Enhancing Cell Injection Systems By Real Time Confirmation Of Cytoplasmic Penetration.

Visual assessment of true cell penetration with a microinjection pipette is not always feasible due to cloudy solutions, adjacent cells, and light microscopy wavelength limitations. This is especially true for microinjection in bovine oocytes and zygotes due to their cytoplasmic opacity. We hypothesized that an increase in electrical resistance upon bovine zygote plasmatic membrane piercing can serve as a real-time tool to confirm cell penetration and embryo viability. Experimental study. In the first part of the study, the minimal electrical resistance increase (minΔR [MΩ]) that occurs when penetrating visually viable zygotes (compared to non-viable ones) was determined. Bovine zygotes were produced by in vitro fertilization. Electrical resistance of the microinjection pipette tip was measured continuously throughout the procedure. ROC analysis was performed. In the second part of the study, the ability of the minΔR [MΩ] (identified in the first part of the study) to predict cell penetration and viability was tested by the microinjection of in vitro transcribed (IVT) mRNA coding for the fluorescent ‘mCherry’ nuclear protein into zygotes. Cleavage embryos showing nuclear fluorescence 20 hours post injection were considered viable. ROC analysis showed minΔR > 4 MΩ to identify visually viable embryos (n=67) versus non-viable ones (n=15) (97% sensitivity, 100% specificity, AUC 0.99 (CI 0.95 – 1.00)). In the second part of the study, 11 zygotes had minΔR > 4 MΩ at the time of IVT mRNA injection. Seven of them (64%) cleaved and showed positive nuclear fluorescence 20 hours post injection. Eight zygotes had minΔR ≤ 4 MΩ and none of them (0%) showed nuclear fluorescence 20 hours post injection. When attempting cytoplasmic zygote microinjection, electrical resistance increase can serve as a reliable tool to confirm successful cell penetration and embryo viability, independent of optical visualization. This technology can potentially be integrated into a manual or robotic cell injection system.