High-throughput Sequencing In Preimplantation Genetic Screening Of Human Early Embryo

Objective: To study the safety and effectivity of high-throughput sequencing technologies in preimplantation genetic screening.Methods: 41 normally fertilized cryopreserved good-quality embryos of D3 and 9 blastocysts have been donated by the couples who have signed a consent form and already given birth to healthy baby by IVF-ET in our assisted reproductive medical centre. After thawing, single cell and 5-10 cells trophectoderm were biopsied from the embryos of D3 and blastocysts for whole genome amplification. The biopsied D3 embryos were cultured sequentially and biopsied again if blastocyst have been developed.Results: 1. Biopsy: 41 embryos of D3 and 9 blastocysts were all survived after biopsy. 5 blastocysts have been formed after D3 biopsy. A total of 55 embryos(41 embryos of D3 and 14 blastocysts) were biopsied and whole genome amplified. 2. Whole genome amplification results: 42 embryos has been amplified successfully(28 embryos of D3 and 14 blastocysts), the successful rate of whole genome amplification is about 76.4%(42/55). 13 embryos has been amplified failure, and the successful rate of single cell from D3 embyro is 68.3%(28/41). 14 blastocysts were all successfully amplified. 3. High-throughput sequencing results of D3 embryos: 11 in 28 embryos of D3 did not detect the chromosome abnormal. 2 embryos were detected as chromosome numerical abnormality. 5 embryos were detected as more than 16 MB deletion and duplication. 10 embryos were detected as both chromosome numerical abnormality and more than 16 MB deletion and duplication. 4. High-throughput sequencing results of blastocysts: 8 embryos of blastocyst in 14 did not detect the chromosome abnormal. 5 embryos were detected as chromosome numerical abnormality.1 embryos were detected as more than 16 MB deletion and duplication. 5. The overall chromosome numerical abnormality or more than 16 MB chromosome deletion and duplication rate was 52.4%(22/42), among which D3 embryos and blastocyst were 60.7%(17/28) and 42.9%(6/14) respectively.6. The results showed that the genetic information of single cell from D3 embryo and 5~10 trophectoderm from D5 blastocyst of the same embryo is consist with each other. 7. The chromosome numerical abnormality or more than 16 MB chromosome deletion and duplication rate of the patient with advanced age, serious male factors, repeated implantation failure and adverse pregnancy history was 76%,33.33%, 33.33% and 28.57% respectively.Conclusion: 1. Single-celled whole genome amplification and high-throughput sequencing of D3 embryo can apply single-cell to PGS/PGD, solving the problem of low blastocyst formation rate, expanding the scope of application of high-throughput sequencing, and accelerating the clinical application and popularization. 2. There is a certain proportion of chromosome abnormal even in the good quality embryos in IVF-ET. It is likely to be one reason of pregnancy loss in ART. 3. The patient with the advanced age, serious male factors, repeated implantation failure and adverse pregnancy history has the high risk of embryo genetic abnormal. PGS may help to improve the clinical pregnancy rate and take baby home rate of ART. 4. The rapid development of molecular biology techniques provides more means for PGS/PGD. It provides more eloboratly and widely genetic information of the embryo. But it needs extensive research to detect the relationship between the genetic segment and its function, phenotype, and disease. PGS must be based on a fully informed with the patients and signed informed consent.Prenatal diagnosis and follow-up of birth must be combined to ensure its safety and effectiveness in improving the success rate and live birth rates of assisted reproduction treatment.