Hello, my name is Marina and now Im going to present you a short review about embryo and

oocyte cryopreservation, with its methods, problems and indications.

Cryopreservation is a technique that is achieved by lowering the temperature of the
maintenance and survival of oocytes and embryos. This exposes the oocytes and embryos to
cryoprotectants, freezing at subzero temperatures, then storage, thawing and finally going
back to the same physiological conditions.

In this temperature reduction, the major obstacle is the transition from liquid water to ice. This
process produces intracellular and extracellular ice, solution effects and osmotic shock that
produces cell damage.

Hence, the fundamental principle of cryopreservation is: Reduce the harm caused by the
formation of intracellular ice.

There are two factors let us reduce cryoinjuries: cooling rates and cryoprotectants.
Currently, the most used cryopreservation techniques are:
- Slow-freezing method and
- Vitrification
Basically differs in cryoprotectants application and concentration, and cooling rates

The first one consists in minimizing intracellular ice formation to achieve a balance
between the speed of dehydration, and the slow decrease in temperature. This is done by
cooling rates of 0.3C/minut, controlling the crystal ice formation through manual seeding and
with low concentration of cryoprotectant which is associated with a low toxicity.
Programmable freezers are needed for a strictly control in temperature conditions.

However, vitrification consists in very high cooling rates between 2000 and 20,000 C/ min.
and directly immersed into liquid nitrogen. It is caused by the solidification of a solution
without crystallization, due to the high cryoprotectant concentracions used. There is an
extreme elevation in viscosity of the solution. But it is associated with a high toxicity. Hence, in
vitrification, the volume of solution and time of exposure to cryoprotectant should be
minimum. We need appropriate carrier systems, like cryotops, cryotips
(Es una presentacin, no un artculo ;) puedes usar la tercera forma plural ;)
If we compare both methods well found that vitrification is superior to the slow-freezing
method. This leads to an improved oocyte survival rate, cleaved and embryo quality rates, and
blastocysts development in vitro. These results may be related to vitrified human oocytes
incurring less damage to spindle integrity and chromosome alignment.
NEGATIVE EFFECTS
There are several problems associated with oocyte or embryo cryopreservation:
- Low rates of survival and fertilization by inappropriate methodology probably.
- Impaired function of the zona pellucida because of premature exocytosis of cortical
granules
- Increased rate of polyploidy because of Polyspermy or No extrusion the 2nd Polar
body.
- Alteration of cytoskeleton elements, for example in a Meiotic Spindle.
- parthenogenetic activation.
- ultrastructural damage, for example vacuolization.

INDICATIONS
The indications for this type of techniques are varied:
- Increases the chances of pregnancy without go through another cycle of stimulation /
puncture

- For Delayed maternity: there are many women that delay maternity till thirties and
forties (30 40) due to her profession expectations, and other reasons. This
represents a decrease in ovarian reserve, it makes going to the receipt of donated
embryos or oocytes, or freezing their oocytes when their ovarian reserve has not yet
been relegated to use much later.

- Also Creation a Oocyte bank:
o Avoids the donor-receiver synchronization.
o Useful in case of unavailability of donors in a given time.
o Availability of specific phenotypes.
o Travel-Programming foreign patients.
o Oocyte-leftovers when no receiver available.

- For Fertility preservation too: in cancer cases is important cryopreserve oocytes,
embryos or ovarian tissue, depends on the case: if chemo can be delayed or not and
hormonal therapy is contraindicated or not, depends on the male partner too.

- In case of failure to obtain the semen sample on the day of follicular puncture: When
this happens everything freezes until obtaining the sample. Although usually frozen
semen in these cases before puncture.

- For Elective Single embryo transfer: if we choose to transfer only one embryo and
freeze the rest, reduces the risk of multiple pregnancy, prematurity, low birth weight
newborns (when compared to transfers of 2 embryos), but this method also reduces
the pregnancy rate by transfer.

- In case of risk of ovarian hiperstimulation: is an abnormally high ovarian response after
hCG administration. Transfer is not recommended in these cases. Therefore, in these
cases it is recommended to freeze all

- For best pregnancy rates: There are some facultative, as the Dr Checa from Hospital
del Mar in Barcelona, who recommends freezing all, in his studies showed that the
probability of a clinical pregnancy is significantly higher from freeze-all cycles than in
fresh embryo transfers. This approach avoids the ill effects fertility drugs may have on
the lining of the uterus (endometrium) and its ability to receive the embryo for
implantation. [Roque M, Lattes K, Serra S, Sol I, Geber S, Carreras R, Checa MA. Fresh
embryo transfer versus frozen embryo transfer in in vitro fertilization cycles: a
systematic review and meta-analysis. Fertil Steril. 2013 Jan;99(1):156-62.].

CONCLUSIONS
As a conclusion
- The cryopreservation technique is quite complex, with many parameters to consider but
allows more advantages than disadvantages, as preserving fertility or increase the chances of
pregnancy without go through another cycle of stimulation / puncture.
-Vitrification is currently the most efficient method of oocyte freezing in obtaining a
pregnancy. (Smith GD, Serafini PC, Fioravanti J,et.al. Prospective randomized comparison of
human oocyte cryopreservation with slow-rate freezing or vitrification. Fertil Steril. 2010
Nov;94(6):2088-95 )
- And new protocols suggest that freeze-all action is a good way to get better pregnancy rates.
So they are finding more advantages than disadvantages in these techniques that are testing
and developing day by day.
Thank you very much. Questions?