Abstract
There have been 60 births after transplantation of cryopreserved ovarian tissue: 58 using the slow freezing method, and two using the vitrification method. DMSO and EG are widely used as cryoprotectants. However DMSO is a known epimutagen, and EG has been reported to be toxic in high concentrations. In this study, we measured residual DMSO and EG in ovarian tissue after vitrification and slow freezing. Cryoprotectants remained at a high concentration in the vitrified/warmed ovarian tissue just before transplantation (DMSO: 9.8 mg/g, EG: 9.8 mg/g). We must consider the impact of the cryoprotectants on the mother and the baby.
Keywords
Introduction
There have been 60 births after transplantation of cryopreserved ovarian tissue (
Donnez, Dolmans, 2015
): 58 using the slow freezing method, and two using the vitrification method. In the vitrification method, the concentration of cryoprotectants is four times higher and warming time is four times shorter than in slow freezing. Therefore, with the warming protocols in vitrification, there is concern that all the cryoprotectants, which may be toxic in the human body, should be washed out at the time of transplantation (Iwatani et al, 2006
).Our aim was to measure residual EG and DMSO concentration in ovarian tissue during warming steps after storage following vitrification and in the thawing steps following slow freezing.
Materials and methods
In this study, five pieces of donated ovarian tissue from four gender identity disorder (GID) patients and one uterine body cancer patient were used. Informed consent was obtained by the clinic which removed the patients' ovaries, and we did not have those patients' personal information. This study was approved by the Kyono ART Clinic Ethics Committee on 29th September 2016. Serum AMH level was 3.4 ± 2.1 ng/ml in the four GID patients and 6.4 ng/ml in the one uterine cancer patient.
Four ovarian tissue pieces (10x10x1mm) were cryopreserved by the vitrification method using 6.4 mol/l EG and DMSO (
Kagawa et al, 2009
) and one piece of ovarian tissue (8x4x1mm) was frozen by the slow freezing method using 1.5 mol/l DMSO (Isachenko et al, 2012
).Warming procedure following vitrification
The warming protocol of Cryotissue kit (Kitazato Corporation, Japan) was performed in 5 steps for a total of 16 min (
Kagawa et al, 2009
) and prepared for transplantation: 1] warming solution (immersion of ovarian tissue in solution of 1.0 mol/l sucrose at 37°C: TS) for 1 min; 2] dilution solution (immersion of ovarian tissue in solution of 0.5 mol/l sucrose: DS) for 5 min; 3] washing solution 1 for 5 min (WS1); 4] washing solution 2 for 5 min (WS2). And then 5] transplantation medium at room temperature.Thawing procedure following slow freezing
The thawing protocol of slow freezing was performed for a total of 65 min (
Isachenko et al, 2012
). The cryotubes were first taken from liquid nitrogen and exposed for 30 seconds to room temperature and then warmed for 2 min in a 37°C water bath until the ice was melted. The tissue was incubated in DPBS+10%HSA solution, with 0.75M (TS1), 0.375M (TS2), and 0.125M (TS3) of sucrose for each 15-minute step at room temperature. After thawing, the tissue was washed in 0M sucrose twice (WS1 and WS2) for each step of 10 min and transferred into the abdomen.Analysis of the concentration of cryoprotectants in ovarian tissue
DMSO and EG concentrations were quantified by gas chromatography mass spectrometry using GCMS-QP2010 Ultra (Shimadzu) with an InertCap Pure-WAX column (0.25 mm × 30m, 0.50 µm thickness; GL Science) by Shimadzu Techno-Research Inc., Kyoto, Japan. Two samples of ovarian tissue (0.021g-0.065g) were analysed per patient. The ovarian tissue was mixed with methanol, homogenized, and centrifuged for 5 min at 1500 g. Then, the supernatants were analyzed.
Deuterated DMSO (DMSO-d6) and EG (EG-d6) were used as internal standards. The column temperature was held at 80°C for 2 min initially, then raised at 5°C/min to 140°C then at 25°C/min to 240°C. Injector temperature was set at 240°C.
We measured the amount of cryoprotectants before warming (I), after TS (II), after DS (III), and after WS2 (IV) in the vitrification method, and before thawing (I), after TS3 (II), and after WS2 (III) in the slow freezing method.,
Results
For vitrification specimens, the maximum amount of DMSO and EG were 130.0 ± 8.2 mg/g and 112.5 ± 15.0 mg/g. For slow freezing specimens, these were 70 mg/g and < 0.05 mg/g, respectively. In the slow freezing method, most of the cryoprotectants were washed out and the amount reached zero after thawing. However, in the vitrification method, we detected around 10 mg/g DMSO and 10 mg/g EG in ovarian tissue after warming just before transplantation (Table 1).
Table 1The average residual amount of DMSO and EG in ovarian tissue.
| Sample | DMSO (mg/g) | EG (mg/g) |
|---|---|---|
| Vitrification (n = 4) | ||
| I. before warming | 130.0 ± 8.2 | 112.5 ± 15.0 |
| II. after TS | 73.0 ± 8.0 | 63.5 ± 15.0 |
| III. after DS | 37.0 ± 5.4 | 33.3 ± 5.6 |
| IV. after WS2 | 9.8 ± 3.3 | 9.8 ± 3.3 |
| Slow freezing (n = 1) | ||
| I. before warming | 70 | <0.05 |
| II. after TS3 | 0.25 | <0.05 |
| III. after WS2 | 0.39 | <0.05 |
a Values are mean ± SD.
Discussion
DMSO and/or EG have been used as cryoprotectants for human ovarian tissue cryopreservation (
Hashimoto et al, 2011
, Isachenko et al, 2012
, Kagawa et al, 2009
); however, it has been revealed that DMSO, which is known to be an epimutagen, although having no effect on about 99% of the DNA methylation profile, alters the DNA methylation status with methylation and demethylation in some particular loci (Iwatani et al, 2006
). Maternal toxicity was observed in oral intake of EG in animal. However, there were various impacts on fetuses after high oral intake of EG (Price et al, 1985
). In this study, we found most of the residual cryoprotectants in ovarian tissue were washed out and approached the detection limit in the case of the slow freezing method. However, in the vitrification method by Cryotissue kit, around 10 mg/g DMSO and 10 mg/g EG remained in ovarian tissue after warming just before transplantation. Safety is crucial in clinical medicine. We believe that DMSO and EG should be completely washed out when we transplant ovarian tissue into the human body after warming/thawing. Further research for improvement of cryopreservation methods, especially vitrification methods is needed.References
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- Dimethyl sulfoxide has an impact on epigenetic profile in mouse embryoid body.Stem Cells. 2006; 24: 2549-2556
- Successful vitrification of bovine and human ovarian tissue.RBM Online. 2009; 18: 568-577
- The developmental toxicity of ethylene glycol in rats and mice.Toxicol. Appl. Pharmacol. 1985; 81: 113-127
Biography
Yusuke Nakamura, graduated from Tokyo University of Agriculture received his B.Sc. in 2010, is working as an Embryologist at Kyono ART Clinic since 2010. His research interests include studies on ovarian tissue cryopreservation.
Article info
Publication history
Published online: June 06, 2017
Accepted:
May 10,
2017
Received in revised form:
May 8,
2017
Received:
October 15,
2016
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© 2017 The Authors. Published by Elsevier Ltd on behalf of Reproductive Healthcare Ltd.
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