Webinar: Viability of Blastocyst Stage Embryos Developed from Abnormally Fertilized Oocytes

Webinar summary

This webinar explores the relationship between pronuclear assessment and preimplantation genetic testing for aneuploidy (PGT-A), with a focus on embryos initially classified as having abnormal fertilization patterns. Dr. Cengiz Cinnioglu, founder and general manager of Luminary Genetics, reviews the evolution of preimplantation genetic testing and examines how molecular approaches can provide additional information beyond microscopic pronuclear assessment.

The presentation focuses specifically on 0PN, 1PN, 2.1PN, and 3PN embryos and examines the ability of short tandem repeat (STR) and single nucleotide polymorphism (SNP)-based approaches to distinguish haploidy, diploidy, and polyploidy. The webinar also presents research from Luminary Genetics investigating embryos that could otherwise be excluded based on pronuclear assessment.

Presentation overview

Pronuclear assessment is performed after fertilization to evaluate the number of visible pronuclei. A 2PN observation is generally considered consistent with normal fertilization, while deviations such as 0PN, 1PN, or 3PN are considered abnormal and these embryos are typically discarded. The presentation highlights an important limitation of this approach: pronuclear assessment is based on microscopic observation and does not directly establish the genomic composition of an embryo.

The webinar reviews published studies demonstrating discordance between pronuclear morphology and subsequent molecular characterization. Earlier studies used SNP and STR markers to investigate embryos initially classified through pronuclear assessment, identifying diploid, haploid, triploid, and aneuploid outcomes within these groups.

Luminary Genetics subsequently investigated this question in a research cohort of 432 embryos initially classified as 0PN or 1PN. PGT-A using NGS was followed by STR analysis because NGS alone could not distinguish haploid, diploid, and triploid states in this workflow. 

Key findings

Pronuclear assessment and molecular findings may differ

• Embryos classified as 0PN, 1PN, or 3PN by microscopy could subsequently demonstrate diploid genomic profiles
• In one larger dataset, 56% of 0PN embryos were confirmed as diploid
• Among 1PN embryos, approximately one-third were haploid, one-third demonstrated multiple aneuploidies, and one-third were diploid
• Approximately half of embryos classified as 3PN were confirmed as such, while 8.1% were identified as diploid
• Even among embryos classified as 2PN, 0.63% were subsequently identified as triploid

SNP and STR analysis provides additional ploidy information

• Haploidy and polyploidy as limitations of NGS-only PGT-A approaches discussed in the webinar
• STR markers were used to assess parental contribution and distinguish haploid from biparental diploid embryos
• Patterns of homozygosity and heterozygosity across informative markers provided additional information about genomic contribution
• SNP-based NGS was presented as an approach for incorporating this information directly into the sequencing workflow

Luminary genetics research identified biparental diploid embryos

• The research cohort included 432 embryos initially classified as 0PN or 1PN
• NGS-based PGT-A identified 63.2% of these embryos as euploid
• Further STR analysis demonstrated biparental contribution in 96.1% of the evaluated 0PN embryos
• Among 1PN embryos, 42.1% were consistent with the original microscopic assessment, while 56.8% were subsequently identified as diploid with contributions from both parents

Follow-up data examined outcomes after further molecular characterization

• Follow-up analysis of embryos initially classified as 0PN or 1PN but subsequently characterized as diploid and euploid
• Twenty-eight transfers were reported from this group
• The reported live birth rate was 53.57%
• This represents a limited dataset and reports that 124 embryos were identified through the research as embryos that could otherwise have been excluded

SNP-Based NGS extended the PGT-A workflow

• Following the initial research, SNP markers were incorporated into the NGS approach
• Collaborating laboratories continued recording 0PN and 1PN observations to enable comparison between microscopic and molecular findings
• Approximately 15% of embryos were associated with uncertainty or 0PN/1PN classification during pronuclear assessment
• Among this group, molecular analysis identified embryos characterized as euploid or mosaic

The webinar highlights limitations in relying on pronuclear morphology alone to characterize fertilization status. Across the published studies and Luminary Genetics research presented, a proportion of embryos initially classified as 0PN, 1PN, or 3PN demonstrated genomic findings that differed from their microscopic classification.

The research demonstrates how STR and SNP-based approaches can provide additional information on haploidy, diploidy, polyploidy, and parental contribution beyond NGS-based copy number assessment alone. Dr. Cinnioglu concludes that SNP-based NGS can identify biparental diploid embryos among those that might otherwise be excluded following pronuclear assessment, providing a molecular framework for further investigation of abnormal fertilization classifications in PGT research.

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