Incidence of haploidy and triploidy in trophectoderm biopsies of blastocysts derived from normally and abnormally fertilized oocytes

Abstract

Purpose: We aimed to identify the correlation between morphological pronuclear (PN) status and the genetically determined ploidy configuration in preimplantation embryos.

Methods: A retrospective observational study was conducted on 1982 embryos displaying normal fertilization and 380 embryos showing an atypical PN pattern, tested for aneuploidies and ploidy status via preimplantation genetic testing (PGT) between May 2019 and May 2024. Ploidy prediction was performed using a validated targeted-NGS approach and a proprietary bioinformatic pipeline analyzing SNPs B-allele frequency information. Ploidy results were obtained in relation to the morphological PN pattern and further stratified by mode of PN observation, maternal age, and embryo quality parameters.

Results: Abnormal ploidy results in 2PN-derived embryos were 1% (n = 20/1982): 0.8% showed triploidy and 0.2% haploidy. Ploidy results in relation to PN number in atypical fertilization were as follows: 0PN (n = 150/380) associated with 87.3% of diploidy, 8.7% of haploidy, and 4.0% of triploidy; 1PN-derived blastocysts (n = 73/153) were haploid in 47.7% of cases, 6.5% were triploid, and 45.7% diploid; 2.1PN (n = 23/280) and 3PN patterns (n = 54/280) predicted a triploid result in 34.8% and 74.1% of cases, respectively. PN observation with time-lapse increased ploidy status predictivity from 28.3% to 80.4% (p < 0.01) and reduced expected diploid rates to 19.6% (p < 0.01). Diploidy rate was higher for maternal age ≤ 35 years and for morphologically high-grade embryos.

Conclusion: Morphological PN check can be improved by incorporating ploidy analysis within the conventional PGT workflow. Euploid 2PN-derived embryos can be further selected removing haploids and triploids, and some atypical PN pattern can be better classified.

Keywords: Fertilization; NGS; PGT; Ploidy; Pronuclei; Time-lapse.

Fig. 1

Study design. Genetic evaluation of embryos in normal and abnormal fertilization. a Fertilization assessment and pronuclear (PN) annotation. Normal or abnormal fertilization status was defined using microscopic PN examination. 2PN outlines normal fertilization. Atypical PN status, including the absence of PN (0PN), monopronuclear (1PN), or more than a 2PN pattern (2.1 with a smaller additional PN or 3 evenly size PN), indicates abnormal fertilization. b First level of genetic analysis: pre-implantation genetic testing for aneuploidies (PGT-A). All zygotes reaching the blastocyst stage, irrespective of PN status, undergo analysis for aneuploidies detection via trophectoderm biopsy and PGT analysis. c Second level of genetic analysis: ploidy status evaluation. Samples displaying euploidy after PGT-A undergo further analysis using a ploidy test to detect non-diploid configurations

Fig. 2

Global distribution of ploidy results in normally fertilized zygotes. Ploidy status distribution in 1982 2PN-derived blastocysts or presumed normally fertilized embryos

Fig. 3

Interplatform comparison of ploidy results distribution. Ploidy configurations (diploid, haploid, and triploid) obtained with the two technologies employed in the study reported according to embryo pronuclear (PN) status: 0PN, 1PN, 2.1PN, and 3PN. a Top: next-generation sequencing (NGS)-based ploidy results obtained using a 357 SNPs panel reported according to embryo PN pattern. Bottom: examples of NGS profiles displaying variant allele fraction (VAF) distribution obtained for different ploidy configurations reported in the study. b Top: SNP array-based ploidy results obtained by analyzing 300,000 SNPs and reported according to embryo PN status. Bottom: examples of SNP array profiles displaying B-allele frequency (BAF) distributions obtained for different ploidy configurations reported in the study

Fig. 4

Global distribution of ploidy configurations in abnormally fertilized zygotes with different pronuclear statuses. Overview of ploidy results distribution (diploid, haploid, and triploid) reported for each group of embryos concerning the number of pronuclei (PN) detected. Ploidy results in a 0PN-derived embryos, b 1PN-derived embryos, c 2.1PN-derived embryos, and d 3PN-derived embryos

Fig. 5

Ploidy constitution of abnormally fertilized zygotes confirmed by time-lapse observation. Overview of ploidy result distribution (diploid, haploid, and triploid) reported for a 0PN, b 1PN, c 2.1PN, and d 3PN-derived embryos when identifying pronuclear (PN) status with time-lapse instruments

Fig. 6

Maternal age and embryo morphological grade correlation with ploidy results. a Ploidy result distribution in abnormally fertilized zygotes according to a maternal age, above and below a cut-off at 35 years. b Trophectoderm quality and c inner cell mass quality. Samples were divided into grades: A, good; B, fair; C, poor, according to Gardner’s embryo grading system