Relationship between blastocoel cell-free DNA and day-5
Renee J. Chosed
T. Arthur Chang
J. David Wininger
William E. Roudebush
Received: 30 March 2018 /Accepted: 22 May 2018
Springer Science+Business Media, LLC, part of Springer Nature 2018
Purpose Cell-free DNA (cfDNA) which is present in the blastocoel cavity of embryos is believed to result from physiological
apoptosis during development. This study assessed cfDNA content and caspase-3 protease activity in day-5 IVF blastocysts to
determine if there was a correlation with embryo morphology.
Methods Day-5 IVF blastocysts were scored according to the Gardner and Schoolcraft system (modified to generate a numerical
value) and cfDNA was collected following laser-induced blastocoel collapsing prior to cryopreservation in 25 μLofmedia.
cfDNA was quantified via fluorospectrometry and apoptotic activity was assessed via a caspase-3 protease assay using a
fluorescent peptide substrate. Data were compared by linear regression.
Results A total of 32 embryos were evaluated. There was a significant (p < 0.01) and positive correlation (cfDNA = 104.753 +
(11.281 × score); R
= 0.200) between embryo score and cfDNA content. A significant (p < 0.05) and positive correlation
(cfDNA = 115.9 + (0.05 × caspase-3); R
= 0.128) was observed between caspase-3 activity and cfDNA levels. 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 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.
Keywords Cell-free DNA
In vitro fertilization (IVF) accounts for 99% of assisted reproduc-
tive technology (ART) procedures. The main challenge of IVF
ultimately is to select the single best embryo that has the greatest
implantation potential and subsequently, the greatest chance of
developing full-term. To optimize embryo implantation potential,
embryo grading via subjective and objective embryo morpholo-
gy is currently used; however, it has limitations in determining
overall embryonic viability . Some of these limitations include
comparing, grouping, and stratifying embryos statistically in or-
der to determine the most viable embryo for implantation .
To increase the success of selecting a viable embryo, pre-
implantation genetic testing for aneuploidy (PGT-A) assesses
the ploidy status of the embryo(s). However, PGT-A is inva-
sive due to its biopsy of trophectoderm (TE) cells from the
embryo itself and researchers are looking for alternative ways
to determine embryo viability, including cell-free DNA anal-
ysis that has recently emerged as a promising approach.
The presence of cell-free DNA (cfDNA) in the circulation
was first documented in adult serum in 1948 . The source of
the cfDNA was thought to be derived from circulatory lysed
cells as well as apoptotic events . Fetal cfDNA was then
discovered in the maternal circulation, which prompted re-
searchers to look further at cfDNA from the embryo. Cell-
* William E. Roudebush
Department of Biomedical Sciences, University of South Carolina
School of Medicine Greenville, Greenville, SC 29605, USA
Department of Obstetrics and Gynecology, University of Texas
Health Science Center, San Antonio, TX 78229, USA
Premier Fertility, High Point, NC 27265, USA
Atlantic Reproductive Medicine Specialists, Raleigh,
NC 27617, USA
Journal of Assisted Reproduction and Genetics