【作者】 李喜红；

【导师】 卢光琇；

【作者基本信息】 中南大学 ， 基础医学， 2013， 博士

【摘要】 随着妇女生育年龄推迟及不孕症发病率的增高,辅助生殖技术(ART)已广泛应用于临床,而如何在孕早期准确评价胚胎的发育状况一直是广大学者关注的课题。当前高分辨率的经阴道超声(TVS)是孕早期最好的检查方法,对于评估胚胎的正常发育,或者是否存在提示妊娠失败的超声标志具有重要的意义。自然妊娠中,孕龄是根据末次月经(LMP)或头臀长估计,与实际孕龄有一定误差,故检测结果无统一标准,存在一定的局限性。采用体外受精-胚胎移植(IVF-ET)助孕者,由于其受孕日期准确,故本研究经TVS诊断IVF-ET后宫内外复合妊娠(HP)；并通过检测孕囊平均直径(GSD)、头臀长(CRL)、胎心率(HR)、卵黄囊平均直径(YSD)等超声指标；及孕早期经TVS诊断为小孕囊、胚胎生长迟缓等不同超声类型后,因胚胎止发育的流产胚胎,进行比较基因组杂交分析(CGH)及荧光原位杂交分析(FISH)。分别评价经TVS对IVF-ET助孕后HP的诊断价值,及对妊娠结局的预测价值,并探讨孕早期不同超声类型与染色体核型的相关性。本研究分三部分阐述如下：第一部分经阴道超声对IVF-ET后宫内外复合妊娠的诊断价值目的：探讨经阴道超声对IVF-ET后宫内外复合妊娠的诊断价值。方法：选择2005年1月-2011年12月,在本院生殖中心接受IVF-ET助孕后,经TVS于孕早期诊断为临床妊娠的16483例妇女中,184例发生HP,对其中132例经手术和病理确诊的HP患者的临床资料进行回顾性分析。HP的超声诊断标准：宫内妊娠囊合并(1)宫外妊娠囊内可见卵黄囊和／或胚芽,有或无原始心管搏动；或(2)宫外空妊娠囊由厚壁强回声环围绕,形态较规则；或(3)附件区非均质混合性包块,靠近卵巢而与卵巢不在同一个切面上。结果：1.16483例临床妊娠的妇女中,HP发生率1.12%(184/16483),其中174例经TVS诊断为HP,漏诊10例,52例经期待处理的HP患者由于缺乏病理学的诊断依据未纳入本研究。122例经TVS诊断及手术和病理确诊的HP,根据异位妊娠的超声类型分为：孕囊型70例,环状征21例,包块型31例。TVS诊断HP的灵敏度为92.42%,特异度为100%,阳性预测值为100%,阴性预测值为99.94%。2.132例经手术和病理确诊的HP,根据异位妊娠的发生部位分为：输卵管异位妊娠103例和非输卵管异位妊娠29例,其宫内妊娠结局：93例足月分娩(70.45%),37例早期流产(28.03%),2例晚期流产(1.52%),无死亡病例。结论：IVF-ET后HP的发生率明显增加,经TVS对IVF-ET后HP具有重要的诊断价值,有助于在异位妊娠破裂前早期诊断并选择适当的治疗方法,对于维持宫内妊娠的继续发展,防止不良妊娠结局的发生具有重要的意义。第二部分经阴道超声对IVF-ET后妊娠结局的预测价值目的：探讨IVF-ET后孕6-10周,评价经TVS检测GSD、CRL、 HR, YSD等超声指标与孕龄的关系,并结合孕妇年龄和阴道流血对妊娠结局的预测价值。方法：回顾性分析2010年1月-2011年5月,于本院生殖中心接受IVF-ET后孕6-10周,经TVS诊断为单活胎,根据妊娠结局分为继续妊娠组(n=2400)和早期流产组(n=201),其中继续妊娠组均追踪至出生。所有早孕胚胎均经TVS测量GSD、CRL、HR, YSD等超声指标,并记录孕妇年龄和阴道流血等其它指标,评价超声指标联合孕妇年龄和阴道流血对妊娠结局的预测价值。结果：1.继续妊娠组孕妇平均年龄30.60±4.31岁,早期流产组32.67±4.35岁,两组之间具有统计学差异(p<0.05)；早期流产率在不同孕妇年龄组之间具有统计学差异(p<0.05)。2.继续妊娠组GSD、CRL、HR、 YSD均与孕龄呈线性相关(p<0.05)。3.比较不同孕龄两组之间GSD、CRL、HR, YSD,其中GSD、CRL、HR除孕67-69天组样本较少(n=3),其余各孕龄两组之间比较,差异均有统计学意义(p<0.05)；YSD仅在部分孕龄两组之间有统计学意义(p<0.05)。4.孕6-10周活胚后继续妊娠率为92.27%。当GSD、CRL、HR分别低于继续妊娠组正常参考值范围(孕囊偏小、胚芽／头臀长偏短、胎心偏慢),YSD低于或高于继续妊娠组正常参考值范围(过大或过小)时,继续妊娠率分别为57.22%,51.00%,58.90%,71.60%；当所有的4个指标均在正常范围时继续妊娠率为97.32%,均在异常范围时继续妊娠率仅为6.90%。各指标预测流产风险：GSD (OR=14.51), CRL (OR=22.08), HR (OR=22.38), YSD (OR=6.19),孕妇年龄(OR=2.24),阴道流血(OR=5.21)。5.孕6-10周GSD、CRL、HR, YSD等超声指标单因素预测早期流产,其灵敏度分别为：41.29%,50.25%,65.67%,27.86%；特异度分别为：95.38%,95.63%,92.13%,94.13%。6.多因素Logistic回归分析预测妊娠结局：GSD、CRL、HR YSD,以及孕妇年龄(AGE)和阴道流血(BLOOD)为影响妊娠结局的危险因素。Logit P=-5.161+1.401GSD+1.876CRL+2.341HR+1.150YSD+0.530AGE+2.006BLOOD。应用方程预测早期流产,其灵敏度为51.74%,特异度为99.29%,阳性预测值为85.95%,阴性预测值为96.09%,假阳性率为0.71%,诊断正确率为95.62%。结论：1.IVF-ET助孕后孕6-10周,孕妇年龄较大、阴道流血和超声检测发现的孕囊偏小、胚芽(头臀长)偏短、胎心偏慢、卵黄囊过大或过小,与妊娠结局不良密切相关。2.TVS检测孕6-10周GSD、CRL、HR、YSD,并联合孕妇年龄、阴道流血等指标对妊娠结局具有重要的预测价值。第三部分IVF-ET后早期流产胚胎超声类型与染色体核型的相关性目的：探讨IVF-ET后早期流产胚胎超声类型与染色体核型的相关性。方法：回顾性分析2005年1月-2011年12月,793例于我院行IVF-ET助孕后,经TVS诊断单胎妊娠早期流产,并行流产胚胎CGH及FISH分析,探讨超声类型与染色体核型的相关性。超声类型分为空孕囊型、仅见卵黄囊的孕囊型、胚胎生长迟缓型、正常超声类型、小胚芽型、小孕囊型等六种。结果：1.793例IVF-ET后早期流产胚胎中,异常核型率为44.77%(355/793)；其中不同超声类型异常核型率分别为：小孕囊58.33%(14/24),胚胎生长迟缓56.17%(91/162),小胚芽50.49%(52/103),正常超声类型44.67%(67/150),仅见卵黄囊的孕囊44.51%(77/173),空孕囊29.83%(54/181)；与总体相比,胚胎生长迟缓型异常核型率较高,空孕囊型异常核型率较低(p<0.05)。2.IVF-ET后早期流产胚胎最常见的异常染色体核型依次为：16三体(19.44%)、X单体(13.80%)、22三体(11.83%)、21三体(7.04%),13三体(5.07%)、20三体(4.79%),占61.97%(220/355)。3.不同超声类型与异常染色体核型的关系：(1)胚胎生长迟缓型、仅见卵黄囊的孕囊型、小胚芽型常见的异常染色体核型为16三体和22三体；(2)空孕囊型常见的异常染色体核型为X单体和22三体；(3)正常超声类型常见的异常染色体核型为X单体和21三体；(4)小孕囊型不与特定的异常染色体核型相关。结论：1.IVF-ET后孕早期流产胚胎中,超声类型与染色体核型相关,其中,胚胎生长迟缓型染色体异常核型率较高；空孕囊型染色体异常核型率较低。2.早期自然流产胚胎中,16三体、22三体、X单体、21三体、13三体、20三体等六种异常染色体核型发生频率高。3.不同超声类型中特定的异常染色体核型发生频率高。更多还原

【Abstract】 With the postponement of child-bearing age and increased incidence of infertility, assisted reproductive technology (ART) has now been widely used in clinic. How to evaluate the embryonic developmental status accurately in the first trimester has always been a hot subject. The high-resolution transvaginal sonography (TVS) is currently the best way to check early pregnancy and of great importance in evaluating the developmental condition of embryo as well as predicting pregnancy failure.In natural pregnancy, gestational age (GA) is estimated based on the last menstrual period (LMP) or crown-rump length (CRL), which may deviate from the actual GA. However, in the IVF-ET procedure, the date of conception is quite accurate.In this study, we diagnosed heterotopic pregnancy (HP) after in vitro fertilization-embryo transfer (IVF-ET) using TVS, investigated the ultrasonographic markers (gestational sac diameter GSD, crown-rump length CRL, heart rate HR, yolk sac diameter YSD) of embryos, and analyzed aborted embryos by comparative genomic hybridization analysis (CGH) together with fluorescence in situ hybridization technology (FISH), which presenting small gestational sac, early symmetrical arrested growth and other ultrasonic morphological types in early pregnancy. And then we evaluated the diagnostic value of TVS in diagnosing HP as well as the predictive value of ultrasonographic markers for pregnancy outcomes in the first trimester and investigated the correlation between different ultrasonic morphological types and abnormal karyotypes in embryos from patients with early pregnancy loss (EPL) after IVF-ET. This study was divided into three parts as follows: CHAPTER ONEValue of transvaginal sonography in diagnosing heterotopicpregnancy after in vitro fertilization-embryo transferOBJECTIVE:To evaluate the diagnostic value of transvaginal sonography (TVS) in the diagnosis of heterotopic pregnancy (HP) after in vitro fertilization-embryo transfer (IVF-ET).METHODS:From January2005to December2011,16,483women received IVF-ET treatment and were diagnosed as clinical pregnancies by TVS in the reproductive center of our hospital,184out of whom were diagnosed as with a HP. We carried a retrospective review study for the clinical data of132cases, whose HPs were confirmed by surgeries and pathological examinations. The HP diagnosis was made if a visible intrauterine gestational sac was detected by TVS with any of the following indicators:(1)a yolk sac and/or fetal pole±cardiac activity in an extrauterine sac or (2) an empty extrauterine gestational sac observed as a hyperechoic ring; or (3) an inhomogeneous adnexal mass or blob sign adjacent to the ovary that moved separately from the ovary.RESULTS:1. Of the16,483women who had clinical pregnancies,174cases were diagnosed as having a HP by TVS, and10cases were missed, with an incidence of1.12%(184/16,483).52cases who were treated expectantly were excluded from the analysis due to lacking of pathological diagnosis. In the122HPs which were diagnosed by TVS and confirmed by surgeries and pathological examinations, three types of ultrasonographic images of ectopic pregnancy (EP) were seen:with a gestational sac found in70cases, a ring sign in21and an adnexal mass in31. The sensitivity and specificity of TVS in detecting HP were92.42%(122/132) and100%(16351/16351), with positive and negative predictive value of100%(122/122) and99.94%(16351/16361) respectively.2. According to the occurrence site, the132HPs confirmed by surgeries and pathological examinations were comprised of103tubal EPs and29non-tubal EPs. And the intrauterine pregnancy (IUP) outcomes were:93patients (70.45%) resulted in a live birth,37patients (28.03%) suffered an early miscarriage and2patients (1.52%) had a late miscarriage. There was no death in this study.CONCLUSION:The incidence of HP significantly increased in IVF-ET procedure. TVS promotes the early diagnosis and appropriate treatment for the unruptured EPs. And it is of great significance in the maintenance of the IUP till a term delivery and the prevention of the occurrence of adverse pregnancy outcomes. CHAPTER TWOValue of transvaginal sonography in predicting pregnancy outcomes after in vitro fertilization-embryo transferOBJECTIVE:To investigate the gestational sac diameter (GSD), crown-rump length (CRL), heart rate (HR) and yolk sac diameter (YSD) of embryos in6-10gestational weeks using TVS after IVF-ET and to assess the correlation between these ultrasonographic markers and gestational age (GA). And then, to evaluate the predictive value of these markers for pregnancy outcomes combined with maternal age (MA) and vaginal bleeding.METHODS:This was a retrospective review study.2601women were diagnosed with a single live embryo by TVS in the6-10gestational weeks from January2010to May2011in the reproductive center of our hospital. They were divided into two groups:the continue pregnancy group (n=2400) and the early miscarriage group (n=201). Patients in the continue pregnancy group were followed untill born. When the TVS examination was given, we measured the GSD, CRL, HR, YSD and recorded MA, vaginal bleeding and related other markers at the same time. Finally, we evaluated the predictive value of sonographic markers together with MA, vaginal bleeding and other markers.RESULTS:1.There was a statistically significant difference in the MA between the continue pregnancy group and the early miscarriage group (30.60±4.31years VS32.67±4.35years, p<0.05); And the early miscarriage rate differed significantly among different MA groups (p<0.005).2.GSD, CRL, HR and YSD respectively had a linear correlation with GA (p<0.05) in the continue pregnancy group.3.The difference in GSD, CRL, HR and YSD of embryo between continue pregnancy group and the early miscarriage group were significant (p<0.05) in different GA except67-69gestational weeks due to small sample size (n=3); YSD differed significantly between these two groups only in some GAs (p<0.05)4.The ongoing pregnancy rate was92.27%when embryo was alive in6-10gestational weeks. When GSD, CRL, HR respectively below and YSD beyond (too small or too large) the normal reference range of the continue pregnancy group, the ongoing pregnancy rate were57.22%,51.00%,58.90%,71.60%, respectively. Four normal markers contributed to an ongoing pregnancy rate of97.32%; only6.90%when all four markers were abnormal. The value of each marker to predict the risk of miscarriage:GSD (OR=14.51), CRL (OR=22.08), HR (OR=22.38), YSD (OR=6.19), MA (OR=2.24), BLOOD (vaginal bleeding)(OR=5.21).5.The sensitivity of single ultrasonographic marker (GSD, CRL, mHR, YSD) to predict early pregnancy outcomes was41.29%,50.25%,65.67%,27.86%; And the specificity was95.38%,95.63%,92.13%,94.13%in6-10gestational weeks, respectively.6.Multivariate logistic regression analysis to predict pregnancy outcomes:GSD, CRL, YSD, HR, MA, BLOOD were risk factors for pregnancy outcomes.LogitP=-5.161+1.401GSD+1.876CRL+2.341HR+1.150YSD+0.530MA+2.006BLOOD.The efficiency of this equation to predict an early miscarriage was with a sensitivity of51.74%, specificity of99.29%, positive predictive value of85.95%, negative predictive value of96.09%, false-positive rate of0.71%, and the diagnostic accurate rate of95.62%.CONCLUSION:1. In the6-10gestational weeks after IVF-ET, older MA, vaginal bleeding, small gestational sac, short CRL, slow fetal heart rate and abnormal yolk sac (too large or too small) related to adverse pregnancy outcomes.2. Sonographic markers (GSD, CRL,HR, YSD) in early pregnancy together with MA, vaginal bleeding had important value in predicting pregnancy outcomes. CHAPTER THREE Correlation between ultrasonic morphological types and karyotypes in early pregnancy loss after in vitro fertilization-embryo transfer OBJECTIVE:To investigate the correlation between ultrasonic morphological types and karyotypes in embryos from patients with early pregnancy loss (EPL) after in vitro fertilization-embryo transfer(IVF-ET).METHODS:This retrospective analysis included patients who had been diagnosed by TVS as early single pregnancy loss after IVF-ET between January2005and December2011. All793aborted embryos had undergone cytogenetic analysis by comparative genomic hybridization analysis (CGH) together with fluorescence in situ hybridization technology (FISH). Correlation between ultrasonic morphological types and karyotypes was evaluated. Based on different ultrasonic morphological features, we classified the embryos into6types:empty sac, gestational sac with only yolk sac, normal ultrasound type, early symmetrical arrested growth, small embryo-fetus, and small gestational sac.RESULTS:1. In the793EPLs, the abnormal karyotype rate was44.77%(355/793); And the abnormal karyotype rate of different ultrasonic morphological types were as follows:small gestational sac of58.33%(14/24), early symmetrical arrested growth of56.17%(91/162), small embryo-fetus of50.49%(52/103), normal ultrasound of44.67%(67/150), anembryonic pregnancy of44.51%(77/173) and empty gestational sac of29.83%(54/181); The prevalence of chromosomal abnormalities was significantly higher in early symmetrical arrested growth group and markedly lower in empty sac group than that in total (p<0.05)2. Trisomy16(19.44%), monosomy X (13.80%), trisomy22(11.83%),trisomy21(7.04%), trisomy13(5.07%), and trisomy20(4.79%) were in turn the most frequent chromosomal abnormalities in the EPLs after IVF-ET, accounting for61.97%(220/355).3. The correlation between different ultrasonic findings and abnormal karyotypes were: (1) Trisomy16and trisomy22were found to be the most common karyotypes in EPLs presenting early symmetrical arrested growth, anembryonic pregnancy and small embryo-fetus.(2) Majority cases of the empty gestational sac were found to be correlated with monosomy X and trisomy22;(3) The most frequent chromosomal anomalies in EPLs with normal ultrasound were monosomy X and trisomy21;(4) Small gestational sac group had no obvious correlation with specific chromosomal abnormalities.CONCLUSION:1. There was a correlation between ultrasonic morphological types and karyotypes in EPLs after IVF-ET. And the early symmetrical arrested growth group had a higher rate of chromosomal abnormalities than that of the empty gestational sac group.2. Six types of chromosomal abnormalities (trisomy16, trisomy22, monosomy X, trisomy21, trisomy13and trisomy20) had the most frequency in the EPLs after IVF-ET.3. There was a correlation between different ultrasonic findings and specific abnormal karyotypes.更多还原