Chapter 20
Cryopreservation of Mammalian Oocytes: Slow Cooling
and Vitrification as Successful Methods for Cryogenic
Storage
Victoria Keros and Barry J. Fuller
Abstract
Two basic methods for the laboratory-focused cryopreservation of mammalian oocytes are described, based
on work with murine oocytes. One method uses a relatively low concentration of the cryoprotectant
propanediol plus sucrose and requires controlled rate cooling equipment to achieve a slow cooling rate.
This method has also produced live births from cryopreserved human oocytes. The second method, which
is described here, employs a high concentration of the cryoprotectant dimethyl sulfoxide plus a low
concentration of polyethylene glycol. This is a vitrification method, which involves ultra-rapid cooling by
plunging standard straws into liquid nitrogen vapor, hence avoiding the need for specialized equipment,
but requires technical ability to manipulate the oocytes quickly in the highly concentrated cryoprotectant
solutions. Murine oocytes that have been vitrified using this technique have resulted in live births.
Vitrification using other cryoprotectant mixtures is now a popular clinically accepted method for cryobanking of human oocytes.
Key words Oocyte, Slow cooling, Vitrification, Murine, Human
1 Introduction
Successful cryopreservation of mature oocytes has been one area of
reproductive cryobiology, which has challenged our understanding
of cryobiology over the past 30 years, in ways unforeseen at the
beginning of the studies. Much had to be learnt about the specific
ultrastructural and physiological characteristics of these large
unique cells, in order to be able to recover functional oocytes
from cryogenic storage, and these details have been published in
many previous reviews [1–5]. With improved outcomes, in the past
decade, there was a renaissance of interest in applying oocyte cryopreservation [6] and the technology has moved from a largely
research-focused activity into the main stream of clinical reproductive medicine, resulting in the recent designation from the ASRM as
a justified therapy [7].
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_20, © Springer Science+Business Media, LLC, part of Springer Nature 2021
437
Cryopreservation of Mammalian Oocytes: Slow Cooling
and Vitrification as Successful Methods for Cryogenic
Storage
Victoria Keros and Barry J. Fuller
Abstract
Two basic methods for the laboratory-focused cryopreservation of mammalian oocytes are described, based
on work with murine oocytes. One method uses a relatively low concentration of the cryoprotectant
propanediol plus sucrose and requires controlled rate cooling equipment to achieve a slow cooling rate.
This method has also produced live births from cryopreserved human oocytes. The second method, which
is described here, employs a high concentration of the cryoprotectant dimethyl sulfoxide plus a low
concentration of polyethylene glycol. This is a vitrification method, which involves ultra-rapid cooling by
plunging standard straws into liquid nitrogen vapor, hence avoiding the need for specialized equipment,
but requires technical ability to manipulate the oocytes quickly in the highly concentrated cryoprotectant
solutions. Murine oocytes that have been vitrified using this technique have resulted in live births.
Vitrification using other cryoprotectant mixtures is now a popular clinically accepted method for cryobanking of human oocytes.
Key words Oocyte, Slow cooling, Vitrification, Murine, Human
1 Introduction
Successful cryopreservation of mature oocytes has been one area of
reproductive cryobiology, which has challenged our understanding
of cryobiology over the past 30 years, in ways unforeseen at the
beginning of the studies. Much had to be learnt about the specific
ultrastructural and physiological characteristics of these large
unique cells, in order to be able to recover functional oocytes
from cryogenic storage, and these details have been published in
many previous reviews [1–5]. With improved outcomes, in the past
decade, there was a renaissance of interest in applying oocyte cryopreservation [6] and the technology has moved from a largely
research-focused activity into the main stream of clinical reproductive medicine, resulting in the recent designation from the ASRM as
a justified therapy [7].
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_20, © Springer Science+Business Media, LLC, part of Springer Nature 2021
437