促血管生成素评估IVF患者子宫内膜血管形成及内膜容受性

发布时间：2018-08-27 14:24

【摘要】：目的：通过研究体外受精-胚胎移植(in vitro fertilization embryo transfer, IVF-ET)患者预备周期胚胎着床窗口期子宫内膜促血管生成素(angiopoietins, Angs)、子宫内膜微血管密度(microvessel density, MVD)表达及相关性分析,进一步阐明Ang-1、Ang-2评估子宫内膜血管形成及内膜容受性预测妊娠结局的价值。方法：选择2014年8月～2014年12月在河北医科大学第四医院生殖医学科首次行IVF助孕治疗患者。年龄为20-35岁,具有正常月经周期,周期21-35天,不孕原因主要为输卵管因素,新鲜周期移植2个优质胚胎。于IVF预备周期胚胎植入窗口期获取子宫内膜,-80℃冻存样本,部分组织用4%甲醛固定。分别用逆转录聚合酶链反应(Reverse Transcription-Polymerase Chain Reaction, RT-PCR)和免疫组织化学技术(immunohistochemistry)测定子宫内膜中Ang-1、Ang-2含量,用免疫组织化学技术测定子宫内膜MVD。应用免疫组化检测：将子宫内膜组织用4%甲醛固定后石蜡包埋,制备4μm厚连续组织切片。分别进行HE和免疫组化染色。用PBS代替一抗做阴性对照。每张染色片随机选取5个视野,进行结果分析,显微镜观察并拍照。应用RT-PCR检测技术,分别取研究组及对照组子宫内膜组织加入Rezol 1 ml置匀浆器内充分匀浆,提取总RNA,取出0.5μ1,进行反转录得cDNA,继而进行PCR扩增,同时以扩增片段长度为605 bp的GAPDH作为内参照。Ang-1和Ang-2PCR循环条件为：95℃10 min,1个循环；95℃ 45 s,60℃ 45 s,72℃50 s,35个循环；72℃ 10 min,1个循环。产物经1.5%琼脂糖凝胶电泳后,用VDS图像分析系统进行照相和图像分析。IOD值经内参照校正,将校正值进行统计学分析。控制性超促排卵：所有患者均采用黄体期长效长方案,即排卵后6-7天(黄体中期)给予醋酸亮丙瑞林(北京博恩特药业有限公司)1.3mg,达到降调节标准后给予重组人促卵泡激素注射液150-225IU(默克雪兰诺公司,意大利),采用经阴道超声监测子宫内膜变化和卵泡生长情况,当至少有2个卵泡直径大于20mm时,当晚注射艾泽(重组人绒促性素注射液,意大利)250μg,艾泽注射后37h左右取卵,适时加精,体外受精,于取卵后3d,移植优质胚胎2枚,移植后28d经阴道B超见到孕囊为临床妊娠。所有数据采用SPSS13.0统计软件进行统计分析。计量资料采用x±s表示,组间差异采用t检验,P0.05即有显著性差异；等级资料采用秩和检验,P0.05即有显著性差异。对相关的计量资料采用Person相关系数进行相关性分析。结果：1临床妊娠组与未妊娠组一般情况比较临床妊娠组(n=12)与未妊娠组(n=13)比较,在年龄、不孕年限、不孕原因、基础FSH、基础LH及窦卵泡数方面,差异无统计学意义(P0.05)。(见Tablel)2临床妊娠组和未妊娠组之间IVF-ET常规比较两组之间获卵率、优胚率、移植日内膜、移植日E2及移植日P差异无统计学意义(P0.05)。(见Table2)3免疫组织化学检测临床妊娠组和未妊娠组预备周期胚胎着床窗口期子宫内膜Ang-1、Ang-2及MVD表达3.1 Ang-1在预备周期胚胎着床窗口期子宫内膜基质细胞、腺上皮细胞及血管内皮细胞均有表达。临床妊娠组(n=12),“—”表达1例,“+”表达2例,“++”表达5例,“+++”表达4例。未妊娠组(n=13),“—”表达4例,“+”表达5例,“++”表达3例,“+++’’表达1例,差异有统计学意义(P=0.026)。(见Table3)3.2Ang2主要在子宫内膜基质细胞及腺上皮细胞表达。Ang-2在临床妊娠组与未妊娠组均有表达,临床妊娠组(n=13),“—”表达2例,“+”表达3例,“++”表达5例,“+++”表达2例。未妊娠组(n=13),“—”表达3例,“+”表达6例,“++”表达2例,“+++”表达2例,差异无统计学意义(P=0.335)。(见Table4)3.3以CD34特异性标记血管内皮细胞测定MVD。胚胎着床窗口期子宫内膜组织中微血管在腺体周围基质中表达丰富。妊娠组MVD(16.42±4.66)表达量高于未妊娠组(12.31+4.40),差异有统计学意义(P0.05)。(见Table5)4 RT-PCR检测临床妊娠组和未妊娠组预备周期胚胎着床窗口期子宫内膜Ang-1mRNA及Ang-2mRNA表达4.1胚胎着床窗口期临床妊娠组子宫内膜Ang-1mRNA的表达明显高于未妊娠组,Ang-1mRNA在妊娠组的表达为0.72+0.35(n=12),未妊娠组的表达为0.33+0.32(n=1 3),差异有统计学意义(P=0.008)。(见Table6)4.2 Ang-2mRNA在临床妊娠组的表达为0.6510.60(n=12),未妊娠组的表达为0.65±0.41(n=13),差异无统计学意义(P=0.988)。(见Table6)5胚胎着床窗口期子宫内膜Ang-1、Ang-2蛋白表达水平与MVD相关性分析5.1 IVF患者胚胎着床窗口期子宫内膜Ang-1蛋白表达与MVD相关系数0.564,P=0.003,差异有统计学意义。(见Table7)5.2 IVF患者胚胎着床窗口期子宫内膜Ang-2蛋白表达与MVD相关系数0.273,P=0.187,差异无统计学意义。(见Table8)结论：1 Ang-1蛋白在预备周期胚胎着床窗口期子宫内膜基质细胞、腺上皮细胞及血管内皮细胞均有表达。2 Ang-1 mRNA可能为子宫内膜容受性相关基因之一,高表达的Ang-1mRNA更利于胚胎植入,有较好的妊娠结局。IVF患者胚胎着床窗口期子宫内膜Ang-1mRNA可能为预测妊娠结局的指标之一。3 Ang-2蛋白主要在预备周期胚胎着床窗口期子宫内膜基质细胞及腺上皮细胞表达。4 Ang-2 mRNA在临床妊娠组与未妊娠组预备周期胚胎着床窗口期子宫内膜表达无差异,不能预测IVF患者妊娠结局,但其在评估子宫内膜容受性中的作用机制及意义有待进一步探索。5 IVF患者预备周期胚胎着床窗口期临床妊娠组子宫内膜MVD明显高于未妊娠组,子宫内膜组织丰富的微血管更有利于胚胎植入,MVD可能为预测妊娠结局的指标之一。6胚胎着床窗口期子宫内膜Ang-1蛋白表达与MVD显著相关,Ang-2蛋白表达与MVD无相关关系。Ang-1是促进血管生成的因子,子宫内膜丰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.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R714.8

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