The impact of in vitro human embryo fragmentation on blastocyst development and ploidy using Next-Generation Sequencing (NGS)

Introduction The degree of fragmentation has been utilized in non-invasive selection of Day 2 or Day 3 embryos for transfer. Fragmentation happens in both in vitro and in vivo embryo, where anucleate cells of varying size and number are found along with nucleate cells in the embryo. While fragmentation may potentially affect the developmental potential of an embryo, very little evidence has been reported in the literature. This study was carried out to evaluate the effects of embryo fragmentation on the blastulation rate (BR), blastocyst utilization rate (BUR), implantation rate (IR) and euploid rate of the blastocyst. Materials & Methods A total of 438 PGS cycles from January 2017 to December 2017 were retrospectively analyzed. Intracytoplasmic sperm injection (ICSI) was carried out after oocyte retrieval. Normally fertilized (2PN) embryos (n= 3720) were evaluated according to the degree of fragmentation (group 0F: 0 – 5% fragments, group 0/1F: 6 -10% fragments, group 1F: 11 – 15% fragments, group 1/2F: 16 – 20% fragments, group 2F: 21 – 25% fragments and group 2/3F: >25% fragments) at 66 – 68 hours post injection. Following extended culture to blastocyst stage, trophectoderm (TE) biopsy was performed on good or fair graded blastocysts (n=1865) on either Day 5 or Day 6. Pre-implantation Genetic Screening (PGS) using Next-Generation Sequencing (NGS) (Veriseq Protocol, Illumina) was used for aneuploidy screening. A total of 252 euploid or low-level mosaicism blastocysts were transferred and the BR, BUR, IR and the euploidy rate of the blastocyst were evaluated. Results Embryos with higher degree of fragmentation have been found to have a lower BR (81.4%, 73.5%, 60.7%, 47.0%, 31.6% and 12.8%, respectively) and BUR (66.2%, 52.6%, 37.7%, 23.5%, 9.9% and 4.3%, respectively) in our study. We found that embryos with no fragment or lower degree of fragmentation have a higher chance of reaching the blastocyst stage, while most of the embryos with a higher degree of fragmentation were arrested after Day 3. Our study did not show any differences in euploidy rate, mosaicism rate and aneuploidy rate between groups with an average of 67.29%, 8.89% and 25.09% respectively. Transfer of blastocyst with higher degree of fragmentation on Day 3 did not show any significant decrease of in implantation rate. Conclusions In our study, fragmentation has been shown to affect blastocyst development due to unequal allocation of cells. But once a blastocyst develops, there do not seem to be any impact on IR and chromosome abnormalities. Indeed, pregnancies has been reported with the transfer of embryos with a high degree of fragmentation. Our small sample size however, may impact on the validity of this study. As embryo selection solely based on the degree of fragmentation on Day 3 is ineffective, extended culture of all embryos regardless of degree of fragmentation to blastocyst stage is recommended. This will potentially avoid accidental discarding of embryos that are destined to implant and form a pregnancy.