Improved developmental potential of mouse vitrified‐warmed oocytes achieved by culturing in recovery medium with glutathione ethyl ester (GSH‐OEt)

Abstract Purpose The aim of the present study was to investigate the effect of glutathione ethyl ester (GSH‐OEt) in the recovery medium on the developmental competence of mouse vitrified‐warmed MII oocytes. Methods Vitrified‐warmed oocytes were incubated for 1 h in recovery medium in the presence or absence of 0.5 mM GSH‐OEt. The authors examined the effects of GSH‐OEt, first on the levels of glutathione (GSH) and reactive oxygen species (ROS) in vitrified‐warmed oocytes, and second, on in vitro blastocyst development, division speed to blastocysts, and total cell numbers of blastocysts from vitrified‐warmed oocytes fertilized by Intracytoplasmic sperm injection (ICSI). Results Adding GSH‐OEt to the recovery medium significantly (p < 0.05) increased GSH content and decreased ROS levels in vitrified‐warmed oocytes. The blastocyst rate did not differ significantly between the two groups, but the speed of development to blastocysts in the GSH‐OEt (+) group was significantly more rapid. In addition, the total blastocyst cell number was significantly higher in the GSH‐OEt (+) group than in the GSH‐OEt (−) group (92.8 ± 5.1 vs. 71.4 ± 3.5, p < 0.01). Conclusion Adding GSH‐OEt to the recovery medium of vitrified‐warmed mouse oocytes enhances the development potential of oocytes and improves the quality of blastocysts.

On the other hand, the vitrification method requires the exposure of oocytes to a high concentration of cryoprotectants and increased osmolalities. [9][10][11] So that, most intracellular water is replaced by a cell membrane-permeable cryoprotectant. In particular, unfertilized oocytes are likely to be damaged by vitrification. 12 The reason is that there is no change in the oocyte membrane structure that occurs during fertilization, unlike in the zygote. Oocytes are difficult cells to cryopreserve, due to their low surface area to volume ratio and high susceptibility to intracellular ice formation. 13 The developmental potential and implantation capacity of fertilized embryos derived from vitrified oocytes are clearly lower than those of fresh oocytes. 14,15 Cryopreservation by vitrification is associated with an increase in reactive oxygen species (ROS) in oocytes. 16,17 Generally, the increase in ROS in cells is erased by glutathione (GSH)-related GSH/ GSSG redox potential. 18 Alterations in GSH-related GSH/GSSG redox potential induce post-ovulatory aging, compromise male pronuclear formation, and cause embryo development to fail. 19,20 GSH is synthesized during oocyte maturation and reaches its maximum concentration before sperm penetration. 21 Thus, GSH is synthesized via through the γ-glutamyl cycle. Even if GSH is added extracellularly, it cannot penetrate the cell membrane and does not act directly on the oocyte. 18 Glutathione ethyl ester (GSH-OEt) easily penetrates the cell membrane and undergoes hydrolysis by intracellular esterase thereby increasing intracellular GSH concentrations. 22,23 Previous studies with bovine oocytes have reported that the addition of GSH-OEt is effective against ROS production by longterm in vitro cultures such as in IVM. 24 Trapphoff et al 25 reported that supplementation of media with GSH-OEt immediately prior to vitrification of IVM oocytes prevents from oxidative damage and improves freezing damage. Furthermore, in vitrification of MII mouse oocytes, supplementation of GSH-OEt immediately before vitrification was effective to embryo development. 26 These studies show the effectiveness of pre-vitrification GSH-OEt supplementation, but the direct improvement of GSH content in oocytes after vitrification-warming is expected rather than pre-vitrification. Therefore, this study focused on effectiveness of adding GSH-OEt to the recovery medium post-vitrification and warming. To our knowledge, no studies have asked whether there is an additive effect of GSH-OEt in the recovery medium of vitrifiedwarmed oocytes on their in vitro development into blastocysts and on the quality of the blastocysts.
The purpose of this study was to investigate the additive effect of GSH-OEt in the recovery culture medium of vitrified-warmed mouse oocytes on embryo development after ICSI. Blastocyst quality was evaluated using time-lapse analysis as non-invasive assessment. 27,28 2 | MATERIAL S AND ME THODS

| Animals
In this study, we used B6D2F1 female mice, 3-4 weeks of age, obtained from CLEA Japan Inc. (Tokyo Japan). All mice were maintained in a temperature-and light-controlled room at 25°C, with a 12-h light/12-h dark cycle (light starting at 6:00 h

| Vitrification and warming of mouse oocytes, and recovery culture
Oocytes were equilibrated in equilibration solution containing 7.5%

| Sperm collection and freezing
Spermatozoa were collected from the cauda epididymis of B6D2F1 mice at 12-16 weeks of age. As previously reported, for sperm freezing and sonication, we used an EGTA solution containing 10 mM Tris-HCl buffer with 50 mM ethylene glycol-bis (-aminoethyl ether)-N, N, N, N-tetraacetic acid (EGTA solution). 29,30 Spermatozoa were suspended in approximately 1 ml of EGTA solution for up to 5 min at 37.0°C under 5% CO 2 in air. Spermatozoa were centrifuged at 500 g for 5 min, the supernatant was removed, and the spermatozoa were mixed with 1 ml of EGTA solution. This suspension was transferred to ice water (0°C), then sonicated for 5 s at 50% sonicator output (20 kHz, VP-5S; Taitec, Japan). More than 90% of mouse spermatozoa underwent head and tail separation. Finally, 100μl aliquots of sonicated spermatozoa in EGTA solution were placed in 0.25 ml cryostraws and frozen in liquid nitrogen (−196°C).

| Microinjection of sperm heads into oocytes
Piezo-ICSI was performed using a microscope (ECLIPSE Ti-U; Nikon, Tokyo, Japan), equipped with a micromanipulator (Narishige Inc., Tokyo, Japan) and a piezo impact drive (MB-U; Prime tech Ltd., Japan).
The piezo drive unit was driven by a controller (PMAS-ET150; Prime Tech Ltd., Ibaraki, Japan). Injection pipettes had an inner diameter of 5.95 μm and an outer diameter of 7 μm. One drop (15 μl) of frozenthawed sonicated sperm heads was suspended in 10% polyvinylpyrrolidone (PVP: Irvine Scientific) at a 1:1 ratio before injection. The MII oocytes were placed into 5 μl droplets of mHTF that contained 20% SSS, which had been placed next to sperm head-containing droplets (15 μl) covered with mineral oil (Irvine Scientific). The zona pellucida of each oocyte was penetrated by applying several piezo pulses (speed 2, intensity 2), and the oolemma was then broken by applying a single piezo pulse (speed 1, intensity 1). All procedures were performed at room temperature. The piezo-ICSI procedure was completed within 1 h of sperm thawing. In measuring ROS and GSH levels, the average fluorescence intensity of fresh oocytes was set to 1.0, and the reported values represent fold differences in fluorescence intensity.

| Assessment of blastocyst cell numbers
Blastocysts at the end of culture at 96 h were individually stained for total cell number. The blastocysts were rinsed twice with PBS (−), then fixed in ethanol and stained with Hoechst 33342 (20 µg/ml) for 5 min at room temperature. Total blastocyst cell numbers were determined using a fluorescence microscope.

| Statistical analysis
Statistical analyses were performed using GraphPad PRISM 6.03 software (GraphPad Inc., San Diego, CA, USA). Fisher's exact probability test or the chi-square test was used to evaluate differences in 2-cell and blastocyst rates. Means in the three groups were compared using one-way ANOVA and the Tukey-Kramer test.
Differences were considered significant at p < 0.05.

| Effect of recovery culture time on the in vitro embryo development of vitrified-warmed oocytes after ICSI
First, in vitro development of vitrified-warmed oocytes after ICSI at each time (0, 1, and 2 h) was examined to determine the optimal recovery time from warming to ICSI. As shown in Figure 1, when the vitrified-warmed oocytes were cultured for 1 h in GSH non-content recovery medium (global total), the blastocyst rate after ICSI was significantly higher than that after 0 (immediate) or 2 h of recovery culture (72.2% vs. 42.5% and 35.7%, respectively, p < 0.05).
Therefore, the optimal recovery time from warming to ICSI was decided to be 1 h.

| Effects of vitrification and warming and of GSH-OEt (0.5 mM) in the recovery medium on GSH and ROS levels in vitrified-warmed oocytes
As shown in Figure 2, the GSH level in vitrified-warmed oocytes was significantly decreased, and the ROS level in vitrified-warmed oocytes was significantly increased, compared with the GSH and ROS levels in fresh oocytes.

GSH-OEt (−) group with 1-h recovery culture without 0.5 mM GSH-
OEt was significantly lower than that in fresh oocytes (Figure 2A).

In contrast, the ROS level in vitrified-warmed oocytes in the GSH-
OEt (−) group was significantly higher than that in fresh oocytes ( Figure 2B). When 0.5 mM GSH-OEt was added to the recovery culture medium for 1 h, the GSH level in the GSH-OEt (+) group was significantly higher than in the GSH-OEt (−) group (p < 0.05) and was not significantly different from that in the fresh oocytes. That is, after 1-h recovery culture with GSH-OEt, the GSH level in vitrifiedwarmed oocytes was significantly increased. The ROS level in the GSH-OEt (+) group was significantly lower than that in the GSH-OEt Note: Vitrified-warmed oocytes were cultured in recovery medium with or without GSH-OEt for 1 h up to ICSI.
For this treatment, 0.5, 1.0, and 3.0 mM GSH-OEt were added into global total medium. a,b Different superscripts indicate a significant difference (p < 0.05), based on Fisher's exact probability test. 1 Fresh oocytes were cultured in global total medium without GSH-OEt for 1 h up to ICSI, used as controls.

TA B L E 1 Effect of adding of GSH-OEt
to the recovery medium of vitrifiedwarmed oocytes on their in vitro development to blastocysts after ICSI

| Effect of adding of GSH-OEt (0.5 mM) to recovery medium of vitrified-warmed oocytes on their total cell numbers of blastocyst at 96 h from ICSI
Total cell numbers in blastocysts derived from vitrified-warmed oocytes cultured with or without GSH-OEt (0.5 mM) in the recovery medium and from fresh control oocytes were counted (Table 2). They were significantly higher in the control and GSH-OEt (+) groups than in the GSH-OEt (−) group (93.9 ± 3.9 vs. 94.6 ± 4.5 vs. 73.1 ± 3.1,

| DISCUSS ION
Oocyte vitrification is now widely used in human ART. However, whether the ultra-rapid vitrification method adversely affects oocytes is not yet fully known. In this study, we examined the effect of of GSH decreased the blastocyst rates. 33

TA B L E 3
Time-lapse analysis of vitrified and warmed mouse oocytes cultured in the recovery medium with or without GSH-OEt following ICSI hamster oocytes, GSH plays an important role in male pronucleus formation. 38 Whether there is a relationship between intracellular GSH and sperm swelling in human unfertilized oocytes is not known. However, vitrification can alter the division speed of embryos generated after oocyte warming. A recent study shows the relationship between embryo morphokinetics and metabolic activity. 39 Vitrification can cause temporary changes in mitochondrial polarity and ATP levels. 40,41 Further studies are needed to clarify the relationship between these intracellular molecular changes and embryo morphokinetic parameters.
By using a time-lapse system, the exact timing of each embryo's progress to the blastocyst stage was clarified, and the total cell number of blastocysts was seen to increase. The cell number is an important indicator in assessing the quality of blastocysts. Early arrival at the blastocyst stage increased the blastocyst cell number at that time and improved the quality of the blastocyst.
This study did not clarify the mechanism of action of GSH synthesis and ROS removal upon the addition of GSH-OEt. Our results suggest that adding GSH-OEt to the recovery medium can shift the quality of a warmed oocyte closer to the better quality of fresh oocytes, ultimately improving oocyte vitrification outcomes.
In conclusion, our results are evidence that recovery in the presence of GSH-OEt can enhance the developmental potential of mouse MII oocytes after cryopreservation by vitrification. However, further studies are needed to determine whether GSH-OEt is also useful in the vitrification of human oocytes.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

H U M A N R I G HT S S TATE M E NT S A N D I N FO R M E D CO N S E NT
This study did not include human participants.

A N I M A L S TU D I E S
All the experiments were approved by the Committee for Ethics on Animal Experiments at the Prefectural University of Hiroshima, Japan (18A010).