Objective:To explore the application value of single-sperm-based single-nucleotide polymorphism (SNP) haplotyping in preimplantation genetic testing of monogenic disorders (PGT-M) associated with de novo mutations. Methods:Whole genome amplification (WGA) of the isolated single sperm was performed based on the multiple displacement amplification (MDA). WGA products were tested for the pathogenic mutation site and informative polymorphic SNP loci located within 2M upstream or downstream of the target gene to establish a sperm-based SNP haplotype. Biopsy samples obtained from embryos were subjected to WGA and next-generation sequencing (NGS). All embryos were verified via haplotype analysis and normal embryos were selected for transfer.Results:Totally 16 sperm samples were selected. Haplotypes of the affected male in 3 families with monogenic hereditary diseases including primary hyperoxaluria type 1 (PH1), Kabuki syndrome and Epidermolysis bullosa (EB) were successfully constructed using single sperm cell sequencing. PGT-M results showed that there were 10 embryos carrying paternal pathogenic variations, while the other 6 embryos did not carry paternal pathogenic variations, 2 of them had chromosomal copy number variations (CNVs). Four embryos obtained from 2 families were found to be normal after NGS according to single-sperm-based SNP haplotype analysis. However, no successful pregnancy was obtained.Conclusion:For males carrying de novo mutations, single-sperm-based SNP haplotyping can be applied for PGT to construct paternal haplotype.

Objective:To explore the application value of single-sperm-based single-nucleotide polymorphism (SNP) haplotyping in preimplantation genetic testing of monogenic disorders (PGT-M) associated with de novo mutations. Methods:Whole genome amplification (WGA) of the isolated single sperm was performed based on the multiple displacement amplification (MDA). WGA products were tested for the pathogenic mutation site and informative polymorphic SNP loci located within 2M upstream or downstream of the target gene to establish a sperm-based SNP haplotype. Biopsy samples obtained from embryos were subjected to WGA and next-generation sequencing (NGS). All embryos were verified via haplotype analysis and normal embryos were selected for transfer.Results:Totally 16 sperm samples were selected. Haplotypes of the affected male in 3 families with monogenic hereditary diseases including primary hyperoxaluria type 1 (PH1), Kabuki syndrome and Epidermolysis bullosa (EB) were successfully constructed using single sperm cell sequencing. PGT-M results showed that there were 10 embryos carrying paternal pathogenic variations, while the other 6 embryos did not carry paternal pathogenic variations, 2 of them had chromosomal copy number variations (CNVs). Four embryos obtained from 2 families were found to be normal after NGS according to single-sperm-based SNP haplotype analysis. However, no successful pregnancy was obtained.Conclusion:For males carrying de novo mutations, single-sperm-based SNP haplotyping can be applied for PGT to construct paternal haplotype.