, Error bar, SD. Fertility and Sterility 2019 111, 178-185DOI: (10.1016/j.fertnstert.2018.09.014) Copyright © 2018 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 Immunohistochemical staining of αSMA in cultured cells. Streptavidin-biotin peroxidase method applied to smooth muscle-specific protein, αSMA (hematoxylin counterstain) in representative sections of (A, B) MED12 WT leiomyoma (sample 7) and corresponding myometrium; (C, D) MED12 MUT leiomyoma (sample 9) and corresponding myometrium; and (E) negative control, myometrium stained without primary antibody. (200× magnification). Fertility and Sterility 2019 111, 178-185DOI: (10.1016/j.fertnstert.2018.09.014) Copyright © 2018 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 3 Expression of EPO mRNA in cultured leiomyoma cells (hypoxic and estrogen treated). Leiomyoma-derived (n=4) wild-type MED12 cells cultured under normoxia (20%) or hypoxia (1%), with or without 17β-estradiol (10−8 M); EPO mRNA expression levels measured (triplet samples) by real-time RT-PCR, relative to glyceraldehyde-3-phosphate dehydrogenase mRNA control; no significant elevation of EPO mRNA due to hypoxia (Dunnett's test). Fertility and Sterility 2019 111, 178-185DOI: (10.1016/j.fertnstert.2018.09.014) Copyright © 2018 American Society for Reproductive Medicine Terms and Conditions