Goodrich David, Xing Tongji, Tao Xin, Lonczak Agnieszka, Zhan Yiping, Landis Jessica, Zimmerman Rebekah, Scott Richard T, Treff Nathan R
Reproductive Medicine Associates of New Jersey, 140 Allen Rd, Basking Ridge, NJ, 07920, USA.
Foundation for Embryonic Competence, 140 Allen Rd, Suite 300, Basking Ridge, NJ, 07920, USA.
J Assist Reprod Genet. 2017 Aug;34(8):975-981. doi: 10.1007/s10815-017-0924-4. Epub 2017 Jun 2.
A subset of preimplantation embryos identified as euploid may in fact possess both whole and sub-chromosomal mosaicism, raising concerns regarding the predictive value of current comprehensive chromosome screening (CCS) methods utilizing a single biopsy. Current CCS methods may be capable of detecting sub-chromosomal mosaicism in a trophectoderm biopsy by examining intermediate levels of segmental aneuploidy within a biopsy. This study evaluates the sensitivity and specificity of segmental aneuploidy detection by three commercially available CCS platforms utilizing a cell line mixture model of segmental mosaicism in a six-cell trophectoderm biopsy.
Two cell lines with known karyotypes were obtained and mixed together at specific ratios of six total cells (0:6, 1:5, 2:4, 3:3, 4:2, 5:1, and 6:0). A female cell line containing a 16.2 Mb deletion on chromosome 5 and a male cell line containing a 25.5 Mb deletion on chromosome 4 were used to create mixtures at each level. Six replicates of each mixture were prepared, randomized, and blinded for analysis by one of the three CCS platforms (SNP-array, VeriSeq NGS, or NexCCS). Sensitivity and specificity of segmental aneuploidy at each level of mosaicism was determined and compared between each platform. Additionally, an alternative VeriSeq NGS analysis method utilizing previously published criteria was evaluated.
Examination of the default settings of each platform revealed that the sensitivity was significantly different between NexCCS and SNP up to 50% mosaicism, custom VeriSeq, and SNP-array up to 66% mosaicism, and between NexCCS and custom VeriSeq up to 50% mosaicism. However, no statistical difference was observed in mixtures with >50% mosaicism with any platform. No comparison was made between default VeriSeq, as it does not report segmental imbalances. Furthermore, while the use of previously published criteria for VeriSeq NGS significantly increased sensitivity at low levels of mosaicism, a significant decrease in specificity was observed (66% false positive prediction of segmental aneuploidy).
These results demonstrate the potential of NGS-based detection methods to detect segmental mosaicism within a biopsy. However, these data also demonstrate that a balance between sensitivity and specificity should be more carefully considered. These results emphasize the importance of vigorous preclinical evaluation of new testing criteria prior to clinical implementation providing a point of departure for further algorithm development and improved detection of mosaicism within preimplantation embryos.
一部分被鉴定为整倍体的植入前胚胎实际上可能同时存在整条染色体和亚染色体嵌合现象，这引发了人们对当前利用单次活检的全面染色体筛查（CCS）方法预测价值的担忧。当前的CCS方法或许能够通过检测活检组织中节段性非整倍体的中间水平来发现滋养外胚层活检中的亚染色体嵌合现象。本研究利用六细胞滋养外胚层活检中的节段性嵌合细胞系混合模型，评估了三种商用CCS平台检测节段性非整倍体的敏感性和特异性。
获取两个已知核型的细胞系，并以六种总细胞的特定比例（0:6、1:5、2:4、3:3、4:2、5:1和6:0）混合在一起。使用一个在5号染色体上有16.2 Mb缺失的雌性细胞系和一个在4号染色体上有25.5 Mb缺失的雄性细胞系，在每个水平创建混合物。每个混合物制备六个重复样本，进行随机化处理，并对其进行盲法分析，由三种CCS平台之一（SNP阵列、VeriSeq NGS或NexCCS）进行分析。确定每个嵌合水平节段性非整倍体的敏感性和特异性，并在各平台之间进行比较。此外，还评估了一种利用先前公布标准的VeriSeq NGS替代分析方法。
对每个平台的默认设置进行检查发现，在嵌合率高达50%时，NexCCS和SNP之间的敏感性存在显著差异；在嵌合率高达66%时，定制的VeriSeq和SNP阵列之间存在显著差异；在嵌合率高达50%时，NexCCS和定制的VeriSeq之间存在显著差异。然而，在任何平台上，嵌合率＞50%的混合物中未观察到统计学差异。由于默认的VeriSeq不报告节段性失衡情况，因此未对其进行比较。此外，虽然使用先前公布的VeriSeq NGS标准在低嵌合水平时显著提高了敏感性，但观察到特异性显著降低（节段性非整倍体的假阳性预测率为66%）。
这些结果证明了基于NGS的检测方法在检测活检组织中的节段性嵌合现象方面的潜力。然而，这些数据也表明，应更仔细地考虑敏感性和特异性之间的平衡。这些结果强调了在临床应用之前对新检测标准进行严格临床前评估的重要性，为进一步的算法开发和改进植入前胚胎嵌合现象的检测提供了一个出发点。
