臺灣博碩士論文加值系統

在正常的月經週期中，雌激素及黃體素調控子宮內膜的血管新生，而血管新生乃胚胎著床之成功關鍵。儘管已知雌激素能刺激子宮內膜產生血管內皮生長因子（VEGF）及後續之血管新生，但黃體素在輔助雌激素所誘發的血管新生作用所扮演之角色仍待闡明。更甚者，此二者造成的人類體內子宮內膜中血液動力學變化亦尚未被確認。本研究探討雌激素及黃體素在體外實驗中對內膜增生及VEGF產生量的影響，並監測它們在體內實驗中產生的血液動力學變化。為檢視雌激素及黃體素對子宮內膜上皮細胞（EEC）之影響，我們採用具有雌激素接受器（ER）的EEC細胞株Ishikawa及不具有ER的Ishikawa02來進行實驗。授予雌激素及黃體素顯著地刺激Ishikawa EEC增生，但對Ishikawa02則無此效應。十分有趣地，雌激素及黃體素能夠協同性地提昇Ishikawa EEC的VEGF產生量，此效應會被雌激素拮抗劑ICI182780所阻斷，但並不被黃體素拮抗劑RU486所影響。此外，上述雌激素及黃體素對VEGF產生量的提升效應並不呈現於Ishikawa02 EEC。接下來我們研究VEGF對滋養層細胞的水平及垂直移動能力的影響。使用BeWo滋養層細胞株為實驗模型，VEGF對滋養層細胞水平及垂直移動能力皆無法有所影響。最後，我們收集接受週期型荷爾蒙療法的早期更年期婦女，運用三維能量都卜勒超音波監測內膜血液動力學變化。療程包括11天的雌激素(E時期)，之後10天再加上人工合成黃體素（E／P時期)；依人工合成黃體素之不同分為兩組：具雄性素活性的norethisterone acetate及抗雄性素活性的cyproterone acetate。相較於治療前（零時期），子宮內膜體積在E時期及E／P時期均有一上升的趨勢。在接受週期性雌激素複合norethisterone acetate的個案，相較於零時期，在 E／P時期呈現了內膜後方血流指標上升的趨勢，而複合cyproterone acetate 則不然。我們的結果指出雌激素及黃體素經由雌激素接受器媒介的機制協同促進VEGF的產生量，且更一步地運用體內實驗方法展現了雌激素及黃體素的血管新生作用，此項發現可能對於了解子宮內膜如何準備迎接胚胎著床有所助益。

During normal menstrual cycle, estrogen (EB2B) and progesterone (PB4B) regulate endometrial angiogenesis that is important for successful embryo implantation. Although EB2B have been shown to stimulate endometrial production of vascular endothelial growth factor (VEGF) and subsequent angiogenesis, the role of progesterone in facilitating estrogen-induced angiogenesis remains unclear. Moreover, the hemodynamic effects of EB2B and PB4B on the human endometrium have not been confirmed. This study aimed to investigate the effects of EB2B and PB4B on endometrial proliferation and VEGF production in vitro as well as the hemodynamic changes induced by EB2B and PB4B treatment in vivo. To examine the effects of EB2B and PB4B on the endometrial epithelial cells (EEC), we utilized Ishikawa and Ishikawa02 cells that are positive and negative for estrogen receptor (ER) expression, respectively. Treatment with EB2B and PB4B significantly increased cell proliferation in Ishikawa but not Ishikawa02 cells. Interestingly, EB2B and PB4B could synergistically enhance VEGF production in Ishikawa cells. The EB2B/PB4B-induced VEGF secretion could be inhibited by the ER antagonist ICI182780, but not by the progesterone receptor antagonist RU486. Furthermore, the promoting effect of EB2B and PB4B on VEGF expression was not found in Ishikawa02 cells. Then we examined the effects of VEGF on trophoblast expansion and migration. Using BeWo trophoblast line, trophoblast expansion and migration were shown to be unaffected by VEGF treatment. Finally, the hemodynamic effects of cyclic estradiol and progestin treatment in early menopausal women were examined by three-dimensional power Doppler angiography (3D-PDA). The recruited patients received 11 days of estradiol (phase E) followed by 10 days of estradiol and progestin (phase E/P). Two different progestins were used, i.e., the androgenic norethisterone acetate and the anti-androgenic cyproterone acetate. There was a trend toward an increase in the endometrial volume in phase E and phase E/P, as compared with the phase 0 (before treatment). In patients receiving cyclic estradiol and norethisterone acetate, subendometrial vascular indices displayed increasing tendencies in phase E/P as compared with phase 0, which was not the case in patients taking cyclic estradiol and cyproterone acetate. Our results established that estrogen and progesterone synergistically enhanced VEGF production in an ER-dependent manner and further demonstrated the angiogenesis effect of estrogen and progesterone in vivo, findings that may be important for understanding endometrial preparation for implantation.

中文摘要 1
ABSTRACT 3
ACKNOWLEDGEMENTS 5
CHAPTER 1 INTRODUCTION 6
A. Roles of estrogen and progesterone in the proliferation, differentiation and angiogenesis of the endometrium during the menstrual cycle 6
1. Endometrium displays histological and physiological changes during the menstrual cycle. 6
2. Estrogen and progesterone control proliferation and differentiation of endometrium are critical for endometrial angiogenesis. 7
B. Expression and signaling of estrogen receptors (ERs) and progesterone receptors (PRs) in the endometrium. 8
1. Mechanism of estrogen receptors 8
2. Mechanism of progesterone receptors 9
3. States of ERs and PRs differ temporally and spatially 10
C. Role of vascular endothelial growth factor (VEGF) in estrogen/ progesterone- induced endometrial angiogenesis. 12
1. VEGF is an essential angiogenic signaling element 12
2. VEGF expression involved with estrogen and progesterone 12
D. Importance of endometrial angiogenesis preparation for embryo implantation 13
1. Successful embryo implantation is required for proper endometrial receptivity. 13
2. Establishment of microvasculature environment is a delicate process of endometrial angiogenesis. 14
3. Subendometrial zone contributes an important part in implantation. 14
E. Purpose 15
1. To determine the role of estrogen and progesterone receptors in mediating the effects of estrogen and progesterone on endometrial epithelial cells (EEC) 15
2. To investigate the role of VEGF in embryo implantation 15
3. To examine the hemodynamic changes of endometrial/ subendometrial vascular ties in early postmenopausal women taking cyclic estrogen and progestins 16
CHAPTER 2 MATERIALS AND METHODS 18
A. In vitro approaches 19
1. Cell culture 19
2. Trophoblast spheroid formation 20
3. Cell proliferstion 20
4. VEGF measurement 21
5. Trophoblast spheroid expansion assay 22
6. Alamar blue assay for trophoblast migration 23
B. In vivo approaches 24
1. Study design and cases recruitment 24
2. Data acquisition and analysis 25
C. Statistical analysis 27
CHAPTER 3 RESULTS 29
A. Estrogen and progesterone both induce cell proliferation in ER-positive EEC but not ER-negative 29
B. Estrogen and progesterone synergistically enhance VEGF production 29
C. Effects of different progestins on EEC proliferation and VEGF production. 30
D. Effects of VEGF on trophoblast expansion and migration 31
F Suubendometrial vascularity is increased in patients taking cyclic estradiol plus progestin with androgenic activity, but not in those taking estradiol plus progestin with anti-androgenic activity. 31
CHAPTER 4 DISCUSSIONS 33
APPENDIX 66
REFERENCES 67

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