Selection of high‐quality and viable blastocysts based on timing of morula compaction and blastocyst formation

Abstract Purpose The time‐lapse system is a device that allows continuous monitoring without removing embryos from the incubator. Using a time‐lapse system, we retrospectively investigated cleavage speed time points as potential indicators for selecting high‐quality viable blastocysts. Methods This study included 963 zygotes of two pronuclei retrieved from 196 patients between January 2015 and December 2016. All embryos in culture were monitored by time‐lapse after intracytoplasmic sperm injection. Of 492 blastocysts developed in vitro, 128 vitrified‐warmed single blastocyst transfers were classified into pregnancy and non‐pregnancy groups, and the parameters were compared. Results In the pregnancy group, timing of both morula compaction and regular blastocyst formation was significantly faster than in the non‐pregnancy group. Furthermore, the optimal cutoff values for compacted morula (94.9 hours) and regular blastocyst (113.9 hours) were determined using the receiver operator characteristic curve analysis. Embryos that formed compacted morulae within 94.9 hours and developed into regular blastocysts within 113.9 hours were associated with a significantly higher pregnancy rate than those that did not (44.4% vs 16.0%). Conclusion The timing of morula compaction and regular blastocyst formation is important as an indicator of high‐quality blastocysts to increase odds for pregnancy after embryo transfer.


| INTRODUC TI ON
In many in vitro fertilization (IVF) clinics, blastocyst transfers have been performed that involve selection of high-quality embryos, leading to improved implantation rates. In the past, with assisted reproductive technology (ART), pregnancies with multiples have been a serious problem resulting from transfer of several embryos to the uterus, 1,2 but single blastocyst transfer reduces this risk dramatically. Obtaining blastocysts for embryo transfer requires that embryos be cultured for 5-6 days in vitro. 3 If multiple blastocysts are obtained from in vitro culture, high-quality blastocysts must be selected for the best odds of continued embryo fetal development.
Widely used indicators for evaluating blastocyst quality based on developmental stage are the scale of the inner cell mass (ICM) and trophectoderm (TE) cell number. 4 These are morphological evaluations, however, and carry the risk of subjectivity. Objective indicators are needed for better predictive criteria for selecting high-quality blastocysts in ART. In recent years, the usefulness of a time-lapse observation system during embryonic development has been reported. [5][6][7] The time-lapse system allows for monitoring of embryo development at all times without removal of the culture dish from the incubator. Using a time-lapse system, the morphokinetics of embryos and the division speed both can be measured. This approach thus enables the noninvasive objective assessment of high potential embryos and provides a tool for predicting embryo development and implantation.
Many reports have demonstrated the usefulness of time-lapse, but several of these studies have predicted embryo development in early cleavage, [8][9][10][11] with few reports addressing blastocyst selection from in vitro culture. 12 In the embryonic developmental process, compaction of the embryo has an important influence on blastocyst and ICM formation and TE differentiation. The relationship between the precise timing of compaction and blastocyst formation and their mechanisms is not clear. In this study, by targeting embryos fertilized by intracytoplasmic sperm injection (ICSI) for which the sperm injection time is clear, we predicted that the time from cleavage to embryo development could be measured accurately. The purpose of this study was to establish an objective indicator of high-quality blastocysts using time-lapse monitoring. We retrospectively evaluated the relationship between compaction and blastocyst formation timing based on comparison of data from pregnancy and non-pregnancy groups.

| Patients
This study used two pronuclei from each of 963 zygotes fertilized by ICSI, obtained from 194 patients from January 2015 to December 2016. The age (mean ± standard deviation) of patients at the oocyte retrieval cycle was 38.3 ± 4.3 years (range: 26-49). All embryos were monitored by a time-lapse system (Primo Vision; Vitrolife). In addition, we examined the outcome of 128 cycles of vitrified-warmed single blastocyst transfer obtained from the same cycles. This retrospective study was approved by the Ethics Committee of Kinutani Women's Clinic, Hiroshima, Japan.

| Stimulation protocols and oocyte retrieval
Women were treated with GnRH agonist using either a short protocol or long protocol and with GnRH antagonist, according to each patient's ovarian response and medical history of IVF treatment. In some cycles, the minimal stimulation protocol was  Only MII oocytes were used for ICSI.

| In vitro culture of human embryos and analysis of time-lapse imaging
Embryos fertilized by ICSI were cultured in WOW dishes (Vitrolife) with SAGE 1 Step (Origio) or continuous single culture medium

| Blastocyst vitrification, warming, and single embryo transfers
Blastocyst vitrification was done using the method by Hiraoka et al 13 The blastocysts were placed in equilibration solution contain-

| Statistical analysis
Pregnancy, abortion, and live birth rates were compared using the chi-square test. Because the time-lapse data were not normally distributed, we used the nonparametric Mann-Whitney U test instead.
For comparison between the two groups along normal distribution, we used Student's t tests. The statistical analysis was performed using GraphPad PRISM 6.03 software (GraphPad Inc). The receiver operating characteristic (ROC) curve was created to determine the cutoff value using JMP 14.0 (SAS Institute, Inc). Significant differences were assumed to be present at P < .05.

| Comparison of developmental time between pregnant and non-pregnant groups after single blastocyst transfer
The cleavage speed of the transferred blastocyst was compared with or without pregnancy (Table 2, Figure 1C

| Clinical pregnancies and live births based on time-lapse selection criteria: morula compaction within 94.9 hours and regular blastocyst formation within 113.9 hours
Distribution of pregnancy or non-pregnancy was based on cutoff values of time from ICSI to compacted morula and time from ICSI to regular blastocyst ( Figure 2). The optimal cutoff values for compacted morula (94.9 hours) and regular blastocyst (113.9 hours) were determined from the receiver operator characteristic curve. Table 3 shows the pregnancy, abortion, and live birth rates after single blastocyst transfer. When single transfer blastocysts developed to the compacted morula stage within 94.9 hours and reached the regular blastocyst stage within 113.9 hours after culture, the clinical pregnancy rate (44.4%) was significantly higher than for blastocysts that did not meet these criteria (16.0%). Similarly, the live birth rate in the former group was significantly higher than in the group associated with blastocysts that did not meet these criteria (37.5% vs 10.7%; P < .01).

| D ISCUSS I ON
The present study showed that morula compaction and blastocyst   26 Assuming that the blastocyst leading to pregnancy was euploid, their results agree in our study.
The mechanism linking a high-quality embryo and early compaction completion is not clear. Transcription of the embryonic genome begins on day 3 and is activated on day 4 physiologically. 27,28 The intercellular adhesion protein E-cadherin is expressed during compaction, allowing cells to adhere more tightly. 29 Several studies have reported the relationship between compaction and embryo quality using time-lapse monitoring. The pattern of compaction completion time affects the embryo implantation rate 30 and starting compaction before the eight-cell stage is usually associated with aberrant embryonic development. 31 In addition, embryos that progressed in development reached the blastocyst stage early, indicating that embryos that complete compaction early reach blastocyst early, as well. Several reports have addressed the timing of blastocyst formation. In the vitrifiedwarmed cycles, results suggest that day 5 and day 6 blastocysts have similar implantation rates. 13,[32][33][34] On the other hand, delayed blastulation is associated with lower live birth rates in frozen cycles. 12,[35][36][37] The cause of delayed blastocyst formation could be the embryo itself or the result of a less-than-optimal in vitro environment that negatively influences embryonic development. Indeed, the risk of long-term in vitro culture of embryos until expansion into blastocysts is well known.
In addition, the case subject to analysis is a vitrified-warmed cycle. It has been reported that blastocyst survival rate is improved by AS. 13,38 AS was performed in the equilibration solution by glass pipetting. 13 By this method, a survival rate of 99% or more and live births has been acquired for several decades, it is possible to think that there is almost no risk of vitrified and warming.
From these results, we can conclude that the timing of morula compaction can be predictive of an embryo's potential to progress to a high-quality viable blastocyst. However, this study is a retrospective study, and a prospective study is necessary to further clarify In conclusion, the timing of the two-cell stage emerged as an indicator of the development of high-quality blastocysts. The timing of morula compaction completion and regular blastocyst development also were important for selection of high-quality viable blastocysts that progress in development and lead to live birth.

Conflict of interest:
The authors declare no conflict of interest.
Human rights statements and informed consent: All procedures were followed in accordance with ethical standards of the institu-