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Study question Is there a difference in ART cycle results after frozen embryo transfer (FET), depending on whether blastocysts were cryopreserved on day 5 or 6? Summary answer There’s no statistical difference in the clinical pregnancy rate (CPR), life birth rate (LBR), miscarriage rate (MR) between embryos frozen on day 5 and 6. What is known already Currently, opinions differ regarding this topic. Previous studies demonstrated no difference in ongoing pregnancy rates between embryos frozen on day 5 (group A) or day 6 (group B) after FET. However, metanalysis (2019) suggested higher CPR and LBR after transferring embryos from group A rather than group B. It has also been established that ovarian stimulation leads to endometrial changes that result in deleterious effects on the implantation window and endometrial receptivity. Consequently, fresh transfers were excluded. Due to hormonal priming of endometrial receptivity, the same pregnancy outcomes should be expected with frozen-thawed blastocysts (day 5 vs 6). Study design, size, duration Retrospective cohort study was conducted between January 2015 and December 2018 with selected group of patients under 40 years of age. Group A consisted of 2275 cryotransfers of blastocyst expanded on day 5; group B included 170 cryotransfers of blastocyst on day 6. Both groups had an average of 1,52 embryos transferred per patient. Participants/materials, setting, methods Embryos were vitrified and warmed with Cryotop method (Kitazato, BioPharma). Blastocysts were scored according to Gardner and Schoolcraft grading system. Only expanded on day or 6 blastocysts of excellent and good (AA, AB, BA, BB) quality were selected. The embryos were cultured in CSC medium (Irvine Scientific) for 2–4 hours prior intrauterine transfer. The cycles with donor gametes, surrogacy and preimplantation genetic testing (PGT) were excluded. Statistical validity was assessed by Pearson’s chi-squared test. Main results and the role of chance The rates of the CPR, the ongoing pregnancy rate (OPR) and the LBR between group A and B were 50,8% (1157/2275) vs 46,5% (79/170) (p = 0,26), 37,4% (852/2275) vs 37,0% (63/170) (p = 0,91), 36,5% (832/2275) vs 35,2% (60/170) (p = 0,73) respectively and no significant differences were found in each category. Moreover, similarly there were no significant differences in the miscarriage rate 26,0% (301/1157) and 21,5% (17/79) (p = 0,37) as well Limitations, reasons for caution The study is limited due to uneven distribution of patients in both groups and by a low number of participants. The grading of blastocysts’ quality is also subjected to a human factor. Wider implications of the findings: This study confirms that frozen-thawed blastocysts do not seem to exhibit a difference in the CPR, OPR, LBR and MR whether they were expanded on day 5 or day 6. The cryopreservation of day 6 blastocyst can increase the chances of the patient for the positive outcome. Trial registration number Not applicable

In order to increase the effectiveness of assisted reproductive technologies (ART) programs, it is essential to improve and develop conditions of embryo culture prior its transfer or cryopreservation of expanded blastocysts on the day 5 or 6. The aim of the study was to assess the effect of human blastocysts’ expansion timing on clinical pregnancy rate (CPR), miscarriage rate (MR) and take-home baby rate (THBR) in frozen-thawed cycles during ART programs. The study involved 2275 frozen embryo transfers (FET) of blastocysts expanded on the day 5 (group A) and 170 FET of blastocysts expanded on the day 6 (group B). The pregnancy rates in both groups were 50.8% and 46.5% respectively. There were no statistically significant differences in clinical pregnancy rate 37.4% and 37.0%, miscarriage rate 26.0% and 21.5% in both groups, respectively. THBR, as the main indicator of efficiency in the programs with transfer of post thawed expanded blastocysts on the day 5 (group A) or 6 (group B) were 36.5% and 35.2%, respectively (the difference is insignificant). In conclusion, in cryoprotocols the day of blastocyst expansion (day 5 or 6 of development) does not statistically affect PR, MR and THBR. In FET programs the quality of blastocyst (excellent and good) should be prioritized regardless of the day of cryopreservation.