The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
Shanghai Municipal Key Clinical Specialty, Shanghai, China.
Hum Reprod. 2021 Jan 1;36(1):236-247. doi: 10.1093/humrep/deaa269.
Can whole genome sequencing (WGS) offer a relatively cost-effective approach for embryonic genome-wide haplotyping and preimplantation genetic testing (PGT) for monogenic disorders (PGT-M), aneuploidy (PGT-A) and structural rearrangements (PGT-SR)?
Reliable genome-wide haplotyping, PGT-M, PGT-A and PGT-SR could be performed by WGS with 10× depth of parental and 4× depth of embryonic sequencing data.
Reduced representation genome sequencing with a genome-wide next-generation sequencing haplarithmisis-based solution has been verified as a generic approach for automated haplotyping and comprehensive PGT. Several low-depth massively parallel sequencing (MPS)-based methods for haplotyping and comprehensive PGT have been developed. However, an additional family member, such as a sibling, or a proband, is required for PGT-M haplotyping using low-depth MPS methods.
STUDY DESIGN, SIZE, DURATION: In this study, 10 families that had undergone traditional IVF-PGT and 53 embryos, including 13 embryos from two PGT-SR families and 40 embryos from eight PGT-M families, were included to evaluate a WGS-based method. There were 24 blastomeres and 29 blastocysts in total. All embryos were used for PGT-A. Karyomapping validated the WGS results. Clinical outcomes of the 10 families were evaluated.
PARTICIPANTS/MATERIALS, SETTING, METHODS: A blastomere or a few trophectoderm cells from the blastocyst were biopsied, and multiple displacement amplification (MDA) was performed. MDA DNA and bulk DNA of family members were used for library construction. Libraries were sequenced, and data analysis, including haplotype inheritance deduction for PGT-M and PGT-SR and read-count analysis for PGT-A, was performed using an in-house pipeline. Haplotyping with a proband and parent-only haplotyping without additional family members were performed to assess the WGS methodology. Concordance analysis between the WGS results and traditional PGT methods was performed.
For the 40 PGT-M and 53 PGT-A embryos, 100% concordance between the WGS and single-nucleotide polymorphism (SNP)-array results was observed, regardless of whether additional family members or a proband was included for PGT-M haplotyping. For the 13 embryos from the two PGT-SR families, the embryonic balanced translocation was detected and 100% concordance between WGS and MicroSeq with PCR-seq was demonstrated.
LIMITATIONS, REASONS FOR CAUTION: The number of samples in this study was limited. In some cases, the reference embryo for PGT-M or PGT-SR parent-only haplotyping was not available owing to failed direct genotyping.
WGS-based PGT-A, PGT-M and PGT-SR offered a comprehensive PGT approach for haplotyping without the requirement for additional family members. It provided an improved complementary method to PGT methodologies, such as low-depth MPS- and SNP array-based methods.
STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the research grant from the National Key R&D Program of China (2018YFC0910201 and 2018YFC1004900), the Guangdong province science and technology project of China (2019B020226001), the Shenzhen Birth Defect Screening Project Lab (JZF No. [2016] 750) and the Shenzhen Municipal Government of China (JCYJ20170412152854656). This work was also supported by the National Natural Science Foundation of China (81771638, 81901495 and 81971344), the National Key R&D Program of China (2018YFC1004901 and 2016YFC0905103), the Shanghai Sailing Program (18YF1424800), the Shanghai Municipal Commission of Science and Technology Program (15411964000) and the Shanghai 'Rising Stars of Medical Talent' Youth Development Program Clinical Laboratory Practitioners Program (201972). The authors declare no competing interests.
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全基因组测序（WGS）能否为单基因疾病（PGT-M）、非整倍体（PGT-A）和结构重排（PGT-SR）的胚胎全基因组单体型分析和植入前遗传检测（PGT）提供一种相对具有成本效益的方法？
通过 10×父母测序深度和 4×胚胎测序深度的 WGS 可实现可靠的全基因组单体型分析、PGT-M、PGT-A 和 PGT-SR。
基于全基因组下一代测序单体型的减少代表性基因组测序已被验证为自动单体型分析和综合 PGT 的通用方法。已经开发了几种基于低深度大规模并行测序（MPS）的单体型分析和综合 PGT 方法。然而，对于低深度 MPS 方法的 PGT-M 单体型分析，需要额外的家庭成员，如兄弟姐妹或先证者。
研究设计、大小、持续时间：在这项研究中，纳入了 10 个接受传统 IVF-PGT 的家庭和 53 个胚胎，包括 2 个 PGT-SR 家庭的 13 个胚胎和 8 个 PGT-M 家庭的 40 个胚胎。总共共有 24 个卵裂球和 29 个囊胚。所有胚胎均用于 PGT-A。核型映射验证了 WGS 结果。评估了 10 个家庭的临床结局。
参与者/材料、设置、方法：从囊胚中活检一个卵裂球或几个滋养外胚层细胞，进行多次置换扩增（MDA）。使用 MDA DNA 和家庭成员的批量 DNA 构建文库。对文库进行测序，并使用内部管道进行数据分析，包括 PGT-M 和 PGT-SR 的单体型遗传推断以及 PGT-A 的读计数分析。使用先证者和仅父母单体型分析而无需其他家庭成员来评估 WGS 方法。对 WGS 结果与传统 PGT 方法进行一致性分析。
对于 40 个 PGT-M 和 53 个 PGT-A 胚胎，无论是否包括 PGT-M 单体型分析的额外家庭成员或先证者，WGS 与单核苷酸多态性（SNP）-array 结果的一致性为 100%。对于来自 2 个 PGT-SR 家庭的 13 个胚胎，检测到胚胎平衡易位，WGS 与 MicroSeq with PCR-seq 的一致性为 100%。
局限性、谨慎的原因：本研究的样本数量有限。在某些情况下，由于直接基因分型失败，PGT-M 或 PGT-SR 仅父母单体型分析的参考胚胎不可用。
基于 WGS 的 PGT-A、PGT-M 和 PGT-SR 为单体型分析提供了一种全面的 PGT 方法，无需额外的家庭成员。它为 PGT 方法，如低深度 MPS 和 SNP 阵列方法，提供了一种改进的互补方法。
研究资助/利益冲突：这项研究得到了中国国家重点研发计划（2018YFC0910201 和 2018YFC1004900）、广东省科技项目（2019B020226001）、深圳市出生缺陷筛查项目实验室（JZF No. [2016] 750）和中国深圳市政府（JCYJ20170412152854656）的支持。这项工作还得到了国家自然科学基金（81771638、81901495 和 81971344）、中国国家重点研发计划（2018YFC1004901 和 2016YFC0905103）、上海市扬帆计划（18YF1424800）、上海市科委项目（15411964000）和上海市“医苑新星”青年医学人才培养资助计划临床实验室医师项目（201972）的支持。作者没有利益冲突。
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