Abstract Study question What is the technical accuracy and clinical utility of reporting small copy number variants (CNVs below 3Mb) detected by a targeted next-generation sequencing-based PGT-A platform? Summary answer Some pathogenic or likely pathogenic CNVs <3Mb can be accurately detected by this assay, increasing clinical utility of PGT-A for a subset of IVF patients. What is known already CNVs are linked to a wide range of phenotypes, spanning from syndromes that include reduced penetrance and variable expressivity to more severe phenotypes. Prenatally, the prevalence of pathogenic CNVs is approximately 1.5%. Most PGT-A platforms that rely on whole genome amplification and shallow sequencing have a resolution limit of 5-10 Mb, preventing the detection of smaller CNVs. Here, we report an innovative PGT-A assay that interrogates thousands of targeted sites in the genome to provide robust copy number analysis allowing for the identification of some small CNVs (incidental findings, IF), outside the primary scope of detecting whole-chromosome aneuploidies in PGT-A. Study design, size, duration Retrospective observational study performed between 2020-2022, involving 12,157 patients who underwent PGT-A performed by targeted-NGS (PGTseq-A) for whole chromosome and large segmental aneuploidies. If an IF < 3 Mb was detected in multiple embryos, the couple was advised to undergo follow-up analysis by chromosomal microarray (CMA) to confirm the parental origin of the CNV, define its breakpoints and determine whether it is classified as benign-likely benign (B/LB), variant of uncertain significance (VUS) or pathogenic-likely pathogenic (P/LP). Participants/materials, setting, methods The PGTseq-A assay employed amplifies 5,000 amplicons across the genome to evaluate copy number. Validation was performed using 5-cell samples from cell lines with known CNVs, and trophectoderm biopsies from embryos with known parental structural rearrangements. An IF was reported when a gain/loss of at least three consecutive amplicons appeared in at least two embryos from the same cycle. Transfer data was reviewed to determine which embryos were transferred. This study received IRB approval. Main results and the role of chance In 77 out of 12,157 PGT-A patients (0.63%;95%CI:0.5-0.8%), an IF that met reporting criteria was identified. To determine the size and pathogenicity, a CMA follow up was requested and performed on 67 couples. In all cases, one of the partners was confirmed to have the CNV identified in the embryos (100.0%: N 67/67 95%CI:94.6-100). The identified CNV was of maternal origin in 36 cases (53.7%) and of paternal origin in 31 cases (46.2%). A strong correlation was identified between PGT-A-predicted CNVs and the genomic coordinates defined by the CMA on parental DNA (r = 0.86). All CNVs intervals predicted by the PGT-A included the CMA genomic region. Twenty-seven (40.2%) were classified as B/LB, 31 (46.2%) as a VUS, and 9 (13.4%) as P/LP. Six of the nine P/LP cases (66.6%) involved imbalances of 16p. The remaining P/LP cases involved deletions of X, 15q and 18p11.32. From a review of transfer data, patients typically transferred embryos negative for IFs as first option regardless of the morphology and pathogenicity classification of CNV-positive sibling embryos. Specifically, in 8 cycles where a detected CNV was classified as B, LB or as VUS, negative embryos were prioritized for transfer despite their poorer morphology. Limitations, reasons for caution De novo CNVs were not considered by design because the reported CNVs had to be present in at least two embryos from the same cohort. Furthermore, the detection of small CNVs is not uniform in the genome and the negative predictive value of the assay for non-targeted regions is null. Wider implications of the findings This PGT-A assay accurately detects small pathogenic CNVs without prior knowledge of parental inheritance, enhancing clinical utility in a subset of patients. Additionally, reporting of VUS and B/LB findings may impact couples’ decision-making concerning embryo selection, reinforcing that scientific rigor is a necessity when evaluating the capabilities of PGT-A. Trial registration number not applicable