School of Medicine, South China University of Technology, Guangzhou, China.
Department of Obstetrics and Gynecology, The Sixth Medical Center of PLA General Hospital of Beijing, Beijing, China.
Hum Reprod. 2023 Nov 2;38(11):2137-2153. doi: 10.1093/humrep/dead186.
Is the chromosome copy number of the trophectoderm (TE) of a human reconstituted embryos after spindle transfer (ST) representative of the inner cell mass (ICM)?
Single-cell multi-omics sequencing revealed that ST blastocysts have a higher proportion of cell lineages exhibiting intermediate mosaicism than conventional ICSI blastocysts, and that the TE of ST blastocysts does not represent the chromosome copy number of ICM.
Preimplantation genetic testing for aneuploidy (PGT-A) assumes that TE biopsies are representative of the ICM, but the TE and ICM originate from different cell lineages, and concordance between TE and ICM is not well-studied, especially in ST embryos.
STUDY DESIGN, SIZE, DURATION: We recruited 30 infertile women who received treatment at our clinic and obtained 45 usable blastocysts (22 from conventional ICSI and 23 reconstituted embryos after ST). We performed single-cell multi-omics sequencing on all blastocysts to predict and verify copy number variations (CNVs) in each cell. We determined the chromosome copy number of each embryo by analysing the proportion of abnormal cells in each blastocyst. We used the Bland-Altman concordance and the Kappa test to evaluate the concordance between TE and ICM in the both groups.
PARTICIPANTS/MATERIALS, SETTING, METHODS: The study was conducted at a public tertiary hospital in China, where all the embryo operations, including oocytes retrieval, ST, and ICSI, were performed in the embryo laboratory. We utilized single-cell multi-omics sequencing technology at the Biomedical Pioneering Innovation Center, School of Life Sciences, Peking University, to analyse the blastocysts. Transcriptome sequencing was used to predict the CNV of each cell through bioinformatics analysis, and the results were validated using the DNA methylation library of each cell to confirm chromosomal normalcy. We conducted statistical analysis and graphical plotting using R 4.2.1, SPSS 27, and GraphPad Prism 9.3.
Mean age of the volunteers, the blastocyst morphology, and the developmental ratewere similar in ST and ICSI groups. The blastocysts in the ST group had some additional chromosomal types that were prone to variations beyond those enriched in the blastocysts of the ICSI group. Finally, both Bland-Altman concordance test and kappa concordancetest showed good chromosomal concordance between TE and ICM in the ICSI blastocysts (kappa = 0.659, P < 0.05), but not in ST blastocysts (P = 1.000), suggesting that the TE in reconstituted embryos is not representative of ICM. Gene functional annotation (GO and KEGG analyses) suggests that there may be new or additional pathways for CNV generation in ST embryos compared to ICSI embryos.
LIMITATIONS, REASONS FOR CAUTION: This study was mainly limited by the small sample size and the limitations of single-cell multi-omics sequencing technology. To select eligible single cells, some cells of the embryos were eliminated or not labelled, resulting in a loss of information about them. The findings of this study are innovative and exploratory. A larger sample size of human embryos (especially ST embryos) and more accurate molecular genetics techniques for detecting CNV in single cells are needed to validate our results.
Our study justifies the routine clinical use of PGT-A in ICSI blastocysts, as we found that the TE is a good substitute for ICM in predicting chromosomal abnormalities. While PGT-A is not entirely accurate, our data demonstrate good clinical feasibility. This trial was able to provide correct genetic counselling to patients regarding the reliability of PGT-A. Regarding ST blastocysts, the increased mosaicism rate and the inability of the TE to represent the chromosomal copy number of the ICM are both biological characteristics that differentiate them from ICSI blastocysts. Currently, ST is not used clinically on a large scale to produce blastocysts. However, if ST becomes more widely used in the future, our study will be the first to demonstrate that the use of PGT-A in ST blastocysts may not be as accurate as PGT-A for ICSI blastocysts.
STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the National Key R&D Program of China (2018YFA0107601) and the National Key R&D Program of China (2018YFC1003003). The authors declare no conflict of interest.
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经过纺锤体转移（ST）后，人类重构胚胎的滋养外胚层（TE）的染色体拷贝数是否代表内细胞团（ICM）？
单细胞多组学测序显示，与传统的卵胞浆内单精子注射（ICSI）胚胎相比，ST 囊胚中具有中间镶嵌性的细胞谱系比例更高，并且 ST 囊胚的 TE 并不能代表 ICM 的染色体拷贝数。
胚胎植入前遗传学检测（PGT-A）假定 TE 活检是 ICM 的代表，但 TE 和 ICM 来源于不同的细胞谱系，TE 和 ICM 之间的一致性尚未得到很好的研究，尤其是在 ST 胚胎中。
研究设计、规模、持续时间：我们招募了 30 名在我们诊所接受治疗的不孕妇女，并获得了 45 个可用的囊胚（22 个来自传统的 ICSI，23 个来自 ST 后重构胚胎）。我们对所有的囊胚进行单细胞多组学测序，以预测和验证每个细胞的拷贝数变异（CNV）。我们通过分析每个囊胚中异常细胞的比例来确定每个胚胎的染色体拷贝数。我们使用 Bland-Altman 一致性和 Kappa 检验来评估两组中 TE 和 ICM 之间的一致性。
参与者/材料、设置、方法：该研究在中国的一家公立三级医院进行，所有的胚胎操作，包括卵母细胞采集、ST 和 ICSI，都在胚胎实验室进行。我们利用单细胞多组学测序技术在北京大学生命科学学院的生物医学创新中心分析囊胚。转录组测序通过生物信息学分析预测每个细胞的 CNV，并用每个细胞的 DNA 甲基化文库进行验证，以确认染色体正常。我们使用 R 4.2.1、SPSS 27 和 GraphPad Prism 9.3 进行统计分析和图形绘制。
志愿者的平均年龄、囊胚形态和发育率在 ST 和 ICSI 组中相似。ST 组的囊胚有一些额外的染色体类型，这些类型容易发生变异，超出了 ICSI 组囊胚中富集的类型。最后，Bland-Altman 一致性检验和 Kappa 一致性检验均显示 ICSI 囊胚的 TE 和 ICM 之间的染色体一致性良好（Kappa=0.659，P<0.05），但 ST 囊胚的一致性不佳（P=1.000），这表明重构胚胎的 TE 不能代表 ICM。基因功能注释（GO 和 KEGG 分析）表明，与 ICSI 胚胎相比，ST 胚胎中 CNV 产生可能存在新的或额外的途径。
局限性、谨慎的原因：本研究主要受到样本量小和单细胞多组学测序技术限制的限制。为了选择合格的单细胞，一些胚胎的细胞被剔除或未标记，导致这些细胞的信息丢失。本研究的发现具有创新性和探索性。需要更大的人类胚胎样本量（特别是 ST 胚胎）和更准确的用于检测单细胞 CNV 的分子遗传学技术来验证我们的结果。
我们的研究证明了在 ICSI 囊胚中常规使用 PGT-A 的合理性，因为我们发现 TE 是预测染色体异常的 ICM 的良好替代品。虽然 PGT-A 并不完全准确，但我们的数据显示出良好的临床可行性。这项试验能够为患者提供关于 PGT-A 可靠性的正确遗传咨询。对于 ST 囊胚，增加的镶嵌率和 TE 不能代表 ICM 的染色体拷贝数都是与 ICSI 囊胚不同的生物学特征。目前，ST 还没有在临床上大规模用于产生囊胚。然而，如果 ST 在未来得到更广泛的应用，我们的研究将首次表明，在 ST 囊胚中使用 PGT-A 可能不如在 ICSI 囊胚中使用 PGT-A 准确。
研究资助/利益冲突：本研究得到了国家重点研发计划（2018YFA0107601）和国家重点研发计划（2018YFC1003003）的支持。作者没有利益冲突。
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