Abstract Study question Can an embryo re-biopsy for a previous inconclusive result influence clinical pregnancy compared to a successful PGT-M diagnosis from the first biopsy? Summary answer The embryo re-biopsy intervention for PGT-M seems to reduce the implantation potential of a blastocyst. What is known already PGT-M allows embryo genetic profiling prior to transfer in couples with hereditary genetic disorders, as part of in vitro fertilization process. The need of a trophectoderm biopsy for the retrieval of a fragment of about 8-10 blastomeres is generally sufficient for optimal DNA amplification and molecular analysis. However, a proportion of embryos (approximately 10-12%) fails to be properly diagnosed after PGT-M testing due to inconclusive results. Therefore, as an attempt to obtain a genetic diagnosis, the embryo biopsy can be repeated on the same embryos although retesting involves a second round of biopsy and a second round of vitrification. Study design, size, duration Twenty-seven embryos from women undergoing PGT-M at an infertility center and transferred after two biopsies for genetic analysis were matched for age, fertility status, and study period, to embryos transferred after a conclusive PGT-M result after the first biopsy. The main outcome was the clinical pregnancy rate following embryo transfers in cases in whom embryos had a single biopsy, and when a re-biopsy was performed. The secondary outcome was the live birth rate. Participants/materials, setting, methods Ovarian stimulation, oocyte collection and fertilization were performed according to standard protocols. Embryos were cultured to blastocyst stage in groups of up to five, using one-step media. Blastocysts were biopsied on day 5, 6, or 7, employing either the pulling or flicking method based on their appearance. Biopsied cells were stored at -20 °C for genetic analysis. Vitrification was performed using Kitazato protocol. If results were inconclusive, a second biopsy was performed in surviving blastocysts. Main results and the role of chance The median [interquartile range] number of retrieved and injected oocytes was similar between the two groups, as well as fertilization rate and number of blastocysts obtained. No difference between the two groups was also observed pertaining to the day of embryo culture in which the first biopsy was performed. Conversely, the number of non-diagnosticated and transferable blastocysts was lower in patients whose blastocysts were re-biopsied. In 90% of the re-biopsies an informative result was obtained. Clinical pregnancy rate was 52% (95% CI: 34-69) following the transfer of a single-biopsy blastocyst and 30% (95% CI: 16-48) following the transfer of a re-biopsied blastocyst. The likelihood to have a healthy baby was 33% (95% CI: 19-52) following the transfer of a blastocyst biopsied once and 22% (95% CI: 11-41) following the transfer of a re-biopsied blastocyst. Limitations, reasons for caution The matching design, which aligns women’s age and fertility status in the two groups, allowed the assessment of pregnancy rates while avoiding potential confounding factors associated with retrospective analysis. Given that it is the first study with this design, a larger sample size is necessary to further elucidate these findings. Wider implications of the findings Despite the limited sample size, these results are of extreme clinical relevance. They offer valuable insights for guiding decisions about embryos with unclear results, contributing to the improvement of strategies for selecting those with the highest probability of pregnancy. Trial registration number not applicable