| BackgroundAbnormal uterine bleeding occured in 1/3 of reproductive women. Using standard doses of hormone therapy, irregular vaginal bleeding occured in nearly half of the women, the mechanism of which might involve the regulation and expression of local factors, and the disorder of signal processing. These included the changes of the ratio of vascular endothelial growth factor and platelet thrombospondin-1 (preangiogenic factors/anti-angiogenesis factor), the changes of matrix metalloproteinases (MMPs) and its tissue inhibitor (TIMP), the changes of tissue factors, and the increase of endometrial leucocytes, especially the increase of uterine natural killer cells and et al. Extracellular matrix proteins were degraded by a variety of enzymes, but MMPs was generally thought to be the most important enzyme involved in extracellular matrix (ECM) reconstruction in the process of the shedding and implantation of menstrual tissues, whose activity was regulated from three kinds of levels:transcription, activation of proenzyme, inhibition of its specific endogenous inhibitor (TIMPs). MMPs was seemed to be controlled by ovarian steroid hormones, but progesterone didn't inhibit the expression of MMPs directly. Endometrial bleeding related factor Lefty was found to adjust the ECM collapse via synthesis suppression of collagen and synthesis stimulation of MMPs to promote collagen degradation. Lefty was an anti-TGF-βcytokine, and could interfere with the other members of TGF-βfamily to promote the integrity of endometrium, and played a critical role in the tissue reconstruction of implantation and menstrual shedding.ObjectiveIn this study, we chose three clinical oral progesterones, micronized progesterone (MP), dydrogesterone (DYD) and medroxyprogesterone acetate (MPA) to estrogen pretreated, ovariectomized mice, in order to observe the features of endometrial morphology and the expression profiles of 6 kinds of uterine bleeding related genes during sequential administration of estrogen/progesterone and after the withdrawal of estrogen/progesterone treatment. We also observed the effects of filtered sterilized peanut oil injected into uterine cavity of estrogen/progesterone pretreated, ovariectomized mice on endometrial morphology and detected the expression patterns of metalloproteinase and its inhibitor gene in uterine matrix of mice. ContentsPart one:to observe the features of endometrial morphology and the expression profiles of bleeding related genes in ovariectomized mice with sequential subcutaneous injection of estrogen/progesterone.Part two:to observe the features of endometrial morphology and the expression profiles of bleeding related genes in ovariectomized mice with oral administration of different progesterone.Part three:to observe the effects of peanut oil injected into uterine cavity of estrogen pretreated, ovariectomized mice on endometrial morphology and the expression patterns of metalloproteinase and its inhibitor gene.Methods1. Subjects:C57BL/6 mice,8 weeks, female.2. Administration Process:a) For each group of sequential estrogen/progesterone administration: subcutaneously to inject ovariectomized mice with 100ng 17β-estradiol at the day 1,2 and 3. No treatment was applied to the mice at the day 4,5 and 6. Then, subcutaneous injection of 5ng 17β-estradiol was combined with different doses of different progesterones at the day 7th,8th and 9th.b) For the group of solvent applied ovariectomized mice:for ovariectomized mice, the administration processes of day 1st to 9th were the same as above, but the agent was solvent only.c) For the group of solvent applied sham mice:for sham mice, the administration processes of day 1st to 9th were the same as above, but the agent was solvent only.3. Subjects grouping:a) Part one:a total of 36 mice were divided into 4 groups, there were 6 mice in the first three groups, including solvent applied ovariectomized mice group, oil applied sham mice group and 100ng 17β-estradiol applied 72h treatment group, respectively. Group 4th was divided into three cages including 6 mice each with subcutaneous injection of progesterone, and were sampled at three points:24h after first administration of progesterone,24h and 48h after last administration of progesterone.b) Part two:A total of 126 mice were divided into 7 groups as follow:Each group was divided into 3 cages including 18 mice each, and sampling time points were the same as part one.c) Part three:48 mice were divided into 8 groups. Sequential administration of estrogen/progesterone was corresponding to part one and part two. After 4h of last administration of progestogen,20μl filtered sterilized peanut oil was injected into the uterine cavity of mice. Sampling after 48h of the last administration.4. Observation Parameters 4.1 Serous concentrations of estradiol and progesterone; 4.2 Wet weight of uterus; 4.3 The morphological features of endometrium; 4.4 Histological localization of ERa and PR in endometrium; 4.5 Expression of uterine ERa, PR, Lefty, MMP3, MMP9 and TIMP-1.Results1. The level of serous estradiol was about 700pg/ml at 72h after subcutaneous injecting 100ng 17P-E2, and about 120pg/ml while the administration of 5ng 17(3-E2, which was significantly higher than the level at 48h after the last administration and the level in the solvent applied ovariectomized mice. The concentrations of serum estradiol in sham mice ranged from 94.85pg/ml to 364.20pg/ml. The concentrations of serum progesterone maintained at about 150nmol/L during the administration in the 500μg progesterone group with subcutaneous injection and 1.2mg MP group with intragastric administration, while the levels were significantly decreased (P=0.000) at 48h after the last administration. During the administration of 0.4mg MP and 0.8mg MP, progesterone concentrations were from 30 nmol/L to 60nmol/ L.2. After castration, the wet weight of the mice uterus were about 10-12mg, and raised to 32-35mg after the application of estrogen alone or combined estrogen/ progesterone. There were no significant differences about mice uterus wet weights between the administration of high-dose estrogen alone (ie. 100ng 17β-E2) and the combined administration of low-dose estrogen/progestogen (ie.5ngl7p-E2 combined with different doses and types of progestogen). There were no significant difference about mice uterus wet weights among the groups of administration with 5ngl7p-E2 combined with different doses and types of progestogen, except for 1.2mg MP group with lower uterus wet weight. After injection of peanut oil into uterus cavity of a variety of estrogen and progesterone pretreated mice, their uterine wet weights significantly decreased.3. After 72h of administration of high-dose estrogen alone, mice uterine luminal epithelium and glands showed proliferation-like, but more interstitial edema. After 24h of combined administration of low-dose estrogen/progesterone, mice endometrium showed compact stroma, no obvious edema, proliferated luminal epithelium and gland. After 72h of combined administration of low-dose estrogen/ progesterone, most of mouse endometrial glands and stroma showed secretory-like changes. After 72h of administration of higher-dose progesterone, estrogen pretreated endometrium performed that the ratio of proliferation-like change turning into secretory-like or mild atrophy changes were higher than that in the lower dose group, but there were no significant differences about the endometrial morphology in the different progesterone groups. After oil injected into uterine cavity in estrogen/progesterone pretreated, ovariectomized mice, part of the mouse endometrial stromal cells surrounding blood vessels become predecidual changes, but no typical endometrial shedding.4. During sequential administration of estrogen and progesterone, ERa localized in nuclei of epithelial cells and stromal cells. The nuclear staining was the deepest at 24h in combined administration of estrogen and progesterone group, and the numbers of positive cells were more than other groups. In this study, PR localized only in cytoplasm of stromal cell during combined administration of estrogen and progestogen, and the strength of staining was lower.5. During the administration of 0.4mg/0.8mg MP, the expression of ERa in mouse uterus was significantly decreased with the extension of treatment, while in the MP 1.2mg group, MPA 0.025mg group and DYD 0.08mg group, ERa expression sustained lower level in the administration of progestogen, which suggested that these 5 doses of progesterone could inhibit the expression of ERa. After 48h of the last administration of progesterone, ERa expression significantly increased. During administration of MPA4mg, ERa expression showed an increasing tendency.The PR expression had no significant change during administration of progesterone in MP0.4mg, DYD0.04mg groups, while other groups showed a decreasing trend in varying degrees.6. During administration of progestogen, the Lefty expression decreased in MP 0.8mg, DYD 0.08mg groups, but had no significant differences among MP 0.4mg, DYD 0.04mg and MPA 0.016mg groups. After the withdrawal of progesterone in these 5 dose groups, the expression of Lefty significantly increased. For MP 1.2mg and MPA 0.025mg administration and withdrawal, Lefty expression showed no significant difference.7. The expression condition of MMP3/MMP9/TIMP-1 in the part one and part two of this study:during the administration of MP 0.8mg/1.2mg and DYD 0.08mg, MMP3 expression level maintained at a lower level, but significantly increased after the withdrawal. In DYD 0.04mg group and MPA 0.016mg group, the expression of MMP3 increased during combined administration of estrogen/progestogen and sustained at a high level or even continued to rise. MMP3 ranged from 0.61 to 4.7 fold.During the administration of various doses of progesterone, the MMP9 expression was not significantly changed, but increased after withdrawal in MP 0.4/0.8mg group, MPA groups and DYD 0.04mg group. MMP9 ranged from 1.1 to 4.1 fold. During the administration of various doses of progestogen, the TIMP-1 expression was not significantly changed, but increased after withdrawal in DYD0.08 group, MPA group. The expression of TIMP-1 was no significant difference after withdrawal in MP group. TIMP-1 ranged from 0.69 to 6.03 fold.8. The expression condition of MMP3/MMP9/TIMP-1 in the third part of this study:in MPA 0.025mg group and MP group, MMP3 expression levels in uterus oil injection mice were significantly higher than that in control mice. MMP3 expression levels increased in MP 0.4mg/0.8mg group. The increase of TIMP-1 expression only showed in MP 0.8mg group.Conclusions1. Administration of estrogen or estrogen/progesterone could increase the weight of wet uterus in ovariectomized mice.2. Different progestogens were able to change the proliferation of endometrium into secretion or mild atrophy in estrogen pretreated ovariectomized mice. However, this process needed a interval of medication and agent doses after administration.3. ERa and PR expression decreased in the endometrium with the extension of administration of estrogen/progesterone.4. A lower dose of progesterones, such as MP 0.4mg, DYD 0.04mg and MPA 0.016mg in this study, weakly inhibit the expression of ERa, PR, Lefty, MMP3 in mouse uterus during the administration.5. The expression of MMP3 and MMP9 may be more closely related to the progestogen dose, and higher-dose of progestogen inhibit their expression. The association between expression of TIMP-1 and dose of progestogen was unclear.6. In this study, the injection of oil into uterine cavity of estrogen/progestogen pretreated mice could not cause typical deciduas-like change and shedding of endometrium.
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