前动力蛋白-1和孕激素受体在反复种植失败患者黄体中期子宫内膜中的表达
发布时间：2018-08-13 20:18
【摘要】：背景 目前被大多数人接受的反复种植失败（repeated implantation failure，RIF）的定义为：经过2-6个体外受精（IVF）周期，移植≥10个高质量胚胎仍未妊娠。近年来各辅助生殖中心一次移植的胚胎数目减少（一般控制在1-2个之内），使得RIF的定义有些变化。Margalioth等[1]提出：移植高质量胚胎超过3次未妊娠定义为RIF。RIF是当今生殖领域研究中的难题之一,究其发生原因，可概括为三部分：胚胎质量下降、种植期子宫内膜容受性不足、母体激素与免疫紊乱[1]。随着胚胎冷冻技术、控制性促排卵方案、胚胎移植等技术的日益成熟，胚胎质量方面已有显著的提高，然而子宫内膜容受性方面的研究却进展缓慢。据国内外研究表明，在移植失败的原因当中，内膜因素约占66.7%[2]。 子宫内膜容受性是指母体子宫内膜对胚泡的接受能力。在黄体中期，孕酮（progesterone，P）可以使子宫内膜处于一种允许胚泡定位、粘附、侵入的状态并使内膜在胚泡的作用下发生蜕膜性变[3]。在此过程中，新生毛细血管大量形成，各种与子宫内膜容受性、胚胎种植相关的细胞因子相互协调相互作用，为迎接胚胎的到来做好准备。若在子宫内膜向容受状态转变的过程中，血管的生成以及容受性、种植相关因子的表达受阻碍，子宫内膜的容受性将会受到影响。内膜对胚胎的接受能力下降，将会导致种植的失败。 前动力蛋白-1(prokineticin-1，PROK1)是近年来发现的一种分泌性蛋白。既往研究发现[4]：PROK1主要表达于卵巢、睾丸、肾上腺等分泌类固醇激素的器官。近年来研究发现[5]：在某些非类固醇激素分泌的器官，如子宫，也有相当数量的表达。PROK1在子宫中的表达主要位于子宫内膜上，并随着月经周期而发生周期性的改变,在黄体中期表达呈一峰值[6]。研究表明，PROK1是一种新型的子宫内膜容受性标志物[7]，能选择性作用于子宫内膜血管内皮细胞，对子宫内膜毛细血管的生成具有调节作用[8]。其与PROK1受体结合，能调控包括白血病抑制因子[9]、IL-8[10]、IL-11[11]等子宫内膜容受性相关因子的表达，参与胚胎种植时的“母-胎对话”。可见，容受性良好的子宫内膜离不开PROK1的正常表达。 子宫内膜向分泌期（黄体期）转化是着床的首要条件。孕激素通过与其受体结合，使子宫内膜腺上皮向分泌期转化，在分泌中期，子宫内膜腔上皮肥大出现胞饮突，子宫腺体顶部聚集成束的腺上皮肥大，基质细胞蜕膜化，为胚胎着床提供准备[12]。可见，孕激素在促使内膜向容受状态转变和调节胚胎植入的过程中均发挥着重要的作用。孕激素的生物学作用是通过其与孕激素受体结合后发挥的。因此，孕激素及其受体二者缺一均会影响孕激素作用的发挥。在接受IVF-ET治疗的患者于排卵后常规使用孕酮支持的今天，孕激素受体（progesterone receptor，PR）在黄体中期子宫内膜中的表达就显得尤其重要，低表达的PR将会直接影响子宫内膜容受性的建立以及胚胎在子宫内膜的种植。 综上所述，PROK1和PR在子宫内膜容受性的建立以及对胚胎在子宫内膜的种植过程中均发挥着重要的作用。 目的 通过检测RIF患者（实验组）和曾孕妇女（对照组）黄体中期子宫内膜中PROK1和PR的mRNA和蛋白表达水平，并比较其表达差异，初步探讨子宫内膜黄体中期PROK1、PR的表达水平与RIF的关系，为阐明RIF发病机制提供新的理论依据。 方法 1、研究对象 2013年8月至2014年4月在广州医科大学附属省妇儿医院生殖健康与不孕症科行体外受精-胚胎移植（IVF-ET）或卵泡内单精子注射（ICSI）的21例反复种植失败患者（实验组），年龄30-38岁，平均年龄（34.94±2.75）岁，及同期23例因输卵管间质部阻塞和/或男方因素行IVF-ET/ICSI助孕，且有一次临床妊娠史以上的患者（对照组），年龄29-36岁，平均年龄（32.53±3.68）岁。 2、实验方法 （1）所有子宫内膜组织均经HE染色，确定为黄体中期后纳入实验。 （2）采用实时荧光定量PCR法对两组患者子宫内膜中PROK1和PR的mRNA表达水平进行检测,并比较两组数据之间的差异是否有统计学意义。 （3）采用免疫组织化学法对两组子宫内膜中PROK1和PR的蛋白表达水平进行检测，并比较两组数据之间的差异是否有统计学意义。 结果 1、实验组与对照组子宫内膜组织共44例，均显示黄体中期内膜。 2、PROK1和PR的mRNA在44例患者子宫内膜中均有表达。但结果显示实验组中PROK1的mRNA表达水平明显低于对照组（0.21±0.20VS0.36±0.18），异有统计学意义（P＜0.05）；实验组中PR的mRNA表达水平明显低于对照组（0.016±0.006VS0.021±0.004），差异有统计学意义（P＜0.05）。 3、PROK1和PR蛋白在44例患者子宫内膜中均有表达。PROK1主要表达于腺上皮细胞、内皮细胞及基质细胞，且腺上皮细胞上的表达稍强于基质细胞及内皮细胞，定位于胞浆，PR亦表达于腺上皮细胞和间质细胞，但定位于细胞核。实验组PROK1的蛋白半定量表达水平明显低于对照组（0.016±0.006VS0.021±0.004），差异有统计学意义（P＜0.05），实验组PR的蛋白半定量表达水平明显低于对照组（2.49±0.87VS4.28±1.22），差异有统计学意义（P＜0.05）。 结论 1、实验组子宫内膜中PROK1和PR的蛋白的半定量表达水平和mRNA表达水平均明显低于对照组，说明PROK1和PR在RIF患者黄体中期子宫内膜中的相对低表达可能是导致RIF的分子机制之一。
[Abstract]:background
Repeated implantation failure (RIF), which is currently accepted by the majority of people, is defined as: after 2-6 in vitro fertilization (IVF) cycles, transplantation of more than 10 high-quality embryos is still not pregnant. In recent years, the number of embryos transferred at one time at various auxiliary reproductive centers has decreased (generally within 1-2), which has led to some changes in the definition of RIF. Margalioth et al. [1] proposed that the transfer of high-quality embryos more than three times without pregnancy is defined as RIF. RIF is one of the difficult problems in the field of reproductive research. The causes can be summarized as three parts: embryo quality decline, endometrial receptivity deficiency during implantation, maternal hormones and immune disorders [1]. With the maturity of the technology of egg scheme, embryo transfer and so on, the embryo quality has been improved remarkably, but the research on endometrial receptivity has been progressing slowly.
Endometrial receptivity refers to the ability of the maternal endometrium to accept blastocysts. In the mid-luteal phase, progesterone (P) causes the endometrium to be in a state that allows the location, adhesion, invasion of the blastocysts and endometrial decidualization under the action of the blastocysts [3]. Endometrial receptivity, the interaction of embryo implantation-related cytokines, is prepared for the arrival of the embryo. A decline in ability will lead to failure in planting.
Prokinetin-1 (PROK1) is a secretory protein found in recent years. Previous studies have found that [4]: PROK1 is mainly expressed in ovaries, testis, adrenal glands and other organs secreting steroids. Recent studies have found that [5]: in some non-steroid hormone secreting organs, such as the uterus, there are also a considerable number of expression of PROK1. The expression of PROK1 in the uterus is mainly located in the endometrium and changes periodically with the menstrual cycle, showing a peak value in the mid-luteal phase [6]. It can regulate the expression of endometrial receptivity-related factors including leukemia inhibitor [9], IL-8 [10], IL-11 [11], and participate in the "mother-fetal dialogue" during embryo implantation.
Progesterone binds to its receptor to make the glandular epithelium of endometrium transform to secretory phase. In the middle secretory phase, the epithelial hypertrophy of endometrial cavity appears the pinocyte process, the glandular epithelium gathers in bundles at the top of the gland is hypertrophy, and the stromal cells are decidualized, which provides the criterion for embryo implantation. Preparations [12]. It can be seen that progesterone plays an important role in promoting the transition of endometrium to receptive state and regulating embryo implantation. The biological function of progesterone is exerted through its binding to progesterone receptor. Therefore, the absence of progesterone and its receptor will affect the function of progesterone. In IVF-ET treatment, progesterone plays an important role. The expression of progesterone receptor (PR) in the mid-luteal endometrium is particularly important today when patients are routinely supported by progesterone after ovulation. Low PR expression will directly affect the establishment of endometrial receptivity and embryo implantation in the endometrium.
In conclusion, PROK1 and PR play an important role in the establishment of endometrial receptivity and in the implantation of embryos into the endometrium.
objective
By detecting the expression of PROK1 and PR mRNA and protein in the mid-luteal endometrium of patients with RIF (experimental group) and pregnant women (control group), and comparing their differences, the relationship between the expression of PROK1 and PR and RIF in the mid-luteal endometrium was preliminarily explored.
Method
1, the object of study.
From August 2013 to April 2014, 21 patients (experimental group) who failed to implant in vitro fertilization-embryo transfer (IVF-ET) or intrafollicular sperm injection (ICSI) were treated in the Department of Reproductive Health and Infertility, Affiliated Provincial Gynecology and Children's Hospital of Guangzhou Medical University. The age ranged from 30 to 38 years, with an average age of (34.94 (2.75)) and 23 patients with tubal interstitial obstruction and/or intrafollicular sperm injection (ICSI). IVF-ET/ICSI was performed for male patients with more than one clinical pregnancy history (control group), aged 29-36 years, with an average age of (32.53+3.68) years.
2, the experimental method.
(1) all endometrial tissues were stained by HE and identified as mid luteal phase.
(2) Real-time fluorescence quantitative PCR was used to detect the expression of PROK1 and PR mRNA in endometrium of the two groups, and the difference between the two groups was statistically significant.
(3) Protein expression levels of PROK1 and PR in endometrium of the two groups were detected by immunohistochemical method, and the difference between the two groups was statistically significant.
Result
1, there were 44 cases of endometrial tissue in the experimental group and the control group, all showing the middle luteal endometrium.
2. The expression of PROK1 and PR mRNA in the endometrium of 44 patients was significantly lower than that in the control group (0.21.20 VS 0.36.18, P < 0.05). The expression of PR mRNA in the experimental group was significantly lower than that in the control group (0.016.006 VS 0.021.004). (P < 0.05).
PROK1 was mainly expressed in glandular epithelial cells, endothelial cells and stromal cells. The expression of PROK1 in glandular epithelial cells was slightly stronger than that in stromal cells and endothelial cells, and was localized in cytoplasm. PR was also expressed in glandular epithelial cells and stromal cells, but localized in nucleus. The semi-quantitative expression level of PR in the experimental group was significantly lower than that in the control group (0.016.006VS 0.021.004), and the difference was statistically significant (P < 0.05). The semi-quantitative expression level of PR in the experimental group was significantly lower than that in the control group (2.49.87VS 4.28.22), and the difference was statistically significant (P < 0.
conclusion
1. The semiquantitative expression level of PROK 1 and PR protein and the mRNA expression level in the endometrium of the experimental group were significantly lower than those of the control group, indicating that the relatively low expression of PROK 1 and PR in the middle luteal endometrium of RIF patients may be one of the molecular mechanisms leading to RIF.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.8
本文编号：2182070
[Abstract]:background
Repeated implantation failure (RIF), which is currently accepted by the majority of people, is defined as: after 2-6 in vitro fertilization (IVF) cycles, transplantation of more than 10 high-quality embryos is still not pregnant. In recent years, the number of embryos transferred at one time at various auxiliary reproductive centers has decreased (generally within 1-2), which has led to some changes in the definition of RIF. Margalioth et al. [1] proposed that the transfer of high-quality embryos more than three times without pregnancy is defined as RIF. RIF is one of the difficult problems in the field of reproductive research. The causes can be summarized as three parts: embryo quality decline, endometrial receptivity deficiency during implantation, maternal hormones and immune disorders [1]. With the maturity of the technology of egg scheme, embryo transfer and so on, the embryo quality has been improved remarkably, but the research on endometrial receptivity has been progressing slowly.
Endometrial receptivity refers to the ability of the maternal endometrium to accept blastocysts. In the mid-luteal phase, progesterone (P) causes the endometrium to be in a state that allows the location, adhesion, invasion of the blastocysts and endometrial decidualization under the action of the blastocysts [3]. Endometrial receptivity, the interaction of embryo implantation-related cytokines, is prepared for the arrival of the embryo. A decline in ability will lead to failure in planting.
Prokinetin-1 (PROK1) is a secretory protein found in recent years. Previous studies have found that [4]: PROK1 is mainly expressed in ovaries, testis, adrenal glands and other organs secreting steroids. Recent studies have found that [5]: in some non-steroid hormone secreting organs, such as the uterus, there are also a considerable number of expression of PROK1. The expression of PROK1 in the uterus is mainly located in the endometrium and changes periodically with the menstrual cycle, showing a peak value in the mid-luteal phase [6]. It can regulate the expression of endometrial receptivity-related factors including leukemia inhibitor [9], IL-8 [10], IL-11 [11], and participate in the "mother-fetal dialogue" during embryo implantation.
Progesterone binds to its receptor to make the glandular epithelium of endometrium transform to secretory phase. In the middle secretory phase, the epithelial hypertrophy of endometrial cavity appears the pinocyte process, the glandular epithelium gathers in bundles at the top of the gland is hypertrophy, and the stromal cells are decidualized, which provides the criterion for embryo implantation. Preparations [12]. It can be seen that progesterone plays an important role in promoting the transition of endometrium to receptive state and regulating embryo implantation. The biological function of progesterone is exerted through its binding to progesterone receptor. Therefore, the absence of progesterone and its receptor will affect the function of progesterone. In IVF-ET treatment, progesterone plays an important role. The expression of progesterone receptor (PR) in the mid-luteal endometrium is particularly important today when patients are routinely supported by progesterone after ovulation. Low PR expression will directly affect the establishment of endometrial receptivity and embryo implantation in the endometrium.
In conclusion, PROK1 and PR play an important role in the establishment of endometrial receptivity and in the implantation of embryos into the endometrium.
objective
By detecting the expression of PROK1 and PR mRNA and protein in the mid-luteal endometrium of patients with RIF (experimental group) and pregnant women (control group), and comparing their differences, the relationship between the expression of PROK1 and PR and RIF in the mid-luteal endometrium was preliminarily explored.
Method
1, the object of study.
From August 2013 to April 2014, 21 patients (experimental group) who failed to implant in vitro fertilization-embryo transfer (IVF-ET) or intrafollicular sperm injection (ICSI) were treated in the Department of Reproductive Health and Infertility, Affiliated Provincial Gynecology and Children's Hospital of Guangzhou Medical University. The age ranged from 30 to 38 years, with an average age of (34.94 (2.75)) and 23 patients with tubal interstitial obstruction and/or intrafollicular sperm injection (ICSI). IVF-ET/ICSI was performed for male patients with more than one clinical pregnancy history (control group), aged 29-36 years, with an average age of (32.53+3.68) years.
2, the experimental method.
(1) all endometrial tissues were stained by HE and identified as mid luteal phase.
(2) Real-time fluorescence quantitative PCR was used to detect the expression of PROK1 and PR mRNA in endometrium of the two groups, and the difference between the two groups was statistically significant.
(3) Protein expression levels of PROK1 and PR in endometrium of the two groups were detected by immunohistochemical method, and the difference between the two groups was statistically significant.
Result
1, there were 44 cases of endometrial tissue in the experimental group and the control group, all showing the middle luteal endometrium.
2. The expression of PROK1 and PR mRNA in the endometrium of 44 patients was significantly lower than that in the control group (0.21.20 VS 0.36.18, P < 0.05). The expression of PR mRNA in the experimental group was significantly lower than that in the control group (0.016.006 VS 0.021.004). (P < 0.05).
PROK1 was mainly expressed in glandular epithelial cells, endothelial cells and stromal cells. The expression of PROK1 in glandular epithelial cells was slightly stronger than that in stromal cells and endothelial cells, and was localized in cytoplasm. PR was also expressed in glandular epithelial cells and stromal cells, but localized in nucleus. The semi-quantitative expression level of PR in the experimental group was significantly lower than that in the control group (0.016.006VS 0.021.004), and the difference was statistically significant (P < 0.05). The semi-quantitative expression level of PR in the experimental group was significantly lower than that in the control group (2.49.87VS 4.28.22), and the difference was statistically significant (P < 0.
conclusion
1. The semiquantitative expression level of PROK 1 and PR protein and the mRNA expression level in the endometrium of the experimental group were significantly lower than those of the control group, indicating that the relatively low expression of PROK 1 and PR in the middle luteal endometrium of RIF patients may be one of the molecular mechanisms leading to RIF.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.8
【参考文献】
相关期刊论文 前2条
1 朱桂金;胞饮突与子宫内膜容受性[J];国外医学.妇产科学分册;2000年03期
2 吴丽姝;任凤岩;卜素芝;王春晓;;内分泌腺来源的血管内皮生长因子与多囊卵巢综合征的研究进展[J];中国现代药物应用;2011年04期
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