28 Evaluation of cell-free DNA in spent holding media as a noninvasive technique for pre-implantation genetic diagnosis on in vivo-produced equine embryos

Cell-free DNA (cfDNA) in “spent” embryo culture medium has been evaluated as an alternative to biopsy of human embryos for genetic screening for sex determination, detection of aneuploidy and presence of genes for inherited diseases. We hypothesised that cfDNA could be detected in spent medium after holding equine embryos for 24 h. Equine embryos are commonly held for 18 to 24 h at 5°C during shipment to a transfer centre. The specific aim of the study was to determine whether cfDNA of embryonic origin is present in spent equine holding media after 24 h of cooled-storage. Ten mares and 1 stallion were used. Mares were inseminated with 500 million progressively motile spermatozoa and monitored daily by transrectal ultrasonography to document ovulation. Embryo collection was performed 8 days after ovulation on nine cycles and 12 days postovulation on one cycle. The uterus was flushed with Hartmann’s solution containing 2 mL of 10% PVA per litre (ABT360) and embryos washed with holding medium devoid of animal origin components (Adapt Holding with HA, ABT360). Embryos were transferred into 500 µL of the same holding medium in a sterile vial and maintained at 5°C for 24 h. Embryos were then transferred into a sterile 1.5-mL microcentrifuge tube. Embryos, spent holding medium, and control medium not used for holding were frozen at −80°C until evaluated for cfDNA. A total of 10 embryos were collected, including nine Day 8 embryos (250 to 1000 μm) and one Day 12 embryo (~8 mm). Whole-genome amplification (WGA) products were used in two independent polymerase chain reaction (PCR)-based genotyping assays. One PCR reaction assayed 17 ISAG microsatellite markers plus two sex identification markers, amelogenin (AMEL) and sex determining region Y (SRY), while the second was restricted to the sex markers. A precipitation procedure was performed on spent and control media samples to concentrate cfDNA. The WGA evaluation of the 10 embryos revealed 2 were male (SRY positive) and 8 were female (AMEL positive and SRY negative). No cfDNA was detected in any of the control media samples. The cfDNA was detected in spent holding medium of the large embryo recovered on Day 12 postovulation and yielded results for sex determination (AMEL positive) and 16 of 17 microsatellite markers. Spent holding media from a single Day 8 postovulation embryo yielded results for sex determination (AMEL positive) but not for the autosomal identification markers. No other cfDNA was recovered in spent holding media of the eight smaller embryos collected on Day 8 postovulation. In conclusion, cfDNA was not consistently detected in spent media after holding of Day 8 in vivo-produced equine embryos for 24 h at 5°C.