|
Middle East Fertility Society Journal
Middle East Fertility Society
ISSN: 1110-5690
Vol. 10, Num. 3, 2005, pp. 205-206
|
Middle East Fertility Society Journal, Vol. 10, No. 3, 2005, pp.
205-206
DEBATE
Vitrification
versus conventional cryopreservation technique - Comment by: Imam El-Danasouri,
D.V.M., Ph.D.
& Helmy Selman Ph.D.
Imam El-Danasouri1, D.V.M., Ph.D., Helmy Selman2,
1Endokrinilogikum, Ulm, Germany,
2University of Perugia, Perugia, Italy
Code Number: mf05034
Related Articles:
Comment by: Imam El-Danasouri,
D.V.M., Ph.D. & Helmy Selman Ph.D.
Comment by: Yasser Orief,
M.D., Askan Schultze-Mosgau, M.D. & Safaa
Al-Hasani, M.D.
Comment by: Khaled Elnomrosy,
MRCOG, MD., Bassam Elhelw, MRCOG & Mostafa
El Sadek, M.D.
During in vitro
fertilization treatment (IVF), a surplus of oocytes is produced which results
in a surplus of embryos which must be preserved. Due to the recent tendency in
ART treatment to transfer fewer embryos in order to avoid multiple pregnancies,
partly a result of new laws and guidelines aimed at controlling the number of
embryos to be transferred and the number of oocytes to be fertilized, the need
for simpler, more effective methods for the cryopreservation of embryos and
oocytes is increasing. Currently, zygotes and embryos are typically
cryopreserved by means of the traditional slow-rate freezing protocol, while
oocyte freezing is still in the experimental phase.
However, during the slow
freezing method, ice crystals are formed or produced, which has a deleterious
effect on the cells. Intracellular ice crystals can damage the cell wall and
structure, while the extracellular precipitation of water as ice crystals
increases the salt concentration to levels that cause damage to the cell. A
delicate balance between these potentially harmful factors must be maintained throughout
the slow freezing process to ensure survival of the cells.
In 1985, the ultra-fast
freezing method through vitrification was introduced based on inducing
solidification of the cells and the surrounding vitrification solution
(glass-like or vitreous) without formation of any ice crystallization. This can
be achieved through the combination of the following factors:
Increasing the concentration of
cryoprotectant: The cryoprotectant is usually used in much higher
concentration than in the slow freezing method. To avoid the toxic effect of
the cryoprotectant, cells are equilibrated in a low concentration solution
before immersion in the high concentration solution (vitrification solution).
The equilibration solution contains between 10 to 15% of the cryoprotectant
while the vitrification solution contains between 35 and 40% (around 5.5 Molar)
of the cryoprotectant. Selecting the optimal cryoprotectant is critical. It
should be of high permeability (low molecular weight) and as non-toxic to the cells
as possible. Ethylene Glycol (EG) has these characteristics and, to lesser
extent, also DMSO.
- Dehydration of the cells: This is achieved
by using a high concentration of nonpermeating cryoprotectant of a large
molecular weight such as disaccharide sugars: sucrose or galactose at a
concentration up to 1 Molar. The end concentration of the nonpermeating
cryoprotectant is added to the vitrification solution. We cannot overemphasize
the importance of minimizing the cells' exposure time in the highly
concentrated vitrification solution; successful vitrification procedures
must limit such exposure to a few seconds.
- Very
fast freezing rate: The vitrification device containing the cells is directly
plunged into liquid nitrogen which can achieve a cooling rate of more than
20,000°C/ minute. The rate depends on the vitrification device used and on the
freezing volume. No special equipment is needed to achieve such a freezing
rate. When the vitrification device enters the liquid nitrogen, a coat of liquid
nitrogen vapor is generated around it, creating an insulation layer that can
lower the cooling rate. Moving the device continuously upon its entry into
the liquid nitrogen until complete solidification occurs easily eliminates
this
effect.
- Increasing the viscosity of the vitrification
solution: The viscosity of the vitrification solution increases during the
cooling process until the solution solidifies. The increased viscosity of the
vitrification solution is automatically achieved through the rapid cooling
process. The viscosity can also be increased through the use of high
concentrations of both the permeating and nonpermeating cryoprotectants and the
addition of macromolecules such as PVP or Ficoll.
- Minimizing the vitrification volume: Several
devices have been developed and used successfully for the vitrification of
oocytes and embryos. Using a "plastic loop" reduces the
vitrification volume to a liquid film within the loop and the "open pulled
straws" allow for a very small vitrification volume which does not exceed
2µl. In this context, the loading of the cells in the vitrification device and
the handling of the device are critical since this is done during the cells'
exposure to the highly concentrated vitrification solution. The choice of the
device is a personal decision depending on the skill and preference of the
individual operator. However, speed and dexterity are crucial as the goal is
to minimize the cells' exposure to the high concentration solution prior to
solidification.
- Rapid warming rate: The vitrified cells
should be warmed rapidly to avoid crystallization as in the fast cooling
process. The vitrified material is immersed directly in the warmed thawing
solution and the rehydration of the cells is usually performed in two or three
steps.
Advantages of vitrification
- The vitrification process is both simple and
fast; it can be completed in less than 10 minutes.
- The prevention of ice crystal formation
during the vitrification process successfully eliminates the major factors
that can cause cell damage during the slow freezing method.
- The
process is easy to master: Vitrification involves simply moving the cells
between two dilutions, loading them into the vitrification device, and plunging
them into liquid nitrogen.
- One of the advantages of vitrification is
that it allows the operator to observe the cells during the vitrification
process. None viable zygotes and blastomeres can be recognized since they do
not contract upon placement into the first or the second vitrification
solution. The presence of contracted cells during the warming process is a
useful marker for cell survival with the exception of oocytes which might
survive the vitrification and warming processes, but show degenerative signs
approximately 10 to 15 minutes after the warming is completed.
- The fact that no special equipment or
investments are needed for implementation of the vitrification procedure
makes it extremely cost-effective, and offers a tremendous economic advantage
over
traditional methods.
Vitrification is a very
simple effective process and the skill needed to perform it can be acquired in
a short time through training on materials to be discarded. Several IVF
programs have adopted the vitrification method as the sole procedure for day-3
human embryos and for human blastocysts, with excellent survival and pregnancy
rates (2, 3). The challenge now is to find a protocol to successfully vitrify
human oocytes, for which the slow freezing method has yet to produce acceptable
survival and development rates.
REFERENCE
- Rall
WF, Fahy GM. Ice-free cryopreservation of mouse embryos at 196°C by vitrification. Nature
1985;313:573-5.
- El-Danasouri,
I. and Selman, H. Successful pregnancies and deliveries after a simple
vitrification protocol for day-three human embryos. Fertil Steril.
76: 400-2, 2001
- Takahashi
K, Mukaida T, Goto T, Oka C: Perinatal outcome of blastocyst transfer
with vitrification using cryoloop: a 4-year follow-up study. Fertil Steril.
2005 Jul;84 (1):88-92.
© Copyright 2005 - Middle East Fertility Society
|