This study provides further evidence that cfDNA is present in blastocoel fluid, can be quantified, and positively correlates with embryonic morphology. There is also evidence that at least a portion of the cfDNA present is from intracellular contents of embryonic cells that underwent apoptosis. Additional studies are warranted to determine other physiological sources of the cfDNA in blastocyst fluid and to determine the relationship with cfDNA content, embryo morphology, and chromosomal ploidy status plus implantation potential.
OBJECTIVE: Cell-free DNA (cfDNA), which is present in the blastocoel cavity of embryos, is believed to result from either physiological apoptosis and, or cellular (re)modeling during development. Additionally, cfDNA has been investigated as a potential reference source for preimplantation genetic screening (PGS). This study assessed cfDNA content in day 5 IVF blastocysts to determine if there was a correlation with embryo morphology. Additionally, this study investigated if the physiological source of the cfDNA is apoptotic in nature.DESIGN: Retrospective study in academic assisted reproductive technology programs.MATERIALS AND METHODS: Day 5 IVF generated blastocysts were scored according to the Gardner system (modified to generate a numerical value) and cfDNA was collected from laser-induced blastocoel collapsing prior to cryopreservation in 25mL of media. cfDNA was quantified via fluorospectrometry (AccuBlue NextGen dsDNA Quantification Kit; Thermo-Scientific NanoDrop 3300 Fluorospectrometer) and apoptosis activity was assessed via a caspase-3 fluorescence assay (Enzo Life Sciences Caspase-3 Cellular Assay; Tecan Infinite M1000 fluorescence reader). Data were compared by linear regression (SigmaPlot for Windows v13.0.0.83).RESULTS: A total of 32 embryos were evaluated. Embryo morphology scores ranged from a low of 15 to a high of 27; with a mean of 21.1. cfDNA content (ng/ mL) ranged from a low of 32.3 to a high of 315.3; with a mean of 133.6. Caspase-3 activity (AFU detected with 30 mM DEVD-AMC substrate) ranged from undetectable levels to a high of 2,226.6; with a mean of 383.8. The maximum embryo score of 27 had a cfDNA concentration of 315.3 ng/mL and the minimum embryo score of 15 had a cfDNA concentration of 32.3 ng/mL. There was a significant (p<0.01) and positive correlation (cfDNA ¼ -104.753+(11.281*score); R 2 ¼0.200) between embryo score and cfDNA. There was a significant (p<0.05) and positive correlation (cfDNA ¼ 115.9+(0.05*caspase-3); R 2 ¼0.128) between caspase-3 activity and cfDNA. There was no significant relationship between caspase-3 activity and embryo morphology score.CONCLUSIONS: This study provides further evidence that cfDNA is present in blastocoel fluid, can be quantified, and positively correlates with embryonic morphology. There is also evidence that at least a portion of the cfDNA is due to apoptotic activity. Additional studies are warranted to determine other physiological sources of the cfDNA in blastocyst fluid and to determine the relationship of cfDNA content with embryo morphology, ploidy status (PGS) and, or implantation potential.
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