66 Developmental potential of single blastomeres within individual embryos presenting bipolar or multipolar divisions

During multipolar zygotic divisions (direct cleavage into three or more blastomeres), entire parental genomes can segregate into distinct blastomeres, leading to mixoploid or chimeric embryos. Still, embryo arrest occurs frequently in those embryos, suggesting that blastomeres resulting from whole-genome segregation errors might have lower viability than normal, diploid blastomeres. To investigate this further, we compared the developmental potential of single blastomeres within an individual embryo after either bipolar (cleavage into two blastomeres) or multipolar division. To do so, bovine cumulus–oocyte complexes were collected from slaughterhouse-derived ovaries and matured separately per ovary (n = 11 cows, 7 replicates) in 500 μL maturation medium for 22 h. Since multipolar divisions are often triggered by polyspermic fertilization, oocytes were fertilized with semen of a bull with known high polyspermy rate. The presumed zygotes were denuded and transferred to 25 μL of culture medium droplets at 21 h postfertilization (hpf), and monitored from 26 to 36 hpf every 30 min to identify zygotes with bipolar and multipolar divisions. Immediately upon the first division, the zona pellucida was dissolved with pronase in 58 embryos and single blastomeres were isolated and cultured individually in well-of-the-well culture dishes. The development of the single blastomeres was checked at 72, 121, and 170 hpf. To evaluate the relation between the type of division and the developmental potential, a chi-squared test of independence was performed. At 72 hpf, the 2-cell stage was reached in a higher number of blastomeres from multipolar divisions (32.1%) than in blastomeres from bipolar divisions (0%; P = 0.018), while no difference was observed among groups in the percentage of blastomeres from embryos with multipolar or bipolar divisions that did not cleave any further (21.4% and 31.2%, respectively; P = 0.469). Interestingly, although there was no statistical difference, only 46.4% of blastomeres from multipolar divisions developed to the 3- to 5-cell stage compared to 68.8% (P = 0.215) in those from bipolar divisions. At 121 hpf, more blastomeres arrested at 1 cell or at the stage of 2–4 cells in the multipolar group (27.5% and 35%) than in the bipolar group (0%, P = 0.023; and 6.3%, P = 0.036), while more advanced development (5–10 cells) was reached in a higher proportion of blastomeres from bipolar divisions (87.5%) than from multipolar divisions (37.5%; P < 0.01). At 170 hpf, a similar number of blastomeres remained uncleaved in both groups (bipolar = 21.4%, multipolar = 41.1%; P = 0.098) or started the first divisions but arrested in 2–5 cells (bipolar = 7.1%, multipolar = 35.7%; P = 0.122), but blastomeres from bipolar divisions showed the highest developmental potential (71.4% blastocyst rate) compared to the multipolar group (26.8%; P = 0.003). To conclude, we showed that blastomeres resulting from bipolar divisions exhibit a higher developmental potential than those from multipolar divisions; and we developed a model to isolate and selectively grow cell lines with distinct parental contribution.