Abstract Study question To compare euploid and mosaicism rate (high or low) among blastocysts derived from fresh and frozen ejaculate sperm in donated oocytes. Summary answer No statistically significant differences were found on blastocyst euploid and mosaicism rate between blastocysts derived from fresh versus frozen. What is known already Even though 5%-10% autosomal aneuploidies and 5%-100% sex chromosomes aneuploidies are paternally derived, the clinical relevance of paternal contribution to chromosome aneuploidy is frequently overlooked. Besides, male infertility could contribute to the generation of chromosomal abnormalities in the resulting embryos. Sperm cryopreservation is a widely used technique in assisted reproduction for different reasons. However, little is known regarding the potential effect of sperm freezing on embryo aneuploidies. Study design, size, duration Retrospective, observational and multicenter study of patients undergoing Preimplantation Genetic Testing for Aneuploidies (PGT-A) from January 2017 to December 2022 in IVIRMA Madrid, Valencia and Barcelona. To elucidate the effect of advance maternal age, 1,780 biopsied blastocysts coming from the egg donation program were analyzed. Autologous sperm samples with less than 2 million spermatozoa per milliliter and male partners with altered karyotype, abnormal spermatozoa fish or any genetic disease were excluded from the study. Participants/materials, setting, methods Embryos were cultured in regular incubators, with an atmospheric concentration of 5% O2, 6.5% CO2 and a temperature of 37ºC. Blastocyst biopsy was performed either on day 5 or day 6 of embryo development and analyzed through Next Generation Sequencing (NGS). Sperm freezing was done following manufacturer’s instructions (Sperm freeze, Origio). Continuous variables were compared with a student´s t-test and categorical variables with a Chi-square using SPSS statistical program. Main results and the role of chance Mean paternal age (44.3±5.20 years old vs 43.2±5.20 years old), sperm concentration (46.67±33.05 mill/ml vs 35.1±32.92 mill/ml) were not significantly different among fresh versus frozen sperm group (p > 0.05). A total 1780 blastocysts tested for PGT-A were included: 1267 blastocysts coming from fresh ejaculate sperm (n = 194 patients) and 513 blastocysts from frozen sperm (n = 81 patients). No statistically significant differences were found in blastocyst euploid rate between fresh versus frozen ejaculate sperm (64.5% versus 67.2%, p = 0.278 ;) respectively. Blastocyst mosaicism rate: High mosaicism (>50% of aneuploidy trophectoderm cells) and low mosaicism (30-50% of aneuploidy trophectoderm cells) was comparable between fresh versus frozen sperm (2.7% versus 4.5%; p = 0.90) and (3.9% versus 3.7%), respectively. Limitations, reasons for caution This study is based on a retrospective data. Wider implications of the findings According to our findings, there are no statistically significant differences in the blastocyst euploid and mosaicism rate between fresh and frozen ejaculate sperm. Trial registration number Not Applicable