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Preservation of human ovarian follicles within tissue frozen by vitrification in a xeno-free closed system using only ethylene glycol as a permeating cryoprotectant.

Fertility and sterility (2013-04-10)
Mona Sheikhi, Kjell Hultenby, Boel Niklasson, Monalill Lundqvist, Outi Hovatta
ABSTRACT

To study the preservation of follicles within ovarian tissue vitrified using only one or a combination of three permeating cryoprotectants. Experimental study. University hospital. Ovarian tissue was donated by consenting women undergoing elective cesarean section. Ovarian tissue was vitrified in closed sealed vials using either a combination of dimethyl sulfoxide, 1,2-propanediol, and ethylene glycol (EG), or only EG as permeating cryoprotectants. Ovarian tissue was vitrified with the use of two vitrification methods. Tissue from the same donor was used for comparison of two different solutions. The morphology of the follicles was evaluated after vitrification, warming, and culture by light microscopy and transmission electron microscopy. Apoptosis was assessed by immunohistochemistry for active caspase-3 in fresh and vitrified tissue. Light and electron microscopic analysis showed equally well preserved morphology of oocytes, granulosa cells, and ovarian stroma when either of the vitrification solutions was used. No apoptosis was observed in primordial and primary follicles. Using only EG as a permeating cryoprotectant in a closed tube gives as good ultrastructural preservation of ovarian follicles as a more complicated system using several cryoprotectants.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Ethylene glycol, anhydrous, 99.8%
Sigma-Aldrich
Ethylene glycol, ReagentPlus®, ≥99%
Supelco
Ethylene glycol, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Ethylene glycol, spectrophotometric grade, ≥99%
Supelco
Ethylene glycol, analytical standard
Sigma-Aldrich
Ethylene glycol, BioUltra, ≥99.5% (GC)
Sigma-Aldrich
Ethylene glycol 5 M solution
Supelco
Ethylene glycol solution, NMR reference standard, 80% in DMSO-d6 (99.9 atom % D), NMR tube size 5 mm × 8 in.
Supelco
Ethylene glycol solution, NMR reference standard, 80% in DMSO-d6 (99.9 atom % D), NMR tube size 3 mm × 8 in.