Pre-Implantation Genetic Diagnosis

Question 1

Sperm Cryopreservation

Answer 1

• Sperm can be frozen “indefinitely” with cryoprotectant and stored in liquid nitrogen o First human sperm cryopreservation in 1950s o First baby born from frozen sperm in 1953 • How long is sperm viable? o Baby born in 2009 from sperm frozen for more than 20 years

Question 2

Sperm Banking

Answer 2

Saving your sperm for your own future use • Cancer-related therapy • Testicular or prostate surgery • Vasectomy • High risk occupational exposure • IUI or assisted reproductive technology • Time when partner’s schedule does not permit availability

Question 3

Sperm Donation

Answer 3

Allowing your sperm to be used by someone else • Used by couple where male is infertile or carries genetic abnormality, single women, lesbians • Anonymous donors are selected from sperm banks that recruit and screen donors o Generally age 20-39 o Psychological, genetic and medical screening (including STD’s) • Donor sperm are frozen and quarantined for 6 months to permit re-testing for STDs

Question 4

Financial Compensation for sperm donation

Answer 4

• $75 per donation • must donate 2-3 times per week (with 24-48 hours abstinence between samples) for minimum of 6 months • 3/week * 4weeks/month * 6 months * $75= $5,400 • 1099 tax form (miscellaneous income for independent contractor) ** Egg donors receive $3,500-10,000 per donation and can donate up to 6-9 times in their lifetime

Question 5

Cryopreservation

Answer 5

a process where cells or whole tissues are preserved by cooling to low sub-zero temperatures in liquid nitrogen (77K; -196C; -321F) • at low sub-zero temperatures biological activity, including biochemical reactions that would lead to cell death, is effectively stopped • Cells are protected from freezing injury by: o Controlled cooling (& thawing) rate o Cryoprotectants (a form of anti-freeze)

Question 6

Slow freezing danger zone: 0 to -60 C

Answer 6

• 0 to -5 C o cells and surrounding medium remain unfrozen and super-cooled • -5 to -15 C o ice forms in external medium spontaneously or by controlled seeding o contents of cells remain unfrozen and super-cooled • -15 to -60 C o contents of cell freeze

Question 7

Causes of damage to cells during slow freezing

Answer 7

1. Extracellular ice formation 2. Solution effects 3. Dehydration 4. Intracellular ice formation 5. Rate of cooling

Question 8

1. Extracellular Ice Formation

Answer 8

• Ice crystals form in extracellular space • cell membrane is permeable to liquid water but not to ice • Water expands as it freezes so extracellular ice crystals physically compress cells • Extracellular ice can cause mechanical damage to cell membrane (sharp)

Question 9

2. Solution Effects

Answer 9

• As ice crystals grow in freezing extracellular water, electrolytes and other solutes are excluded, o causing them to become more concentrated in the remaining liquid water • Extracellular fluid becomes hypertonic to cell

Question 10

3. Dehydration

Answer 10

• Water migrates out of cell due to extracellular ice formation coupled with increasing solute concentration in liquid water

Question 11

4. Intracellular ice formation

Answer 11

• while some organisms and tissues can tolerate some extracellular ice, any appreciable intracellular ice is almost always fatal to cells

Question 12

5. Rate of cooling

Answer 12

• The slower the cooling rate, the longer intracellular water has the opportunity to move out of the cell by osmosis due to the increasing osmolarity of the extracellular environment as water is incorporated into ice crystals. • Very slow cooling may lead to excessive cell dehydration, solute concentration and cell death • Maximum cell viability is usually achieved at an intermediate cooling rate that balances osmotic dehydration and the risk of intracellular ice formation • Rapid cooling permits intracellular ice formation and usually leads to cell death

Question 13

Cryoprotectants

Answer 13

Substances used to protect biological tissue from freezing damage • Permeating • Non-permeating

Question 14

Permeating Cryoprotectants

Answer 14

• Small molecules that can diffuse across membranes • Form hydrogen bonds with water to slow or prevent both intra- and extra-cellular ice crystallization • Protect cell from solution effects by diluting electrolytes which can be toxic to cell • E.g, propylene glycol, glycerol, DMSO

Question 15

Non-permeating Cryoprotectants

Answer 15

• Larger molecules that remain extracellular • slow or prevent ice formation • can assist controlled dehydration of cells • used in combination of permeating cryoprotectants • e.g, sucrose (disaccharide)

Question 16

Sperm Cryopreservation Standard Protocol

Answer 16

1. Semen analysis of fresh sample 2. Divide entire specimen into vials containing equal number of sperm • Average ejaculate produces 5 vials 3. Add cryoprotectant and slowly freeze in liquid nitrogen 4. Thaw one aliquot, remove cryoprotectant and repeat semen analysis

Question 17

Post-thaw semen analysis/viability

Answer 17

• Sperm can take on abnormal forms from freezing • Fresh is better than frozen • Post-thaw motility of human sperm can range from 20-65% o Loss of motility after cryopreservation is believed to be caused by several factors;  decreased integrity of membranes  Cryo-damage to membranes of intracellular compartments  Altered energy metabolism and synthesis

Question 18

Embryo Freezing

Answer 18

• Embryo crypreservation is well-established • Thaw survival rates o Day 5 blastocyst- about 90% • Frozen embryo transfer success rates o Similar to fresh transfer *first baby born from frozen embryo in 1984 *Italy banned embryo freezing in 2004

Question 19

Oocyte (Egg) Freezing

Answer 19

Sperm: • 5 m by 60 m • Head is approximately 5m X 3 m Blastomere • Single cell from early embryo • 40 m diameter Oocyte • 130 m diamter • Eggs are much bigger than sperm or embryo and are harder to freeze

Question 20

Leidenfrost Effect

Answer 20

Large size of egg (smaller surface area to volume ratio) makes it subject to Leidenfrost effect • Gas barrier forms between hot surface (warm egg) and boiling liquid (liquid nitrogen) if temperature different is great enough • Gas barrier greatly slows heat transfer between the two which allows liquid to last longer and consequently hot surface to remain hot longer (it takes longer for heat to escape from center of egg)

Question 21

Oocytes are susceptible to freezing damage

Answer 21

Meiotic spindle is highly sensitive to temperature • Microtubule and microfilament damage may lead to abnormal division of chromosomes • Spindle re-forms after thaw and may reform in abnormal way Zona Pellucida hardening through premature cortical granule release • Cortical reaction normally occurs after fertilization to prevent polyspermy • Frozen oocytes require ICSI (intra-cytoplasmic sperm injection)

Question 22

Egg Banking

Answer 22

• Cancer patients • Women who want to delay reproduction • Failure to obtain sperm on retrieval day • Ethical objections to embryo cryopreservation (Italy) *world’s first egg bank- 1994 in Australia

Question 23

Egg Donation

Answer 23

Currently, most donor egg cycles involve fresh donation (no egg freezing but may involve embryo freezing) • Generally ages 20-30 • Psychological, genetic and medical screening (including STDs) • Compensation is $3,500-10,000 per donation • May donate 6-9 times in their lifetime Frozen eggs are immediately available • Donor-recipient coordination is not necessary • Would permit quarantine and STD re-screening

Question 24

Vitrification

Answer 24

• Process of converting material into glassy, amorphous solid free from crystalline structure • Uses ultra-rapid freezing process (100-10,000C/min) that is so fast it literally allows no time for ordered ice crystals to form o Cells/tissues with larger surface area relative to volume permit more rapid heat transfer o Minimizes trauma to cell/tissue * preferred method of freezing oocytes

Question 25

Oocyte freezing survival & success rates

Answer 25

• Vitrified eggs are about as successful as fresh eggs in many parameters including: fertilization rate, # of embryos transferred, and ongoing pregnancy rate • ~30% embryos (fertilized eggs) survive to blastocyst stage • “experimental” label was removed in 2013 • Current recommendation is to purchase frozen eggs in cohorts of 6 to get 1-2 embryos for transfer

Question 26

Auto-transplantation

Answer 26

transplant tissue back into same individual

Question 27

Allo-transplantation

Answer 27

transplant tissue into same species but different individual

Question 28

Xeno-transplantation

Answer 28

transplant tissue from one species to another

Question 29

Heterotropic

Answer 29

transplant tissue so it is not in its normal position • Spontaneous conception is not possible

Question 30

Orthotropic

Answer 30

transplant tissue back into natural position • Allows for possibility of natural conception, longevity of transplant is uncertain

Question 31

Vascular anastomosis

Answer 31

connect two cut or separate blood vessels to for continuous channel (blood supply to transplanted tissue)

Question 32

Concerns for ovarian cryopreservation and transplantation

Answer 32

• Risk of additional surgical procedure • Tissue composed of large size and many cell types complicated freezing and thawing process • Risk of cancer cell transmission from cryopreserved tissue by auto- or allo-transplantation • Recurrence of cancer (impact on child/family) • Age of patient (who makes decision for minors?) • Ethical and safety issues with xeno-transplantation o Creation of chimeric organisms o Transmission of retroviruses, prions