Jinji Mizuno

Mouse zona pellucida dynamically changes its elasticity during oocyte maturation, fertilization and early embryo development

A change in the elasticity of mouse zona pellucida was quantitatively evaluated during oocyte maturation, fertilization and early embryo development. Young’s modulus of zona pellucida of germinal vesicle (GV), metaphase-II (MII), pronuclear (PN), 2cell, 4cell, 8cell, morulae (M) and early blastocyst (EB) stages was measured using a micro tactile sensor (MTS) and a chamber exclusively designed for the measurement. The MTS has very high sensitivity and a deformation of only 5 μm was sufficient to calculate the Young’s modulus and the oocyte/embryo maintained its original spherical shape during the measurement. The Young’s modulus of GV, MII, PN, 2cell, 4cell, 8cell, M and EB was 22.8 ± 10.4 kPa (n = 30), 8.26 ± 5.22 kPa (n = 74), 22.3 ± 10.5 kPa (n = 66), 13.8 ± 3.54 kPa (n = 41), 12.6 ± 3.34 kPa (n = 19), 5.97 ± 4.97 kPa (n = 6), 1.88 ± 1.34 kPa (n = 8) and 3.39 ± 1.86 kPa (n = 4), respectively. Experimental results clearly demonstrated that the mouse zona pellucida hardened following fertilization. Interestingly, once the zona pellucida hardened at the PN stage, it gradually softened as the embryo developed (i.e. it was found that the zona hardening is a transient phenomenon). Furthermore, the zona pellucida of the GV oocyte was as hard as that of the PN embryo and became soft as it matured to the MII stage. In addition, the safety of the MTS measurement for oocytes and embryos was discussed both theoretically and experimentally.

Elasticity Measurement of Zona Pellucida Using a Micro Tactile Sensor to Evaluate Embryo Quality

ABSTRACT To enable improved success rates of IVF, we developed the technology to optimize embryo selection with the highest implantation potential while ensuring no damage to embryos using zona elasticity as the selection criterion. In this communication we outline the biomechanics and safety of zona pellucida (ZP) elasticity measurement using the micro tactile sensor (MTS) system and demonstrate the specific changes in ZP elasticity during oocyte maturation, fertilization, and early embryo development. Zona hardening was demonstrated mechanically following fertilization at the pronuclear (PN) stage, and Youngs modulus decreased gradually as the embryo developed. Evaluation of the quality of expanded blastocysts (EPB) showed that the quality of EPBs could also be evaluated from elasticity parameters. Furthermore, the observations indicated that ZPs of embryos generated in vivo were significantly harder than those of embryos generated in vitro at each stage. Preliminary results also indicated that denuded oocytes matured in vitro did not show zona hardening following parthenogenetic activation by strontium chloride, suggesting that sufficient maturation and consequent oocyte activation may be evaluated by increases in ZP elasticity. We conclude that MTS-elective single embryo transfer can be applied to human assisted reproductive technology to enable embryo quality evaluation in both early embryos and EPBs.

Single embryo-coupled gate field effect transistor for elective single embryo transfer.

In this study, we have proposed and demonstrated experimentally a novel monitoring device of single mouse embryo activity after in vitro fertilization (IVF) using a semiconductor-based field effect transistor (FET). The FET biosensor realized to detect it noninvasively, quantitatively, and continuously by change of hydrogen ions with positive charges, which were induced by dissolved carbon dioxide due to cellular respiration activity during cleavage. The electrical signal of FET biosensor should become an effective indication to evaluate objectively single embryo activity as its morphology is observed subjectively after IVF. The platform based on the FET biosensor will contribute to promote elective single embryo transfer (eSET) in human assisted reproductive technology (ART).

Softening of the mouse zona pellucida during oocyte maturation

A change in the elasticity and the resistance to dissolution of the mouse zona pellucida (ZP) was quantitatively evaluated at immature germinal vesicle (GV), mature metaphase II (MII) and fertilized pronuclear (PN) stages. Youngs modulus of the ZP was measured using a micro tactile sensor (MTS), a highly sensitive resonator-based sensor for a micro scale elasticity measurement. 0.25% α-chymotrypsin was used for the ZP dissolution assay. The results of measuring the ZP elasticity and the dissolution time clearly showed that the ZP softened during oocyte maturation and the ZP hardened after fertilization. The results indicate that the amount of the zona softening can be a criterion to evaluate oocyte quality for the selection of top quality mature oocyte before in vitro fertilization (IVF) treatment.

New embryo co-culture system for human Assisted Reproductive Technology (ART): Verification of the effects of mechanical stress to embryo co-culture system

In vitro fertilization (IVF) and in vitro culture (IVC) are important technics for human ART. Especially, IVC is most important factor to achieve good/high pregnancy rate. In order to reach this goal, we have to develop in vivo mimic/like human embryo co-culture system. We presented/introduced a new stretch embryo co-culture system for human ART