Prematuration Culture with Phosphodiesterase Inhibitors After Vitrification May Induce Recovery of Mitochondrial Activity in Vitrified Mouse Immature Oocytes.

This study investigates the possible causes for low development of blastocysts in vitrified immature oocytes by evaluating the changes of mitochondrial membrane potential and reactive oxygen species (ROS) production and finds a recovery mechanism for these conditions in vitrified immature oocytes. To recover from the cryoinjury, we cultured vitrified immature oocytes in milrinone containing medium for 1, 3, and 5 hours and then extended the culture for oocyte maturation. There was no difference in in vitro maturation and fertilization rate between fresh and vitrified/warmed oocytes. However, the development rate of blastocysts in vitrified/warmed oocytes was significantly lower than that in fresh oocytes (p < 0.05). The development rate of blastocysts was recovered if these oocytes were cultured for 3 hours in milrinone. Vitrified/warmed oocytes incubated in milrinone for 0 and 1 hour showed a significantly higher level of ROS (p < 0.05) and a significantly lower mitochondrial membrane potential (p < 0.05) than fresh oocytes. However, there was no significant difference (p > 0.05) between vitrified oocytes incubated in milrinone for 3 hours and fresh oocytes in terms of ROS level and mitochondrial membrane potential. In conclusion, alteration of highly polarized mitochondria distribution in vitrified oocytes may have an effect on mitochondrial activity, including ROS production during fertilization and further development. Preincubation in milrinone before in vitro maturation of immature vitrified/warmed oocytes may help the redistribution of highly polarized mitochondrial inner membrane potential and in reducing ROS and enhance the further embryonic development after fertilization.

Alterations in calcium oscillatory activity in vitrified mouse eggs impact on egg quality and subsequent embryonic development.

Cryopreservation of mature eggs is a useful technique that can be applied in assisted reproductive technology. However, the method has some limitations, such as cryodamage induced by biophysical modifications during the cryopreservation process. To assess these biophysical damage, we analyzed the relationship between intracellular calcium ([Ca2+]i) oscillatory activity via type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) distribution after vitrification and efficiency of cryopreservation according to cryoprotectant (CPA) composition. In immunostaining, results of IP(3)R1 with eggs after the vitrification performed using ethylene glycol (EG) alone or EG + dimethylsulfoxide (DMSO) as CPAs, CPA-treated, and fresh eggs displayed a homogeneous IP(3)R1 distribution which is spread uniformly throughout cytoplasm with clusters on the cortex. However, after vitrification and warming process, more than 60% of eggs displayed a heterogeneous distribution which is non-uniform distribution with patches and disconnection of IP(3)R1. In 90-min incubation for recovery from cryodamage, eggs from the EG + DMSO group recovered from with a heterogeneous IP(3)R1 distribution to the homogeneous distribution, but not in EG alone group. In ICSI experiments, vitrified eggs in the EG-alone group presented significantly low blastocyst formation compared to those of the fresh and EG + DMSO groups. These results suggest that the vitrification process influences IP(3)R1 distribution, and subsequently, [Ca2+]i oscillatory activity and embryonic development. Accordingly, we propose that IP(3)R1 distribution and [Ca2+]i oscillatory activity are correlated with egg quality and developmental potential after vitrification, and may thus be applied as an effective indicator to evaluate the efficiency of oocyte cryopreservation methods.