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Abstract Study question Which is the likelihood of having a euploid/diploid embryo considering female age and the morphological pronuclear (PN) configuration? Summary answer 2.1 and 3PN embryos showed higher risk of aneuploidy. Advanced female age reduced the chance of having a euploid/diploid embryo for all PN categories. What is known already Testing preimplantation embryos and confirming their ploidy status via Single Nucleotide Polymorphisms (SNP)-based preimplantation genetic testing (PGT) methodologies is becoming a common alternative to morphological PN check. Using these advanced methods, we previously characterized the genetic constitution of 2PN-derived embryos and atypically-pronucleated zygotes (APZ), showing their expected genome-wide ploidy abnormalities. However, little is known about the impact of female age on ploidy status in relation to PN configuration. To guide reproductive counselling and improve decision-making in the use of APZ, we present a detailed overview of the chromosomal composition of preimplantation embryos highlighting specific trends in relation to maternal age. Study design, size, duration Retrospective observational study evaluating the impact of maternal age on aneuploidy and ploidy results in IVF embryos, in relation to their PN status. Embryos were cultured in conventional or time-lapse incubators until blastocyst stage, regardless of their PN configuration. PGT for aneuploidies (PGT-A) and ploidy assessment were performed on 2830 2PN-derived zygotes and 812 APZ, testing trophectoderm biopsies collected between January 2021-November 2024. Genetic results were stratified according to female age and morphological PN composition. Participants/materials, setting, methods International study involving consenting patients undergoing PGT-A with ploidy analysis. Combined genetic testing was performed using a validated NGS copy number analysis to detect aneuploidies and a validated targeted NGS approach composed of a custom panel of 357 SNPs and a proprietary pipeline of analysis. Reliable ploidy classification was obtained using a combined algorithm strategy based on SNPs allele frequencies. Categorical variables were compared using Chi-square. Nominal variables were analysed using Cochran-Armitage test for trends. Main results and the role of chance We firstly looked at aneuploidies incidence and found significant differences among different PN configurations (p < 0.001). When stratified according to female age, APZ (0PN, 1PN, 2.1PN, 3PN) showed 58.5% (n = 38/65;95%CI=45.56-70.56), 58.0% (n = 105/181;95%CI=50.46-65.29), 83.3% (n = 50/60;95%CI=71.48-91.71) and 70.1% (n = 157/224;95%CI=63.63-76.01) aneuploidy for female age >38 years. All of them were significantly higher compared to female age ≤38 years, where aneuploidy was 35.1% (n = 66/188;95%CI=28.30-42.39), 38.7% (n = 92/238;95%CI=32.43-45.16), 64.9% (n = 24/37;95%CI=47.46-79.79) and 61.2% (n = 35/63;95%CI=52.52-69.30) respectively. Interestingly, in young females (≤35 years), significantly higher aneuploidy for 2.1PN (66.7%,n=12/18) and 3PN (55.6%,n=35/63) were reported, compared to 2PN embryos (37.7%,n=780/2071)(p < 0.02). Secondly, we confirmed a significant association between diploidy, haploidy, triploidy and PN status (p < 0.05). In details, ploidy assessment of 731 euploid APZ showed collectively 90.2% (n = 240/266;95%CI=86.01-93.52), 50.8% (n = 151/297;95%CI=45.00-56.66), 66.7% (n = 16/24;95%CI=44.68-84.37) and 19.6% (n = 28/143;95%CI=13.42-27.04) of diploidy for 0PN, 1PN, 2.1PN and 3PN respectively. Overall diploidy rates decreased with maternal age in APZ (OR = 0.53;p-value=0.001), however only higher triploidy in 0PN (5.3%,n=7/132) and higher haploidy in 1PN embryos (51.5%,n=99/192) significantly contributed to this decrease in older patients (p < 0.03). Differently, 2PN embryos displayed 1.2% (n = 34/2830;95%CI=0.83-1.68) triploidy and 0.2% (n = 7/2830;95%CI=0.10-0.51) haploidy. Interestingly, only triploidy was significantly impacted by female age, going from 0.7% (n = 15/2071;95%CI=0.41-1.19) for age ≤35 years, to 4.5% (n = 14/309;95%CI=2.5-7.5) for age >40 years (p < 0.0001). Limitations, reasons for caution Despite we have previously demonstrated higher accuracy of time-lapse systems in predicting PN status, here we didn’t distinguish between static and time-lapse observations. Moreover, the potential paternal age contribution on ploidy abnormalities requires further investigation. Wider implications of the findings APZ embryos have different diploid/aneuploid risk compared to 2PN embryos. Indeed, the presence of an additional PN is associated with higher aneuploidy in young females. Moreover, considering the higher risk of triploidy in advanced female age, and the higher incidence in miscarriages, PGT should always incorporate ploidy analysis. Trial registration number No