Authors: Kathryn Wozniak, MS; Amy Jones, MS, ELD(ABB); Minjae Kwon, MS, MB(ASCP)CG; Ping Zou, PhD, HCLD(ABB); Mitchel C. Schiewe, MS, PhD, HCLD(ABB)

Materials and Methods:

Fertilization was assessed ~16-18 hours post-ICSI for the presence of pronuclei (PN). Zygotes were subdivided according to PN status (0PN, 1PN, 2PN). A retrospective analysis of blastocyst formation and utilization (biopsied/cryopreserved) was contrast for development on days 5-7. Genetic outcomes were evaluated from trophectoderm (TE) samples analyzed for aneuploidy using next generation sequencing (NGS). Whole genomic amplification DNA from TE samples and DNA extracted from the saliva of each embryo’s biological parents was compared with GeneMapper to assess the ploidy status of female 1PN blastocysts. Proportional analyses were statistically assessed using Chi-squared testing (P<0.05).

Results:

We obtained 291 1PN zygotes from 210 fresh/frozen ICSI PGT-A cycles, representing 24.06% of the total fresh/frozen ICSI cycles, in contrast to 518 0PN and 2787 2PN zygotes. Blastocysts formed from 60 (20.62%) 1PN, 22 (4.25%) 0PN and 1935 (69.43%) 2PN zygotes, of which 32 (10.97%), 13 (2.51%) and 1,621 (58.16%) were biopsied/cryopreserved, respectively. 1PN blastocyst formation and utilization rates were lower than 2PN but higher than 0PN (P<0.05). Interestingly, 71.88% of biopsied 1PN blastocysts were euploid, being higher (P<0.05) than 0PN (38.46%) and 2PN (51.51%). No difference in euploidy rates was determined between 0PN and 2PN blastocysts. The 1:1 female-to-male ratio observed in the 2PN group was skewed towards female embryos (2.5:1) in the 1PN group. The ploidy constitutes of 12 female 1PN blastocysts were analyzed; 2 (16.67%) were diploid with a biparental genome, and the others (83.33%) were haploid with maternal-only genomes.

Conclusions:

Previously, 0PN and 1PN zygotes have been considered to arise from failed/abnormal fertilization and thus discarded. Recent work has shown their potential to give rise to healthy live births. Although at a lower rate than 2PN zygotes, our data confirm that ICSI-derived 0PN and 1PN zygotes can develop into euploid blastocysts. Unfortunately, NGS-PGT-A technology is unable to discern uniparental disomy or haploidy in female embryos, as shown in our study. The low rate of biparental chromosome inheritance observed in 1PN ICSI-derived female blastocysts suggests a need for verification of ploidy status. Ploidy evaluation and 0PN- and 1PN- derived embryo transfers are ongoing, as we seek to determine a suitable selection criterion for the transfer of euploid ICSI-derived 0PN and 1PN blastocysts.

Impact Statement:

ICSI-derived 0PN and 1PN zygotes may not be failed/abnormally fertilized oocytes. However, their blastocysts should undergo additional genetic testing, particularly 1PN female blastocysts, in which confirmation of biparental chromosome inheritance is recommended to reduce unwanted pregnancy losses.