PGT-A reveals triploid embryos among normally fertilized oocytes: frequency and blastocyst features

With the widespread adoption of blastocyst culture and advances in genetic testing technologies enabling reliable determination of ploidy status, the traditional concept of normal fertilization is being re-evaluated. Although the presence of two pronuclei (2PN) is conventionally considered indicative of normal fertilization, not all 2PN-derived embryos are euploid. A proportion of trophectoderm (TE) biopsies analyzed by next-generation sequencing (NGS) for preimplantation genetic testing for aneuploidy (PGT-A) are identified as triploid.

To determine whether specific morphological and morphokinetic features can differentiate 2PN-derived triploid blastocysts from 2PN-derived euploid blastocysts.

Retrospective study including 3369 cycles from 2666 patients aged 18-45 years who underwent PGT-A with NGS and single-nucleotide polymorphism (SNP) genotyping of TE biopsies at our IVF center from 2019 to 2023. Among 12,395 analyzed blastocysts, 59 were classified as triploid, originated from 57 PGT-A cycles and belonged to 54 patients. Fertilization assessment was reevaluated. Triploid embryos were analyzed according to second polar body extrusion, day of biopsy (day 5 vs. day 6), the quality of the TE and the inner cell mass (ICM), and morphokinetic parameters (cleavage times from 2- to 5-cell stage). Categorical variables were expressed as proportions. Student's t-test was performed for continuous variables, with a P-value < 0.05 considered statistically significant.

Genetic analysis identified 50 triploid blastocysts derived from 2PN zygotes. Two subpopulation of triploid blastocysts were distinguished: 2 PN 2 polar bodies (PB)-derived and 2PN/1 PB-derived blastocysts. Compared to their euploid sibling embryos, triploid embryos demonstrated (a) significant pronuclear dimorphism, characterized by one PN being significantly larger than the other; (b) delayed morphokinetic parameters, exhibiting slower cell division rates starting from the first cleavage division (t2: 28.59 ± 3.74 h vs. 25.70 ± 2.90 h; t3: 39.76 ± 3.48 vs. 36.75 ± 3.03 h; t4: 40.75 ± 4.07 vs. 37.25 ± 3.66 h; t5: 54.85 ± 8.07 vs. 46.34 ± 5.54 h, respectively; P < 0.05); and (c) reduced embryo quality, with a lower proportion of blastocysts exhibiting high quality TE.

The findings primarily support increased awareness of triploidy risk among embryos derived from apparently normal (2PN) fertilization. Morphological and morphokinetic parameters, although associated with triploidy, do not provide sufficient discriminatory accuracy to replace direct genetic assessment of ploidy, which remains the only reliable method to exclude triploidy. Most 2PN-derived triploid embryos appear to result from retention of the second polar body, highlighting the importance of accurate fertilization checks with special consideration given to the extrusion of the second PB. This consideration is especially relevant in non-PGT-A cycles or when the testing platform does not permit ploidy determination, as triploidy is the most common form of aneuploidy observed in first-trimester miscarriages. The incidence of triploidy in 2PN zygotes may vary across IVF laboratory settings. Given the limited number of triploid embryos identified, these findings should be interpreted with caution.