Progesterone responsiveness is not downregulated in adenomyosis.
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1 Progesterone responsiveness is not downregulated in adenomyosis. Takehiro Hiraoka Yasushi Hirota Tomoko Saito-Fujita Hirofumi Haraguchi Miyuki Harada Tetsuya Hirata Kaori Koga Osamu Hiraike Tomoyuki Fujii Yutaka Osuga Department of Obstetrics and Gynecology, Graduate School of Medicine, The University of Tokyo, Japan.
2 Background Ø P 4 resistance is involved in the pathophysiology of endometriosis. Hirota Y, et al. Am J Pathol 2008 Ø Although progestins (including oral contraceptives) have been widely used for the symptoms of adenomyosis, there still remains a population of the patients who do not sufficiently respond to progestins, suggesting that progesterone (P 4 ) resistance may be involved in the pathophysiology of adenomyosis. Ø However, its exact involvement in adenomyosis still remains unclear.
3 Aim of the study Research questions: Ø How is P 4 responsiveness of adenomyosis? Ø Is P 4 resistance involved in the pathophysiology of adenomyosis? Ø If so, how is it? If not, why do some patients show poor response to progestins? The aim of this study is to clarify these questions
4 Progesterone receptor expression is comparable between adenomyosis and endometrium PR immunostaining Human tissues Endometrium Adenomyosis Proliferative phase Secretory phase
5 Establishment of human adenomyosis-derived cells Non-adenomyosis patients ex. Uterine fibroids Endometrium Adenomyosis patients Adenomyotic lesions Tissue collection Control cells: ESCs (Endometrial stromal cells) DMEM/F12 5%FBS Cell culture Cell isolation -Mincing, Collagenase, Filtration(100µm, 70µm) Adenomyosis-derived cells: AdSCs (Adenomyosis-derived stromal cells)
6 Progesterone treatment of human adenomyosis-derived cells Day 1 Day 3 Day 5 Day 6 Control Estradiol-17β (E2) (10-8 M) Vehicle Cell lysis & RNA isolation AdSCs (Adenomyosis derived stromal cells) ESCs (Endometrial stromal cells) Estradiol-17β (E2) (10-8 M) Progesterone (P4) (10-6 M) P4 treatment Quantitative RT-PCR
7 P4-responsive genes is comparably induced in adenomyosis and endometrium ZBTB16 Hand2 Control P4 Control P4 Control P4 Control P4 FKBP5 PAI-1 Control P4 Control P4 Control P4 Control P4
8 P4 treatment significantly suppresses the expression of pro-inflammatory cytokine IL-6 in adenomyosis, but the suppression is not complete. *** IL-6 * ** IL-8 * Control P4 Control P4 Control P4 Control P4 ESCs AdSCs ESCs AdSCs u Basal levels of IL-6 were significantly high in AdSCs compared to ESCs. u P 4 treatment eliminated IL-6 expression both in ESCs and AdSCs, and however, IL-6 levels were higher even in P 4 -treated AdSCs than control ESCs.
9 Ø P 4 responsiveness is normal in adenomyosis. Ø Heightened levels of IL-6 in adenomyosis suggest that fundamental inflammatory environment may be associated with failure of progestin treatment in some adenomyosis patients. IL-6 expression is basically upregulated in adenomyosis. How is the status of STAT3, a downstream signaling pathway of IL-6?
10 Adenomyosis pstat3 immunostaining Endometrium Human tissues STAT3, a downstream signaling pathway of IL-6, is activated in adenomysis, regardless of menstrual phases. Proliferative phase Secretory phase
11 STAT3 is constantly expressed in human adenomyotic lesions pstat3 immunostaining Adenomyosis (Human tissues) Progestin treatment (Dienogest) GnRH agonist treatment u pstat3 expression is not fully suppressed with progestin treatment and -. GnRH agonist in lesions of adenomyosis. u Adenomyosis may have activated pro-inflammatory pathways even under anti-estrogen therapies.
12 Establishment of mouse adnomyosis model adenomyosis mouse model Lumen S Injury Needle Anti-mesometrial side of the uterus Myo Adenomyosis-like lesion
13 STAT3 is activated in mouse adnomyosis-like lesions Adenomyosis-like lesions (Mouse tissues) pstat3 immunostaining Myo Myo Lumen Lumen Five days after injury Fourteen days after injury
14 STAT3 promotes epithelial cell proliferation in the post-surgical uterus : Possible involvement of STAT3 in the pathogenesis of adenomyosis u Cell proliferation was down-regulated in the surgical site of the uterus in STAT3 cko mice Surgical sites of the uterus WT STAT3 cko Hiraoka T, Hirota Y, et al. JCI insight 2016, in press.
15 Hypothesis from our results Adenomyosis IL-6 Progestins Constant activation STAT3 Next questions: Maintenance of lesions?cell proliferation?cell survival?fibrosis Inflammation? pro-inflammatory cytokines Chronic inflammatory response Amplification mechanism?
16 Conclusions Ø P 4 responsiveness was not downregulated in adenomyosis. Ø IL-6/STAT3 signaling might be involved in the pathophysiology of adenomyosis. Ø Heightened levels of IL-6 and pstat3 in adenomyosis suggested that fundamental inflammatory environment may be associated with failure of progestin treatment in some adenomyosis patients.
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