Preimplantation genetic testing, including screening for aneuploidy (PGS or PGD-A) and diagnostic testing (PGD) for monogenic diseases or unbalanced translocations, is a tool for embryo testing aimed at identifying the euploid and/or non-affected embryos produced during an IVF cycle. When the technique is optimally applied, it can prevent the transfer of an embryo with a genetic disease and significantly reduce the risks of transferring chromosomally abnormal embryos that may result in implantation failure or miscarriage. A critical aspect of this technology is the embryo biopsy procedure. Both the biopsy itself and preparation of the biopsied cells can have negative influences on the test results and on the embryo’s developmental potential.
The stage at which the biopsy is performed (Day 3 vs. Day 5) has been reported to exert significant effects. During a D3 biopsy, a single cell is removed at the cleavage (8 cell) stage rather than a group of cells (5-10) taken from a D5, trophectoderm biopsy. The single-cell, D3 biopsy sample is prone to much higher rates of amplification failure per embryo relative to D5 samples that may contain as much as 10X more input DNA. In relation to aneuploidy detection, mosaicism has been reported to be a predominant feature of D3, cleavage-stage biopsies; with rates reported as high as 75% (Taylor TH et al., Hum Reprod Update, 2014). Additionally, the results of any single-cell biopsy are more likely to be affected by incomplete DNA replication during S-phase of the cell cycle. Dimitriadou reported an increased false positive CNV detection rate, genome-wide in S-phase cells (Mol Cytogenet, 2014). Importantly, the authors point out that the risk of a false negative is also higher, and this may have a significant impact for translocation carriers using PGD.
Following the biopsy, the cells must be carefully rinsed before transferring them to PCR tubes for DNA amplification. This process seems straightforward, but it is essential that samples be washed thoroughly to reduce the risk of contaminating DNA. Recent studies have shown that embryonic DNA is easily amplified from the fluid within the blastocyst cavity of D5 embryos. The trophectoderm biopsy procedure necessarily ruptures the cavity of the blastocyst and releases this DNA around the resulting biopsy samples. Tobler and colleagues recently reported discordance between the blastocoel fluid and the associated biopsy samples in >50% of cases (Fertil Steril, 2015). Such contamination risks are not limited to D5 trophectoderm biopsy samples. Various reports have also demonstrated measurable quantities of DNA in spent, D3-embryo culture media, and the concentration of DNA has been shown to be inversely related to embryo quality (Sigliani S et al., Hum Reprod/, 2013). The media surrounding embryo biopsy samples from all embryo stages therefore represents an important potential source of contamination, which may lead to elevated noise levels in many diagnostic platforms.
Lastly, the developmental potential of the embryo may also be negatively impacted by the embryo biopsy procedure. In a 2013 well-designed, randomized controlled trial, Scott and colleagues demonstrated a dramatic 39% reduction in sustained implantation for embryos biopsied on D3. Importantly, they were unable to detect any corresponding impact on embryos biopsied on D5 (Fertil Steril, 2013). However, this does not mean that D5 embryo biopsy cannot affect development or implantation. Earlier this year, Neal and colleagues demonstrated that trophectoderm biopsy samples with the highest relative DNA concentration generally had the lowest implantation and live–birth rates. The authors suggest one likely explanation is that such samples result from the removal of too many trophectoderm cells and thus compromise the developmental potential of the embryo.
Overall, the potential negative impacts of embryo biopsy are best mitigated by the careful training and education of practitioners, a devotion to trophectoderm biopsy in preference over cleavage-stage biopsy, and the careful handling, washing, and storage of biopsy samples before DNA amplification. The implementation and monitoring of each of these elements will help ensure that the fullest potential of the technology can be realized for every patient.