STUDY QUESTION:Are the fetal outcomes of singleton pregnancies that result from cleavage stage embryo transfer (ET) different from the outcomes from Day 5/6 blastocyst stage ET? SUMMARY ANSWER:There was a significantly higher risk of preterm birth (<37 weeks) in singletons after extended embryo culture (Day 5/6) compared with cleavage stage (Day 3) transfer. WHAT IS KNOWN ALREADY:Two recent studies, from Sweden and the USA, reported an increased risk of preterm birth in singleton pregnancies after Day 5/6 ET compared with Day 3 ET. The US study also showed increased early preterm births and the Swedish study showed increased fetal malformations in this group. STUDY DESIGN, SIZE AND DURATION:A retrospective cohort study was performed. Data were collected from the Canadian ART Register database for all singleton births after fresh IVF/ICSI ET cycles (2001-2009). PARTICIPANTS/MATERIALS, SETTING, METHODS:A total of 12 712 singleton births were included. Of these, 9506 resulted from a Day 3 ET and 3206 resulted from a blastocyst (Day 5/6) ET. MAIN RESULTS AND THE ROLE OF CHANCE:Preterm birth rate <37 weeks (unadjusted by potential confounding factors) was higher with Day 5/6 versus Day 3 transfers (17.2 versus 14.1%, P < 0.001). Using logistic regression analysis to adjust for confounding factors, preterm birth rate <37 weeks was the only outcome significantly increased after Day 5/6 compared with Day 3 transfer (odds ratio 1.32, 95% confidence interval 1.17-1.49). The following confounding factors were adjusted for: year of treatment (2001-2009), maternal age (continuous), parity (0 versus ≥1 birth), diagnosis category, number of oocytes retrieved [≤20 versus >20 (high responder group)], insemination method (IVF versus ICSI), number of embryos transferred (1, 2 or ≥3) and the presence of a vanishing twin (≥1 fetal heart on the initial ultrasonographic examination). LIMITATIONS, REASONS FOR CAUTION:Post-natal follow-up studies will be required to determine if this difference we observed translates into adverse long-term effects on these offspring. The rate of early preterm births (<32 weeks) was higher in Day 5/6 versus Day 3, but the low number of cases in this category did not have the power to show a difference (3.0 versus 2.7%, P = 0.34). WIDER IMPLICATIONS OF THE FINDINGS:We found a significantly higher risk of preterm birth (<37 weeks) in singletons after extended embryo culture (Day 5/6) compared with cleavage stage (Day 3) transfer, even when adjusting for confounding factors. Our findings are in agreement with the previous two studies; however, we did not show a difference in the very preterm deliveries (unlike the US study) or in fetal malformations (as in the Swedish study). We hypothesize that there may be a deleterious effect of prolonged in vitro embryo culture on subsequent placentation. Longer term follow-up studies will be required to determine if prolonged in vitro culture to the blastocyst stage has an adverse effect on the long-term health of offspring when compared with shorter cleavage stage culture. STUDY FUNDING/COMPETING INTEREST(S):None.