Better late than never: the clinical value of Day 7 blastocysts

The advancement of in vitro culture media has improved the efficacy of ARTs in the treatment of infertility (Chronopoulou and Harper, 2015). Despite this progress, embryos may demonstrate delayed development and reach the blastocyst stage on Day 7. The prevalence of these slow embryos has been reported as 5% and even though they are considered to be less competent and reveal a lower euploidy rate than faster growing embryos, they are still capable of achieving high implantation and clinical pregnancy rates (Hammond et al., 2018). These slow-developing embryos could have clinical value in patients with reduced chance to achieve a live birth (LB) with their own oocytes. This clinical value has been demonstrated by the increased number of patients that obtain at least one transferable embryo following preimplantation genetic testing for aneuploidy (PGT-A) with the extension of culture to Day 7 (Capalbo et al., 2014; Cimadomo et al., 2018; Tiegs et al., 2019). Studies thus far exploring the extension of culture to Day 7 lack uniformity in defining the Day 7 embryo, as well as providing context toward the parameters for proceeding with extended culture, culture conditions, local regulations and clinical setting. The October edition of the ESHRE Journal Club (#ESHREjc) discussed an observational study by Cimadomo et al. (2022b), in which 1966 blastocysts obtained from 681 patients were cultured to the full expanded blastocyst stage up to Day 7 and before undergoing embryo biopsy and PGT-A. The study defined Day 7 based on hours post insemination (h.p.i.) and assessed embryo quality using Gardner’s criteria, confirming with artificial intelligence (AI)-powered software. Euploidy, miscarriage and live birth rates (LBRs) were also assessed per vitrified-warmed euploid single blastocyst transfer for different groups based on the time of biopsy (from <120 h.p.i. and at 12 h intervals up to >168 h.p.i.). The #ESHREjc discussion focused on the definition, utilization and safety of Day 7 embryos and their grading/ranking using AI software. The Twitter event hosted 27 participants along with experts Nick Macklon, Aïda Pujol, Antonio Capalbo, Marcos Meseguer and author Danilo Cimadomo. The discussion resulted in 882 tweets and ∼1.3 M impressions generated over a 24-h period. Figure 1 summarizes the key points discussed. Graphical summary of the #ESHREjc discussion. Cimadomo et al., reported the use of ICSI, continuous culture with Day 5 media refresh, trophectoderm (TE) biopsy without assisted hatching and PGT-A for all cases. These lab procedures allowed them to define Day 7 embryos as those reaching the blastocyst full expansion stage at a specific time-lapse imaging observational timing reported in h.p.i. Since there is not yet an internationally standardized definition for a Day 7 blastocyst, the Journal Club participants agreed with the authors on their definition and the use of the time point 144 h.p.i. to characterize Day 6 embryos as the threshold to which Day 7 embryos can be defined. However, participants also acknowledged that clinical and laboratory disparities could result to a few hour differences in different clinical settings. The study was conducted in Italy, where Law 40/2004 imposes the clinical use of all viable embryos produced. The Journal Club discussion continued with a question on whether there is any value behind extended culture over performing another cycle in countries where there are no stringent policies around the use of all viable embryos. Journal club participants agreed that, even though we cannot generalize for all patients, poor prognosis patients such as advanced maternal age and those with less than three embryos would benefit more in terms of cumulative live birth rate (cLBR) increase per intention to treat. Furthermore, patients that cannot consider donor cycles and those that experience financial difficulties could also benefit from the extended culture. The emotional impact of each failed cycle should also be considered as IVF failure can result in subsequent psychological distress (Pasch et al., 2012). Furthermore, it was highlighted that patient preference to optimize the chances of one cycle instead of undergoing additional cycles should also be considered. With regard to the optimal window to transfer slow developing embryos, Journal Club experts, explained that slower blastocysts may breach the epithelium beyond the ‘window of selectivity’ which means that the first maternal check point for quality would fail (Teklenburg et al., 2010). Transferring slow-developing embryos in a fresh cycle could therefore lead to higher miscarriage rate of endometrial origin. Journal club participants agreed that slow-developing blastocysts should not be transferred fresh, but after vitrification and frozen embryo transfer (FET) on a synchronous endometrium. Even further, Journal Club experts pointed out that despite slow-developing blastocysts taking longer to reach the stage of initiating implantation, the number of days of exposure to progesterone does not need to be adjusted in subsequent FET cycles. Therefore, Day 7 blastocysts should be considered as Day 5 embryos in regard to the planning of the FET cycle in order to optimize synchronization with the window of implantation of the endometrium. Since Cimadomo et al., claimed that euploidy is imputable to quality and age, but not to a slower development, another question focused on the factors that could be contributing to the higher rate of aneuploidy seen in Day 7 blastocysts in this study. Journal Club experts explained that, to the best of our knowledge, meiotic aneuploidies are just mildly associated with a developmental arrest in the preimplantation period and certainly do not prevent blastulation. Nonetheless, aneuploidies have been associated with a higher prevalence of mild defects such as slower development, poorer morphological quality and more frequent spontaneous collapses (Bamford et al., 2022). In the future, it would be interesting to investigate whether, which and to what extent certain hallmarks of embryo development might be impaired by chromosomal aneuploidies. Cimadomo et al., reported that miscarriage was not statistically associated with slow development after adjusting for euploidy in their study. However, miscarriage rate was as high as 40% when biopsy was performed beyond 144 h.p.i. (compared to 13% in the control at <120 h.p.i.). In terms of the clinical value of these findings, Journal Club participants and experts agreed that these correlations need to be taken with caution because of the retrospective observational origin of the study. It was highlighted that the sample size beyond 144 h.p.i. is small for robust conclusions, simply because few patients had transfers of euploid blastocysts in this group. In clinical practice and workflow, slow-developing blastocysts are considered the last option and are therefore transferred in those women with no other available euploid embryos. This practice can introduce a selection bias toward poor prognosis patients receiving slow-developing embryos for transfer, who perhaps failed to get pregnant with faster and better quality euploid blastocysts. Furthermore, euploid blastocysts beyond 144 h.p.i. only account for a minority of embryos. A larger dataset will thus be required to corroborate these data. Overall, participants and experts agreed that retrospective associations do not provide evidence of causation or estimation of the clinical gain. To understand if and to what extent extended culture could impact embryo selection in clinical practice, randomized control trials (RCTs) would need to be conducted where slow developing euploid embryos are not de-prioritized for transfer. In certain countries, including Italy, extended embryo culture is used, as the law mandates the use of all viable embryos for clinical purposes. While many other clinics are not subject to laws or regulations like those in Italy, they continue to culture embryos beyond Day 5, particularly for couples with poor prognosis cycles and those who are unable to consider donor cycles. With this approach, LBR per embryo transfer (ET) might decrease because the embryos are less competent, but more patients would have embryos available for ET and therefore an increase in the cLBR might be expected. From experience in centers that utilize extended culture, it was noted that not all patients have extended culture and it often involves only a limited extension of the time of culture. Therefore, not much disruption of the laboratory workflow is anticipated from a cost-benefit perspective when employing extended culture. However, data and knowledge are both lacking regarding the safety of extended culture systems. In the early stages of embryonic development, the external environment and in vitro culture conditions can have profound effects (Behr and Wang, 2004; Duranthon and Chavatte-Palmer, 2018). Culture conditions can have an impact on the epigenome, gene expression and embryo metabolism. These effects for instance can have a variety of effects such as increasing chances of monozygotic twinning and influencing fetal growth and birth weight (Cassuto et al., 2003; Behr and Wang, 2004; Nelissen et al., 2013; Spangmose et al., 2020). The incubator environment has also been shown to impact embryo metabolism with implications for embryos in prolonged culture and for the potential importance of changing the culture medium (Kermack et al., 2022). The number and timeframes of changing the culture medium in extended culture can therefore be of added importance and requires validation. Concerning obstetric and perinatal risks on children conceived after fresh blastocyst transfer, a Danish study found an increased risk of placental and perinatal complications when compared to children born after fresh cleavage-stage ETs or spontaneous conception (Spangmose et al., 2020). If part of this effect could be due to the extended culture, it might be assumed that culturing to Day 7 could worsen these effects. So far there has been limited evidence for safety for extended embryo culture. Day 7 blastocysts have been shown to result in healthy LBs with comparable perinatal outcomes to the ones obtained by transferring Days 5 and 6 blastocysts (Hiraoka et al., 2009). Similar perinatal outcomes were also reported in a more recent retrospective study that examined perinatal outcomes from Day 7 vitrified-warmed blastocyst transfer cycles (Huang et al., 2020). The study by Huang et al., showed a higher risk of very large for gestational age of Day 7 blastocysts when compared with Day 3 cleavage-stage embryos, while blastocysts with diverse growth rates demonstrated similar developmental viability regardless of the day of vitrification. Having in mind the complexity in terms of the number of confounding variables with known effects on obstetric outcomes such as the type of embryo (fresh vs frozen), stimulation protocol, fertilization method (IVF vs ICSI), cause of infertility, maternal and paternal age, co-morbidities, adjacent treatments, and type of pregnancy (single vs twin/multiple), execution of an RCT becomes a mountainous task (Kondapalli and Perales-Puchalt, 2013). Regarding the plausible biological factors undermining delayed attainment of blastocyst stage in good quality embryos, several factors have been attributed (Hammond et al., 2018). An investigation of the different embryological events such as the first cleavage, genome activation and morula formation in blastocysts with slow development will shed light on factors affecting the speed of development. Potential issues with maternal to zygotic transition could for example lead to a delayed formation of inner cell mass (ICM) and TE. Journal club participants indicated that there could be some embryos more sensitive to culture conditions that delay their development when the environment is sub-par or maybe there is a correction machinery in vivo that we still don’t know. Omics will also be useful to attain information regarding this ‘black box’ of embryo development. ESHREjc participants agreed that this is just the beginning of threading the needle of slow-developing blastocysts. The factors affecting blastocyst expansion dynamics are not fully elucidated. Time lapse (TL) monitoring systems are well suited to describe these dynamic features of blastocyst expansion. Since the first report of a successful pregnancy following blastocyst selection by TL monitoring (Pribenszky et al., 2010), several studies have provided useful data in terms of blastocyst features useful for selection algorithms (Marcos et al., 2015; Huang et al., 2016). For instance, it has been shown that expansion may be relatively restricted in embryos blastulating at relatively later times (Huang et al., 2016). The feature of time of blastulation has since been integrated by AI to rank embryos, with embryos blastulating late having a lower score and diminished clinical outcomes (Bori et al., 2022). One of the features monitored by TL systems, blastocyst collapse, has also been identified as an embryo marker of low implantation potential (Sciorio et al., 2020). In particular, it was shown that human blastocysts that collapse spontaneously during development are less likely to implant and generate a clinical pregnancy compared with embryos that do not. This was also observed in a recent study, where collapsing blastocysts were reported less euploid even when adjusting for morphological quality (Cimadomo et al., 2022a). As Journal Club experts pointed out, blastocyst collapse is one of those features that should be implemented by all TL users, as morphology assessment alone may limit implantation potential for embryos exhibiting a strong collapse. It is therefore reasonable to consider that expansion and spontaneous collapse dynamics are indicative of reproductive fitness. The question, however, remains on what the clinical gain is from identifying these features. Prospective trials would be valuable to investigate if any of these features result in improvement of cycle outcomes. Even though Day 7 embryos are less competent than faster-growing ones, Cimadomo et al., demonstrated that ending embryo culture on Day 6 would result in a 4.4% relative reduction in LBR. Since safety studies for extended culture systems are generally lacking, the choice of extending culture to Day 7 should be evaluated individually for each couple based on their financial and emotional status along with their prognosis and reproductive history in mind. Future advances in AI can potentially identify slow-developing embryos with the ability to reach the full expansion stage. No datasets were generated or analyzed in the current manuscript. The authors would like to thank Danilo Cimadomo and all the participants of ESHRE journal club on Twitter for their contribution in the discussion. This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. All authors declare no conflict of interest.