Abstract目的： 我国出生缺陷的发生率为4%~6%，染色体非整倍体是导致新生儿出生缺陷的主要原因之一。目前国内外对该类疾病均无有效的治疗方法，其患儿给家庭和社会带来了沉重的精神和经济负担，因此在孕期及时诊断出胎儿染色体异常，适时终止妊娠，是降低出生缺陷的重要手段。现有产前筛查存在的假阳性、产前诊断方法的创伤性是我国染色体疾病产前诊断存在的关键问题。高通量测序技术（high-throughput sequencing, HTS）为当前的产前筛查-产前诊断模式提供了新的选择，虽然国内外指南均已推荐HTS用于高危人群的初筛，但仍需要对更多人群，尤其是普通人群的检测能力进行评价，以明确此方法对非整倍体低危人群的检测能力。为此，本课题对进行HTS检测的样本进行回顾性研究，探讨传统血清学筛查、超声筛查及HTS检测应用于染色体非整倍体产前检测的检测效率，评价HTS及联合筛查对胎儿染色体非整倍体疾病检测的灵敏度和特异度及临床可行性，并从中发现适合双胎孕妇的检测手段，解决产前诊断的难题。 方法： 1. 按照“知情同意及知情选择”原则，本实验收集2013.01.01~2013.12.31进行HTS检测的孕妇资料2351例。入选条件为：①孕11周以上孕妇；②自然受孕；③孕妇体重＜100kg；④孕妇知情同意，并签署知情同意书。排除标准为：①孕妇为染色体疾病患者；②孕妇近期接受过异体输血、移植手术、干细胞治疗等。本研究在研究前已取得医院伦理委员会认可。 2. 根据预产期年龄，将单胎样本分为低龄组和高龄组。低龄单胎组孕妇行血清学筛查、超声筛查和高通量测序；高龄单胎组孕妇及双胎妊娠孕妇行超声筛查和高通量测序。本研究以核型分析为金标准，血清学筛查高风险、超声筛查异常或HTS检测阳性者在知情同意基础上进行核型分析。其余病例随访至胎儿出生后。 3. 采用SPSS23.0软件进行统计分析，对血清学筛查、超声筛查及HTS的检测效力进行对比分析，分别计算其检测21、18、13及性染色体非整倍体的灵敏度、特异度、阳性预测值等，并对上述指标进行卡方检验，比较各筛查方法检测效率之间的差异。 结果： 1. 本研究共收集2351例样本，其中5例未有妊娠结局随访结果(0.21%)，12例未通过测序前的质控(0.51%)，因此有效样本数为2334例。 2. 对于低龄单胎孕妇胎儿染色体非整倍体疾病的产前检测，血清学筛查、超声筛查、HTS的灵敏度分别为65%（13/20）、24%（6/25）、92%（23/25），特异度分别为42.52%（608/1430）、91.50%（1442/1576）、99.56%（1569/1576），阳性预测值分别为1.56%（13/835）、4.29%（6/140）、76.67%（23/30）。超声联合血清学筛查、超声联合HTS的检测灵敏度分别为68%（17/25）、96%（24/25），特异度分别为40.55%（647/1576）、91.24%（1438/1576），阳性预测值分别为1.78%（17/954）、14.81%（24/162）。 3. 对于高龄单胎孕妇胎儿染色体非整倍体疾病的产前检测，超声筛查、HTS及超声联合HTS的灵敏度分别为56.25%（9/16）、100%（16/16）、100%（16/16），特异度分别为97.13%（677/697）、99.43%（693/697）、96.56%（673/697），阳性预测值分别为31.03%（9/29）、80%（16/20）、40%（16/40）。 4. 超声筛查单胎胎儿染色体非整倍体疾病的灵敏度为36.59%（15/41），特异度为93.22%（2119/2273），阳性预测值为8.88%（15/169） 5. HTS检测单胎胎儿染色体非整倍体疾病的灵敏度为95.12%（39/41），特异度为99.52%（2262/2273），阳性预测值为78%（39/50）。HTS对21、18、13及性染色体非整倍体的检测灵敏度分别为90.48%（19/21）、100%（9/9）、0（0/0）、100%（11/11），特异度分别为99.96%（2292/2293）、100%（2305/2305）、99.91%（2312/2314）、99.65%（2295/2303）。 结论： 1. HTS检测T21、T18的灵敏度、特异度及阳性预测值高，假阳性率低，证明了HTS检测应用于胎儿染色体非整倍体的产前筛查具有临床可行性。 2. HTS可以检测13、X、Y染色体非整倍体等目前血清学筛查无法检测的染色体疾病。 3. 与血清学筛查、超声筛查相比，HTS检测效率较高，且无年龄和孕周限制，可避免因较高假阳性而造成的不必要的侵入性产前诊断。 4. 相比于超声联合血清学筛查，超声联合HTS具有更高的灵敏度、特异度及阳性预测值，更低的假阳性率，可以缓解产前诊断工作的压力。 5. 相比于HTS，超声联合HTS检测的特异度等方面有所降低，但总检出率有所提高，有利于避免出生缺陷患儿的出生。
Objective: Chromosome aneuploidy is the main cause of birth defects, the incidence of which in China is 4% to 6%. Currently there is no effective treatment for the diseases at home and abroad. The children bring heavy mental and economic burden to their families and society. Therefore, prompt diagnosis during pregnancy and timely termination of pregnancy are important means to reduce birth defects. Crucial problems in prenatal diagnosis of chromosomal diseases are false positives of prenatal screening and invasive prenatal diagnostic methods. High-throughput sequencing technology provides a new choice for the current prenatal screening - prenatal diagnosis mode. Although international guidelines have recommended HTS for the primary screening of high-risk populations, we still need to evaluate the detection capability in more people, especially in the general population in order to identify the detection capability in low-risk populations. Therefore, the subject performs a retrospective study about the HTS screening in single pregnancy. We discuss the detection efficiency of traditional serological screening, ultrasound screening and HTS in the prenatal testing of chromosome aneuploidy. Meanwhile, sensitivity, specificity and clinical feasibility of HTS and combined screening for fetal chromosomal aneuploid are evaluated. Finally, we try to find suitable means of detection in twin-pregnant women to solve the problem of prenatal diagnosis. Methods: 1. According to the principle "informed consent and informed choice", the subject collect informations of 2351 cases who have HTS testing during 2013.01.01 and 2013.12.31. Inclusion criteria : ① gestational age≥11 weeks; ② natural fertilization; ③ maternal weight <100kg; ④ pregnant women consented and signed the medical informed consent document. Exclusion criteria: ① pregnant women have chromosomal diseases; ②recently pregnant women have had allogeneic transfusion, transplantation or stem cell therapy. This research has obtained approval of the hospital ethics committee. 2. Singleton pregnant women were divided into two groups: younger age group and advanced age group. Singleton pregnant women of younger age group had serological screening, ultrasound screening and high-throughput sequencing; while those of advanced age group and twin pregnancy women had ultrasound screening and high-throughput sequencing. In this study, the gold standard is karyotype analysis. On the basis of informed consent, karyotype analysis was taken in pregnant women who had abnormal serological screening result, abnormal ultrasound screening or HTS positive. The remaining cases were followed up until after birth. 3. Using SPSS23.0 software, we compared and analyzed testing effectiveness of serological screening, ultrasound screening and HTS. Respectively, we calculated the sensitivity, specificity and positive predictive value of these screening methods for 21,18,13 and sex chromosome aneuploidy, etc. Finally, chi-square test was used to compare the efficiency differences of these screening methods. Results: 1. We totally collected 2351 cases of pregnancy samples. Among these, 5 cases had no follow-up of pregnancy outcomes (0.21%) and 12 cases failed in the quality control before sequencing (0.51%). Therefore, the number of effective samples was 2334. 2. For the prenatal detection of fetal chromosomal aneuploidy in younger pregnant women, sensitivities of serological screening, ultrasound scanning and HTS respectively was 65% (13/20), 24% (6/25) and 92% (23/25); specificities respectively was 42.52%（608/1430）, 91.50% (1442/1576) and 99.56%（1569/1576）; meanwhile, positive predictive values respectively was 1.56% (13/835), 4.29% (6/140) and 76.67% (23/30). Sensitivities of ultrasound combined with serological screening and ultrasound combined with HTS separately was 68% (17/25), 96% (24/25); specificities separately was 40.55% (647/1576), 91.24% (1438/1576); and positive predictive values separately was 1.78% (17/954), 14.81% (24/162). 3. For the prenatal detection of fetal chromosomal aneuploidy in advanced pregnant women, sensitivities of ultrasound screening, HTS and ultrasound combined with HTS respectively was 56.25% (9/16), 100% (16/16) and 100% (16/16); specificities separately was 97.13% (677/697), 99.43% (693/697) and 96.56% (673/697); and positive predictive values separately was 31.03% (9/29), 80% (16/20) and 40% (16/40). 4. The sensitivity, specificity and positive predictive value of ultrasound screening for fetal chromosomal aneuploidy respectively was 36.59% (15/41), 93.22% (2119/2273) and 8.88% (15/169). 5. The sensitivity, specificity and positive predictive value of HTS for fetal chromosomal aneuploidy separately was 95.12% (39/41), 99.52% (2262/2273) and 78% (39/50). Sensitivities of HTS for 21,18,13 and sex chromosome aneuploidy respectively was 90.48% (19/21), 100% (9/9), 0（0/0） and 100% (11/11); and specificities separately was 99.96% (2292/2293), 100% (2305/2305), 99.91% (2312/2314) and 99.65% (2295/2303). Conclusion: 1. The sensitivity, specificity and positive predictive value of HTS for T21,T18 are higher, while false positive rate is lower, which prove that HTS used in the prenatal screening of fetal chromosomal aneuploidy is clinically feasible. 2. HTS can detect 13, X, Y chromosome aneuploidy which currently serological screening can not detect. 3. Compared with serological screening and ultrasound screening, HTS has higher detection efficiency, with no gestational age and age limit; it can avoid unnecessary invasive prenatal diagnosis caused by high false positive. 4. Compared with ultrasound combined with serological screening, the sensitivity, specificity and positive predictive value of ultrasound combined with HTS are higher, while false positive rate is lower, which can relieve the pressure of prenatal diagnosis. 5. Compared with HTS, specificity of ultrasound combined with HTS is lower, but total detection rate is higher, which can help to avoid the birth of children with birth defects.