CRYOPRESERVATION…… HOPE IN A TANK ???

DR. YASMIN MAGDI ABD-ELKREEM

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.

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

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.

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 parameters7. The quality and embryonic stage.8. 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.

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.

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?

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.

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?

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.

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 surgeriesIV. 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.

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?

• Embryo, sperm, oocyte are generally use and achieve by frozen-thawed programs in Human ART program.

Vitrification has superior outcomes over slow freezing.

Conclusions…………

Email: Yas.magdi@hotmail.com