The Clinical Utility Of Next Generation Sequencing For Identifying Chromosome Disease Syndromes In Human Embryos

BackgroundChromosome diseases include ploidy changes, aneuploidy, structural variations, micro-duplication and micro-deletion and so on, which are associated with embryo implantation failure, early pregnancy lossing, abortions duo to abnormal in prenatal diagnosis even laboring defect infants in clinic procedure, so it is significant to launch preimplantation genetic test (PGT) for couples having healthy infants. In past decade, most reproductive medicine centers adopted chip technique, such as array comparative genomic hybridization (aCGH) and single nucleotide polymorphism array (SNP array) as primary method to proceed PGT. Now next generation sequencing emerged as frontline technique, such as copy number variation sequencing (CNV-Seq), and there are more and more clinic trial reports. It has been validated that CNV-Seq has a significant value in preimplantation genetic screening (PGS, for aneuploidy exclusion) and preimplantation genetic diagnosis (PGD). Meanwhile, some small copy number variations (CNVs,<10 Mb) related to abnormality in early pregnancy abortion duo to prenatal diagnosis attracted our attentions, and there is rare report on CNV-Seq reproducibility and resolution at cell level for chromosome disease observation in PGT clinic procedure. So we set up our study to clear CNV-Seq reproducibility and stability at cell level and explore its value for chromosome diseases comprehensive test in PGT.Objective1. Validate the value of CNV-Seq for detecting chromosome disease at genome DNA (gDNA) level through comparing with aCGH, and stability of CNV-Seq using 33 pg diluted gDNAand single cell whole genome amplification (WGA) products through comparing with gDNA level;2. Clear the reproducibility, CN cut-off value for detecting abnormal regions and resolution of CNV-Seq at five cell and single cell levels;3. To construct the cell model for PGT biopsy;4. Taking CNV-Seq as primary method to proceed PGT with comprehensive chromosome disease test before embryos transfer.Methods1. We compared the concordance of CNV-Seq and aCGH for detecting 6 CNVs from 6.52 Mb to 93.02 Mb at gDNA level, and explored CNV-Seq stability through constructing library with 33 pg diluted gDNA and single cell WGAproducts for sequencing and compared with gDNA diagnosis;2. Then chose smallest deletion 6.52 Mb and duplication 14.76 Mb in former part to proceed triplicates with gDNA,15 times 5-cell and 15 times 1-cell WGA products sequencing. Made a summary of 2 regions copy number value (CN value) in form of Mean ±SD at different levels, and analyzed difference among 3 three levels using one way ANOVA and LSD-T test between every two levels using SPSS 17.0 to clear CN cut-off value. Took a=0.05 as significant difference;3. Took 5 frozen blood contained 6 gradient small CNVs from 0.56 Mb to 5.78 Mb (diagnosed at gDNA level using CNV-Seq) as cell source, isolated 5-cell and 1-cell for WGA, and respectively do triplicates of CNV-Seq analysis for every CNV and every level. Did a summary to explore the CNV-Seq resolution at cell level;4. At last, selected 7 couples with PGT indications in reproductive medicine center of Chinese People’s Liberation Army General Hospital and signed consent. Took CNV-Seq as primary method to make comprehensive chromosome diseases test for 35 blastocysts from 7 couples, including aneuploidy, unbalanced translocation and mico-CNVs (≤10 Mb) for selected balanced embryos transfer in latter.Results1. Firstly, the CNV-Seq result agreed with aCGH diagnosis on 6 CNVs detecting at gDNA level, and it showed significant up and down of abnormal regions with accurate location information. It also had a robust performance with 33 pg diluted gDNA and single cell WGA products;2. Secondly, we successfully detected the abnormal regions in triplicates at gDNA level and 30 times sequencing of cell WGA products. The Mean ±SD of two regions CN value respectively were 1.01±0.03 and 2.96±0.03 (gDNA control,3 replicates),1.16±0.06 and 2.75±0.10 (5-cell), and 1.13±0.10 and 2.69±0.13 (1-cell). The difference was statistic significance among three levels (p<0.05), between gDNA level and five cell level (p<0.05), between gDNA level and single cell level (p<0.05), but two cell levels (p>0.05);3. CNV-Seq successfully detected 31 times out of 36 times observing items for 6 CNVs from 0.56 Mb to 5.78 Mb at five cell level and single cell level. The failures contains 4 times 0.56 Mb and 1 time 2.32 Mb high repeating region;4. We totally got 35 blastocysts from 7 couples. Application of CNV-Seq to the analysis of 34 blastocysts duo to 1 WGA failure undergoing PGT, detected 18 with major aneuploidies,1 with an aneuploidy and a 4.98 Mb 5q35.2-qter deletion associated with Sotos syndrome,1 with a 6.66 Mb 7pter-p22.1 deletion associated with 7p terminal deletion syndrome and 14 with no detectable abnormalities that were suitable for transfer. Totally,5 out of 7 couples had balanced embryos to transfer.Conclusions1. CNV-Seq was highly consistent with aCGH, and it had a good reproducibility and stability at cell level;2. We set up PGT biopsy model;3. We precluded 2.6 (>2.6 duplication) and 1.3 (<1.3 deletion) as CN cut-off value to make diagnosis at cell level;4. CNV-Seq had a high resolution at cell level, and it might around 1.5 Mb;5. CNV-Seq displayed the hallmark of a comprehensive chromosome disease test including aneuploidy, unbalanced translocation and small CNVs (≤10 Mb) in PGT for decreasing pathogenic embryos transfer.