Application of preimplantation genetic diagnosis in 91 equine ICSI embryos

Abstract

Preimplantation genetic diagnosis (PGD) of in vitro-produced (IVP) equine embryos has been reported in research and clinical settings. In the previous reports, 4-16% biopsied samples failed to yield PCR results of disease-causing genes. There is little information on the feasibility of a second biopsy and refreezing of those PGD-failed frozen IVP equine embryos. In this report, we evaluated biopsy results from 91 IVP equine embryos produced from 2018 to 2022 and tested for hereditary equine regional dermal asthenia (HERDA), glycogen branching enzyme deficiency (GBED) and hyperkalemic periodic paralysis (HYPP) genes. We also examined the pregnancy and foaling rates of twice biopsied-frozen (2BF) embryos by comparing them with fresh or once biopsied-frozen (1BF) embryos. IVP embryos were produced by intracytoplasmic sperm injection and in vitro culture of shipped immature oocytes. Biopsy of embryos was performed as previously reported with minor modifications (Choi et al., Reprod Fertil Dev 2016;28:1382-1389). Biopsies were kept frozen for a maximum of 7 days and shipped to the University of California, Davis for genetic analysis. A total of 15 fresh, 39 1BF and 5 2BF IVP embryos were transferred to recipients at embryo transfer facilities. The non-transferred vitrified embryos, 12 1BF and 1 2BF IVP embryos, were kept frozen accordingto owners’ instructions. Among the 91 IVP biopsies, 7 (8%) failed to yield PCR results and 84 samples were genetically analyzed; 14 (24%) were homozygous for HERDA in 58 samples, 4 (21%) were homozygous for GBED in 19 samples and 0 were homozygous for HYPP in 7 samples. The molecular sexes of 84 samples were 44 males and 40 females. Among the 7 2BF IVP biopsies, 1 failed to yield PCR results and 6 were genetically analyzed; 1 was homozygous for GBED. The molecular sexes of 6 2BF samples were 1 male and 5 females. The pregnancy rates of fresh, 1BF and 2BF IVP embryos were not significantly different among groups (60% (9/15), 64% (25/39) and 80% (4/5), respectively, Fisher's exact test). Of these, 3 pregnancies from 1BF were removed from the foaling rate calculation because of unknown and ongoing pregnancies. The foaling rates of fresh, 1BF and 2BF IVP embryos were not significantly different among groups (47% (7/15), 33% (12/36) and 60% (3/5), respectively, Fisher's exact test). Out of 22 foals analyzed, all matched with molecular genders. In summary, PGD for disease-causing genes can be further improved by a second biopsy and refreezing of frozen IVP equine embryos. In addition, two biopsy-freezing cycles of IVP equine embryos were not detrimental to in vivo embryo development.