Impact of cleavage morphokinetics on blastocyst development

Authors: SCHETTINI, GUSTAVO - Virginia Tech; WALSH, ALLIE - Virginia Tech; MARRELLA, MACKENZIE - Virginia Tech; KAPS, MARTIM - University Of Veterinary Medicine; Miles, Jeremy; RHOADS, MICHELLE - Virginia Tech; Snider, Alexandria; BIASE, FERNANDO - Virginia Tech

Submitted to: International Embryo Society
Publication Type: Abstract Only
Publication Acceptance Date: 7/20/2024
Publication Date: 1/18/2025
Citation: Schettini, G., Walsh, A., Marrella, M., Kaps, M., Miles, J.R., Rhoads, M., Snider, A.P., Biase, F. 2025. Impact of cleavage morphokinetics on blastocyst development [abstract]. In proceedings: International Embryo Technology Society 51st Annual Conference. January 18-22, 2025, Fort Worth, TX. Poster 64.

Interpretive Summary:

Technical Abstract: In vitro embryo production has revolutionized the cattle industry. However, there are still critical limitations to our understanding of embryo competence. The use of time-lapse imaging has significantly enhanced our ability to evaluate embryo development by assessing cleavage patterns, abnormal events, and morphology. Here, we aimed to determine cleavage patterns and morphological features during early embryonic development in cattle associated with blastocyst development. Putative zygotes (n=1218) were individually cultured in two experimental sites using synthetic oviduct fluid medium (5%CO2, 5%O2, 38.5°C). All embryos were produced using semen from one sire. Time-lapse imaging allowed the recording of cleavage timing and patterns, such as ruffling/blebbing, asynchronous cleavage, reversal, or direct cleavage patterns. Overall, 21.3%, 34.3%, 18.9% embryos halted development at 2-4 cells, 5-8 cells or morula, whereas 8.1% developed to blastocysts. Categorical variables were analyzed using logit models that included ruffling, direct, synchronous reverse, and symmetric cleavage as fixed effects and date and plate as random variables, while development was a dependent variable. Quantitative variables were analyzed using linear mixed-effect models that included developmental stage and location as fixed effects and date and plate as random variables. Each tested variable was included as a dependent variable. Cell ruffling increased developmental arrest at 2-4 cell (P<0.01). Reverse cleavage increased developmental arrest at 2-4 cells and morula stage (P<0.05). Direct and asynchronous cleavage increased developmental arrest at 2-4 cells, 5-8 cells, and at the morula stage (P<0.05). Most blastocysts developed out of synchronous (P<0.01), symmetric (P<0.1) cleavages, with no reversed cleavages (P<0.01). Diameter of putative zygotes and time of first cleavage were associated with developmental arrest at 2-4 cells stage (P<0.01). Time to reach 4-cells and 8-cells were associated with blastocyst development (P<0.01). Next, we combined all variables into a machine learning model, which predicted 94% (255/272) and 21% (6/29) of the embryos that failed or successfully developed to blastocyst, respectively. In parallel, image analysis of 8-cell embryos using a deep convolutional neural network predicted 73.3% (11/15) and 83.3% (10/12) of the embryos that failed or successfully developed to blastocyst, respectively. Furthermore, we cryopreserved 107 blastocysts followed by thawing and individual culture. Thirty-five blastomeres survived cryopreservation as evaluated by the expansion of the blastocoele. None of the categorical or quantitative variables were significantly associated with competence to survive cryopreservation. We conclude that morphokinetic features were correlated with embryonic competence and are useful for identification of early developmental arrest.