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

Clinical-grade vitrification of human ovarian tissue in fertility preservation, with the use of only one permeating cryoprotectant, ethylene glycol, in a closed system results in intact follicular ultrastructure.


Mona Sheikhi, M.Sc., Kjell Hultenby, Ph.D., Boel Niklasson, R.N.M., Monalill Lundqvist, Ph.D., Outi Hovatta, M.D., Ph.D.

Volume 100, Issue 1, Pages 170-177.e2, July 2013



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.

Main Outcome Measure(s):

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.

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