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Study question What are the clinical outcomes following transfer of day 7 embryos; could day 7 embryo culture become part of standard embryology practice? Summary answer Although clinical outcomes remain low, success has been seen following transfer of day 7 embryos. Extended culture plays an important role in standard embryology practice. What is known already In vitro culture conditions and cryopreservation techniques have improved greatly in recent years, allowing for extended culture, and freezing of embryos at the blastocyst stage. Optimally, embryos should develop to the blastocyst stage on day 5, however deviation from normal development can sometimes occur due to problems in either the embryonic intrinsic factors or imprinting timings. Live births have been reported from slower developing day 7 embryos. Extended culture to day 6 is standard practice in most IVF laboratories, however the aim of this study is to investigate the outcomes following prolonged embryo culture to day 7. Study design, size, duration This is a retrospective analysis study of 47 patients who underwent frozen embryo transfers (FETs) with a cryopreserved day 7 embryo at a UK licensed centre between 2017-2021. Patients were split into two groups: those transferring untested (non-PGT-A) day 7 embryos, and those transferring euploid PGT-A tested day 7 embryos. Clinical pregnancy (CPR) was analysed to study the clinical outcome of day 7 embryo culture followed by a frozen transfer. Participants/materials, setting, methods All patients who had FET with a day 7 embryo between 2017-2021 were included in this study. A total of 49 FETs (48 eSET,1 DET) involving 47 patients took place: 38 with untested embryos and 11 with euploid embryos. Clinical pregnancy rate (CPR) and live birth (LBR) was analysed to study the efficacy in transfer of day 7 embryos and the two groups of patients (untested and PGT-A tested) were compared. Main results and the role of chance The overall CPR per embryo transfer was 8% (47 patients, 49 ETs, 50 embryos transferred) and the LBR per embryo transfer was 4%. In the PGT-A group, a total of 201 day 7 embryos were biopsied over the 5-year study period; 28 were euploid following testing (14% euploidy rate). Of these, 11 have been transferred; 4 resulted in a pregnancy and 2 in a live birth. (LBR 18%) There is a significant difference in CPR between the PGT-A group (11 patients, 11 FETs; 11 embryos transferred, 4 foetal hearts seen, CPR=36%) and the untested group (36 patients, 38 ETs; 39 embryos transferred, CPR=0%) Chi square test performed and confirmed (p < 0.001). The mean age of the patient at the time of embryo transfer was 38.6 ± 0.5 and the mean age of the embryo transferred was 35.6± 0.5 (this was the patient’s age at the time of embryo freezing). Limitations, reasons for caution Limiting factors include the retrospective study design and the small sample size. It is also worth noting the high number of day 7 embryos required to undergo PGT-A testing to result in a low number of euploid embryos suitable for embryo transfer. Wider implications of the findings This study indicates the value of day 7 embryo culture as it has resulted in some patients achieving an ongoing pregnancy and a live birth. However, care must be taken to counsel patients appropriately around the chance of success when transferring day 7 embryos, particularly patients using untested embryos. Trial registration number Not Applicable

Study question The aim of this study was to evaluate the influence of blastocoele re-expansion time of warmed vitrified blastocysts on clinical pregnancy outcome. Summary answer Clinical pregnancy rate was significantly higher after transfer of warmed vitrified blastocysts that were fully expanded within 2 hours post thaw. What is known already The number of blastocysts being vitrified worldwide has increased dramatically over recent years. A combination of factors has led to this including the introduction of vitrification, an increase in freeze-all policies, single embryo transfer and an increase in preimplantation genetic testing. Currently, blastocyst re-expansion after thawing is used to indicate the survival status of the blastocyst and when combined with the morphology of blastocyst can predict its reproductive potential. While time taken for blastocoele re-expansion has been proposed to be a biomarker of viability, its value in clinical practice remains unclear. Study design, size, duration This retrospective study analysed outcomes in patients who had frozen embryo transfers between June-December 2020. 233 embryos were reviewed with time-lapse to assess their blastocoele expansion post-warming and three groups were identified. The first included fully expanded blastocysts post-warming. The second group included partially expanded blastocysts and the third non-expanded blastocysts. In addition, the groups were subcategorised into two further categories depending on whether they took less or more than 2 hours to complete expansion. Participants/materials, setting, methods 233 vitrified/warmed embryos from 216 patients were analysed using time-lapse incubators. The first group included 134 blastocysts, of which 70 were fully expanded within 2 hours and 64 after 2 hours post thaw. The second group had 70 embryos of which 45 expanded partially within 2 hours and 25 after 2 hours. The third had 28 embryos that had no expansion within the first 2 hours (n = 20) or after 2 hours (n = 8). Main results and the role of chance Blastocysts were collapsed by laser prior to vitrification. Single blastocyst transfer was performed for all patients. The mean transferred embryo age was 32.1± 5.5 and the recipient’s was 37.5± 5.9. Fully expanded blastocysts (n = 70) within 2 hours demonstrated a clinical pregnancy rate (CPR) of 57% compared with 38% from those that expanded fully after 2 hours (n = 64) (p = 0.02). Blastocysts with some form of expansion (full or partial) within 2 hours post-warming (n = 115) were associated a significantly higher CPR compared to those expanding after 2 hours (n = 89). The CPR was 55% and 39% respectively (p = 0.02). Embryos that showed no expansion (n = 20) within the first 2 hours post thaw resulted in CPR of 28%. Interestingly, embryos that showed no expansion after 2 hours resulted in no pregnancy. When combining morphology as a selection criterion, expansion within 2 hours of thawing was associated with a CPR of 62.5% for ≥4AB embryos, 50% for BB embryos and 45% for poorer embryos ≤CB.In conclusion, failure of blastocoele expansion post 2 hours reduced by half the chances of clinical pregnancy (p = 0.03). Combination of the degree of re-expansion and embryo morphology is an important predictor tool to improve clinical outcomes in frozen embryo transfers. Limitations, reasons for caution This study uses a small sample size of patients. The data are observational and were retrospectively analysed so unknown confounders could not be assessed. The addition of more cycles and further multivariate analysis, is essential for confirmation of the findings. However, initial results are very reassuring. Wider implications of the findings: The degree of speed of re-expansion post warming should be used as a predictor for prioritisation of embryos for transfer. Owing to these preliminary findings there is rationale for a larger scale study combining other morphological indicators that could further assess implantation indicators and assist patient counselling Trial registration number Not applicable