Karyomapping in preimplantation genetic testing for ß-thalassemia combined with HLA matching: a systematic summary.

PURPOSE: To investigate the validity, accuracy, and clinical outcomes of Karyomapping in preimplantation genetic testing (PGT) for ß-thalassemia combined with human leukocyte antigen (HLA) matching. METHODS: A total of 128 cycles from January 2014 to December 2017 were identified, and 1205 embryos were biopsied. The case group included 88 cycles using Karyomapping for PGT-HLA, compared with 40 cycles using polymerase chain reaction-short tandem repeat (PCR-STR) as the control group. RESULTS: There were significant differences in the HLA matching rate (21.34 vs. 14.37%), the matched transferable embryo rate (9.79 vs. 14.07%), the clinical pregnancy rate (65.08 vs. 41.86%), and the spontaneous miscarriage rate (2.44 vs. 22.22%) between the case and control groups. In the case group, nearly 1/3 (33.37%) of the embryos showed aneuploidy. According to the results of single nucleotide polymorphism (SNP) haplotype analysis, the recombination rates of HBB (hemoglobin subunit beta) and HLA were 11.46% and 5.61% respectively. HLA gene recombination was mostly distributed between HLA-A and HLA-B and the downstream region of HLA-DQB1. In addition, STR analysis could be considered in the case of copy-neutral loss of heterozygosity (LOH) in the region where the HLA gene is located. CONCLUSION: Karyomapping contributes to accurate selection of matched embryos, along with aneuploidy screening. However, STRs assist identification in cases of LOH in the target region.

Preimplantation genetic testing of Robertsonian translocation by SNP array-based preimplantation genetic haplotyping.

OBJECTIVES: The present study attempted to confirm a method that distinguishes a balanced Robertsonian translocation carrier embryo from a truly normal embryo in parallel with comprehensive chromosome screening (CCS). METHODS: Comprehensive chromosome screening was performed in 107 embryos from 11 couples carrying Robertsonian translocations. Among them, embryos from 2 families had been transferred before the diagnosis of translocation, which resulted in successful pregnancies; embryos from the remaining families were transferred after the identification of translocations. The single nucleotide polymorphism (SNP) genotypes were acquired on a genome-wide basis, and breakpoint regions and flanking were assessed by establishing haplotypes. The predicted karyotypes from the transferred embryos were confirmed by prenatal diagnosis. RESULTS: Among the 9 families finally undergoing translocation diagnosis, the amniotic cell karyotypes of 3 families were concordant with the results predicted by preimplantation genetic haplotyping, revealing a good consistency rate. After CCS, the euploid embryos from 2 other families could not be further detected because of the absence of abnormal embryos as probands. CONCLUSIONS: Molecular karyotypes and haplotypes could be established with SNP microarray simultaneously in each embryo. SNP array-based PGT can simultaneously complete the CCS and identify Robertsonian translocation carriers, thus making it possible to prevent Robertsonian translocations from being passed to subsequent generations.

Genotyping single-sperm cells by universal MARSALA enables the acquisition of linkage information for combined pre-implantation genetic diagnosis and genome screening.

PURPOSE: This paper aims to investigate the feasibility of performing pre-implantation genetic diagnosis (PGD) and pre-implantation genetic screening (PGS) simultaneously by a universal strategy without the requirement of genotyping relevant affected family members or lengthy preliminary work on linkage analysis. METHODS: By utilizing a universal Mutated Allele Revealed by Sequencing with Aneuploidy and Linkage Analyses (MARSALA) strategy based on low depth whole genome sequencing (~3x), not involving specific primers' design nor the enrichment of SNP markers for haplotype construction. Single-sperm cells and trephectoderm cells from in vitro fertilized embryos from a couple carrying HBB mutations were genotyped. Haplotypes of paternal alleles were constructed and investigated in embryos, and the chromosome copy number profiles were simultaneously analyzed. RESULTS: The universal MARSALA strategy allows the selection of a euploid embryo free of disease mutations for in uterus transfer and successful pregnancy. A follow-up amniocentesis was performed at 17 weeks of gestation to confirm the PGD/PGS results. CONCLUSION: We present the first successful PGD procedure based on genotyping multiple single-sperm cells to obtain SNP linkage information. Our improved PGD/PGS procedure does not require genotyping the proband or relevant family members and therefore can be applicable to a wider population of patients when conducting PGD for monogenic disorders.

[Preimplantation genetic diagnosis for carriers of thalassemia and chromosomal abnormality].

OBJECTIVE: To provide preimplantation genetic diagnosis(PGD) for two couples carrying thalassemia mutations and chromosomal abnormalities. METHODS: Couple 1 were both carriers of ß 41/42 thalassemia mutations, while the husband has carried a reciprocal translocation with a karyotype of 46,XY,inv(9)(p11;q13),t(11;22)(q25;q13). Couple 2 were both carriers of α (-SEA) thalassemia mutation. Their chromosome karyotypes were both normal, but had two spontaneous abortions. The couples had received 1 and 3 blastocysts respectively through in vitro fertilization(IVF) cycles. Following the biopsy, the cells underwent whole genome amplification, and the amplified DNA from each embryo was subjected to genetic testing and a 23-chromosome single nucleotide polymorphism(SNP) microarray assay. RESULTS: The embryo of couple 1 was diagnosed as carrier of ß 41/42 thalassemia with euploid chromosomes. The embryo was transferred and resulted in intrauterine pregnancy. Similarly, an embryo of couple 2 was verified as carrier of α (-SEA) thalassemia with euploid chromosomes. CONCLUSION: PGD for aneuploidy coupled with testing for single gene disorders via trophectoderm biopsy and whole genome amplification is feasible. The approach can attain diagnosis with minimal damage with sound clinical outcome.