Lecture 15: Gamete and Embryo Cryopreservation Flashcards
Sperm can be frozen “INDEFINTELY” with cryoprotectant and stored in liquid nitrogen?
T/F
True
When was the first human SPERM cryopreservation?
When was the first baby born from frozen sperm?
- 1950’s
- 1953
What reasons to people freeze sperm in a sperm bank?
6
- Cancer-related therapy
- Testicular or Prostate surgery
- Vasectomy
- High-risk occupational exposure
- IUI or ART
- Varied time intervals of partners
What is “Sperm Banking?”
Saving sperm for your own future use
What is “Sperm Donation?”
Allow your sperm to be used by someone else
What reasons do people use frozen sperm from donations?
3
- Used by couples who have male infertilities
- Couples who have genetic abnormalities
- Single Women or Lesbians
What are the qualifications of donor sperm?
3
- Around ages 20-39
- Pyschological, genetic and medical screening
- STD screening
Donor sperm are frozen and quarantined for ___ months to permit _______ of STD’s
- 6
- re-testing
What are the financial compensations for sperm donation?
- $125 per donation
- Donate 2-3 times per week (24-48 abstinence period)
- Expected Participation is 9-12 months
- 1099 tax form
What is “cryopreservation?”
Process where cells or whole tissues are preserved by cooling to low sub-zero temps in liquid nitrogen
What is the temperature they keep the sperm in liquid nitrogen?
- (77K)
- (-196 C)
- (-321 F)
What 2 ways are cells protected by freezing injury?
- Controlled cooling (& thawing) rate
- Cryoprotectants (a form of anti-freeze)
What is the Slow Freezing Danger zone between?
- 0 to -60 C
What happens between 0 to -5 C?
Cells and surrounding medium remain unfrozen and super-cooled
What happens between -5 to -15 C?
- Ice forms in external medium spontaneously or by controlled seeding
- Contents of cells remain unfrozen and super-cooled
What happens between -15 to -60 C?
Contents of cell freeze
What happens during Extracellular Ice Formation?
- Ice crystals form in EC space
- Cell membranes is permeable to liquid water but not ice
- Water expands as it freezes, EC ice crystals physically compresses cells
Why can Extracellular Ice cause mechanical damage to cell membrane?
Because the ice crystals are sharp
What happens during Solution Effects?
- Ice Crystals grow in freezing EC water
- Electrolytes and other solutes are excluded, causes them to become concentrated in reaming liquid water
During Solution Effects, Extracellular Fluid becomes HYPOtonic to cell.
T/F
False, it becomes HYPERtonic
What is “Osmolality?”
Concentration of particles dissolved in a fluid
What happens during Dehydration?
- Water migrates out of cell due to EX ice formation coupled with increasing solute concentration in liquid water
What is the fate of the cell during Dehydration?
What is the fate of the cell during Cytolysis?
- Cells shrinks from efflux of water
- Cell ruptures from influx of water
What happens during Intracellular Ice Formation?
- Intracellular ice is always fatal to cell, because organisms and tissues can tolerate some EC ice
What happens during The Rate of Cooling?
- Water moves out of the cells by osmosis due to increasing osmolality of EC environment as water is incorporated into ice crystals
The SLOWER the cooling rate the ____ intracellular water has to move out
Longer
What are “Cryoprotectants?”
Substance(s) used to protect biological tissue from freezing damage
What Permeates when in Cryoprotectants?
3
- Small molecules diffuse across membranes
- Form hydrogens bones with water to slow or prevents both IC and EC ice crystallization
- Protecting cells from solution effect by diluting electrolytes which are toxic to cells
What are some electrolytes that are toxic to cells?
Hint: there are at least 3 on this list
- Propylene glycol
- Glycerol
- DMSO
What Non-Permeates when in Cryoprotectants?
- Large molecules remain in EC
- Slow or prevent EC ice formation
- Can assist controlled dehydration of cells
- Used in combination with premating molecules
Optimal rate differs between _____ of differing _____ and ____ permeability.
- cells
- sizes
- water
What are the 4 steps of the Sperm Cryopreservation Standard Protocol.
- Semen analysis of fresh sample
- Divide entire specimen into vials containing equal number of sperm
- Add cyroprotectant and slowly freeze in liquid nitrogen
- Thaw one aliquot, remove cyroprotectant and repeat semen analysis
Frozen is better than Fresh Sperm.
T/F
False. Fresh is better
What is the post-thaw motility of humans sperm range?
20-65%
What reasons are their loss of motility by post-thaw?
3
- Decreased integrity of membranes
- Cryo-damage to membranes of IC compartments
- Altered energy metabolism and synthesis
When was the first baby born from frozen embryo born?
What did Italy banned embryo freezing?
- 1984
- 2004
Frozen embryo transfer rate is similar to or better than fresh transfer.
T/F
True
With Oocyte freezing:
1) What is the size of the sperm and head?
2) What is the size of the Blastomere?
3) What is the size of the Oocyte?
1) 5-60 picometers; 5x3 picometers
2) 40 picometers in diameter
3) 130 picometers in diameter
Gas barrier forms between a hot surface (_____) and boiling liquid (______) if temperature it great enough.
Warm egg; liquid nitrogen
Gas barrier greatly _____ heat transfer between the two which allows liquid to ____ _______ and consequently hot surface to remain hot longer
slow; last longer
Meitotic Spindle is highly sensitive to temperature.
T/F
True
Microtubules and Microfilaments damage may lead to ____ division of ______
Abnormal; Chromosomes
Zona Pellucida hardening through a ______cortical granule release
premature
Cortical reaction normally occurs BEFORE fertilization to prevent polyspermy.
T/F
False; occurs AFTER fertilization
Why do women freeze their eggs for Egg Banks?
4
- they are cancer patients
- women who want to delay reproduction
- failure to obtain sperm on retrieval day
- ethical objections to embryo cryopreservation
Egg donation involve fresh egg donation more than embryo freezing.
T/F
True
What are the criteria for egg donation and compensation?
- ages 20-30
- physiological, genetic and medical screening
- $3,500-$10,000 per donation
- May donate 6 times in their lifetime
What is “Vitrification?”
Process of converting material into glassy, amorphous solid free from crystalline structure
What is the difference between Slow freeze and vitrification in regards to Saline solution?
Vitrified solution is clear
What is “Autotransplanation?”
transplant tissue back into same individual
What is “Allotransplantation?”
Transplant tissue into the same species but a different individuals
What is “Xenotransplantation?”
Transplant tissue from one species to another
What is “Heterotopic?”
Transplant tissue so it is not in its normal position
What is “Orthotopic?”
Transplant tissue back into natural position
What is “Vascular Anastomosis?”
Connect two cut or separate blood vessels to form a continuous channel
