Overview on Embryos Cryopreservation slow cryo and rapid cryo & Vitrification, cryoprotectants work, cryoprotectatns mechanism, rapid thaw, ovarian tisssue cryopreservation

1. An Overview on Embryos Cryopreservation
& Vitrification
Mohamed Fadel Al Mohr
Embryologist
IVF Lab Director
B.Sc. in Biological Science (Zoology)
MSc in Embryo culture
Fadell20@gmail.com

2. Cryopreservation
Use of very low temperatures to preserve structurally
intact living cells, tissues and organs at the ultra-low
temperature of liquid nitrogen (-196°c).
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3. Why liquid Nitrogen?
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4. At this temperature, the vegetative cells enter in a
state of “Absolute Quiescence”, as all the physical
and biochemical reactions are practically halted.
In this particular condition, conservation time
becomes unlimited.
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5. Disadvantage Of Cryopreservation
The major disadvantage to using low temperature
that lead to crystallization of water, this can create
new and unwanted physical and chemical events
that may injure the cells that are being preserved.
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6. Applications of cryopreservation in
IVF
1- Ovarian Tissue 2- Oocytes and embryos 3-Sperm , semen and testicular
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7. Ovarian Tissue Cryopreservation
Indication
 Strategy for fertility conservation.
 Option is best suited for patients
who are less than 30 years old and
have had no previous chemo- or
radiotherapy.
Criteria
The uterus must be functional and the
patient should have a high probability
of long-term survival after treatment.
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9. Oocyte Cryopreservation
Indication
 Prior to cancer treatments like chemo/radiotherapy.
 Prior to organ transplant therapy (liver transplant,
bone marrow transplant, etc. ;)
 In severe Poly Cystic Ovarian Syndrome cases.
 In cases where male partner fails to produce semen
sample on the day of IVF after egg pickup and
ICSI/IVF needs to be postponed.
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10. Single Women Wanting To Preserve The Fertility
(Social Egg Freezing).
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11. Embryos Cryopreservation
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12. Indication
 Reduced multiple pregnancies.
 Storage of good quality surplus embryos for later use.
 In case of ovarian hyper-stimulation syndrome.
 Thin endometrium.
 Thick endometrium.
 Fluid in endometrial cavity.
 Bleeding in cycle.
 Polyp.
 Difficult embryo transfer.
 PGD or PGS.
 Preservation of fertility. Future offspring's.
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13. Stage of freeze embryos
 PN Stage
 Cleavage Stage
 Blastocyst Stage
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14. Cryopreservation Requirements
 Liquid nitrogen
 Device
 Cryo-protectants
Mechanism of Cryopreservation
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15. Cryoprotectants
Cryoprotectants are substances that are used to protect biological tissue
from freezing damage by achieving the required intracellular
dehydration.
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16. Characteristics Of Cryoprotectants:
 Higher degree of cell survival during freezing.
 Lower the freezing point.
 Protect cell membrane from freeze-related injury.
 High solubility.
 Low toxicity at high concentrations.
 Low molecular weight.
 Ability to interact with water via hydrogen bonding.
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17. How CRYOPROTECTANTS work?!
Cryoprotectants acts by salt buffering action
(bind water and less ice crystal formation).
1-Gives more time to cell for de-hydrate.
2- Interact with cell membrane to make it less
brittle during freezing.
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18. Types of cryoprotectants
Based on their ability to diffuse across cell membranes
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19. A- Permeating cryoprotectants
 Lower the freezing point of the solution.
 increase the viscosity and thus reduce diffusion in solutions.
 Replace intracellular water to prevent the formation of
large ice crystals intracellularly.
 Ability to reduce the amount of water which freezes as ice.
EX(Dimethyl sulphoxide DEMSO, Glycerol, Propylene glycol,
Methanol, Ethylene glycol and Dimethyl acetamide.)
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20. B- Non-permeating cryoprotectants
 LOW MOLECULAR WEIGHT
It changes the water balance of cells by shrinking cells before
freezing.
Sucrose, Maltose, Trehalose, Sorbitol and Acetamide.
 HIGH MOLECULAR WEIGHT
Closing the cell membrane defect. Repair damaged cell
membrane post thawing.
Polyvinyl Pyrorolidone (PVP), Dextran, Serum.
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21. Types of Cryopreservation
1- Slow-freezing
2- Rapid freezing (vitrification)
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22. Slow Freezing
 Low levels of cryoprotectants.
 Slow controlled rates of cooling (0.3o C/min).
 Slow dehydration to minimize ice-crystal formation.
 takes hours.
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23. Rapid Freezing (Vitrification)
 High levels of cryoprotectants
 Very fast cooling rates (~20,000o C/min)
 Fast cooling rates result in solidification of solution into glass-like
structure (no crystallization)
 takes seconds
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25. Slow-cryo or Rapid-cryo protocols both satisfy the fundamental cryo-biological
principles for reduction of damage by ice crystal formation during cooling and
warming in six principal steps:
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26. 1) Initial exposure to the cryoprotectant (intracellular water
has to be removed by gradual dehydration)
2) Cooling (slow/rapid) to subzero temperatures (-196°C),
3) Storage at low temperature,
4) thawing/warming by gradual rehydration,
5) Dilution and removal of the cryoprotectant agents and
replacement of the cellular and intracellular fluid at precise
rate.
6) Recovery and return to a physiological environment
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27. So that vitrification is the future
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28. What Is Vitrification Meaning?
Vitrification (decrystalization). It completely avoids ice crystal
formation in cryopreserved cells during warming to recover the
cells for biological applications.
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29. Scientific Technique Of Vitrification
To achieve this glass-like solidification of living
cells for cryostorage:
A- high cooling rates
B- in combination with high concentrations of
cryoprotectants are used.
A primary strategy for vitrifying cells and tissue is to
increase the speed of thermal conductivity (Open
and Closed System) , while decreasing the
concentration of the vitrificants to reduce their
potential toxicity.
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30. There Are Two Main Ways To Achieve The
Vitrification Of Water Inside Cells Efficiently:
 1) to increase the cooling rate by using special
carriers that allow very small volume sizes
containing the cells to be very rapidly cooled;
and
 2) Find materials with rapid heat transfer.
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31. Types Of Vitrification
1- Closed System M FADEL

32. 2- Open System
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33. Steps Of vitrification :
1- Equilibrium Solution
(DMSO, ethylene glycol and HAS)
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35. 2- Vitrification Solution (Sucrose ,
DEMSO , ethylene glycol and HAS)
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38. Open System
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39. Closed system
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40. Thawing
 1. TS ( High concentration with low CP)
 2. DS (low concentration with low CP)
CP in thawing solution mainly Extracellular
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41. Factors That Can Affect The
Vitrification Outcome
• Embryo Selection
• Post-thaw blastocyst selection
• Blastocoele collapse
• Media protocols
• Freezing rate
• Warming rate
• Assisted hatching
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44. Thank You
Mohamed Fadel Al Mohr
Embryologist
IVF Lab Director
B.Sc. in Biological Science (Zoology)
MSc in Embryo culture
Fadell20@gmail.com