The results of our study demonstrate that different post-thaw culture durations had similar clinical outcomes on blastocyst development on D5. However, the IR, CPR, and LBR after a short period of warming (2 to 6 h) were statistically higher than those after a long period of warming (18 to 20 h) in frozen-vitrified D6 embryo transfer cycles. In terms of LBR, no difference was noticed between three different short culture intervals (2 h, 4 h, or 6 h).
Currently, the subject of which post-thaw culture duration is best continues to be debated. In 2003, one retrospective study compared the effects of a short or a long period after warming (4 h vs. 20 h) on the clinical outcomes of frozen blastocyst transfer cycles [11]. In contrast to our study, a longer culture interval increased the CPR three-fold (27.0 vs. 8.0%) and the IR four-fold (23.4 vs. 6.1%) after a 20-h incubation period compared to a 4-h incubation period. However, applying the results of this study should be interpreted with caution, as there were some major flaws. In addition, a study which involved frozen embryo transfer cycles of D5, D6, and D7 blastocysts showed that pregnancy outcomes were comparable for blastocysts thawed and cultured overnight 1 day before transfer and those thawed and transferred on the same day [8]. In another study, Hwang et al. [15] enrolled patients having both vitrified D5 and D6 blastocysts for transfer and demonstrated that a 2 to 4 h culture interval yielded similar IR, CPR, AR, and LBR with a 20 to 24 h culture interval. These two studies collectively examined blastocysts from two different embryo developmental stages, neglecting the fact that D5 and D6 embryos may have metabolic or epigenetic differences, which can lead to some other conclusions [19]. Here, we found that D5 blastocysts could be transferred at any culture duration, with a preference for a short culture duration in D6 blastocyst transfer cycles. The discrepancy between the two previously published studies [8, 15] and our study may be related to the inclusion of embryos at different developmental stages. For instance, Hwang and colleagues reported that the majority (~ 80%) of enrolled cycles were D5 transfer cycles, with the remaining enrolled cycles consisting of D6 embryo transfer cycles [15].
Although the molecular mechanisms associated with our findings, namely that the transfer of D6 blastocysts after short-term culture is associated with better pregnancy outcomes compared to long-term culture, are unclear, they may be explained as follows. The synchronization of the embryo and the endometrium is paramount for successful clinical pregnancy, although it is important to note that the optimized embryo-endometrial synchrony is often vague and obscure. Blastocyst transfers are normally performed on the sixth day of P administration [20]. For blastocysts developed on D6, an additional 24 h culture period is present before vitrification compared with D5 blastocysts. If embryo thawing is scheduled one day before transfer, another 16 to 24 h culture period will ensue, which might contribute to asynchrony between the embryo and the endometrium, leading to a decreased IR and a higher AR. Moreover, many events occur between thawing and hatching, such as blastocyst expansion, blastocoel collapse, zona pellucida thinning, and zona escape, which collectively can affect embryo development [21]. The prolonged culture time after warming can increase the degree of blastocoel expansion, indicating that a higher proportion of embryos can develop to stage 5 or 6 [13, 15]. Marcos et al. [21] utilized a time-lapse system and observed that human blastocysts reaching stage 5 could experience one or more collapse-expansions of the blastocoel cavity. Their findings revealed that the collapse of blastocysts adversely affected the IR; the IRs for patients with collapsed blastocysts compared to those without collapsed embryos were 48.5% and 35.1%, respectively. The collapse-expansion phenomenon was further confirmed in subsequent publications [22, 23]. Single and multiple collapses occurred in 22% (61/277) and 24% (66/277) of the examined blastocysts, respectively, and the live birth rate was significantly lower if multiple collapses occurred [22]. Meanwhile, blastocysts with spontaneous collapse in vitro were less likely to implant compared to embryos without collapse [23]. In our D6 embryo transfer cycles, 16.6% (130/781) of blastocysts reached the hatching or hatched stage before transfer in the short culture group compared to 82.1% (400/487) of blastocysts in the long culture group. Based on the available evidence, the incidence of collapse-expansion and multiple collapses is expected to be much higher after subjecting slowly developed D6 embryos to a longer post-thaw culture. We, therefore, postulate that poor transfer outcomes for D6 blastocyst transfers might be due to more mechanical stress and excessive energy consumption with regards to the repeated collapse-expansion events [21].
In addition to investigate that the short post-thaw duration results better pregnancy outcomes for D6 blastocysts, the best specific interval remains a debatable subject that would be meaningful for the laboratory practice. There were three short post-thaw culture durations of 2 h, 4 h, and 6 h in our study. Multivariable logistic regression analysis revealed that the LBR did not differ, regardless of the culture duration. A review of the literature shows no clear agreement on the time of blastocele re-expansion after warming. In a study by Ahlstrom et al. [3], most of the blastocysts were assessed for re-expansion 1 to 5 h after warming, and the results indicated that the time had little contribution on the re-expansion degree once an interval of 2 h was reached. Hwang et al. [15] also reported that 90% of blastocysts completed re-expansion within an average of 1.4 to 3.5 h after warming. A longer in vitro culture protocol might increase metabolic or mechanical stress based on the already compromised D6 blastocyst viability. Compared with the 2-h culture group, the 6-h culture group showed no detrimental effects on the LBR (aOR 1.01, 95% CI 0.68-1.49, P = 0.974), indicating that a culture period up to 6 h could still reliably ascertain embryo viability and developmental competency. Nonetheless, the sample size of this subgroup was relatively small for effective statistical analysis. Thus, further investigation in a large-scale clinical trial is warranted.
No statistically significant reduction in the LBR after long-term culture was observed for blastocysts vitrified on D5. Since the number of good-quality transferred embryos was equally distributed between the two groups, our results were in line with a recent study that enrolled 162 blastocysts transfer cycles with only good-quality D5 blastocysts [13]. The authors investigated whether the post-thaw culture duration (1 h vs. 18 h) could influence the FET results. Their prospective randomized study demonstrated that the IR, CPR, and AR were similar, regardless of the post-thaw culture duration. As such, both warming protocols can be applied to patients with good-quality D5 blastocysts.
To date, several studies have suggested that there is a correlation between blastocyst developmental competence and a higher degree of blastocele re-expansion after thawing [10, 13, 15]. It is understandable, compared with the short duration group, that the long culture group showed a better ability to promote full expansion of blastoceles. As shown in a prospective study [13], the group with a post-thaw interval of 16 to 22 h had a higher proportion of B5/6 grade at the time of transfer compared with an interval of 0.5 to 5 h (38.6 vs. 12.7%, P < 0.001). Additionally, B5/B6 grade blastocysts in the long culture protocol demonstrated a significantly higher IR than B4 grade blastocysts in the short culture protocol (52.9 vs. 32.9%, P = 0.048). Consequently, the implantation potential of blastocysts cannot be always evaluated after a short post-thaw culture interval. Maezawa et al. [24] applied time-lapse imaging and revealed that blastocysts that failed to expand 5 h after thawing could not develop to the hatched stage and 25% of blastocysts remained shrunken after 6 h. Thus, an overnight culture protocol in D5 blastocyst transfer cycles can not only select embryos with re-expansion, but also maintain their developmental ability until hatching. Currently, data have shown the priority of transferring D5 blastocysts to D6 blastocysts both in fresh [25, 26] and frozen [2, 27] embryo transfer cycles. The underlying reasons can be partly explained by better embryo quality [2], decreased abnormal spindle rate [28], and low aneuploidy incidence [29, 30]. All these reasons lead to the fact that blastocysts developed on D5 might have higher quality and improved implantation potential than D6 embryos to cope well with extended warming period. To conclude, the abovementioned reasons indicate that a long post-thaw culture period is harmless to the clinical outcomes of D5 blastocyst transfer cycles compared with a short post-thaw culture period.
To the best of our knowledge, the current study is the first to use a larger sample size to examine the effects of post-thaw culture duration on frozen blastocyst transfer outcomes involving both D5 and D6 embryos. A major strength of this study was that we evaluated both vitrified/warmed D5 and D6 blastocyst transfer cycles and observed that post-thaw culture duration plays an important role in predicting the LBR for D6 blastocysts. Our study also firstly examined frozen D6 blastocyst transfer outcomes from the viewpoint of three short culture strategies after warming. Data showed that LBR had a similar trend after a shorter culture regimen in D6 blastocyst transfer cycles, regardless of the post-thaw duration. Another strength was that PSM analysis was used to control for potential covariates, thereby ensuring that the outcomes were independent of the different patient characteristics in some way. Finally, our study was conducted in the past 1.5 years, which means that the laboratory conditions, culture media, and embryo selection criteria remained consistent. Given the undisputed high performance of vitrification compared with slow cooling protocols, the need of a longer culture period to allow for survival and additional morphology evaluation of thawed blastocysts seems non-existent. Some may argue that it is a common procedure to culture D6 blastocysts for a shorter post-thaw interval; however, there is a lack of consensus in the previous literature, and some laboratories continue to culture thawed D6 blastocysts for many hours for logistic reasons. Thus, we believe our study is still clinically relevant and worthy of interest.
Despite our efforts, there were some shortcomings in this study. Although PSM analysis was applied to match the confounders, this was a retrospective study and selection bias could not be excluded. Our results revealed that transferring D6 blastocysts after a short culture interval after thawing yielded better FET outcomes; however, we could not determine which specific interval was the best. A prospective randomized control study with a larger sample size is needed to confirm our results.