Pre-fertilization genetic testing to select healthy gametes in a mammalian Marfan syndrome model

Abstract Study question Is it possible to identify disease-free gametes before insemination to prevent vertical transmission of an autosomal dominant disease? Summary answer In a mammalian Marfan syndrome model, we successfully generated and identified healthy androgenotes to be utilized as male gametes to produce unaffected conceptuses. What is known already In patients with autosomal dominant genetic disorders such as Marfan syndrome, germline heterozygosity poses a risk to offspring health. For these patients, preimplantation genetic testing for monogenic disorders (PGT-M) allows us to select unaffected conceptuses for embryo replacement. However, PGT-M is inherently inefficient and can lead to embryo wastage. This has sparked interest in replicating the genome of individual gametes for (1) pre-fertilization genetic testing and (2) subsequent use as genotyped gamete substitutes during IVF. Study design, size, duration For this study, we selected 4 male B6 mice that were heterozygous for the Fbn1tm1Hcd mutation and displayed classic manifestations of Marfan syndrome. Spermatozoa from these mice were used to inseminate enucleated wildtype mouse oocytes and generate haploid androgenetic embryos for (1) Fbn1tm1Hcd genotyping and (2) use as unaffected male gamete substitutes. Participants/materials, setting, methods Using tail clippings and embryo biopsy specimens, a Fbn1tm1Hcd allele-specific PCR primer was developed and validated. Haploid androgenetic embryos were allowed to progress to the 8-cell stage. Subsequently, a single-haploid pseudo-blastomere was isolated for genotyping, while the remainder were individually grafted into activated recipient wildtype oocytes and fused using inactivated Sendai virus to generate biparental conceptuses. Resulting diploid conceptuses were cultured in a time-lapse incubator and then genotyped to confirm the healthy paternal haplotype. Main results and the role of chance A total of 175 wildtype B6 oocytes were enucleated yielding 169 ooplasts (96.6% survival rate). The ooplasts were inseminated with spermatozoa from mutant Fbn1tm1Hcd mice, yielding 144 (85.2%) monopronucleated haploid androgenetic embryos. Of those, 100 (69.4%) progressed to the 8-cell stage. In 47 (47.0%) of them, genetic analysis by PGT-M on an individual pseudo-blastomere characterized the embryo as unaffected because it harbored the wildtype Fbn1 allele. The remaining 7 haploid pseudo-blastomeres from 5 Fbn1 wildtype androgenetic embryos were individually isolated and grafted into 35 recipient oocytes to generate 31 (88.6%) biparental conceptuses with the desired paternal haplotype. These achieved a 61.3% (19/31) blastocyst development rate after 96 hours, displaying normal embryo morphokinesis. A blastocyst biopsy was carried out (n = 19), confirming the unaffected paternal haplotype void of the Fbn1tm1Hcd mutation. Limitations, reasons for caution Although this technique is feasible for pre-fertilization genetic testing, it needs to be reproduced with the same efficiency in humans. A major concern is related to the dynamics and roles of the sperm centrosome in the first mitotic division of human zygotes, which may be missing in the pseudo-gametes. Wider implications of the findings This approach to pre-fertilization genetic testing will benefit patients with heritable genetic conditions. In addition, the inherited decondensation of the male genome by the ooplasm may enable genome editing experiments to be performed. Trial registration number Not applicable