We discuss here all essential principles of cryoperservation in assisted reproductive techniques: 1. Oocye cryoperservation 2. Embryo cryopersevration 3. Sperm cryopersevration

1. CRYOPRESERVATION…… HOPE IN A
TANK???
DR. YASMIN MAGDI ABD-ELKREEM

2. What is Cryopreservation?
• Enable to interfere biological clock and to stop it for a while until it is
thawed.
• Cryopreservation is the freezing of cells or tissues to sub zero temperatures,
typically -196 º C (boiling point of liquid nitrogen).
• Basically, cryopreservation is a procedure to preserve the gamete,
embryonic cells or tissues by arresting or slowing metabolic activity until
the subsequent thawing procedures.
• Cryopreservation era arised with the accidentally understanding of the value
of cryoprotectants in 1949 by Christopher Polge.

3. Intracellular ice formation(IIF):
Cooling to storage temperature is accomplished by formation of ice in the intracellular as
well as extracellular compartments. Extensive IIF is incompatible with the preservation of
cell viability, causing widespread physical/structural damage.
Prevention of IIF is achieved by:
• Use of cryoprotective agents(CPAs), chemicals that
interfere with the water and interact with
biomolecules.(Osmotic gradient based on kinetic
bases)
• Permeating or Intracellular CAPs: are oligohydroxy
compounds of relative low toxicity which can enter
cell across the plasmamembrane.
• Impermeating or extracellular CAPs: are
polyhydroxy compounds and are unable to diffuse
intracellular compartment
• The cell is cooled down to subzero slowly (normally to a
rate of -2 ͦC/min) to avoid thermal shock. (Control ice
nucleation)
A] Water replacement: B] Slow cooling:
Essential principles of controlled rate cooling

4. Two basic techniques currently rule the field of
cryopreservation
Slow freezing
• Lower cryoprotectant (less
toxic) concentration.
• Longer exposure time.
• Longer to perform.
• Fast cooling rates results in
solidification of solution into
glass-like structure(no
crystalization).
• Technically easier.
• Closed system.
• Freezing machine.
• Greater chance of ice formation.
• Not cost effective.
Vitrification
• Higher cryoprotectant
concentration.
• Shorter exposure time.
• Shorter to perform.
• Slow dehydration to minimize
ice-crystal formation.
• More clinical expertise.
• Open or closed system.
• No machine needed.
• Less chance of ice formation.
• Cost effective.

6. How to maximize the outcome of vitrification?
1. Technical parameters
1. Type and concentration of cryoprotectants, even all cryoprotectants are toxic.
2. Temperature of vitrification solution at exposure.
3. Lenght of time embryos are exposed to the final cryoprotectant before plunging into liquid
nitrogen.
4. Variability in the volume of cryoprotectant solution surrounding the embryos
5. Technical proficiency of the embryologists
6. Cooling speed of vitrification sample size and type of carrier.
2. Embryonic parameters
1. The quality and embryonic stage.
2. Inherent fecundity.
Vitrification is associated with a significantly lower cellular trauma and higher post-warming
survival, pregnancy, and implantation rates than with other cryopreservation techniques.

7. Why is Oocyte Cryopreservation?
I. Women at risk of losing ovarian function
II. Women desiring fertility preservation (e.g. delayed maternity).
III. Eliminating ethical concerns of embryo cryopreservation.
IV. Solving the dilemma of abandoned frozen embryos in the IVF laboratory.

8. First pregnancy after oocyte
cryopreservation was reported by Chen in
1986.
Less than 200 pregnancies have been
achieved worldwide.
Survival rate of 80% and fertilization rates
of 83%, however low pregnancy rates.
Although pregnancy rates might be
improving, rates appear to be significantly
less than those seen with standard IVF.
Procedure abandoned for approx. 10 years
due to poor results
 Low fertilisation rate
 Low survival rates
 Hardening of zona
 Possible spindle damage
Oocyte Freezing:
Where do we
stand?

9. Why is Embryo Cryopreservation?
I. Large number of good-quality embryos or blastocyst available for transfer.
II. Female uterus is not ready to receive embryos.
III. Reduction in the incident of multiple pregnancies.
IV. Decrease in the need of repeated cycles of ovarian stimulation.
V. Embryo donation.
VI. Thaw cycle is cost-effective, approximately 1/5 of normal ICSI cycle.
VII. Detect infectious disease and genetic abnormalities
VIII. Ovarian hyper-stimulation syndrome.

10. By the 1980s, the freezing of human
embryos emerged as a common procedure in
the treatment of infertile couples.
First pregnancy after embryo
cryopreservation was reported by Trounson
and Mohr in 1983.
Two year later, Cohen published new
procedure for human blastocyst stage
embryo freezing.
Nowadays, 31% of embryo transfers utilize
embryo cryopreserved/warmed transfer
cycles.
8 % of all ART babies are born from
cryopreserved embryos.
Embryo Freezing:
Where do we
stand?

11. When embryos are cryopreserved?
• PN stage : (18-22 hours after ICSI)
• Cleaved stage: Day 2, 3 or 4 after ICSI.
• Blastocyst stage: Day 5, 6 or 7 after ICSI.

13. Why is Sperm Cryopreservation?
I. Semen containing a very limited number of spermatozoa or abnormal
semen parameters.
II. For cancer patients to preserve their fertility prior to gonadotoxic
chemotherapy or radiation.
III. Patients undergoing certain types of pelvic or testicular surgeries
IV. Patients who suffer from degenerative illnesses such as diabetes or multiple
sclerosis; spinal cord disease or injury.
V. Persons in occupations where a significant risk of gonadotoxicity prevails.
VI. Surgical sterilization such as vasectomy.
VII.Donor semen samples .
VIII.Used in combination with ART techniques.

14. 1950, the methods for cryopreserving
human semen and artificial insemination
were refined, resulting in the first human
birth.
Today, conservative estimates prove that
more than 300,000 births have been
achieved through artificial insemination with
cryopreserved semen.
cryopreserved human semen maintained for
over 30 years, no limit has been established
for how long human semen can be frozen .
Sperm cryopreservation and banking has
become a widely accepted procedures.
Sperm Freezing:
Where do we
stand?

15. • Embryo, sperm, oocyte are generally use and achieve by frozen-thawed
programs in Human ART program.
Vitrification has superior outcomes over slow freezing.
Conclusions…………

16. Email: Yas.magdi@hotmail.com