Quality of Blastocysts Created by Embryo Splitting: A Time-Lapse
Monitoring and Chromosomal Aneuploidy Study
Omidi M, Khalili MA, Halvaei I, Montazeri F, Kalantar SM. Quality of blastocysts created by embryo splitting: a time-lapse monitoring and chromosomal aneuploidy study. Cell J. 2020; 22(3): 367-374. doi: 10.22074/cellj.2020.6717.
The aim of this study was to screen the potential of human embryos to develop into expanding blastocysts
Materials and Methods
In this experimental study, a total of 82 good quality cleavage-stage donated embryos (8-
14 cells) were used (24 embryos were cultured to the blastocyst stage as controls and 58 embryos underwent
This study showed that while no chromosomal abnormalities were seen,
Identical twins resulting from natural splitting of human
embryos are accepted by society which are comparable
with non-identical twins. Successful pregnancies
Successful pregnancy and live birth of healthy animals
as well as morphologically normal adequate human
Materials and Methods
The embryos were donated without any financial incentive. Informed consent was obtained from each couple. The Ethical Committee of our institute approved this experimental study since the embryos would not be transferred to the uterus after experimental procedures (IR.SSU.MEDICINE.REC.1395.93).
All day-2 or day-3 embryos were cryopreserved
from 2011 to 2016 by vitrification using RapidVit™
Cleave kit (Vitrolife, Sweden). Donated embryos were
warmed using RapidWarm™ Cleave kit (Vitrolife,
Sweden) according to the manufacturer’s instructions.
The warmed embryos were cultured
Embryo micromanipulation and time-lapse monitoring
The good quality 8-14-cell embryos were preincubated in 5 µL microdroplets of Ca-Mg-free
culture medium (PGD medium, Vitrolife, Sweden)
prior to biopsy and covered with mineral oil for 3
minutes at 37˚C in order to facilitate the separation of
blastomeres. A 1480 nm infrared diode laser (OCTAX
Laser Shot®, MTG, Germany) was used to open a 35-
40 μm diameter hole in the zona pellucida (ZP). Half
of the blastomeres were taken out using a micropipette
with a 30 μm inner diameter (Sunlight Medical,
Jacksonville, FL, USA) regardless of the presence or
absence of the nucleus. The biopsied blastomeres were
then inserted one by one from donor embryos (twin
A) into a previously prepared empty ZP to create the
recipient embryos (twin B). In this study, the empty
ZPs were derived from immature oocytes or discarded
embryos (14). After
The developmental stages after
Blastocyst morphology was assessed using the images acquired from the time-lapse system. At the blastocyst stage, embryo quality was assessed based on Gardner’s classification, which takes into account the expansion grade and the development of the ICM and TE (15). According to this classification, we defined three blastocyst quality classes for full and expanded blastocysts: A) good- (AA, AB, BA and BB), fair- (AC, CA, BC and CB) and poor-quality blastocysts (CC).
The diameter (in micrometers) of the expanded blastocysts was measured by EmbryoViewer. The measurements were taken on the images of the blastocysts. The diameter of each blastocyst was calculated as the average of the distance between the outside borders of the TE measured in two directions (vertical and horizontal).
Cytogenetic screening procedures Trophectoderm biopsy
Embryo biopsies were performed on a pre-warmed stage in a dish prepared with 5 µL droplets of HEPES buffered medium (G-MOPS, Vitrolife, Sweden) overlaid with pre-equilibrated mineral oil. The herniated TE cells were biopsied in the expanded blastocysts developed from A and B twins, through the previously created hole in the ZP. In the control embryos, a 10-20 µm hole was made in the ZP directly opposite the ICM of the blastocysts using a diode laser. Blastocysts were incubated for a further 4 hours to allow blastocoel expansion and herniation of the TE cells. After herniation, 5-10 TE cells were drawn into the biopsy pipette followed by laser-assisted cutting of the target cells.
The biopsied TE cells were washed in a hypotonic solution (6 mg/mL bovine serum albumin in 0.1% sodium citrate), then placed in a hypotonic solution for 3 minutes. The TE cells were then placed on a prewashed (with 100% ethanol) microscope slide. After that, an aliquot of fixative (methanol: acetic acid, 3:1) was dropped onto the specimen. Air was then blown across the sample to evaporate the fixative (16).
in situ hybridization
The biopsied TE cells were fixed on glass slides as previously described (17). FISH assays of the fixed TE cells took place using two sequential hybridizations. The first hybridization contained probes for chromosomes 13, 18, 21, and X (MetaSystems, Altlussheim, Germany) and the second round was performed using probes for chromosomes 15, 16, 22, and Y (MetaSystems, Altlussheim, Germany). The prepared slides were examined under a fluorescence microscope (Olympus BX51, GSL-10 with BX61, Japan). Classification of embryos after FISH assay results was done according to the criteria published by f Delhanty et al. (18). In this classification, the embryos were categorized into four groups: normal, abnormal non-mosaic, diploid mosaic, and abnormal mosaic.
Statistical analysis was performed using SPSS (SPSS version 20, Chicago, IL) and/or GraphPadPrism (GraphPad Software, San Diego, CA, USA). The quantitative and qualitative data were presented as mean ± SD and percentages, respectively. The Shapiro-Wilk test was applied to evaluate the normal distribution of data. t test was used for independent samples and one-way ANOVA (followed by Tukey’s test) as parametric and Mann-Whitney U and Kruskal-Wallis as nonparametric were used tests wherever appropriate. The chi-squared test was applied for comparison between qualitative data. P<0.05 was considered as significant.
Developmental potential to expanding blastocyst is unaffected following embryo splitting
After warming, there were 82 good quality cleavagestage embryos. Among these, 58 embryos were split into two groups: group 1 (n=37), including embryos with 8- 9 blastomeres; and group 2 (n=21), including embryos with 10-14 blastomeres. The remaining 24 embryos in the same condition were used as the controls. In general, from 116 resulting twin embryos, 80 (69%) of them were developed to the EBL stage compared to 21 (87.5%) embryos in the control group. Moreover, developmental potential of A and B twins was similar regardless of their groups (70.7% vs. 67.2%, P= 0.688). Furthermore, when comparing twin and control embryos, the number of starting blastomeres appeared to have no significant effect on them reaching each stage.
Next, we compared the developmental potential of the embryos of different origins i.e. control, twin A or twin B. Although overall more embryos in the group 2 were developed to each stage compared to group 1, the only significant difference was in the number of embryos reaching the SB stage between twin B embryos: 73% of embryos in group 1 versus 95.2% of embryos in group 2 (P= 0.038).
Dynamic pattern of twin embryos
Assessment and comparison of the developmental dynamics between twin and control embryos that reached the EBL stage was done regarding two parameters; time of reaching each stage and the duration between the stages. In comparing the time of reaching each stage, there was no significant difference between the control and twin embryos, except for time of reaching more than 9 blastomeres (t9+) in the group 1 (Fig .2A,). The time these embryos took to get to this stage was significantly lower in the control embryos (9.80 ± 3.51 hours) compared to twins (twin A: 19.70 ± 7.05 hours and twin B: 20.54 ± 7.03 hours, P˂0.0001). In a different way, regarding the origin, the differences between the embryos in groups 1 and 2 were significant for the time the embryos took to reach all developmental stages (Fig .2B,).
Comparison of twins and control embryos did not reveal a pronounced rhythm in their developmental dynamics regards to the duration of critical stages in embryo development. Although some significant differences were found between twin and control embryos at the compaction and expansion stages (Fig .3A,). A and B twins belonging to groups 1 and 2, did not differ in duration between the different stages (Fig .3B,).
Blastocyst morphology and inner cell mass quality following splitting
The findings showed that the proportion of blastocysts with good morphology was significantly higher in the control group (71.4%) compared to A twins (39.6%, P=0.015) and B (28.6%, P=0.001). Although, the rate of fair quality embryos increased in the twins (A: 39.6% and B: 40.5%) after the splitting procedure compared to the control group (23.8%, Table 1,). Furthermore, the sub-group analysis displayed an increased rate of grade C ICM and grade B TE in twin embryos (Table 1,). Two (4.2%) ICMs in the twin A group were grade A. However, no grade A ICMs were noticed in the B twins.
Decreased size of blastocysts developed from twin embryos
Morphometric analysis showed a significant decrease in the overall size of twin expanded blastocysts compared to controls (mean ± SD (µm): 102.35 ± 5.19 vs. 120.92 ± 4.55, P˂0.0001). Regardless of the number of starting blastomeres, the average diameter of blastocysts in A and B twins was 103.53 µm and 101.11 µm, respectively, whereas the average diameter for control embryos was 120.92 µm.
No significant difference in the prevalence of aneuploidy or mosaicism in twin embryos
As presented in Table 2, the aneuploidy prevalence of each chromosome was assessed in total cells of embryos (Fig .4,). The blastocysts originated in all groups were similar in total abnormal cells (P=0.179). There was no significant difference between different groups regarding the rate of chaotic genomes (the cells with more than one chromosomal abnormality).
Next, we compared chromosomal abnormalities in the whole blastocysts developed from each group. Our data revealed no significant differences in the abnormality status between twins and control embryos (P=0.845). However, there was a statistically insignificant trend towards a decrease of normal embryos in twins (twin A: 60% and twin B: 57.1%) compared to the controls (71.4%, P>0.05).
|Variables||Control||Twin A||Twin B||P value|
|A||9 (42.9)||2 (4.2)||0||˂0.0001|
|B||8 (38.1)||18 (37.5)||14 (33.3)|
|C||4 (19)||28 (58.3)||28 (66.7)|
|A||16 (76.2)||12 (25)||8 (19)|
|B||2 (9.5)||25 (52.1)||19 (45.2)|
|C||3 (14.3)||11 (22.9)||15 (35.7)|
The values are presented as the number of embryos (%).
|Chromosome||Control||Twin A||Twin B||P value|
|13||8 (2.9)||9 (1.8)||17 (3.5)||0.237|
|15||1 (0.4)||3 (0.6)||1 (0.2)||0.621|
|16||11 (4)||23 (4.6)||26 (5.4)||0.682|
|18||10 (3.7)||23 (4.6)||24 (5)||0.71|
|21||5 (1.8)||19 (3.8)||24 (5)||0.097|
|22||0||4 (0.8)||1 (0.2)||0.17|
|X||2 (0.7)||5 (1)||0||0.1|
|Chaotic cells||1 (0.4)||6 (1.2)||3 (0.6)||0.401|
|Total abnormal cells||36 (13.2)||83 (16.5)||89 (18.5)||0.179|
The values are presented as the number of embryos (%).
Successful experiments in the development of human
twin embryos to the blastocyst stage following
Morphokinetic assessments revealed no significant difference in the length of time twin embryos took to reach the EBL stage compared to controls. Interestingly, the blastulation time showed a decreasing trend in the twin embryos in group 2 (10-14 blastomeres) compared to the controls. Moreover, A twins reached each stage faster than B twins; however, the differences were not significant. We hypothesized that manipulated blastomeres need extra time for recovering in order to continue the cell cycle. This hypothesis can be supported by some events, especially in recipient embryos during TLM, such as cytoplasmic waves without sign of division, and blastomere displacing and rotation. Furthermore, all embryos in group 2, regardless of being twins or controls, significantly grew faster to the EBL stage. Since the embryos in group 2 had extended culture from pronuclear stage, they needed less time to develop to the blastocyst stage compared to the group 1. These findings demonstrated a similarity in total time needed for blastocyst development for embryos in either of the groups 1 or 2. There was a wide range between the minimum and maximum times of reaching each stage in the twins compared to controls. Twins exhibited a significantly shorter time duration for the compaction (9+ to Mor) and the start of blastulation (Mor to SB) stages than the control embryos. This result is in accordance with findings of Noli et al. (8), suggesting a kind of ‘compensation’ for the lower cell number in twin embryos. In a different assessment, twins from both groups did not differ regarding the duration between the stages.
Based on data from the quality assessment of human
twin embryos, splitting resulted in smaller blastocysts
with a lower quality of ICM and TE compared to nonmanipulated embryos. A previous study demonstrated
that in spite of increasing the number of blastocysts
after splitting, the percentage of good quality blastocysts
significantly decreased in the mice model (6). In line with
our results, Noli and associates had detected a significant
difference in size between twins compared to the
controls. In addition, they found that the decreased size
of blastocysts developed after
To the best of our knowledge, this is the first study that
evaluates the impact of human embryo
Recent studies have introduced comparative genomic
hybridization (CGH) and microarray-CGH as more
optimal strategies for aneuploidy detection (39), in spite of
some of their limitations (40). It is suggested that further
studies be conducted with a higher number of donor
The current study shows that some developmental timepoints were affected by
This study was extracted from the Ph.D. thesis of Marjan Omidi. The authors appreciate Yazd Research and Clinical Center for Infertility for all its support. The authors declare no conflicts of interest.
M.O., M.A.K., S.M.K.; Contributed to conception and design. M.O.; Contributed to all experimental work. M.O., S.M.K., F.M.; Contributed to data analysis and conducted. cytogenetic analysis. M.O., I.H.; Contributed to data analysis. M.O., I.H., M.A.K., S.M.K.; Contributed extensively in interpretation of the data and the conclusion. M.O, I.H.; Contributed to write the manuscript. All authors read and approved the final manuscript.