Purpose <p>Embryos release cell-free DNA into spent culture medium (SCM), mostly from day 5 to day 6, making extended culture to day 6 (something done in IVF only for slow embryos) a preferred option in non-invasive preimplantation genetic testing for aneuploidy (niPGT-A). The impact of day 6 culture on embryo quality remains unclear. This study investigates whether prolonged culture to day 6 in niPGT-A improves informativity without compromising embryo quality and live birth rates (LBR).</p> Methods <p>In this retrospective cohort study, we investigate the impact of day 6 culture on embryo quality and LBR. After preliminary concordance assessment on 18 embryos, a total of 336 SCM from day six blastocysts were collected. According to the prioritisation criteria applied, 103 embryos were transferred in 91 frozen embryo transfer cycles (January 2021–February 2024). Informativity rates in day 5/6, levels of expansion/quality, female age impact and effects of prolonged culture to day 6 on embryo quality were assessed.</p> Results <p>Higher informativity rate was observed in day 6 versus day 5 blastocysts (<i>p</i> &lt; 0.001). Informativity was unaffected by different age groups (<i>p</i> = 0.22), blastocyst expansion (<i>p</i> = 0.51), blastocyst quality (<i>p</i> = 0.67), or incubator setting (<i>p</i> = 0.74). Day 6 prolonged culture did not compromise embryo viability and quality and better quality embryos had higher probability of euploidy (<i>p</i> = 0.01). We report a LBR of 33.3%, higher than the reported 10% by HFEA for a similar age group (40–42&#xa0;years).</p> Conclusions <p>Day 6 extended culture in niPGT-A improves informativity and does not compromise embryo viability and quality. LBR significantly increased compared to international benchmarks of non-tested embryos.</p>