| BackgroundA s a safe, highly effective, long acting and reversible contraceptive method, intrauterine device (IUD) is widely used in China. The main side effect of IUD is abnormal uterine bleeding which includes menorrhagia, prolonged menstrual period or spotting/bleeding. This is also the main reason hampered its continue using. Previous reports indicated there are many changes in the endometrium of women with abnormal bleeding induced by IUD. These include abnormal synthesis of prostanoid, increasing of abnormal fibrinolysis activity and regional inflammatory reaction, etc. But medicine of antiprostaglandin, antifibrinolysis and anti-inflammatory can't cure all these patients satisfactorily. There is periodical growing and shedding in human endometrium which followed with cyclical angiogenesis. This course is regulated by several vascular growth factors. Previous studies indicated that in the endometrium of women with unacceptable uterine bleeding induced by IUD, spiral artery dilated accompanied with degeneration of vessel wall and endothelial cells, expression of VEGF and mean vessel density increased. Therefore we presumed the abnormal structure and function of vascular regulated by several growth factor may be one reason of unacceptable uterine bleeding induced by IUD.Angiopoietin (Ang) is a new family of angiogenic growth factor. The main subfamily expressed in human is Ang-1 and Ang-2, both of whom are ligands for the endothelial cell-specific receptor tyrosine kinase Tie-2. They all participate inmodulating angiogenesis and vascular stability during inflammation, so they play an important role in biological and psychological angiogenesis. After binding and inducing autophosphorylation of Tie-2, Ang-1 promotes endothelial cell migration, sprouting, and survival in vitro; and increases new vessel growth, branching, maturation, and integrity in vivo. Ang-2 acts as a natural antagonist of Tie-2 signaling and subsequently leads to vessel destabilization and neovascularization in the presence of vascular endothelial cell growth factor (VEGF), or vascular regression in its absence. Both Ang-1 and Ang-2 are expressed in human endometrium. Expression of Ang-1 is progesterone dependent, and increased in the secretory phase. Ang-1 expression was decreased in women with menorrhagia. Expression of Ang-2 has no apparente changes through the menstrual cycle, but hypoxia and inflammatory factor and up-regulate Ang-2 expression in human endometrium endothelial cells. All these indicated that Ang-1 and Ang-2 may be involved in biological and psychological angiogenesis in endometrium.We collected the endometrium of women with abnormal bleeding after useing IUD then detected changes of Ang-1, Ang-2, and Tie-2 in mRNA level, and Ang-1, Ang-2 in protein level, so to investigate the bleeding mechanism in angiogenesis aspect and provide new idea for treatment of abnormal bleeding induced by IUD. MethodsThe patients were selected from outpatients of Women's Hospital, School of Medicine, Zhejiang University. Patients in the experimental group meet requirements as follows: ①ages range: 25～45 years old; ②haven't used steroid hormone in the last three months; ③with normal menstrual cycle before used IUD, and complaining of menorrhagia or prolonged menstrual period after IUD was inserted; ④exclude menorrhariga induced by hysteromyoma, adenomyosis or endometrial polyp through B-mode ultrasonography; ⑤the time limit of IUD using: 0.5~10 years; ⑥have no hepatopathy or hematopathy history; ⑦exclude LNG-IUS or IUD that has indometacin. Among 33 cases in the experimental group, 23 cases were during proliferative phase and 10 cases were during secretory phase. The control was normal endometrium, 12 cases were during proliferative phase and 16 cases were duringsecretory phase. There is comparability in ages and the phase of endometrium between the experimental group and control. The samples were divided into two portions, one part was storied in liquid nitrogen and another was fixed in 10% neutral formalin. Expression of Ang-1, Ang-2 and Tie-2 mRNA were determined by RT-PCR and Ang-1, Ang-2 protein were determined by immunohistochemistry. The degree of staining was semiquantitatively indexed using H scores. Differences in relative optical density and H-Score were analyzed by Independent Sample T-test and Nonparametric Test. Results1. Expression of Ang-1, Ang-2, Tie-2 mRNA in each groupThe expression of Ang-1, Ang-2, Tie-2 mRNA can be detected in human endometrium. Ang-1 mRNA decreased in both proliferative and secretory phase in the endometriun of women with IUD (p<0.05). In IUD secretory group, expression of Tie-2 and the ratio of Ang-1 /Ang-2 is also decreased when compared with normal (p＜0.05).2. Expression of Ang-1 proteinAng-1 has strong expression in gland epithelium, luminal epithelium, endothelial cells, and weak expression in stromal cells. During secretory phase, Ang-1 expression is increased, especially in decidualized stromal cells around spiral artery. The H-Score of Ang-1 in every group indicated that Ang-1 is increased in normal secretory phase (p<0.001). Ang-1 expression is decreased in both gland and stromal of IUD proliferative group (p＜0.001). Ang-1 expression is also decreased in the gland of IUD secretory group (p<0.001).3. Ang-2 expression in the endometriumAng-2 is mainly expressed in gland epithelium, luminal epithelium and endothelial cells. Expression of Ang-2 in stromal cells is weaker than Ang-1. Ang-2 expression is decreased from normal proliferative to secretory phase (p<0.05). In the IUD secretory group, Ang-2 expression in gland is increased when compared with normal secretory phase (p<0.05).Conclusion1. Ang-1 protein is increased while Ang-2 protein is decreased from normal proliferative to secretory phase. All these may promote vascular stability in secretory phase.2. Ang-1 mRNA and protein, Tie-2 mRNA decreased accompanied with Ang-2 protein increased in the endometrium of women with unacceptable bleeding that induced by IUD. These may affect the function of vessels and may be one reason for IUD induced abnormal uterine bleeding.
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