Reproductive Medical Center, Department of Obstetrics and Gynecology, General Hospital of Tianjin Medical University, Tianjin, 300052, China.
J Assist Reprod Genet. 2019 Dec;36(12):2505-2513. doi: 10.1007/s10815-019-01603-w. Epub 2019 Nov 15.
To perform a preliminary exploration of a new embryo rank in clinical practice by combining the embryo chromosome copy number and mitochondrial copy number analysis of DNA extracted from embryo culture medium and blastocoel fluid.
Eighty-three ICSI embryos from day 2 or day 3 were cultured to day 5 or day 6. Thirty-two blastocysts of 3 cc or above were obtained. Culture medium and blastocoel fluid were collected at 24 h before blastocyst formation. The genomic DNA and mitochondrial DNA (mtDNA) from the culture medium combined with blastocoel fluid and the whole blastocyst were amplified and sequenced by MALBAC-NGS. We compared the chromosomal information generated by the new protocol from the culture medium and the information employed by the whole embryo method. A multivariable linear regression was performed to study the impact of the blastocyst morphological score, chromosomal abnormality, embryo mtDNA copy number, and female age on the culture medium mtDNA copy number.
(1) The DNA from 31 blastocysts was successfully amplified, and the successful amplification rate was 96.9% (31/32). The success rate of the amplification of genomic DNA extracted from the culture medium was 87.5% (28/32). (2) There were 18 blastocysts in which the less invasive method and the whole embryo method revealed the same results. The consistency rate was 66.7% (18/27). (3) The culture medium mitochondrial DNA copy number (MCN) had a significantly positive correlation with the blastocyst mitochondrial DNA copy number (P = 0.001), female age (P = 0.012), and blastocyst score (P = 0.014), but there was no obvious correlation with blastocyst chromosome (P = 0.138).
The preliminary exploration result of the less invasive approach for having an embryo rank was not satisfying, which still awaits further long-term evaluation.
通过联合分析胚胎培养液和囊胚腔液中提取的 DNA 的胚胎染色体拷贝数和线粒体拷贝数，在临床实践中对胚胎等级进行初步探索。
对第 2 天或第 3 天的 83 个 ICSI 胚胎进行培养，直至第 5 天或第 6 天。获得 3 cc 或以上的 32 个囊胚。在囊胚形成前 24 小时收集培养液和囊胚腔液。通过 MALBAC-NGS 对来自培养液和整个囊胚的培养基质 DNA 和线粒体 DNA（mtDNA）进行扩增和测序。我们比较了新方案从培养液中生成的染色体信息和整个胚胎方法所使用的信息。采用多元线性回归分析来研究囊胚形态评分、染色体异常、胚胎 mtDNA 拷贝数和女性年龄对培养液 mtDNA 拷贝数的影响。
（1）31 个囊胚的 DNA 成功扩增，扩增成功率为 96.9%（31/32）。培养基质中提取的基因组 DNA 扩增成功率为 87.5%（28/32）。（2）有 18 个囊胚采用非侵入性方法和整个胚胎方法的结果相同，符合率为 66.7%（18/27）。（3）培养液线粒体 DNA 拷贝数（MCN）与囊胚线粒体 DNA 拷贝数（P=0.001）、女性年龄（P=0.012）和囊胚评分（P=0.014）呈显著正相关，但与囊胚染色体无明显相关性（P=0.138）。
这种非侵入性方法对胚胎等级进行初步探索的结果并不令人满意，仍需要进一步的长期评估。
