【摘要】：背景 目前被大多數(shù)人接受的反復(fù)種植失敗（repeated implantation failure，RIF）的定義為：經(jīng)過(guò)2-6個(gè)體外受精（IVF）周期，移植≥10個(gè)高質(zhì)量胚胎仍未妊娠。近年來(lái)各輔助生殖中心一次移植的胚胎數(shù)目減少（一般控制在1-2個(gè)之內(nèi)），使得RIF的定義有些變化。Margalioth等[1]提出：移植高質(zhì)量胚胎超過(guò)3次未妊娠定義為RIF。RIF是當(dāng)今生殖領(lǐng)域研究中的難題之一,究其發(fā)生原因，可概括為三部分：胚胎質(zhì)量下降、種植期子宮內(nèi)膜容受性不足、母體激素與免疫紊亂[1]。隨著胚胎冷凍技術(shù)、控制性促排卵方案、胚胎移植等技術(shù)的日益成熟，胚胎質(zhì)量方面已有顯著的提高，然而子宮內(nèi)膜容受性方面的研究卻進(jìn)展緩慢。據(jù)國(guó)內(nèi)外研究表明，在移植失敗的原因當(dāng)中，內(nèi)膜因素約占66.7%[2]。 子宮內(nèi)膜容受性是指母體子宮內(nèi)膜對(duì)胚泡的接受能力。在黃體中期，孕酮（progesterone，P）可以使子宮內(nèi)膜處于一種允許胚泡定位、粘附、侵入的狀態(tài)并使內(nèi)膜在胚泡的作用下發(fā)生蛻膜性變[3]。在此過(guò)程中，新生毛細(xì)血管大量形成，各種與子宮內(nèi)膜容受性、胚胎種植相關(guān)的細(xì)胞因子相互協(xié)調(diào)相互作用，為迎接胚胎的到來(lái)做好準(zhǔn)備。若在子宮內(nèi)膜向容受狀態(tài)轉(zhuǎn)變的過(guò)程中，血管的生成以及容受性、種植相關(guān)因子的表達(dá)受阻礙，子宮內(nèi)膜的容受性將會(huì)受到影響。內(nèi)膜對(duì)胚胎的接受能力下降，將會(huì)導(dǎo)致種植的失敗。 前動(dòng)力蛋白-1(prokineticin-1，PROK1)是近年來(lái)發(fā)現(xiàn)的一種分泌性蛋白。既往研究發(fā)現(xiàn)[4]：PROK1主要表達(dá)于卵巢、睪丸、腎上腺等分泌類(lèi)固醇激素的器官。近年來(lái)研究發(fā)現(xiàn)[5]：在某些非類(lèi)固醇激素分泌的器官，如子宮，也有相當(dāng)數(shù)量的表達(dá)。PROK1在子宮中的表達(dá)主要位于子宮內(nèi)膜上，并隨著月經(jīng)周期而發(fā)生周期性的改變,在黃體中期表達(dá)呈一峰值[6]。研究表明，PROK1是一種新型的子宮內(nèi)膜容受性標(biāo)志物[7]，能選擇性作用于子宮內(nèi)膜血管內(nèi)皮細(xì)胞，對(duì)子宮內(nèi)膜毛細(xì)血管的生成具有調(diào)節(jié)作用[8]。其與PROK1受體結(jié)合，能調(diào)控包括白血病抑制因子[9]、IL-8[10]、IL-11[11]等子宮內(nèi)膜容受性相關(guān)因子的表達(dá)，參與胚胎種植時(shí)的“母-胎對(duì)話”�？梢�(jiàn)，容受性良好的子宮內(nèi)膜離不開(kāi)PROK1的正常表達(dá)。 子宮內(nèi)膜向分泌期（黃體期）轉(zhuǎn)化是著床的首要條件。孕激素通過(guò)與其受體結(jié)合，使子宮內(nèi)膜腺上皮向分泌期轉(zhuǎn)化，在分泌中期，子宮內(nèi)膜腔上皮肥大出現(xiàn)胞飲突，子宮腺體頂部聚集成束的腺上皮肥大，基質(zhì)細(xì)胞蛻膜化，為胚胎著床提供準(zhǔn)備[12]�？梢�(jiàn)，孕激素在促使內(nèi)膜向容受狀態(tài)轉(zhuǎn)變和調(diào)節(jié)胚胎植入的過(guò)程中均發(fā)揮著重要的作用。孕激素的生物學(xué)作用是通過(guò)其與孕激素受體結(jié)合后發(fā)揮的。因此，孕激素及其受體二者缺一均會(huì)影響孕激素作用的發(fā)揮。在接受IVF-ET治療的患者于排卵后常規(guī)使用孕酮支持的今天，孕激素受體（progesterone receptor，PR）在黃體中期子宮內(nèi)膜中的表達(dá)就顯得尤其重要，低表達(dá)的PR將會(huì)直接影響子宮內(nèi)膜容受性的建立以及胚胎在子宮內(nèi)膜的種植。 綜上所述，PROK1和PR在子宮內(nèi)膜容受性的建立以及對(duì)胚胎在子宮內(nèi)膜的種植過(guò)程中均發(fā)揮著重要的作用。 目的 通過(guò)檢測(cè)RIF患者（實(shí)驗(yàn)組）和曾孕婦女（對(duì)照組）黃體中期子宮內(nèi)膜中PROK1和PR的mRNA和蛋白表達(dá)水平，并比較其表達(dá)差異，初步探討子宮內(nèi)膜黃體中期PROK1、PR的表達(dá)水平與RIF的關(guān)系，為闡明RIF發(fā)病機(jī)制提供新的理論依據(jù)。 方法 1、研究對(duì)象 2013年8月至2014年4月在廣州醫(yī)科大學(xué)附屬省婦兒醫(yī)院生殖健康與不孕癥科行體外受精-胚胎移植（IVF-ET）或卵泡內(nèi)單精子注射（ICSI）的21例反復(fù)種植失敗患者（實(shí)驗(yàn)組），年齡30-38歲，平均年齡（34.94±2.75）歲，及同期23例因輸卵管間質(zhì)部阻塞和/或男方因素行IVF-ET/ICSI助孕，且有一次臨床妊娠史以上的患者（對(duì)照組），年齡29-36歲，平均年齡（32.53±3.68）歲。 2、實(shí)驗(yàn)方法 （1）所有子宮內(nèi)膜組織均經(jīng)HE染色，確定為黃體中期后納入實(shí)驗(yàn)。 （2）采用實(shí)時(shí)熒光定量PCR法對(duì)兩組患者子宮內(nèi)膜中PROK1和PR的mRNA表達(dá)水平進(jìn)行檢測(cè),并比較兩組數(shù)據(jù)之間的差異是否有統(tǒng)計(jì)學(xué)意義。 （3）采用免疫組織化學(xué)法對(duì)兩組子宮內(nèi)膜中PROK1和PR的蛋白表達(dá)水平進(jìn)行檢測(cè)，并比較兩組數(shù)據(jù)之間的差異是否有統(tǒng)計(jì)學(xué)意義。 結(jié)果 1、實(shí)驗(yàn)組與對(duì)照組子宮內(nèi)膜組織共44例，均顯示黃體中期內(nèi)膜。 2、PROK1和PR的mRNA在44例患者子宮內(nèi)膜中均有表達(dá)。但結(jié)果顯示實(shí)驗(yàn)組中PROK1的mRNA表達(dá)水平明顯低于對(duì)照組（0.21±0.20VS0.36±0.18），異有統(tǒng)計(jì)學(xué)意義（P＜0.05）；實(shí)驗(yàn)組中PR的mRNA表達(dá)水平明顯低于對(duì)照組（0.016±0.006VS0.021±0.004），差異有統(tǒng)計(jì)學(xué)意義（P＜0.05）。 3、PROK1和PR蛋白在44例患者子宮內(nèi)膜中均有表達(dá)。PROK1主要表達(dá)于腺上皮細(xì)胞、內(nèi)皮細(xì)胞及基質(zhì)細(xì)胞，且腺上皮細(xì)胞上的表達(dá)稍強(qiáng)于基質(zhì)細(xì)胞及內(nèi)皮細(xì)胞，定位于胞漿，PR亦表達(dá)于腺上皮細(xì)胞和間質(zhì)細(xì)胞，但定位于細(xì)胞核。實(shí)驗(yàn)組PROK1的蛋白半定量表達(dá)水平明顯低于對(duì)照組（0.016±0.006VS0.021±0.004），差異有統(tǒng)計(jì)學(xué)意義（P＜0.05），實(shí)驗(yàn)組PR的蛋白半定量表達(dá)水平明顯低于對(duì)照組（2.49±0.87VS4.28±1.22），差異有統(tǒng)計(jì)學(xué)意義（P＜0.05）。 結(jié)論 1、實(shí)驗(yàn)組子宮內(nèi)膜中PROK1和PR的蛋白的半定量表達(dá)水平和mRNA表達(dá)水平均明顯低于對(duì)照組，說(shuō)明PROK1和PR在RIF患者黃體中期子宮內(nèi)膜中的相對(duì)低表達(dá)可能是導(dǎo)致RIF的分子機(jī)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.
【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R714.8

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