Liu WeiQiang, Liu JianQiao, Du HongZi, Ling JiaWei, Sun XiaoFang, Chen DunJin
a Department of Clinical Laboratory of Gynecology and Obstetrics, Key Laboratory for Reproduction and Genetics of Guangdong Higher Education Institutes, Key Laboratory for Major Obstetric Diseases of Guangdong Province , Third Affiliated Hospital of Guangzhou Medical University , Guangzhou , PR China.
b Department of Reproductive Medicine , Third Affiliated Hospital of Guangzhou Medical University , Guangzhou , PR China.
Ann Med. 2017 Jun;49(4):319-328. doi: 10.1080/07853890.2016.1254816. Epub 2016 Dec 14.
Cell-free nuclear DNA has been isolated from spent embryo culture medium. Whether this small amount of DNA can be amplified at the whole genome level and the concordance rate of karyotypes and specific alleles between biopsied cells and media has not been evaluated.
Seven couples were recruited, 88 donated embryos and their corresponding media were collected for whole genome amplification (WGA). The efficiency of WGA, the concordance of chromosome status, and the HBB gene IVSII654 allele between biopsied cells and media were investigated.
After WGA, the DNA detection rate was 90.90% with a mean concentration of 26.15 ng/μl. The full chromosome concordance rate between biopsied cells and medium was 64.52%, and it increased to 90.00% for diploid blastocyst samples. Analysis of the mutated IVSII654 locus and SNP linkage verified that the DNA present in the medium originated from embryonic cells.
We confirmed that nuclear DNA is present in spent culture medium and that the majority of this DNA can be amplified for subsequent analysis. Our results showed that non-invasive embryo genetic testing at the chromosomal-level using medium can concordant to the biopsied cells, but it needs further optimized before use in clinical applications. KEY MESSAGES The aggressive biopsy step during PGD/PGS procedure would have a negative effect on the future development of the embryo. Cell-free nuclear DNA has been observed in spent embryo culture medium, which holds promise for the development of non-invasive PGD/PGS approaches. The presence of DNA in medium, its efficiency for WGA, and the concordance between chromosome status and the HBB gene IVSII654 allele as diagnosed from biopsied cells or medium were investigated. Non-invasive embryo genetic testing at the chromosomal-level and allele site using medium can concordant to the biopsied cells, but it needs further optimized before use in clinical applications.
已从废弃胚胎培养基中分离出游离核DNA。但这少量DNA能否在全基因组水平上进行扩增，以及活检细胞与培养基之间的核型和特定等位基因的一致性率尚未得到评估。
招募了7对夫妇，收集了88个捐赠胚胎及其相应的培养基进行全基因组扩增（WGA）。研究了WGA的效率、染色体状态的一致性以及活检细胞与培养基之间的HBB基因IVSII654等位基因。
WGA后，DNA检测率为90.90%，平均浓度为26.15 ng/μl。活检细胞与培养基之间的全染色体一致性率为64.52%，对于二倍体囊胚样本，该率增至90.00%。对突变的IVSII654位点和SNP连锁的分析证实，培养基中存在的DNA源自胚胎细胞。
我们证实废弃培养基中存在核DNA，并且大部分这种DNA可以扩增用于后续分析。我们的结果表明，使用培养基进行染色体水平的非侵入性胚胎基因检测与活检细胞结果一致，但在临床应用前还需要进一步优化。关键信息：PGD/PGS程序中的侵入性活检步骤会对胚胎的未来发育产生负面影响。已在废弃胚胎培养基中观察到游离核DNA，这为非侵入性PGD/PGS方法的发展带来了希望。研究了培养基中DNA的存在情况、其WGA效率以及从活检细胞或培养基诊断出的染色体状态与HBB基因IVSII654等位基因之间的一致性。使用培养基进行染色体水平和等位基因位点的非侵入性胚胎基因检测与活检细胞结果一致，但在临床应用前还需要进一步优化。
