Non-invasive pre-implantation aneuploidy screening and diagnosis of beta thalassemia IVSII654 mutation using spent embryo culture medium

<b>Background:</b> 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. <b>Methods:</b> 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. <b>Results:</b> 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. <b>Conclusion:</b> 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 MESSAGESThe 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. 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.

背景：细胞游离核DNA已从废弃胚胎培养液中分离获得。目前尚不明确该微量DNA能否在全基因组水平完成扩增，以及活检细胞与培养液之间的核型和特定等位基因的一致性水平。方法：本研究招募7对夫妇，收集88枚捐赠胚胎及其对应培养液用于全基因组扩增（Whole Genome Amplification, WGA）。我们探究了WGA的扩增效率、活检细胞与培养液的染色体状态一致性，以及二者间HBB基因IVSII654等位基因的一致性情况。结果：经WGA扩增后，DNA检出率达90.90%，平均浓度为26.15 ng/μl。活检细胞与培养液的全染色体一致性为64.52%；在二倍体囊胚样本中，该一致性提升至90.00%。对突变IVSII654位点与单核苷酸多态性（Single Nucleotide Polymorphism, SNP）连锁的分析证实，培养液中的DNA来源于胚胎细胞。结论：本研究证实，废弃培养液中存在核DNA，且其中大部分可通过WGA扩增用于后续分析。结果表明，基于培养液的染色体水平非侵入式胚胎基因检测结果与活检细胞检测结果具有一致性，但该方法仍需进一步优化后方可应用于临床。核心要点：1. 胚胎植入前基因诊断（Preimplantation Genetic Diagnosis, PGD）/胚胎植入前染色体筛查（Preimplantation Genetic Screening, PGS）流程中的侵入性活检步骤，会对胚胎后续发育产生负面影响。2. 已有研究证实废弃胚胎培养液中存在细胞游离核DNA，这为非侵入式PGD/PGS技术的发展提供了应用前景。3. 本研究针对培养液中DNA的存在情况、WGA扩增效率，以及基于活检细胞与培养液诊断的染色体状态、HBB基因IVSII654等位基因的一致性展开了探究。4. 基于培养液的染色体水平及等位基因位点水平非侵入式胚胎基因检测结果与活检细胞检测结果具有一致性，但该方法仍需进一步优化后方可应用于临床。