【摘要】：目的：通過研究體外受精-胚胎移植(in vitro fertilization embryo transfer, IVF-ET)患者預(yù)備周期胚胎著床窗口期子宮內(nèi)膜促血管生成素(angiopoietins, Angs)、子宮內(nèi)膜微血管密度(microvessel density, MVD)表達(dá)及相關(guān)性分析,進(jìn)一步闡明Ang-1、Ang-2評(píng)估子宮內(nèi)膜血管形成及內(nèi)膜容受性預(yù)測妊娠結(jié)局的價(jià)值。方法：選擇2014年8月～2014年12月在河北醫(yī)科大學(xué)第四醫(yī)院生殖醫(yī)學(xué)科首次行IVF助孕治療患者。年齡為20-35歲,具有正常月經(jīng)周期,周期21-35天,不孕原因主要為輸卵管因素,新鮮周期移植2個(gè)優(yōu)質(zhì)胚胎。于IVF預(yù)備周期胚胎植入窗口期獲取子宮內(nèi)膜,-80℃凍存樣本,部分組織用4%甲醛固定。分別用逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(Reverse Transcription-Polymerase Chain Reaction, RT-PCR)和免疫組織化學(xué)技術(shù)(immunohistochemistry)測定子宮內(nèi)膜中Ang-1、Ang-2含量,用免疫組織化學(xué)技術(shù)測定子宮內(nèi)膜MVD。應(yīng)用免疫組化檢測：將子宮內(nèi)膜組織用4%甲醛固定后石蠟包埋,制備4μm厚連續(xù)組織切片。分別進(jìn)行HE和免疫組化染色。用PBS代替一抗做陰性對(duì)照。每張染色片隨機(jī)選取5個(gè)視野,進(jìn)行結(jié)果分析,顯微鏡觀察并拍照。應(yīng)用RT-PCR檢測技術(shù),分別取研究組及對(duì)照組子宮內(nèi)膜組織加入Rezol 1 ml置勻漿器內(nèi)充分勻漿,提取總RNA,取出0.5μ1,進(jìn)行反轉(zhuǎn)錄得cDNA,繼而進(jìn)行PCR擴(kuò)增,同時(shí)以擴(kuò)增片段長度為605 bp的GAPDH作為內(nèi)參照。Ang-1和Ang-2PCR循環(huán)條件為：95℃10 min,1個(gè)循環(huán)；95℃ 45 s,60℃ 45 s,72℃50 s,35個(gè)循環(huán)；72℃ 10 min,1個(gè)循環(huán)。產(chǎn)物經(jīng)1.5%瓊脂糖凝膠電泳后,用VDS圖像分析系統(tǒng)進(jìn)行照相和圖像分析。IOD值經(jīng)內(nèi)參照校正,將校正值進(jìn)行統(tǒng)計(jì)學(xué)分析�？刂菩猿倥怕眩核谢颊呔捎命S體期長效長方案,即排卵后6-7天(黃體中期)給予醋酸亮丙瑞林(北京博恩特藥業(yè)有限公司)1.3mg,達(dá)到降調(diào)節(jié)標(biāo)準(zhǔn)后給予重組人促卵泡激素注射液150-225IU(默克雪蘭諾公司,意大利),采用經(jīng)陰道超聲監(jiān)測子宮內(nèi)膜變化和卵泡生長情況,當(dāng)至少有2個(gè)卵泡直徑大于20mm時(shí),當(dāng)晚注射艾澤(重組人絨促性素注射液,意大利)250μg,艾澤注射后37h左右取卵,適時(shí)加精,體外受精,于取卵后3d,移植優(yōu)質(zhì)胚胎2枚,移植后28d經(jīng)陰道B超見到孕囊為臨床妊娠。所有數(shù)據(jù)采用SPSS13.0統(tǒng)計(jì)軟件進(jìn)行統(tǒng)計(jì)分析。計(jì)量資料采用x±s表示,組間差異采用t檢驗(yàn),P0.05即有顯著性差異；等級(jí)資料采用秩和檢驗(yàn),P0.05即有顯著性差異。對(duì)相關(guān)的計(jì)量資料采用Person相關(guān)系數(shù)進(jìn)行相關(guān)性分析。結(jié)果：1臨床妊娠組與未妊娠組一般情況比較臨床妊娠組(n=12)與未妊娠組(n=13)比較,在年齡、不孕年限、不孕原因、基礎(chǔ)FSH、基礎(chǔ)LH及竇卵泡數(shù)方面,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。(見Tablel)2臨床妊娠組和未妊娠組之間IVF-ET常規(guī)比較兩組之間獲卵率、優(yōu)胚率、移植日內(nèi)膜、移植日E2及移植日P差異無統(tǒng)計(jì)學(xué)意義(P0.05)。(見Table2)3免疫組織化學(xué)檢測臨床妊娠組和未妊娠組預(yù)備周期胚胎著床窗口期子宮內(nèi)膜Ang-1、Ang-2及MVD表達(dá)3.1 Ang-1在預(yù)備周期胚胎著床窗口期子宮內(nèi)膜基質(zhì)細(xì)胞、腺上皮細(xì)胞及血管內(nèi)皮細(xì)胞均有表達(dá)。臨床妊娠組(n=12),“—”表達(dá)1例,“+”表達(dá)2例,“++”表達(dá)5例,“+++”表達(dá)4例。未妊娠組(n=13),“—”表達(dá)4例,“+”表達(dá)5例,“++”表達(dá)3例,“+++’’表達(dá)1例,差異有統(tǒng)計(jì)學(xué)意義(P=0.026)。(見Table3)3.2Ang2主要在子宮內(nèi)膜基質(zhì)細(xì)胞及腺上皮細(xì)胞表達(dá)。Ang-2在臨床妊娠組與未妊娠組均有表達(dá),臨床妊娠組(n=13),“—”表達(dá)2例,“+”表達(dá)3例,“++”表達(dá)5例,“+++”表達(dá)2例。未妊娠組(n=13),“—”表達(dá)3例,“+”表達(dá)6例,“++”表達(dá)2例,“+++”表達(dá)2例,差異無統(tǒng)計(jì)學(xué)意義(P=0.335)。(見Table4)3.3以CD34特異性標(biāo)記血管內(nèi)皮細(xì)胞測定MVD。胚胎著床窗口期子宮內(nèi)膜組織中微血管在腺體周圍基質(zhì)中表達(dá)豐富。妊娠組MVD(16.42±4.66)表達(dá)量高于未妊娠組(12.31+4.40),差異有統(tǒng)計(jì)學(xué)意義(P0.05)。(見Table5)4 RT-PCR檢測臨床妊娠組和未妊娠組預(yù)備周期胚胎著床窗口期子宮內(nèi)膜Ang-1mRNA及Ang-2mRNA表達(dá)4.1胚胎著床窗口期臨床妊娠組子宮內(nèi)膜Ang-1mRNA的表達(dá)明顯高于未妊娠組,Ang-1mRNA在妊娠組的表達(dá)為0.72+0.35(n=12),未妊娠組的表達(dá)為0.33+0.32(n=1 3),差異有統(tǒng)計(jì)學(xué)意義(P=0.008)。(見Table6)4.2 Ang-2mRNA在臨床妊娠組的表達(dá)為0.6510.60(n=12),未妊娠組的表達(dá)為0.65±0.41(n=13),差異無統(tǒng)計(jì)學(xué)意義(P=0.988)。(見Table6)5胚胎著床窗口期子宮內(nèi)膜Ang-1、Ang-2蛋白表達(dá)水平與MVD相關(guān)性分析5.1 IVF患者胚胎著床窗口期子宮內(nèi)膜Ang-1蛋白表達(dá)與MVD相關(guān)系數(shù)0.564,P=0.003,差異有統(tǒng)計(jì)學(xué)意義。(見Table7)5.2 IVF患者胚胎著床窗口期子宮內(nèi)膜Ang-2蛋白表達(dá)與MVD相關(guān)系數(shù)0.273,P=0.187,差異無統(tǒng)計(jì)學(xué)意義。(見Table8)結(jié)論：1 Ang-1蛋白在預(yù)備周期胚胎著床窗口期子宮內(nèi)膜基質(zhì)細(xì)胞、腺上皮細(xì)胞及血管內(nèi)皮細(xì)胞均有表達(dá)。2 Ang-1 mRNA可能為子宮內(nèi)膜容受性相關(guān)基因之一,高表達(dá)的Ang-1mRNA更利于胚胎植入,有較好的妊娠結(jié)局。IVF患者胚胎著床窗口期子宮內(nèi)膜Ang-1mRNA可能為預(yù)測妊娠結(jié)局的指標(biāo)之一。3 Ang-2蛋白主要在預(yù)備周期胚胎著床窗口期子宮內(nèi)膜基質(zhì)細(xì)胞及腺上皮細(xì)胞表達(dá)。4 Ang-2 mRNA在臨床妊娠組與未妊娠組預(yù)備周期胚胎著床窗口期子宮內(nèi)膜表達(dá)無差異,不能預(yù)測IVF患者妊娠結(jié)局,但其在評(píng)估子宮內(nèi)膜容受性中的作用機(jī)制及意義有待進(jìn)一步探索。5 IVF患者預(yù)備周期胚胎著床窗口期臨床妊娠組子宮內(nèi)膜MVD明顯高于未妊娠組,子宮內(nèi)膜組織豐富的微血管更有利于胚胎植入,MVD可能為預(yù)測妊娠結(jié)局的指標(biāo)之一。6胚胎著床窗口期子宮內(nèi)膜Ang-1蛋白表達(dá)與MVD顯著相關(guān),Ang-2蛋白表達(dá)與MVD無相關(guān)關(guān)系。Ang-1是促進(jìn)血管生成的因子,子宮內(nèi)膜豐富的血流灌注有利于胚胎植入。
[Abstract]:AIM: To investigate the expression of angiopoietins (Angs) and endometrial microvessel density (MVD) during the implantation window stage of embryos in vitro fertilization embryo transfer (IVF-ET) and to elucidate the relationship between Ang-1 and Ang-2 assessors. Methods: From August 2014 to December 2014, IVF assisted pregnancy patients were selected from the Department of Reproductive Medicine, the Fourth Hospital of Hebei Medical University. They were 20-35 years old and had normal menstrual cycle with a period of 21-35 days. Endometrium was harvested at the stage of IVF embryo implantation window. Samples were frozen at - 80 C and some tissues were fixed with 4% formaldehyde. Ang-1 and Ang-1 in endometrium were determined by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively. Endometrial MVD was determined by immunohistochemical staining. Immunohistochemical staining: Endometrial tissues were embedded in paraffin fixed with 4% formaldehyde to prepare 4 micron thick serial sections. HE and immunohistochemical staining were performed respectively. PBS were used as negative control. Each staining sheet was randomly selected from 5 visual fields and the results were analyzed. The endometrial tissues of the study group and the control group were added to the Rezol 1 ml homogenizer for full homogenization, total RNA was extracted, 0.5 mu 1 was retrieved, and the cDNA was obtained by reverse transcription. The amplified fragment length of GAPDH was 605 BP as internal reference. Ang-1 and Ang-2 PCR cycle strips The products were photographed and analyzed by VDS image analysis system after 1.5% agarose gel electrophoresis. The IOD value was corrected by internal reference, and the corrected value was statistically analyzed. Controlled hyperstimulation: All patients were used. Leuprorelin acetate (Beijing Boente Pharmaceutical Co., Ltd.) was administered 6-7 days after ovulation (mid-luteal phase) at a dose of 1.3 mg. The recombinant human follicle-stimulating hormone injection 150-225IU (Merck-Sherano, Italy) was administered at a reduced regulatory level. Transvaginal ultrasound was used to monitor endometrial changes and follicular growth. At least two follicles with a diameter greater than 20 mm were injected with AIZE 250 UG at night. The eggs were taken about 37 hours after AIZE injection and fertilized in vitro. Three days after oocyte retrieval, two high-quality embryos were transplanted. The pregnancy sacs were detected by transvaginal ultrasound 28 days after transplantation. All data were analyzed by SPSS13.0 statistical software. Statistical analysis. Measurement data were expressed by X There was no significant difference in age, duration of infertility, causes of infertility, basal FSH, basal LH and number of sinus follicles between the two groups (P 0.05). Significance (P 0.05). (See Table 2)3 Immunohistochemical detection of Ang-1, Ang-2 and MVD expression in endometrial stromal cells, glandular epithelial cells and vascular endothelial cells of pregnant and non-pregnant embryos in preimplantation window stage. There were 1 case of expression, "+" expression in 2 cases, "++" expression in 5 cases, "++" expression in 4 cases. Ang-2 was mainly expressed in endometrial stromal cells and glandular epithelial cells in clinical pregnancy. There was no significant difference in the expression of'-', 2'+', 3'+,'+', 5'+', 2'++', 3'-,'+', 6'+,'+', 2'++', 2'++', 2'+++'and 2'++++' in clinical pregnancy group (n = 13), 3.3.3 in non-pregnancy group (n = 13), 6 in'+', 2 in clinical pregnancy group (P = 0.335). MVD was detected by endothelial cells. The expression of microvessels in the endometrium was abundant in the periglandular matrix during the embryo implantation window. The expression of MVD (16.42 + 4.66) in pregnant group was higher than that in non-pregnant group (12.31 + 4.40), and the difference was statistically significant (P Ang-1 mRNA and Ang-2 mRNA expression in endometrium of clinical pregnancy group at implantation window stage were significantly higher than those of non-pregnancy group. Ang-1 mRNA expression was 0.72+0.35 (n=12) in pregnancy group and 0.33+0.32 (n=13) in non-pregnancy group. The difference was statistically significant (P=0.008). (See Table6) 4.2 Ang-2 mRNA expression in clinical pregnancy group. The expression of Ang-1 and Ang-2 was 0.6510.60 (n=12) in the endometrium of 5.1 IVF patients during implantation window, and the correlation between Ang-1 protein expression and MVD was 0.564 (P=0.003), respectively. The correlation coefficient between Ang-2 protein expression and MVD was 0.273, P = 0.187 in the endometrium of 5.2 IVF patients at implantation window stage, and there was no significant difference (see Table 8). Conclusion: Ang-1 protein was expressed in endometrial stromal cells, glandular epithelial cells and vascular endothelial cells at implantation window stage of preimplantation cycle. Ang-1 mRNA may be one of the endometrial receptivity-related genes. High expression of Ang-1 mRNA is more conducive to embryo implantation and has a better pregnancy outcome. Ang-1 mRNA may be one of the predictors of pregnancy outcome in IVF patients during implantation window. 3 Ang-2 protein is mainly expressed in endometrial stromal cells and endometrial stromal cells during implantation window. The expression of 4 Ang-2 mRNA in the endometrium of pregnant and non-pregnant embryos during the implantation window period was not different, and could not predict the pregnancy outcome of IVF patients. However, the mechanism and significance of Ang-2 mRNA in the evaluation of endometrial receptivity need to be further explored. MVD in the endometrium of pregnant women was significantly higher than that of non-pregnant women. MVD may be one of the predictors of pregnancy outcome. Ang-1 protein expression in the endometrium during implantation window was significantly correlated with MVD, but Ang-2 protein expression was not correlated with MVD. Endometrial rich blood perfusion is beneficial to embryo implantation.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R714.8

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8 王s，

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