Abstract Study question Does the reproductive potential, especially live birth probability, of embryos decrease when blastocyst development takes longer than the traditionally accepted 5 and 6 days?. Summary answer The reproductive potential decreases with blastulation time. Day 7 had the worst live birth (LB), aneuploidy and miscarriage rates compared to Day 5 and 6. What is known already Ending culture on Day 6 is current practice in most clinics, it is important to elucidate whether or not this is a good practice. It has been reported that Day 7 blastocysts represent only 5% of useable blastocysts, although slower growing compared to Day 5 and 6 blastocysts.Day 7 blastocysts can be viable, they can be of top morphological grade, euploid and result in a LB. Furthermore, the effects of prolonged culture to Day 7 in regards to embryo development and their reproductive potential are not well established. Study design, size, duration Retrospective cohort study of patients from US RMA clinics attempting conception through ICSI and utilizing preimplantation genetic testing for aneuploidy screening (PGT-A) from January 2022 to January 2023. A total of 6,132 ICSI PGT-A cycles were included in the analysis; three thousand six hundred and eighty frozen single embryo transfer (SET) from those resulting ICSI cycles occurred during that period of time were included in the study. Participants/materials, setting, methods Patients that performed an ICSI cycle with autologous oocytes were included in the study. Patients with frozen oocytes, donor egg recipients, non PGT-A cases, and conventional insemination were excluded. The main outcome measures include euploidy rates, miscarriage rates and LB rates between Days 5, 6 and 7. Statistical tests were conducted through Chi-Square Test and logistic regression methods adjusted for maternal age at time of transfer and morphological quality of the blastocyst. Main results and the role of chance Euploidy rate was worse on D7 than D5 or D6, with 45.5%95%CI(43,46%-47,50%), 78.1% 95%CI(77,02%-79,13%), and 63.2%95%CI(62,42%-63,96%) respectively, p>0.0001. LB rate per ET (embryo transfer) was also worse on D7, 21.6%95%CI(15.05-28.09), than D5, 62.3%95%CI(59.73-64.95), and D6, 53.3%95%CI(51.23-55.40). We found D5 and D6 statistically significant vs D7, p<0.0001. When adjusted for potential confounders, and establishing D7 as reference, for LB AdjORD5=4.127,95%CI(2.671-6.378), and AdjORD6=3.241, 95%CI(2.145-4.898). Clinical miscarriage was 20.8%, 95%CI(9.84-31.67) on D7, and 11.2%, 95%CI(9.38-13.09) and 14.1%, 95%CI(12.41-15.75) for D5 and D6 respectively, with AdjORD5=0.478, 95%CI(0.230-0.994) and AdjORD6, 95%CI(0.619, 95%CI(0.310-1.239), being statistically different only for AdjORD5 (p=0.048). Finally, when we analyzed the quality of the inner cell mass (ICM) and the trophectoderm among Day 7 ETs, we found that blastocysts with an A quality ICM had a higher chance of LB (40%) when compared with B (25%) and C (7.1%) ICM. The adjusted OR comparing A vs C revealed significant difference (p=0.005), when A ICM is established as reference. Also, ET of blastocysts graded with an A trophectoderm had higher probability of a LB (44%) when compared with B and C (17.3% and17%, respectively). The adjusted OR, with A trophectoderm as reference, B and C trophectoderm revealed significant difference (p=0.012 and p=0.007, respectively). Limitations, reasons for caution The primary limitation is the low number of Day 7 blastocyst transfers. To determine whether Day 7 blastocysts are intrinsically worse from Day 5/6 embryos, prospective randomized studies with patients who have at least one embryo developed on day 5, 6 and 7 suitable for transfer are needed. Wider implications of the findings Expanding blastocyst culture increases the number of useable embryos per cycle and provides further opportunity of LB for patients, especially those with a few or low-quality blastocysts. This study demonstrates that Day 7 blastocysts can achieve live births and challenges the predominant practice of ending culture before Day 7. Trial registration number 2206-SFR-079-MC