Preimplantation genetic haplotyping: 127 diagnostic cycles demonstrating a robust, efficient alternative to direct mutation testing on single cells.

Preimplantation genetic diagnosis using whole genome amplification and a haplotyping approach (PGH) was first described in 2006 and suggested as an efficient alternative to single-cell PCR for monogenic disorders. DNA from single cells was amplified using multiple displacement amplification; the resulting products were then tested using disease-specific PCR multiplexes applied under standard laboratory conditions to determine the haplotypes in the embryo. This study reports on a total of 127 completed biopsy cycles for 101 couples at risk of: autosomal recessive disease (71 cycles, 53 couples including one germ-line mosaic carrier), autosomal dominant disease (31 cycles, 26 couples including one germ-line mosaic carrier), X-linked recessive disease (18 cycles, 16 couples including one germ-line mosaic carrier), X-linked dominant disease (six cycles, five couples) and a double inheritance of both autosomal and X-linked recessive diseases (one cycle, one couple). Of these, 107 cycles reached embryo transfer. Overall success rates were: fetal heart beat-positive pregnancies (FHB+)/biopsy cycle=28%; FHB+/embryo transfer=34%; FHB+/couple=36%; 26 babies born, 13 ongoing pregnancies. These data demonstrate that PGH provides a robust, efficient and successful alternative to single-cell PCR for monogenic diseases.

Strategy for the creation of clinical grade hESC line banks that HLA-match a target population.

Here, we describe a pre-derivation embryo haplotyping strategy that we developed in order to maximize the efficiency and minimize the costs of establishing banks of clinical grade hESC lines in which human leukocyte antigen (HLA) haplotypes match a significant proportion of the population. Using whole genome amplification followed by medium resolution HLA typing using PCR amplification with sequence-specific primers (PCR-SSP), we have typed the parents, embryos and hESC lines from three families as well as our eight clinical grade hESC lines and shown that this technical approach is rapid, reliable and accurate. By employing this pre-derivation strategy where, based on HLA match, embryos are selected for a GMP route on day 3-4 of development, we would have drastically reduced our cGMP laboratory running costs.

Proof of principle and first cases using preimplantation genetic haplotyping--a paradigm shift for embryo diagnosis.

Preimplantation genetic haplotyping (PGH) proof-of-principle was demonstrated by multiple displacement amplification (MDA) of single buccal cells from a female donor and genotyping using 12 polymorphic markers within the dystrophin gene; the known paternal genotype enabled identification of the paternal haplotype in the MDA products despite 27% allele dropout. MDA amplified DNA from 49 single human blastomeres with 100% success. The MDA products were genotyped using a total of 57 polymorphic markers for chromosomes 1, 7, 13, 18, 21, X and Y; 72% of alleles amplified providing results at 90% of the loci tested. A PGH cycle was carried out for Duchenne muscular dystrophy. One embryo was biopsied: PGH showed a non-carrier female, which was transferred with no resulting pregnancy. A PGH cycle was carried out for cystic fibrosis. Seven embryos were biopsied and PGH allowed the exclusion of 2 affected embryos; a carrier and a non-carrier embryo were transferred resulting in an on-going twin pregnancy. PGH represents a paradigm shift in embryo diagnosis, as one panel of markers can be used for all carriers of the same monogenic disease, bypassing the need for development of mutation-specific tests, and widening the scope and availability of preimplantation genetic testing.

Investigation of chromosomal imbalance in human embryos using comparative genomic hybridization.

Studies of cleavage-stage human embryos using fluorescence in-situ hybridization (FISH) to identify sub-sets of chromosomes have indicated that the incidence of chromosomal abnormalities is high. Whole genome amplification (WGA) and comparative genomic hybridization (CGH) to investigate the full chromosome complement applied to a small number of human embryos suggested an even higher rate of abnormality. WGA and CGH were used to identify genomic imbalance in individual blastomeres from human embryos, and the results were correlated with FISH analysis of sibling blastomeres. Forty embryos were analysed; 17 (42.5%) had a normal diploid karyotype and 23 (57.5%) were abnormal, with a chromosome imbalance in one or more cells including three (7.5%) that had a chaotic chromosome complement. Of the abnormal embryos, only three showed consistent aneuploidy. FISH results obtained from sibling blastomeres were in agreement with the CGH results in all 22 of the embryos where both tests were informative. It is concluded that rates of meiotic aneuploidy in human embryos may be lower than previous estimates. The results indicate that chromosomally abnormal embryos were more likely to have arisen as a result of cultural artefact or inadequate cell cycle surveillance, rather than meiotic error.

Preimplantation genetic diagnosis as a novel source of embryos for stem cell research.

The generation of human embryonic stem (hES) cells has captured the public and professional imagination, largely due their potential as a means of overcoming many debilitating and degenerative diseases by cell replacement therapy. Despite this potential, few well-characterized hES cell lines have been derived. Indeed, in the UK, despite several centres having been active in this area for more than 2 years, there are as yet no published reports of human embryonic stem cells having been generated. Part of the reason for this lack of progress may relate to the quality of embryos available for research. Embryos surplus to therapeutic requirements following routine assisted reproduction treatment are often of poor quality and a large proportion may be aneuploid. This study reports a new approach to hES cell derivation. Embryos surplus to therapeutic requirements following preimplantation genetic diagnosis were used. Although unsuitable for embryo transfer due to the high risk of genetic disease, these embryos are from fertile couples and thus may be of better quality than fresh embryos surplus to assisted reproduction treatment cycles. Embryos donated after cryopreservation were also used, and putative hES lines were derived from both sources of embryos. The cell lines described here are thought to be the first reported hES cell lines to have been derived in the UK.