Selecting embryos with the highest developmental potential for transfer is the key to improving clinical outcomes of IVF. Our results showed that in the vitrified-warmed SBT cycles, the clinical pregnancy and LBRs increased with the increase in D3 cell number of the transferred blastocyst. This finding suggests that D3 cell number may be an effective indicator for predicting the clinical outcome of blastocyst transfer cycles.
The cell cycle duration is approximately 10–12 h [11]. By D3, ‘normal’ embryos are assumed to reach 7–9 cells, have the highest proportion of chromosomal euploidy, and exhibit the best clinical implantation potential [10]. However, a low number of cells in the embryo (< 8 cells) generally results in a prolonged cell cycle, developmental arrest, and cellular debris. Therefore, such embryos have reduced clinical implantation potential [12]. In this study, the high-quality blastocyst formation, clinical pregnancy, and LBRs were lower for the 3–7 cell embryo group than for those of the other three groups, consistent with previous research results [9, 12].
The effect of the ‘accelerated’ embryos on clinical outcomes has been controversial. Some studies reported that D3 embryos with higher cell numbers (> 8 cells) have poorer clinical outcomes [13, 14]. In 2015, Kroener et al. [7] reported a significant increase in the rate of chromosomal aneuploidy in embryos with D3 cell numbers > 9. However, their study had certain limitations; for instance, embryo biopsies were performed on D3 rather than on D5. The proportion of chromosomal chimerism is high in D3 embryos and they undergo self-repair mechanisms during blastocyst formation. Therefore, the chromosomal status of D3 cleavage-stage embryos is not equivalent to the chromosomal status after blastocyst formation. In addition, for patients < 35 years old, a D3 cell number > 13 was associated with a relatively low aneuploidy rate in this study [7].
A recent study retrospectively analyzed the data of 3543 patients who underwent vitrified-thawed SBT and reported comparable clinical pregnancy rate and LBR between the > 8 cell group and 8 cell group [9]. However, the overall development rate of the embryos was low; on D3, 60.7% of embryos had ≤ 6 cells, and only 3.33% of embryos had > 8 cells. Therefore, studies with large sample sizes are needed to confirm these findings.
‘Accelerated’ embryos reportedly have a high potential for successful clinical implantation. For instance, Kong et al. collected data from 799 embryos cultured in a time-lapse incubator and compared the effects of cell number, developmental time, and division behavior on embryonic developmental potential [12]. Their results showed that for embryos with normal cleavage patterns, developmental potential, implantation, and LBRs increased with cell number. Moreover, Tian et al. [15] found that the LBRs were significantly higher in patients transplanted with 10-cell embryos (OR 1.62, 95% CI 1.03–2.53; P = 0.035) and ≥ 11-cell embryos (OR 2.14, 95% CI 1.47–3.11; P < 0.001) than in those transplanted with 8-cell embryos. These results are consistent with those of our study.
A high development rate indicates a strong growth ability of an embryo. Such embryos can usually form blastocysts at stage 4 and above on day 5, and their ICM and TE cell number are relatively high. The morphological score of these blastocysts is similar to or even better than that of embryos with a moderate cleavage rate [6]. However, most centers still prefer blastocysts with 7–9 cells on D3 when selecting blastocysts for transfer, which may result in a potential waste of embryos and possibly prolong the time to pregnancy (TTP).
Moreover, abnormal cleavage behavior of direct division may increase the number of blastomeres. However, such chromosomally abnormal embryos often cannot undergo normal activation of the embryonic genome; thus, they cannot continue to develop to compaction and blastocyst stages [16]. Since the size of the fragments and blastomeres is similar on D3, embryologists may misjudge the fragments as blastomeres, thereby incorrectly identifying these embryos as ‘accelerated’ embryos. However, expectedly, the probability of these embryos developing into usable blastocysts is relatively low. Therefore, most embryos with direct division or a high degree of fragmentation can be excluded using the blastocyst culture technology.
Additionally, differences in culture conditions may influence embryo cleavage and affect embryonic metabolic activity. Kasterstein et al. showed that the cell number of embryos incubated under 5% O2 exceeded that of those grown under 20% O2 conditions [17]. Moreover, culture conditions and male factors can affect the duration of the synthetic phase (S phase) and ooplasmic maturity [18, 19]. Besides, IVF centers worldwide use various culture media and environments, which may account for the inconsistent conclusions of various studies conducted so far.
Setting the inclusion criteria in this study corrected bias as much as possible to reduce the interference of confounding factors. However, the limitations of our study include its retrospective design and the data source being limited to the Department of Reproductive Medicine, Women and Children’s Hospital, affiliated with Xiamen University. Therefore, our findings should be verified in a future prospective multicenter clinical study.
In conclusion, during the vitrified-warmed SBT cycle, the LBR and clinical pregnancy rate increased with an increase in cell number on D3. The clinical outcome of blastocysts formed from embryos with > 9 cells on D3 was better than that of blastocysts formed from embryos with 8–9 cells on D3. Therefore, in cases with the same blastocyst morphological score, those derived from > 9 cells on D3 can be preferentially selected for transplantation to reduce the TTP considerably. The findings of this study provide insights to optimize blastocyst grading and provide evidence for improving the reference indicator used for blastocyst selection.