孕激素不同种类及不同剂量对小鼠子宫内膜形态及出血有关基因表达影响的比较研究
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摘要
背景
1/3的育龄妇女可发生子宫异常出血,妇女应用标准剂量雌孕激素序贯治疗初期,有将近一半出现不规则阴道出血。子宫内膜出血的机制可能涉及局部介质的调节、表达和信号处理的紊乱,包括血管内皮生长因子与血小板凝血酶敏感蛋白-1的比值改变(前血管生成因子/抗血管生成因子)；基质金属蛋白酶(MMPs)及组织基质金属蛋白酶抑制剂(TIMP)的改变；组织因子的改变；内膜白细胞尤其是子宫自然杀伤细胞的增加等等。细胞外基质蛋白被多种酶降解,但一般认为MMPs是内膜组织脱落和胚胎种植过程中细胞外基质(ECM)降解和重建中最重要的酶之一,其活性在3个水平上被调控：转录、酶原的激活、被TIMPs的抑制。MMPs受卵巢性甾体激素控制的,但是,这种调控作用并不是直接的。内膜出血相关因子(Lefty)通过抑制胶原合成、刺激MMPs生成以促进胶原降解,来调节ECM的崩解。Lefty是一个抗TGF-β的细胞因子,可干扰TGF-β家族的其他成员的促进子宫内膜完整性作用,与胚胎植入和月经期内膜组织脱落过程中的组织重建关系密切。已有研究提示异常出血时内膜Lefty的表达增强,使MMPs的生成过量,继而ECM与血管基底膜不适时的崩解。
不同种类及剂量的孕激素对于各种出血问题的治疗效果,以及不同种类及剂量的孕激素应用于性激素治疗方案时出血模式存在细微的差别,而造成这种差别的原因尚不明了。本研究观察不同种类及剂量的孕激素应用期间和撤退后内膜的形态学表现,检测与出血有关基因的表达情况,试图发现各项观察指标在不同孕激素处理组中可能存在的差异,以期将这些差异与临床出血问题相联系,在一定程度上辅助临床工作者选择孕激素种类和剂量。
目的
本研究以目前临床常用的三种口服孕激素——微粉化黄体酮(MP)、地屈孕酮(DYD)和醋酸甲羟孕酮(MPA)应用于雌激素预处理的去势小鼠,观察序贯应用雌孕激素期间以及雌孕激素撤退后,子宫内膜形态学表现、子宫组织与出血相关的6种基因的表达情况。向雌孕激素预处理的去势小鼠子宫腔注射过滤除菌的花生油,观察不同孕激素组小鼠内膜形态学特点,检测此期间小鼠子宫MMPs及TIMP-1的表达情况。
内容
1.第一部分观察序贯经皮下给予雌孕激素过程中去势小鼠子宫内膜形态学表现,检测与出血有关基因的表达情况
2.第二部分观察不同种类不同剂量的口服孕激素作用于雌激素预处理的去势小鼠时子宫内膜的形态学表现,检测与出血有关的基因表达的情况
3.第三部分向雌激素与不同孕激素预处理的去势小鼠子宫腔内注射花生油,观察内膜形态学表现,检测MMPs及TIMP-1基因表达的情况
方法
1.实验对象：8周龄雌性C57BL/6小鼠
2.给药流程：
2.1各个序贯给予雌孕激素组：去势成功小鼠,第1、2、3天皮下注射17β-雌二醇(17β-E2)100nng,第4、5、6天不予处理,第7、8、9天皮下注射17β-E25ng合并不同剂量不同种类的孕激素；
2.2溶剂对照组1(去势溶剂组)：去势成功小鼠,第1-9天操作流程与2.1所述相同,但仅注射或灌胃溶剂。
2.3溶剂对照组2(假去势溶剂组)：假去势小鼠,第1-9天操作流程与2.1所述相同,但仅注射或灌胃溶剂。
3.实验对象分组：
3.1第一部分：共36只小鼠,分4组,1-3组每组6只,分别为去势溶剂组、假去势油剂组和100ng17β-E272h组,第4组为序贯经皮下给予雌、孕激素(孕酮)组,又分为3亚组,每组6只,分别为皮下注射孕酮一次后24h取材组(皮下首次24h组)；每天一次、连续3天、末次注射后24h取材组(皮下末次24h组)；每天一次、连续3天、末次注射后48h取材组(皮下末次48h组)。
3.2第二部分：共126只小鼠,分组如下：
·微粉化黄体酮0.4mg/只组(MP0.4mg组)
·微粉化黄体酮0.8mg/只组(MP0.8mg组)
·微粉化黄体酮1.2mg/只组(MP1.2mg组)
·地屈孕酮0.04mg/只组(DYD0.04mg组)
·地屈孕酮0.08mg/只组(DYD0.08mg组)
·醋酸甲羟孕酮0.016mg/只组(MPA0.016mg组)
·醋酸甲羟孕酮0.025mg/只组(MPA0.025mg组)
每组再分3个亚组,分别为灌胃1次后24h取材组(灌胃首次24h组)；每天一次、连续3天、末次灌胃后24h取材组(灌胃末次24h组)；每天一次、连续3天、末次灌胃后48h取材组(灌胃末次48h组)。
3.3第三部分：共48只小鼠,分8组,每组6只。序贯给予雌孕激素方案及给药途径与研究第1、2部分一致,在末次应用孕激素后4h,向小鼠子宫腔注射20μ1过滤除菌的花生油,并在末次应用孕激素后48h取材(皮下末次48h组(打油)或灌胃末次48h组(打油))。
4.观察指标：
4.1子宫湿重
4.2血清E2和孕酮浓度
4.3子宫内膜形态学表现
4.4 ERa和PR在内膜的组织学定位
4.5子宫ERα、PR、Lefty、MMP3、MMP9及TIMP-1的表达
结果
1.子宫湿重：取材时,鼠龄约11-12周,体重约20g。
1.1研究第一部分：去势溶剂组、假去势溶剂组和100ng17β-E2 72h组的小鼠子宫湿重平均为11.28mg、32.03mg和34.46mg,经皮注射雌孕激素组的三个亚组子宫湿重分别为34.92mg、32.78mg和30.83mg,去势溶剂组小鼠子宫湿重显著低于其余各组(P=0.000),而其余各组间无显著差异(P>0.05)。
1.2研究第二部分：7个口服孕激素组的21个亚组小鼠子宫湿重在28.51-39.04mg之间,MP1.2组中的灌胃末次24h亚组的小鼠子宫湿重为28.51 mg,显著低于其他各亚组(P<0.01),而其余各亚组间无显著差异(P>0.05)。
1.3研究第三部分：皮下末次48h组(打油)和灌胃末次48h组(打油)小鼠子宫湿重在20.8-24.94之间,组间无显著差异(P=0.225),但显著低于各自对应的未打油的末次应用孕激素48h组小鼠子宫湿重(P<0.001)。
2.血清雌孕激素水平：
2.1血清E2：100ng17β-E2 72h组小鼠血清雌二醇水平在700pg/ml左右,而应用5ng17β-E2期间,则雌二醇维持于120pg/ml左右,显著高于5ngl7β-E2末次应用48h后(29.66pg/ml,P=0.000)和去势溶剂组(15.8pg/ml,P=0.000)。假去势组小鼠的血清雌二醇浓度在94.85-364.20pg/ml之间。
2.2血清孕酮：在皮下注射500μg孕酮组及MP1.2mg灌胃组,用药期间小鼠血清孕酮在150nmol.L左右,应用MP0.4mg和MP0.8mg期间,小鼠血清孕酮在30-60nmol/L之间,末次用药48h孕酮水平均显著下降。应用DYD和MPA的各组小鼠血清孕酮均低于10nmol/L.
3.子宫内膜形态学表现：
3.1研究第一部分：100ng17β-E272h组小鼠子宫内膜腔上皮和腺体均呈增殖样表现,间质较水肿明显；皮下首次24h组小鼠内膜腔上皮和腺体为增殖样表现,间质致密,其中1只小鼠内膜出现分泌样改变(1/6)；皮下末次24h组小鼠子宫内膜腺体和间质均呈分泌样改变(5/5)。
3.2研究第二部分：孕激素末次灌胃24h时,MP1.2mg组雌激素预处理的内膜由增殖样转化为分泌样的比例为2/5,另3只小鼠内膜大部分呈分泌样改变而局部有轻度萎缩表现；MP0.8mg组和DYD0.08mg组小鼠内膜均由增殖样转化为分泌样表现；MP0.4mg组、MPA0.016mg组、DYD0.04mg组内膜转化比例分别为2/5、4/6和5/6。分泌样内膜的形态学表现在不同种类孕激素组间未呈现明显差别。
3.3研究第三部分：向雌孕激素预处理的去势小鼠子宫腔注射油剂后,部分小鼠内膜局部血管周围基质细胞呈现前蜕膜样改变,其中,皮下注射孕酮组5只小鼠中有3只局部出现前蜕膜样细胞,MP0.8mg组、MP1.2mg组和DYD 0.08mg组均有2/5小鼠内膜出现前蜕膜样细胞,而其余各组仅有1/5-1/6出现前蜕膜样细胞。各组均未见典型的内膜脱落表现。
4.序贯应用雌孕激素过程中,ERa定位于上皮细胞和基质细胞胞核,孕激素首次应用24h时胞核着色最深,阳性细胞最多；而PR仅在联合应用雌孕激素过程中的基质细胞胞浆着色,且着色强度较低。
5.子宫组织ERa基因的表达情况：
5.1研究第一部分：ERa表达量在100ng17β-E2 72h组最低,而在去势溶剂组最高,而假去势溶剂组和序贯经皮注射雌孕激素(孕酮)组的三个亚组ERa的表达水平之间无显著差异(P>0.05)。
5.2研究第二部分：ERa在MP1.2mg、MPA0.025mg和DYD0.08mg应用期间的表达量持续较低,与经皮孕酮组相近,而ERa表达量在MP0.4mg/0.8mg应用期间,随用药时间延长而显著降低(P=0.000 vs P=0.018),上述5组孕激素撤退后ERa的表达量均升高；MPA0.016mg组ERa表达量呈现升高趋势(P=0.013)。
6.子宫组织PR基因的表达情况：
6.1研究第一部分：PR在去势溶剂组、假去势溶剂组和100ng17β-E2 72h组的表达量均较低,组间无显著差异(P>0.05)；PR在皮下首次24h组表达水平最高,随孕酮应用72h时PR表达量下降,但差异无显著意义(P=0.522),至皮下末次48h时PR表达量显著下降(P=0.020)。
6.2研究第二部分：PR在MPA0.8mg、MPA0.016mg和MPA0.025mg应用及撤退期间的表达模式与皮下孕酮组相似,撤药后表达量显著降低(P值分别为0.004、0.000和0.000)；在MP1.2mg和DYD0.08mg应用期间,PR表达量显著下降(P=0.029 vs P=0.003)；MP0.4mg和DYD0.04mg组,用药及撤药期间PR表达量均无显著改变(P>0.05)。
7.子宫组织Lefty基因的表达情况：
7.1研究第一部分：Lefty在假去势溶剂组表达量最低,与之相比,100ng17p-E272h组、皮下首次24h组和皮下末次24h组的表达量较高,但后3组间无显著差异(P>0.05)。孕酮撤退后,Lefty表达量进一步显著升高(P<0.05)。经皮孕酮撤药后Lefty的表达量是末次24h时Lefty表达量的1.54倍。
7.2研究第二部分：Lefty在MP1.2mg和MPAO.025mg应用期间和撤药后的表达量均无显著改变(P>0.05)。应用MP0.8mg和DYDO.08mg期间Lefty的表达量显著下降(P=0.008 vs P=0.049),而应用MP0.4mg、DYDO.04mg和MPAO.016mg期间Lefty表达量无显著改变(P值分别为0.985、0.275和0.277),但这5个剂量的孕激素撤退后,Lefty表达均显著升高(P<0.01)。MPO.8mg、DYDO.08mg和MPAO.016mg撤药后Lefty表达量是灌胃末次24h时的2.31倍、3.99倍和2.81倍；而MP1.2mg组和MPA0.25mg组为1.15倍和0.97倍；MP0.8mg和DYD0.04mg组分别为1.59倍和1.67倍。
8.子宫组织MMP3/MMP9/TIMP-1基因的表达：
8.1研究第一部分：
(A)MMP3在去势溶剂组、假去势溶剂组和100ng17β-E2 72h组的表达量均较低(P>0.05),皮下首次24h组和皮下末次24h组与之相比,MMP3表达量显著升高(P<0.05),而孕酮撤退后,MMP3的表达量显著高于本部分研究中其他各组(P<0.01)。经皮孕酮撤退后MMP3表达量是经皮末次24h时的1.95倍。
(B)假去势溶剂组、100ng17β-E2 72h组及皮下注射孕酮期间的MMP9表达量均较低,组间无显著差异(P>0.05),均显著低于孕酮撤退后的MMP9表达水平(P值分别为0.000、0.002、0.000和0.001)。经皮孕酮撤退后MMP9表达量是经皮末次24h的1.78倍。经皮孕酮撤退后MMP3表达量是经皮末次24h的1.95倍。
(C)去势溶剂组和假去势溶剂组小鼠子宫TIMP-1的表达量均较低,而应用性甾体激素后,包括单雌激素或雌孕激素联合应用,TIMP-1表达量升高,且随孕激素应用时间延长而进一步升高(P=0.022)。
8.2研究第二部分：
(A)MMP3基因的表达在MP0.8mg组、MP1.2mg组和DYD0.08mg组用药期间和撤药后的表达模式一致,即应用期间表达水平无显著改变(P值分别为0.527、0.480和0.303)而撤药后表达水平显著升高(P值分别为0.002、0.006和0.000)；MMP3的表达量随着应用MPA0.016/0.025mg的时间延长而显著升高(P=0.000 vs P=0.043),而撤药短期内MMP3表达量的改变并不显著。MPl.2mg撤退后MMP3表达量是灌胃末次24h的4.73倍；MP0.8mg组和DYD0.08mg组分别为2.07倍和2.25倍；MP0.4mg组和MPA0.025mg分别为1.36倍和1.14倍。
(B)各种剂量孕激素应用期间MMP9表达量均无显著改变,但MP0.4mg/0.8mg组、MPA组和DYDO.04mg组,停药后表达量升高；而MP1.2mg和DYDO.08mg组停药后MMP9的表达量亦无显著改变。MPO.8mg/1.2mg、MPAO.016mg/0.025mg撤退后MMP9表达量是灌胃末次24h的2.93倍、2.99倍、2.32倍和2.39倍；MP1.2mg、DYDO.04mg和DYDO.08mg分别为1.24倍、1.14倍和1.28倍。
(C) TIMP-1的表达量各种类、各剂量口服孕激素应用期间均无显著改变,但DYDO.08mg、MPAO.016mg、MPAO.025mg撤药后TIMP-1的表达量显著升高(P值分别为0.000、0.007和0.002)。
8.3研究第三部分：宫腔打油后小鼠子宫MMP3表达量升高出现在MPA 0.025mg组、MP0.4mg/0.8mg/1.2mg组,MMP9表达升高出现在MP0.4mg/0.8mg组,而TIMP-1表达量的升高仅出现在MP0.8mg组。
结论
1.不同种类及剂量的孕激素与雌激素联合应用均可使去势小鼠子宫湿重增加,但较高剂量MP组子宫湿重低于其他孕激素组。
2.不同种类及剂量的孕激素应用72h时均能够使雌激素预处理的去势小鼠子宫内膜由增殖转为分泌或轻度萎缩,较高剂量组的转化比例高于较低剂量组。
3.孕激素首次应用24h时ERα和PR在子宫内膜的免疫染色最强,随着用药时间延长,ERa和PR的染色均降低。
4.较低剂量孕激素应用期间对小鼠子宫组织ERα、PR、Lefty、MMP3基因表达的抑制作用较弱；而较高剂量对内膜基因表达的抑制作用较强,以至于未出现撤药后相关基因(如Lefty、MMP3、MMP9)表达升高的表现。孕激素对Lefty和MMPs表达抑制作用的强弱可能与内膜不规则出血发生情况有关,推测应用较低剂量孕激素可能更易发生出血。
5.向雌孕激素方案预处理后的小鼠子宫腔注射油剂内膜出现蜕膜样细胞,但程度及范围均很小,应用较高剂量孕激素组小鼠内膜出现蜕膜样细胞的比例稍高。
Background
Abnormal 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.
Objective
In 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.
Contents
Part 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.
Methods
1. 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.
Results
1. 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.
Conclusions
1. 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.
1/3的育龄妇女可发生子宫异常出血,妇女应用标准剂量雌孕激素序贯治疗初期,有将近一半出现不规则阴道出血。子宫内膜出血的机制可能涉及局部介质的调节、表达和信号处理的紊乱,包括血管内皮生长因子与血小板凝血酶敏感蛋白-1的比值改变(前血管生成因子/抗血管生成因子)；基质金属蛋白酶(MMPs)及组织基质金属蛋白酶抑制剂(TIMP)的改变；组织因子的改变；内膜白细胞尤其是子宫自然杀伤细胞的增加等等。细胞外基质蛋白被多种酶降解,但一般认为MMPs是内膜组织脱落和胚胎种植过程中细胞外基质(ECM)降解和重建中最重要的酶之一,其活性在3个水平上被调控：转录、酶原的激活、被TIMPs的抑制。MMPs受卵巢性甾体激素控制的,但是,这种调控作用并不是直接的。内膜出血相关因子(Lefty)通过抑制胶原合成、刺激MMPs生成以促进胶原降解,来调节ECM的崩解。Lefty是一个抗TGF-β的细胞因子,可干扰TGF-β家族的其他成员的促进子宫内膜完整性作用,与胚胎植入和月经期内膜组织脱落过程中的组织重建关系密切。已有研究提示异常出血时内膜Lefty的表达增强,使MMPs的生成过量,继而ECM与血管基底膜不适时的崩解。
不同种类及剂量的孕激素对于各种出血问题的治疗效果,以及不同种类及剂量的孕激素应用于性激素治疗方案时出血模式存在细微的差别,而造成这种差别的原因尚不明了。本研究观察不同种类及剂量的孕激素应用期间和撤退后内膜的形态学表现,检测与出血有关基因的表达情况,试图发现各项观察指标在不同孕激素处理组中可能存在的差异,以期将这些差异与临床出血问题相联系,在一定程度上辅助临床工作者选择孕激素种类和剂量。
目的
本研究以目前临床常用的三种口服孕激素——微粉化黄体酮(MP)、地屈孕酮(DYD)和醋酸甲羟孕酮(MPA)应用于雌激素预处理的去势小鼠,观察序贯应用雌孕激素期间以及雌孕激素撤退后,子宫内膜形态学表现、子宫组织与出血相关的6种基因的表达情况。向雌孕激素预处理的去势小鼠子宫腔注射过滤除菌的花生油,观察不同孕激素组小鼠内膜形态学特点,检测此期间小鼠子宫MMPs及TIMP-1的表达情况。
内容
1.第一部分观察序贯经皮下给予雌孕激素过程中去势小鼠子宫内膜形态学表现,检测与出血有关基因的表达情况
2.第二部分观察不同种类不同剂量的口服孕激素作用于雌激素预处理的去势小鼠时子宫内膜的形态学表现,检测与出血有关的基因表达的情况
3.第三部分向雌激素与不同孕激素预处理的去势小鼠子宫腔内注射花生油,观察内膜形态学表现,检测MMPs及TIMP-1基因表达的情况
方法
1.实验对象：8周龄雌性C57BL/6小鼠
2.给药流程：
2.1各个序贯给予雌孕激素组：去势成功小鼠,第1、2、3天皮下注射17β-雌二醇(17β-E2)100nng,第4、5、6天不予处理,第7、8、9天皮下注射17β-E25ng合并不同剂量不同种类的孕激素；
2.2溶剂对照组1(去势溶剂组)：去势成功小鼠,第1-9天操作流程与2.1所述相同,但仅注射或灌胃溶剂。
2.3溶剂对照组2(假去势溶剂组)：假去势小鼠,第1-9天操作流程与2.1所述相同,但仅注射或灌胃溶剂。
3.实验对象分组：
3.1第一部分：共36只小鼠,分4组,1-3组每组6只,分别为去势溶剂组、假去势油剂组和100ng17β-E272h组,第4组为序贯经皮下给予雌、孕激素(孕酮)组,又分为3亚组,每组6只,分别为皮下注射孕酮一次后24h取材组(皮下首次24h组)；每天一次、连续3天、末次注射后24h取材组(皮下末次24h组)；每天一次、连续3天、末次注射后48h取材组(皮下末次48h组)。
3.2第二部分：共126只小鼠,分组如下：
·微粉化黄体酮0.4mg/只组(MP0.4mg组)
·微粉化黄体酮0.8mg/只组(MP0.8mg组)
·微粉化黄体酮1.2mg/只组(MP1.2mg组)
·地屈孕酮0.04mg/只组(DYD0.04mg组)
·地屈孕酮0.08mg/只组(DYD0.08mg组)
·醋酸甲羟孕酮0.016mg/只组(MPA0.016mg组)
·醋酸甲羟孕酮0.025mg/只组(MPA0.025mg组)
每组再分3个亚组,分别为灌胃1次后24h取材组(灌胃首次24h组)；每天一次、连续3天、末次灌胃后24h取材组(灌胃末次24h组)；每天一次、连续3天、末次灌胃后48h取材组(灌胃末次48h组)。
3.3第三部分：共48只小鼠,分8组,每组6只。序贯给予雌孕激素方案及给药途径与研究第1、2部分一致,在末次应用孕激素后4h,向小鼠子宫腔注射20μ1过滤除菌的花生油,并在末次应用孕激素后48h取材(皮下末次48h组(打油)或灌胃末次48h组(打油))。
4.观察指标：
4.1子宫湿重
4.2血清E2和孕酮浓度
4.3子宫内膜形态学表现
4.4 ERa和PR在内膜的组织学定位
4.5子宫ERα、PR、Lefty、MMP3、MMP9及TIMP-1的表达
结果
1.子宫湿重：取材时,鼠龄约11-12周,体重约20g。
1.1研究第一部分：去势溶剂组、假去势溶剂组和100ng17β-E2 72h组的小鼠子宫湿重平均为11.28mg、32.03mg和34.46mg,经皮注射雌孕激素组的三个亚组子宫湿重分别为34.92mg、32.78mg和30.83mg,去势溶剂组小鼠子宫湿重显著低于其余各组(P=0.000),而其余各组间无显著差异(P>0.05)。
1.2研究第二部分：7个口服孕激素组的21个亚组小鼠子宫湿重在28.51-39.04mg之间,MP1.2组中的灌胃末次24h亚组的小鼠子宫湿重为28.51 mg,显著低于其他各亚组(P<0.01),而其余各亚组间无显著差异(P>0.05)。
1.3研究第三部分：皮下末次48h组(打油)和灌胃末次48h组(打油)小鼠子宫湿重在20.8-24.94之间,组间无显著差异(P=0.225),但显著低于各自对应的未打油的末次应用孕激素48h组小鼠子宫湿重(P<0.001)。
2.血清雌孕激素水平：
2.1血清E2：100ng17β-E2 72h组小鼠血清雌二醇水平在700pg/ml左右,而应用5ng17β-E2期间,则雌二醇维持于120pg/ml左右,显著高于5ngl7β-E2末次应用48h后(29.66pg/ml,P=0.000)和去势溶剂组(15.8pg/ml,P=0.000)。假去势组小鼠的血清雌二醇浓度在94.85-364.20pg/ml之间。
2.2血清孕酮：在皮下注射500μg孕酮组及MP1.2mg灌胃组,用药期间小鼠血清孕酮在150nmol.L左右,应用MP0.4mg和MP0.8mg期间,小鼠血清孕酮在30-60nmol/L之间,末次用药48h孕酮水平均显著下降。应用DYD和MPA的各组小鼠血清孕酮均低于10nmol/L.
3.子宫内膜形态学表现：
3.1研究第一部分：100ng17β-E272h组小鼠子宫内膜腔上皮和腺体均呈增殖样表现,间质较水肿明显；皮下首次24h组小鼠内膜腔上皮和腺体为增殖样表现,间质致密,其中1只小鼠内膜出现分泌样改变(1/6)；皮下末次24h组小鼠子宫内膜腺体和间质均呈分泌样改变(5/5)。
3.2研究第二部分：孕激素末次灌胃24h时,MP1.2mg组雌激素预处理的内膜由增殖样转化为分泌样的比例为2/5,另3只小鼠内膜大部分呈分泌样改变而局部有轻度萎缩表现；MP0.8mg组和DYD0.08mg组小鼠内膜均由增殖样转化为分泌样表现；MP0.4mg组、MPA0.016mg组、DYD0.04mg组内膜转化比例分别为2/5、4/6和5/6。分泌样内膜的形态学表现在不同种类孕激素组间未呈现明显差别。
3.3研究第三部分：向雌孕激素预处理的去势小鼠子宫腔注射油剂后,部分小鼠内膜局部血管周围基质细胞呈现前蜕膜样改变,其中,皮下注射孕酮组5只小鼠中有3只局部出现前蜕膜样细胞,MP0.8mg组、MP1.2mg组和DYD 0.08mg组均有2/5小鼠内膜出现前蜕膜样细胞,而其余各组仅有1/5-1/6出现前蜕膜样细胞。各组均未见典型的内膜脱落表现。
4.序贯应用雌孕激素过程中,ERa定位于上皮细胞和基质细胞胞核,孕激素首次应用24h时胞核着色最深,阳性细胞最多；而PR仅在联合应用雌孕激素过程中的基质细胞胞浆着色,且着色强度较低。
5.子宫组织ERa基因的表达情况：
5.1研究第一部分：ERa表达量在100ng17β-E2 72h组最低,而在去势溶剂组最高,而假去势溶剂组和序贯经皮注射雌孕激素(孕酮)组的三个亚组ERa的表达水平之间无显著差异(P>0.05)。
5.2研究第二部分：ERa在MP1.2mg、MPA0.025mg和DYD0.08mg应用期间的表达量持续较低,与经皮孕酮组相近,而ERa表达量在MP0.4mg/0.8mg应用期间,随用药时间延长而显著降低(P=0.000 vs P=0.018),上述5组孕激素撤退后ERa的表达量均升高；MPA0.016mg组ERa表达量呈现升高趋势(P=0.013)。
6.子宫组织PR基因的表达情况：
6.1研究第一部分：PR在去势溶剂组、假去势溶剂组和100ng17β-E2 72h组的表达量均较低,组间无显著差异(P>0.05)；PR在皮下首次24h组表达水平最高,随孕酮应用72h时PR表达量下降,但差异无显著意义(P=0.522),至皮下末次48h时PR表达量显著下降(P=0.020)。
6.2研究第二部分：PR在MPA0.8mg、MPA0.016mg和MPA0.025mg应用及撤退期间的表达模式与皮下孕酮组相似,撤药后表达量显著降低(P值分别为0.004、0.000和0.000)；在MP1.2mg和DYD0.08mg应用期间,PR表达量显著下降(P=0.029 vs P=0.003)；MP0.4mg和DYD0.04mg组,用药及撤药期间PR表达量均无显著改变(P>0.05)。
7.子宫组织Lefty基因的表达情况：
7.1研究第一部分：Lefty在假去势溶剂组表达量最低,与之相比,100ng17p-E272h组、皮下首次24h组和皮下末次24h组的表达量较高,但后3组间无显著差异(P>0.05)。孕酮撤退后,Lefty表达量进一步显著升高(P<0.05)。经皮孕酮撤药后Lefty的表达量是末次24h时Lefty表达量的1.54倍。
7.2研究第二部分：Lefty在MP1.2mg和MPAO.025mg应用期间和撤药后的表达量均无显著改变(P>0.05)。应用MP0.8mg和DYDO.08mg期间Lefty的表达量显著下降(P=0.008 vs P=0.049),而应用MP0.4mg、DYDO.04mg和MPAO.016mg期间Lefty表达量无显著改变(P值分别为0.985、0.275和0.277),但这5个剂量的孕激素撤退后,Lefty表达均显著升高(P<0.01)。MPO.8mg、DYDO.08mg和MPAO.016mg撤药后Lefty表达量是灌胃末次24h时的2.31倍、3.99倍和2.81倍；而MP1.2mg组和MPA0.25mg组为1.15倍和0.97倍；MP0.8mg和DYD0.04mg组分别为1.59倍和1.67倍。
8.子宫组织MMP3/MMP9/TIMP-1基因的表达：
8.1研究第一部分：
(A)MMP3在去势溶剂组、假去势溶剂组和100ng17β-E2 72h组的表达量均较低(P>0.05),皮下首次24h组和皮下末次24h组与之相比,MMP3表达量显著升高(P<0.05),而孕酮撤退后,MMP3的表达量显著高于本部分研究中其他各组(P<0.01)。经皮孕酮撤退后MMP3表达量是经皮末次24h时的1.95倍。
(B)假去势溶剂组、100ng17β-E2 72h组及皮下注射孕酮期间的MMP9表达量均较低,组间无显著差异(P>0.05),均显著低于孕酮撤退后的MMP9表达水平(P值分别为0.000、0.002、0.000和0.001)。经皮孕酮撤退后MMP9表达量是经皮末次24h的1.78倍。经皮孕酮撤退后MMP3表达量是经皮末次24h的1.95倍。
(C)去势溶剂组和假去势溶剂组小鼠子宫TIMP-1的表达量均较低,而应用性甾体激素后,包括单雌激素或雌孕激素联合应用,TIMP-1表达量升高,且随孕激素应用时间延长而进一步升高(P=0.022)。
8.2研究第二部分：
(A)MMP3基因的表达在MP0.8mg组、MP1.2mg组和DYD0.08mg组用药期间和撤药后的表达模式一致,即应用期间表达水平无显著改变(P值分别为0.527、0.480和0.303)而撤药后表达水平显著升高(P值分别为0.002、0.006和0.000)；MMP3的表达量随着应用MPA0.016/0.025mg的时间延长而显著升高(P=0.000 vs P=0.043),而撤药短期内MMP3表达量的改变并不显著。MPl.2mg撤退后MMP3表达量是灌胃末次24h的4.73倍；MP0.8mg组和DYD0.08mg组分别为2.07倍和2.25倍；MP0.4mg组和MPA0.025mg分别为1.36倍和1.14倍。
(B)各种剂量孕激素应用期间MMP9表达量均无显著改变,但MP0.4mg/0.8mg组、MPA组和DYDO.04mg组,停药后表达量升高；而MP1.2mg和DYDO.08mg组停药后MMP9的表达量亦无显著改变。MPO.8mg/1.2mg、MPAO.016mg/0.025mg撤退后MMP9表达量是灌胃末次24h的2.93倍、2.99倍、2.32倍和2.39倍；MP1.2mg、DYDO.04mg和DYDO.08mg分别为1.24倍、1.14倍和1.28倍。
(C) TIMP-1的表达量各种类、各剂量口服孕激素应用期间均无显著改变,但DYDO.08mg、MPAO.016mg、MPAO.025mg撤药后TIMP-1的表达量显著升高(P值分别为0.000、0.007和0.002)。
8.3研究第三部分：宫腔打油后小鼠子宫MMP3表达量升高出现在MPA 0.025mg组、MP0.4mg/0.8mg/1.2mg组,MMP9表达升高出现在MP0.4mg/0.8mg组,而TIMP-1表达量的升高仅出现在MP0.8mg组。
结论
1.不同种类及剂量的孕激素与雌激素联合应用均可使去势小鼠子宫湿重增加,但较高剂量MP组子宫湿重低于其他孕激素组。
2.不同种类及剂量的孕激素应用72h时均能够使雌激素预处理的去势小鼠子宫内膜由增殖转为分泌或轻度萎缩,较高剂量组的转化比例高于较低剂量组。
3.孕激素首次应用24h时ERα和PR在子宫内膜的免疫染色最强,随着用药时间延长,ERa和PR的染色均降低。
4.较低剂量孕激素应用期间对小鼠子宫组织ERα、PR、Lefty、MMP3基因表达的抑制作用较弱；而较高剂量对内膜基因表达的抑制作用较强,以至于未出现撤药后相关基因(如Lefty、MMP3、MMP9)表达升高的表现。孕激素对Lefty和MMPs表达抑制作用的强弱可能与内膜不规则出血发生情况有关,推测应用较低剂量孕激素可能更易发生出血。
5.向雌孕激素方案预处理后的小鼠子宫腔注射油剂内膜出现蜕膜样细胞,但程度及范围均很小,应用较高剂量孕激素组小鼠内膜出现蜕膜样细胞的比例稍高。
Background
Abnormal 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.
Objective
In 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.
Contents
Part 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.
Methods
1. 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.
Results
1. 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.
Conclusions
1. 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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7. Cornet PB PC, Lemoine P, Osteen KG, Bruner-Tran KL, Tabibzadeh S, Courtoy PJ, Eeckhout Y, Marbaix E, Henriet P Regulation and function of LEFTY-A/EBAF in the human endometrium. mRNA expression during the menstrual cycle, control by progesterone, and effect on matrix metalloproteinases. J Biol Chem.2002;277:42496-42504.
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1. Finn CA PM. Vascular and cellular changes in the decidualized endometrium of the ovariectomizedmouse following cessation of hormone treatment: a possible model for menstruation. J Endocrinol.1984;100:295-300.
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