Synchrony of the first division as an index of the blastocyst formation rate during embryonic development

Abstract Purpose To devise an uninvasive selection system for human embryos with high developmental potential after a single oocyte retrieval cycle by comparing the in vitro and in vivo effectiveness of first division synchrony against subsequent embryonic developmental stages. Methods The effects of using assisted reproductive technology on 948 embryos that were produced in 137 cycles were examined by dividing the embryos into “early cleavage” (first division within 25.90 hours) and “late cleavage” (first division at or after 25.90 hours) groups and comparing the blastocysts and good‐quality blastocyst formation rates between the two groups. These two groups were each divided further into “high synchrony” (first division synchrony within 3.96 hours) and “low synchrony” (first division synchrony at or after 3.96 hours) groups. The blastocysts, good‐quality blastocyst formation rates, and pregnancy rates were compared among these four groups. Results Both the blastocysts and good‐quality blastocyst formation rates were significantly higher in the early‐cleavage groups than in the late‐cleavage groups. The blastocyst formation rate of the latter was also significantly increased in the high‐synchrony, compared with the low‐synchrony, group. Conclusion First division synchrony in a single oocyte retrieval cycle could be a useful assessment of the blastocyst formation rate that enables the selection of viable embryos at an early stage of culture.

Using this instrument, embryos and embryonic development can be assessed and monitored at all times without removal from the incubator, eliminating the risks that are posed by external stressors, such as temperature change, light exposure, high-level oxygen exposure, and pH changes in the culture medium. In this manner, more information can be obtained by analyzing embryonic growth at various time points, rather than morphological observation at a single time point. The concurrent application of a single-step culture medium enables a continuous blastocyst culture system to be established.
Eliminating the need for the direct observation or exchange of culture medium guarantees undisturbed growth conditions and increased success of embryogenesis.
Most importantly, the continuous observation of embryo development in a time-lapse system allows the optimal selection and prioritization of the order of embryos to be transferred. Recent assisted reproductive technology (ART) includes improvements in cryopreservation techniques 7-10 that have increased the priority for the culture and transfer of blastocysts, compared with fresh embryo transfer. [11][12][13] Limiting embryo transfer to blastocysts alone is not always recommended, as low blastocyst formation rates can terminate the embryo transfer process. Therefore, the most reliable indicator of embryo quality is whether or not embryos reach the blastocyst stage.
Embryos need to be cultured for 5-6 days to reach the blastocyst stage. In early-stage embryo transfer (day 2 or 3), the selection of good embryos during the early stage of culture is crucial in order to determine the optimal culture duration and timing for embryo transfer. Previous studies have demonstrated the methods for selecting good embryos, with a focus on the division speed and morphology of early-stage embryos. 11,[14][15][16] Although the early-stage transfer of embryos that are selected by using a time-lapse system does not increase pregnancy rates, blastocyst transfer does so. 17 Thus, the use of a time-lapse system is important in order to determine whether embryos have reached the blastocyst stage at an early stage of culture.
Various studies attempting to identify the indicators of the blastocyst formation rate have found: correlations between the time from fertilization to the first division and the number of blastomeres and embryo implantation potential on day 2; 18 an association between the times of first and second division and embryo implantation potential on day 3; 19 relationships between synchrony of the second and third divisions and the rate of formation of good-quality blastocysts; 20 and correlations between synchrony of the second division and the potential of embryos to develop into blastocysts. 21 Several other studies also have found that the time until the first division is an important factor for blastocyst formation rates. [22][23][24][25][26][27] From this perspective, it was examined whether synchrony and the average time until the first division were indicators of blastocyst formation rates in order to determine the characteristics of the embryos that developed into transferable blastocysts during the early culture period.

| MATERIALS AND METHODS
All the study's participants were required to provide written informed consent and the study design was approved by the appropriate ethics review boards. Ovarian stimulation followed a short protocol that under 5% CO 2 , 5% O 2 , and 90% N 2 for a maximum of 6 days. All the oocytes were cultured in the EmbryoScope immediately after microinjection. Cryopreservation of the blastocysts was performed by established methods 9,10 by using a Cryotop ® (Kitazato, Shizuoka, Japan). The blastocysts in which both the inner cell mass and trophectoderm were graded as B or higher were regarded as "good quality," 28 whereas the blastocysts that were graded as C were discarded.

| Experimental designs
The treatment cycles in which four or more fertilized oocytes were cultured in the EmbryoScope were targeted. The time until division was measured from the point at which ICSI was performed. The study used 25.90 hours as a reference value for the definition of early-cleavage embryos in the first division, according to the authors' previous study. 17 The average time of synchrony of the first division in a single oocyte retrieval cycle was 3.96 hours for 137 cycles.

| Experiment I
The embryos were divided into two groups: those in which the first division occurred within 25.90 hours (early-cleavage group) and those in which the first division occurred at or after 25.90 hours (late-cleavage group). The blastocyst and good-quality blastocyst formation rates were compared between these two groups.

| Experiment III
Blastocysts were transferred to 92 patients for 134 cycles after the next hormone replacement therapy cycle. After thawing, a single blastocyst transfer was performed, and 3 weeks after transfer, the clinical pregnancy rates were determined by using ultrasound to detect the presence of a gestational sac.

| Statistical analysis
The statistical analysis was performed by using the chi-square test with continuity correction (experiment I) or Bonferroni corrected chisquare test with continuity correction (experiments II and III). P< .05 was considered to be statistically significant.

| RESULTS
Age, maturation, fertilization, blastocyst formation and good-quality blastocyst formation rates, and the average synchrony time are shown in Table 1.

| Experiment I
As shown in Table 2, the blastocyst formation rate in the satisfactory group (74.1%) was significantly higher (P<.01) than in the unsatisfactory group (56.1%). The good-quality blastocyst formation rate in the satisfactory group (47.5%) was also significantly higher (P<.01) than in the unsatisfactory group (26.8%).

| Experiment II
As shown in Table 3, the blastocyst formation rate in group A (75.6%) was significantly higher (P<.01) than in groups C (62.9%) and D (49.3%), while the blastocyst formation rates in groups B and C F I G U R E 1 A detailed view of synchrony observed in embryos undergoing first division (70.3 and 62.6%, respectively) were significantly higher (P<.01) than in group D (49.3%). The good-quality blastocyst formation rates in group A (49.1%) and group B (43.5%) were also significantly higher (P<.01) than in groups C (30.0%) and D (23.5%).

| Experiment III
As shown in Table 4, there was no significant difference in the pregnancy (36.0-63.6%) and miscarriage (11.1-35.7%) rates among groups.

| DISCUSSION
The blastocyst and good-quality blastocyst formation rates were high for the embryos that exhibited an early first division (before 25.90 hours). Even for the embryos that displayed a later first division, those featuring high synchrony of the first division in a single oocyte retrieval cycle had a high probability of reaching the blastocyst stage. Moreover, the embryos that displayed an early first division and shorter average time of synchrony showed the highest blastocyst formation rate of all the embryo groups that were examined in this study. Therefore, it has been hypothesized that high synchrony of the first division in a single oocyte retrieval cycle was a useful criterion for predicting the blastocyst formation rates. However, the embryos that had a late first division and shorter average time of synchrony showed lower blastocyst and good-quality blastocyst formation rates than the embryos that exhibited an early first division and either a shorter-or longer-than-average time of synchrony.
These results suggested that the first division time (25.90 hours) was a more effective indicator of blastocyst formation, compared with synchrony of the first division.

F I G U R E 2 Embryos showing synchrony of the first division. (A)
The embryos that displayed synchrony within 3.96 hours were recognized as "high synchrony" and (B) the embryos that displayed synchrony at or after 3.96 hours were recognized as "low synchrony" T A B L E 1 Embryological outcomes of intracytoplasmic sperm injection with respect to the study's parameters Previous reports have shown that early-cleavage embryos had strong developmental potential. [22][23][24][25][26] Some studies classified all embryos into "early-cleavage" and "non-early-cleavage" groups and compared them. [23][24][25][26][27] Other studies also grouped the embryos into those that ultimately resulted in pregnancy and those that did not. 22,26 In contrast, this study compared the embryos that were retrieved from a single oocyte retrieval cycle and measured the synchrony of the first division, thereby predicting the subsequent embryo development more accurately than the retardation time until first division. The stronger correlation for synchrony, rather than the retardation time until division with the blastocyst formation rate, might be attributed to the high synchrony of the first division in a single oocyte retrieval cycle and therefore a more homogeneous quality of retrieved oocytes. In the present study, cryopreserved-thawed blastocyst transfer achieved good pregnancy rates for all groups, suggesting that this was an effective transfer technique. Once the embryos reach the blastocyst stage, a certain pregnancy rate can be expected. Therefore, it is important to determine whether the embryos will reach the blastocyst stage. The synchrony of the first division, as well as the first division time, can be indicators of whether the embryos reach the blastocyst stage. In cases in which the embryos show poor synchrony of the first division and are unlikely to reach the blastocyst stage, alternatives such as early embryo transfer or the cancellation of embryo transfer should be adopted as early as possible, if cryopreservation of all the blastocysts was chosen as a treatment modality. The decision to perform an early embryo transfer must be taken by at least day 2 of culture, as a delay in starting luteal support can reduce the pregnancy rate. 32 If synchrony of the first division, which is observed on day 1 of culture, is used as an indicator of subsequent embryo development, luteal support through oral administration, injection, or transvaginal administration can be started on day 1. This enables the treatment to be changed, if necessary. This method also required a shorter observation time than those that were used in previous studies to determine whether embryos have high developmental potential.
In conclusion, synchrony of the first division in a single oocyte retrieval cycle was found to be a useful indicator of the blastocyst formation rate. In the future, the authors intend to use this method in order to determine the optimal culture duration and to effectively time the embryo transfer process.