148 Microaspiration-assisted electrical impedance spectroscopy used for characterization of bovine oocytes during in vitro maturation

Electrical impedance spectroscopy (EIS) is widely used to characterise electrical properties of different materials like liquids, organic tissue, or macromolecules. Their impedance is measured as resistance units. Microaspiration-assisted electrical impedance spectroscopy (MAEIS) allow label-free and noninvasive measurement of impedance of cells, for example oocytes. The MAEIS system consists of a microfluidic chip with microchannels perforated with apertures in the microchannel’s cover lid. With microelectrodes arranged around the apertures EIS can be carried out to characterise trapped objects at the aperture. The impedance magnitude change ΔZ/Z0 (impedance change ΔZ by trapping an object divided by impedance of an empty aperture Z0) is used to characterise the measured object. The aim of this experiment was to evaluate the electrical changes during IVM of oocytes via MAEIS. Cumulus–oocyte complexes (COCs) were isolated from abattoir-derived bovine ovaries and placed in maturation medium (tissue culture medium 199, TCM-199) supplemented with hCG and eCG. In vitro maturation lasted for 24 h in an incubator with 38.0°C and 5% CO₂. At different time points (t = 0 h, 4 h, 18 h, 20 h, 22 h, 24 h) 4 COCs were denuded and placed individually on the microfluidic chip in the aperture filled with TCM-Air. The chip was positioned on a heating plate of an inverted microscope with stable temperature of 36.0°C to avoid too many temperature changes for the oocytes during handling outside the incubator. The impedance of the oocyte was measured via EIS for 1 minute. Immediately after that, oocytes were stained with Hoechst 33342 and the maturational stage was determined (germinal vesicle = GV; germinal vesicle breakdown = GVBD; meiosis I = MI; meiosis II = MII). In total, 24 COCs were analysed. The high steadiness of the measured impedance values required no statistical analysis. At time point 0 h, 2 oocytes were at the GV and 2 at GVBD stage. Four hours later all oocytes showed GVBD. After 18 h 4 oocytes reached the MI stage, 2 h later most oocytes did so, but 1 oocyte started to be at MII. With 22 h passed, MI and MII were equally distributed and at the end of maturation, after 24 h, most of the oocytes were at the MII stage. According to their maturational stage, the oocytes were split into groups. Oocytes at the GV stage gave an impedance magnitude change of 7.23 ± 0.73% (ΔZ, mean values ± standard deviation) compared with the impedance of the open aperture (Z0). Oocytes at the GVBD did show an increase to 10.02 ± 0.81%, whereas the oocytes at MI and MII displayed impedance changes of 6.60 ± 0.78% and 6.56 ± 0.97%, respectively. These data indicate that maturational stages of oocytes during IVM can be detected noninvasively via MAEIS. Further work has to be done, but we speculate that determining end of maturation is possible via EIS. So fertilization time could be set more precisely depending to the maturational stage of the oocytes.