【作者】 李秋华；

【导师】 全松；

【作者基本信息】 南方医科大学 ， 妇产科学， 2010， 硕士

【摘要】 研究背景目前认为影响IVF/ICSI-ET结局最主要的几个因素为：(1)胚胎质量；(2)子宫内膜容受性(endometrial receptivity, ER); (3)胚胎发育与子宫内膜的同步化。在胚胎质量较难改善的情况下,如何提高ER就显得至关重要。因此,如何准确、及时评价ER,以及怎样提高ER引起了临床医生及学者的广泛关注。子宫内膜容受性是指子宫内膜允许胚胎定位、粘附、穿透,并诱导间质发生改变,从而允许胚胎着床的状态。评价ER最准确的是子宫内膜活检和病理学检查,通过子宫内膜的组织形态学结构评价ER。血管发生是从已经存在的血管形成新的血管,在成人血管发生受到严格调控,胚胎着床、发育与子宫内膜血管周期性增殖密切相关。组织中,血管的发生可以通过测定微血管密度(microvessel density, MVD)来反映,目前血管内皮细胞的标记物很多,有CD34、CD31、Ⅷ因子等。人类白细胞抗原CD34是近年来发现的一种新的与小血管相关的抗原,为血管内皮细胞的特异性标记,其灵敏度及特异性高于内皮细胞的其他标记物。本研究采用鼠抗人CD34单克隆抗体标记子宫内膜内皮细胞,采用Weildner的方法计算MVD。近年来,超声技术越来越多的应用到辅助生殖领域,特别是随着三维超声技术的发展,其用在辅助生殖技术中评价ER的价值已为人们所关注并进行相关研究。超声评价ER的参数包括：子宫内膜容积、子宫内膜厚度、类型；彩色多普勒超声测量子宫动脉血流的阻力指数(RI)、搏动指数(PI)；子宫内膜及内膜下血流3DU-PDA参数(三维超声能量多普勒血管成像)：VI(血管指数)、FI(血流指数)、FVI(血管血流指数)。许多报道提示子宫内膜容积无法预测ER,子宫内膜厚度及类型在评价ER方面也缺乏特异性。Ng等认为三维超声及其衍生技术3DU-PDA可能通过评估子宫内膜容受性-宫腔微环境(ER-UM),提高对IVF-ET治疗结局的预测效能。陈雷宁等发现优质胚胎移植后早期妊娠结局中,持续妊娠组较流产组子宫内膜3DU-PDA参数VI、FI、VFI均偏高。Merce等发现在IVF/ICSI-ET周期中,子宫内膜VI、FI、VFI妊娠患者比非妊娠患者要高,差异具有统计学意义,预测妊娠的ROC(受试者工作特征曲线)曲线下面积为VI (0.724)、FI (0.828)和VFI(0.800),作者认为3DU-PDA参数有利于评价ER。胞饮突被认为是ER的一个标志性结构,其功能可能是直接或间接参与胚胎与子宫内膜的黏附反应。Nikas等对连续活检的内膜进行扫描电镜观察,结果显示：胞饮突出现在排卵后的1周左右,且在2天内发育并退化,平均出现在自然月经周期的第20-21天,持续时间不超过48小时。根据表达有胞饮突的子宫内膜占整个内膜的百分比,划分为丰富(>50%)、适中(20%～50%)和少量；胞饮突发育按Nikas等制定的标准分为发育期、成熟期、衰退期3个发育阶段。越来越多的证据表明这些胞饮突是ER的精确标记,其出现、完全发育及退化的时间与种植窗完全吻合,可用其作为判断子宫内膜种植床的标志。目的探讨子宫内膜微血管密度和三维超声参数之间的关系,以及二者评价子宫内膜容受性和在预测IVF-ET结局方面的价值。方法1、研究对象104例于2009年4月-2009年12月在南方医院生殖医学中心进行IVF/ICSI-ET治疗的患者,平均年龄为31.64±4.09岁。2、纳入标准与排除标准纳入标准：1)受试者愿意并有足够智力签署知情同意书。2)采用黄体中期长方案。3)月经周期规则。4)新鲜周期。排除标准：1)有细菌性阴道炎、急性盆腔炎、附件炎,或其它全身性感染。2)有凝血功能缺陷或出血性疾病。3)取消新鲜胚胎移植。4)卵泡未破裂黄素化综合征(LUFS)。5)周期开始前服用雌激素、孕激素、阿司匹林、复方丹参等药物一个月以上的。3、分组标准追踪IVF/ICSI-ET结局按是否获临床妊娠分成2组：临床妊娠组和未妊娠组。4、助孕方案所有患者均在IVF-ET前一个自然周期的第8-10天进行阴道超声监测卵泡发育,当优势卵泡直径达到14mm时辅助以尿LH峰监测。采用促黄体激素(LH)检测试剂盒(上海凯创生物技术有限公司),每6h测一次,当T≥C是来中心复诊,出LH峰当天或在24h内抽血,测血中LH和E2的浓度。按照LH峰后36h左右监测有无排卵,若排卵,排卵后7天取内膜组织,在同一天给予GnRHa(达菲林)。所有患者均采用黄体中期长方案,具体方法参见我中心常规。5、子宫内膜取材方法患者喝水留尿保持膀胱充盈,服用达利新一片预防感染,术后继续服用达利新,每日两次连用三天。超声仪采用Voluson i(美国GE公司),调整探头在腹部超声模式下。患者采取膀胱截石位,常规消毒铺巾,采用立可灵一次性宫腔组织吸引管(C3.1／30-1S型,上海家宝医学保健科技有限公司),它利用自身负压原理(≥200mmHg)从宫腔吸取子宫内膜组织,主要用于子宫内膜组织病理学检查和细胞学检查。这种管由高分子材料制成,管径只有3.1mm,具有创伤小、安全、简便的优点。超声引导下进管,平均进管深度7.8cm,到宫底后壁(前壁)然后往回抽吸,带出少量子宫内膜组织,然后第二次进管到前壁(后壁),再次抽吸,共抽两次。所取内膜组织分成两部分,部分内膜组织放入10%的甲醛溶液固定,石蜡包埋制片。另一部分用PBS缓冲液冲洗,然后用2.5%的戊二醛液固定至少8h。然后脱水制片行扫描电镜观察。6、免疫组化方法采用鼠抗人CD34单克隆抗体标记不孕症患者子宫内膜内皮细胞,采用Weildner等方法计算子宫内膜组织MVD。7、三维超声检查方法每位患者于IVF前一自然周期围着床期和HCG注射日用Voluson i(美国GE公司)超声仪分别测量子宫内膜的3DU-PDA参数(子宫内膜容积；内膜及内膜下VI、FI、VFI)并记录。8、子宫内膜胞饮突测量应用南方医科大学分析测试中心日本日立S-3000N型扫描电子显微镜(加速电压：0.3～30kV；分辨率：3 nm；放大倍率：×15～300000。对随机抽取的7例患者的子宫内膜组织进行观察,计数胞饮突。9、图片分析MVD测定采用Image J软件(美国国立卫生研究院)自动计算免疫组化图片褐色区域面积占整个视野的百分比。10.统计学分析应用SPSS 13.0统计学软件处理,对计量资料以均数±标准差(X±SD)表示,计数资料以百分率表示。对计量资料采用两独立样本t检验,计数资料采用卡方检验,预测妊娠评价指标采用受试者工作特征曲线(ROC曲线)。微血管密度和三维超声参数的相关分析采用双变量相关分析。微血管密度和三维超声参数联合预测妊娠采用二分类的Logistic回归分析。当P<0.05时,认为差异有统计学意义。结果1、围着床期的确定：随机抽取临床妊娠组(3例)和未妊娠组(4例)共7例患者的子宫内膜组织行扫描电镜检查,均观察到胞饮突,证实所取的子宫内膜组织均在围着床期。2、微血管密度比较临床妊娠组(50例)MVD(4.12±1.84%)高于未妊娠组(54例)MVD(3.46±1.26%),差异有统计学意义(t=-2.127,P=0.036),但MVD预测妊娠的ROC曲线下面积仅为0.598,用于判断妊娠无意义。3、三维超声参数比较临床妊娠组(31例)与未妊娠组(36例)之间围着床期子宫内膜3DU-PDA参数Ⅵ：2.24±4.11%／1.47±1.63%；FI：25.26±5.85／23.41±4.41；VFI：0.72±1.18／0.444±0.509；内膜下Ⅵ：4.18±4.32%／3.20±3.24%；内膜下FI：25.99±6.20／25.15±4.08；内膜下VFI：1.19±1.36／1.00±0.99；子宫内膜容积：3.80±1.84ml／3.75±1.79ml,均无显著性差异(P>0.05)。但临床妊娠组患者HCG日子宫内膜下FI(27.56±4.89%)高于未妊娠组(25.15±4.08%),差异具有统计学意义(t=-2.398,P=0.019)。结论在辅助生殖技术中测量子宫内膜微血管密度和三维超声参数对评价子宫内膜容受性有一定意义,但是特异性不高。更多还原

【Abstract】 BACKGROUNDSuccessful implantation depends on a close interaction between the functional blastocyst and the receptive endometrium.Under certain conditions,we can not improve the quality for embryos significantly,we turn to improve the endometrial receptivity.Histological evaluation, now considered to add little clinically significant information,should be replaced by functional assessment of endometrial receptivity.Angiogenesis,the formation of new blood vessels from existing vessels, plays a critical role in various female reproductive processes such as develoment of a dominant follicle,formation of a corpus luteum;growth of endometrium and implantion. A good blood supply towards the endometrium is usually considered as an essential requirement for implantion. Angiogenesis can be described by a microvessel density(MVD) count. MVD can be measured by staining for CD34. CD34 is a glycosylated transmembrane protein present on endothelial cells and may possibly be involved in endothelial migration during angiogenesis.Potential uterine predictors for implatation that are measurable by sonography include endometrial thickness and volume,endometrial pattern and also blood flow in the uterine and (sub)endometrial arteries.Unfortunately,two-dimensional ultrasound studies on blood flow in (sub)endometrial vessels are limited in their selection of individual vessels and the assumption that these are representative of the subendometrium as a whole. The combination of power Doppler sonography with the three-dimensional ultrasound (3D-PDS) is better suited to the study of the (sub)endometrial vasculature, as it provides a unique tool for examining the blood supply of the whole (sub)endometrial region, as opposed to individual vessels or two-dimensional planes.Ultrasound parameters including:endometrial volume,endometrial thickness, endometrial pattern, uterine artery Doppler pulsatility index (PI) and resistance index (RI),three-dimensional ultrasound parameters such as vascularization index (VI), which measures the ratio of the number of colour voxels to the number of all the voxels, is thought to represent the presence of blood vessels (vascularity) in the endometrium, and this was expressed as a percentage (%) of the endometrial volume;flow index (FI), the mean power Doppler signal intensity inside the endometrium, is thought to express the average intensity of flow;vascularization flow index (VFI) is a combination of vascularity and flow intensity.Previous studies could not demonstrate that the endometrial volume is predictive for pregnancy, and this could be explained by methodologic differences in the volume calculation. A triple-layer endometrial pattern and an endometrial thickness greater than 7 mm have been proposed as markers of endometrial receptivity but have yielded a high percentage of false-positive results.Three-dimensional ultrasonography and 3DU-PDA may predictive IVF-ET outcome by the way of evaluating endometrial receptivity- uterine cavity micro-environment. Chen Lei-ning found the relationship between three-dimensional ultrasonography and power Doppler angiography (3DU-PDA) parameters of endometrium on HCG administration day and on-going pregnancies or early spontaneous abortions after good quality embryos transfer,they found that the the endometrial vascularization index, flow index andvascularization flow index were significantly higher for on-going pregnancy group than the early spontaneous abortion group(P< 0.05).Pinopods are bleb-like protrusions on the apical surface of the endometrial epithelium.These structures are several micrometers wide and project into the uterine lumen above the microvilli level. They were first described in mice, and later in human endometrium.The term’pinopod’, from the Greek’drinking foot’, signifies their pinocytotic function in the mouse.Nikas fond that pinopods can be only fond between days 20 and 21 of a regular menstrual cycle,and that their total lifespan did not exceed 48 h. More and more scholars think that uterine pinopodes as markers of the’window of implantation’ According to expression pinopodes endometrial lining of the percentage of the total, is divided into rich (50%), moderate (20%～50%) and a small amount.Pinopodes were classified as developing,fully developed,orregressing.More and more studies evidenced that pinopodes is the exact marker of implantation window.OBJECTIVEThe aim of this study was to evaluate the relationship between microvessel density and endometrial and subendometrial blood flows measured by 3D power Doppler ultrasoud and their role of assessing endometrial receptivity.METHODS1、Patients Patients undergoing their frist IVF/ICSI-ET cycle in the Assisted Reproduction Unit of the Department of Obsterics and Gynaecology,The University of Southern Medical University between April 2009 to December 2009 were recruited if they meeting the inclusion and exclusion criterias. Finally, a total of 104 patients were recruited, the average age is 31.64±4.09 years old.2、The inclusion criteria①All the patients have enough intelligence to participate the study,and every patient gave a written informed consent prior to participating in the study.②A mid-luteal phase long protocol③Women with regular menstrual cycles④Fresh cycleThe exclusion criteria①Acute cervicitis;vaginitis;bacterial vaginosis;Bartholin’s duct cyst and abscess;urinary tract infection and other pelvic inflammatory disease②Blood diseases and disorders③Failue to embryo transfer④Luteinized unruptured follicle syndrome(LUFS)⑤Application of estrogen,aspirin, salvia, at least a month before the the assisted cycle3. SubgroupThe patients in every part of this study were divided into two groups:the clinical pregnancy group; non-pregnant group.4. IVF protocolAll subjects had ovarian stimulation according to a mid-luteal phase long p rotocol.Follicular monitoring by vaginal ultrasonography was initiated on cycle day 10, and after a dominant follicle sized 14mm was identified, we detection the urinary LH surges every 6 hours daily by urinary luteinizing hormone plate (Chemtrue company,ShangHai). Blood for LH and E2 determination was drawn on the day of the urinary LH surge.36 hours later, vaginal ultrasonography was initiated,if the leading follicle (s) disappeared we gave the patients GnRHa 7 days later.5.Gain endometrium tissueOn the GnRHa day we gain endometrium tissue by disposable uterine cavity suction catheter (C3.1/30-1S, Jiabaocompany,ShangHai) The catheter entered uterine cavity about 7.8cm, aspirating two times. All of the endometrium tissue were divided into two groups,one in formaldehyde(10%) for Imunohistochemistry(IHC); one in glutaraldehyde(2.5%)for scanning electron microscope(SEM).6.ImmunohistochemistryEndometrial microvessel density(MVD) was detected by dyeing vascular endothelial cells with CD34 mouse anti-human monoclonal antibody.7.Three-dimensional ultrasound observation time and Ultrasonic InstrumentAll ultrasound measurements were performed by one ultrasound doctor on the day of GnRHa and HCG. throuth Voluson i (GE Medical Systems).8. Scanning electron microscope(SEM)Hitachi,S-3000N,Japan (AV:0.3-30kV; 2e-image resolution:3nm; Magnification:×15～300000)9. Image analysisMVD count by Image J software (U.S. National Institutes of Health), and the software can calculate immunohistochemistry images brown, size of the area percentage of the total field of vision automately.10. Statistical analysisStatistical analysis was performed using the Statistical Program for Social Sciences (SPSS Inc., version 13.0, Chicago, IL). Statistical.Comparison was carried out by Independent-Samples T test and Chi-Square test. The receiver operating characteristic (ROC) curve analysis and Logistic regression were applied to determine the best predictive variables.The two-tailed value of P<0.05 was considered statistically significant.RESULTS1. Scanning electron microscopy observation endometrial pinopods The clinical pregnancy group (3 cases); non-pregnant group (4cases).we can see pinopods both of the two groups,so we can say that we get the endometria in the period of window of implantation.2.MVDThe MVD for the clinical pregnancy group (50 cases,4.12±1.84%)were significantly higher than non-pregnant group (54 cases,3.46±1.26%)(t=-2.127, P=0.036).3. Three-dimensional ultrasound parametersThe clinical pregnancy group (31 cases), non-pregnant group (36 cases).There was no significant difference in 3DU-PDA parameters on GnRHa day between the two groups, VI:2.24±4.11%/1.47±1.63%; FI:25.26±5.85/23.41±4.41; VFI: 0.72±1.18/0.44±0.509; subendometrial VI:4.18±4.32%/3.20±3.24%; subendometrial FI:25.99±6.20/25.15±4.08; subendometrial VFI:1.19±1.36/1.00±0.99; endometrial volume:3.80±1.84ml/3.75±1.79ml, The patients of the clinical pregnancy group on HCG day in subendometrial FI (27.56±4.89) is higher than that of the non-pregnant group (25.15±4.08%), the difference was statistically significant (t=-2.398, P= 0.019).CONCLUSIONIn the IVF/ICSI-ET cycles, microvessel density and 3DU-PDA parameters is helpful to assessing endometrial receptivity,but the specificity is low.更多还原