Association between the morphokinetics of in-vitro-derived bovine embryos and the transcriptomic profile of the derived blastocysts

Abstract

The time-lapse system is a non-invasive method that enables a continuous evaluation through embryo development. Here, we examined the association between the morphokinetics of the developing embryo and the transcriptomic profile of the formed blastocysts. Bovine oocytes were matured and fertilized in vitro; then, the putative zygotes were cultured in an incubator equipped with a time-lapse system. Based on the first-cleavage pattern, embryos were categorized as normal or abnormal (68.5±2.2 and 31.6±2.3%, respectively; P<0.001). A cleaved embryo was defined as normal when it first cleaved into two equal blastomeres; it was classified as synchronous or asynchronous according to its subsequent cleavages. An abnormal pattern was defined as direct, unequal, or reverse cleavage. Direct cleavage was classified as division from one cell directly into three or more blastomeres; unequal cleavage was classified as division that resulted in asymmetrically sized blastomeres; and reverse cleavage of the first division was classified as reduced number of blastomeres from two to one. Of the normally cleaving embryos, 60.2±3.1% underwent synchronous cleavage into 4, 8, and 16 blastomeres, and 39.7±3.1% cleaved asynchronously (P<0.001). The blastocyte formation rate was lower for the synchronously vs. the asynchronously cleaved embryos (P<0.03). The abnormally cleaved embryos showed low competence to develop to blastocysts, relative to the normally cleaved embryos (P<0.001). Microarray analysis revealed 895 and 643 differentially expressed genes in blastocysts that developed from synchronously and asynchronously cleaved embryos, respectively, relative to those that developed from directly cleaved embryos. The genes were related to the cell cycle, cell differentiation, metabolism, and apoptosis. About 180 differentially expressed genes were found between the synchronously vs. the asynchronously cleaved embryos, related to metabolism and the apoptosis mechanism. We provide the first evidence indicating that an embryo’s morphokinetics is associated with the transcriptome profile of the derived blastocyst, which might be practically relevant for the embryo transfer program.

Embryo morphokinetics derived from fresh and vitrified bovine oocytes predict blastocyst development and nuclear abnormalities

“…Hence, there seems to be an association between blastomere size, nuclear content, and multipolar division. With regard to abnormal cleavage events, we found lower developmental capacity in zygotes with multipolar cleavage or ruffling membrane, which is in line with human studies 29,37 and recent publications in human and bovine zygotes that showed how multipolar division leads to lower embryo development compared to normal cleavers 30,34 . As supported by the higher number of nuclear abnormalities following multipolar zygotic division, multipolar zygotic division seems to be associated with genome segregation errors in embryos leading to a higher frequency of mixoploidy, and chromosomal abnormalities in the resultant embryos 33,39,41 and embryonic arrest 30 .…”

“…In our hands, the average time to reach the first division for fresh cumulus enclosed oocytes in serum free culture was slightly higher (33 h) than in other studies (~ 25 h) 33,36,39,41 , which may be related to the use of fetal calf serum in those studies, as this has been demonstrated to accelerate embryo development 59,60 . On the other hand, our results also matched with studies in bovine 34,41 and humans 61 which showed that differences in blastomere size after zygotic division decrease subsequent developmental potential. Our ROC curve results revealed the ideal cut-point for differentiating between even and uneven blastomeres (< 24% difference in area).…”

“…Fast first cleavage, the presence of a lag-phase, and the absence of abnormal zygotic cleavage were elementary for blastocyst development in for both vitrified and fresh bovine oocytes. Both timing and nature of the first embryonic division are linked to subsequent embryo development 34,52,53 . Our results confirm previous studies demonstrating that zygotes which cleave faster present higher developmental competence 38,54,55 .…”

“…For example, the pace of development of in vitro produced embryos can be linked to embryo quality, and embryos that cleave faster following fertilization are more developmentally competent than are those that cleave relatively late 31,32 . On the other hand, cytokinetic events diverging from the normal division of mammalian zygotes in two equally sized blastomeres have been shown to relate to embryo quality 24,33,34 . Multipolar division-the first division of the embryo directly into three cells 29,30,33,35 , ruffling membranes-motility of the oolemma before the first division 36,37 , the occurrence of a resting period in between the divisions or so-called lag-phase, and heterogeneous size of the blastomeres following the first division can all affect embryo development and pregnancy rates 33,38 .…”

“…Multipolar division-the first division of the embryo directly into three cells 29,30,33,35 , ruffling membranes-motility of the oolemma before the first division 36,37 , the occurrence of a resting period in between the divisions or so-called lag-phase, and heterogeneous size of the blastomeres following the first division can all affect embryo development and pregnancy rates 33,38 . Although some time-lapse studies in cows have reported on the prevalence of abnormal cleavage patterns and on the length of cell cycles and their correlation with developmental capacity 30,33,34,36,[39][40][41] , integration of the effect of all the morphokinetic events mentioned previously in one single study has not yet been performed. Furthermore, the ideal cut-off points for cleavage time or size differences in blastomeres remain to be determined.…”