Simultaneous genome-wide haplotyping and copy number detection enabling PGT-A and PGT-M testing in equine in vitro produced embryos

Abstract

The number of equine embryos being produced increases yearly (37,094 +10% in 2021; newsletter IETS December 2022). The reasons include the growing popularity of in vitro embryo production (IVP), which increases the genetic yield from high quality mares, maximizes the use of expensive straws of semen from high quality stallions and enables IVP to be performed outside the reproductive season. On the 4th October 2022, a record price of €95,000 was paid for an embryo. However, despite the embryos’ promising pedigrees, it remains unknown if transfer of such embryos will result in a healthy foal with the aspired phenotypical characteristics. This is largely because in horses, preimplantation genetic testing for aneuploidies and monogenic diseases or traits (PGT-A and PGT-M) remains underdeveloped especially when compared to humans. In addition, equine IVP has been reported to double early pregnancy losses, possibly because of an increased incidence of genetic errors. Here, we explored the possibility of concurrent genome-wide PGT-A and PGT-M in horses and assessed the incidence of chromosomal aberrations in IVP arrested or blastocyst stage embryos. We used GGP Equine SNP arrays (Illumina) with over 70,000 SNP positions to analyze whole-genome amplified DNA (Repli-G Single cell kit, Qiagen) from 14 trophectoderm biopsies and corresponding whole blastocysts and 26 individual cells from six embryos arrested at cleavage stage following IVP. Haplarithmisis (Zamani–Esteki et al. Am. J. Hum.Genet. 2015; 96:894-912) was applied to simultaneously determine the genome-wide copy number and haplotype. Five biopsies and seven single cells failed to give a result. From six arrested embryos, five had chromosomal errors in most of their cells, including (combinations of) triploidy and one or more monosomies and trisomies of meiotic and / or mitotic origin. Two biopsies showed a loss of maternal chromosome 28 and 31 respectively, which was confirmed in the corresponding blastocysts, suggesting meiotic errors. In one of those biopsies, additional complex aneuploidies not present in the blastocyst were found, suggesting a mitotic origin. Thus, aneuploidy and ploidy errors are highly prevalent in IVP arrested embryos compared to blastocysts. Like other mammals, these errors may contribute to embryonic arrest before or after clinical pregnancy detection. As a proof of principle, we determined the inherited haplotypes for four common disease-associated genes with high carrier frequency in different horse breeds (GBE1, PLOD1, B3GALNT2, MUTYH), and for one color coat-associated gene (STX17) in one biopsy-embryo combination, confirming the potential of generic genome-wide PGT-M concurrent with PGT-A testing. Similar to humans, we postulate that embryo selection will improve embryo transfer success rates in horses and enable selection for traits and against diseases thus accelerating breeding programs.