单细胞高通量测序技术在胚胎植入前遗传学诊断中的应用

The application of single cell high flux sequencing technology in preimplantation genetic diagnosis

目的探讨单细胞高通量测序技术在胚胎植入前遗传学诊断(PGD)中的应用效果。方法选取2012年3月—2017年3月在本院生殖中心进行体外受精-胚胎移植(IVF-ET)并在胚胎种植前需进行遗传学诊断(PGD)的夫妇95例为研究对象,抽取这些病例冻存的第三天卵裂期胚胎细胞50枚、第五天囊胚期囊胚15枚和活检后第三天胚胎形成囊胚8枚,经过单个卵裂球细胞与囊胚期滋养外胚层细胞的活检,以及单细胞全基扩增和高通量测序,分析囊胚扩增成功率及总的扩增成功率、染色体正常检出率及异常率以及胚胎临床资料分析。结果第三天卵裂期胚胎共50枚,扩增成功率为76.0%(38/50);第五天囊胚期囊胚15枚,扩增成功率为93.3%,活检后第三天卵裂期胚胎形成囊胚8枚,扩增成功率为100.0%(8/8)。总的扩增成功率为82.2%(60/73);第三天卵裂期胚胎的总异常率为63.2%、囊胚为45.5%,总体异常率为56.7%。基因扩增成功率因为高龄、性别以及反复植入因素分别为89.0%、49.3%、40.0%;染色体异常检出率因为高龄、性别以及反复植入因素分别为73.8%、30.6%、25.0%。结论单细胞高通量测序技术在胚胎植入前遗传学诊断中的应用效果和价值较高,可以弥补囊胚形成率低无可活检的胚胎用于PGD难题,更有利于高通量测序技术的临床推广和应用。

Objective To investigate the application of single cell high-throughput sequencing in preimplantation genetic diagnosis(PGD).Methods 95 cases were selected from the reproductive center of our hospital from March 2012 to March 2017 in vitro fertilization-embryo transfer(IVF-ET)and genetic diagnosis(PGD)before embryo planting.Fifty cryopreserved third day of cleavage high-quality embryonic cells,fifth day of blastocysts and the embryo formed 8 blastocysts after biopsy were collected from these patients.Ball cell and blastula stage trophectoderm cells biopsy,as well as single-cell total base amplification and high-throughput sequencing,analysis of blastocyst expansion success rate and the total success rate of amplification,chromosomal normal detection rate and abnormal rate and Embryo clinical data analysis.Results A total of 50 third day of cleavage embryos were amplified successfully,38 of which were successfully amplified.The success rate of amplification was 76.0%.Fifth day of blastocysts were successfully amplified and 14 were obtained,the success rate of amplification was 93.3%.Eight blastocysts were formed after third day of cleavage of biopsies,the success rate of amplification was 100.0%.The total success rate of amplification was 82.2%.The total abnormal rate of three generations of embryos was 63.2%,the blastocyst was 45.5%,and the overall abnormal rate was 56.7%.The success rate of gene amplification was 89.0%,49.3%and 40.0%respectively due to age,sex and repeated implant factors.The detection rate of chromosomal abnormalities was 73.8%,30.6%and 25.0%respectively due to age,sex and repeated implant factors.Conclusion The single cell high-throughput sequencing technique is highly effective and valuable in preimplantation genetic diagnosis.It can be used for PGD problems to compensate for the low rate of blastocyst formation.It can be used for the clinical promotion and application of high-throughput sequencing technology.