Embryo quality was assessed during the second and third day [32] of culture and was defined by the number, shape and size of blastomeres, presence of vacuoles and the grade of fragmentation.
Grade III: Fragmentation and fissuring in an area more than half an inch in diameter.
Grade II: Fragmentation and fissuring in an area half an inch or less in diameter.
Quantities and patterns of circulating DNA differ significantly between the groups, with total quantities of nuclear and mitochondrial DNA increasing with pathological grade, and fragmentation patterns changing between normal, adenomatous, and cancer populations.
The cleavage stage embryos were scored based on cell number and the degree of fragmentation, according to the grading system of Hardarson (2001) [ 12].
Furthermore, the PCR-based method for quantifying DNA can be influenced by the fragmentation grade of the DNA in that some degraded β-globin gene copies may not be amplified, resulting in an underestimation of DNA levels.
The analysis was performed with a FACScan® analyzer (Becton Dickinson), where DNA content and grade of DNA fragmentation (apoptosis appearing as a sub-G1 fraction), and distribution of cells in G1, G2/M or S-phase, was determined.
In contrast, because our method of DNA quantification is based on the absorbance at 260 nm, we are able to measure the levels of cfDNA, irrespective of the source or the fragmentation grade of the DNA.
Embryos graded as grade 1 (6–10 cells, no fragmentation and equal blastomere size) or grade 2 (allowing up to 20% fragmentation) were qualified as good quality embryos.
Embryos were examined for cleavage (cell number) and grade, which includes cytoplastmic fragmentation.
