Exam 3 - Cryopreservation

Question 1

Why would someone use sperm banking?

Answer 1

Cancer-related therapy, Testicular or prostate surgery, Vasectomy, High-risk occupational exposure (e.g., military), IUI or ART, Times when partner’s schedule does not permit his availability

Question 2

Why would someone use egg banking?

Answer 2

Cancer patients, Delaying reproduction, Failure to obtain sperm on transfer day, Ethical objections to embryo cryopreservation

Question 3

What screenings do egg and sperm donation require?

Answer 3

psychological, genetic, and medical screening (including STDs)

donor sperm are frozen and quarantined for 6 months to permit re testing for STDs

Question 4

What is cryopreservation?

Answer 4

cells or whole tissues are preserved by cooling them from body temperature (37C) to low temperatures in liquid nitrogen (-196C)

Question 5

Why does cryopreservation work to preserve cells?

Answer 5

at low, sub zero temps, biological activity especially biochemical reactions that would lead to cell death are effectively stopped

Question 6

What two ways are cells protected from freezing injury?

Answer 6

controlled cooling and thawing rate, and cryoprotectants

Question 7

Describe the process of ice formation during cell freezing

Answer 7

• extracellular ice formation (exclude solutes)
• extracellular fluid becomes hypertonic to intracellular fluid (because solutes concentrate)
• cells dehydrate due to the hypertonicity
• some dehydration is ok because less water decreases risk of intracellular ice formation which can be lethal

Question 8

What happens if the cooling rate is too slow or too fast?

Answer 8

too slow: too much dehydration from elevated solute concentrations extracellularly

too fast: rapid freezing injury due to intracellular ice formation (since they couldn’t dehydrate at all)

Question 9

Is the optimal rate of cooling the same for all cells/tissues?

Answer 9

No!

Question 10

How do the sizes of sperm, oocytes, and embryos compare?

Answer 10

sperm - head is approx. 5x3 micrometers

oocyte - 130 micrometers in diameter

embryo - single cell from a D3 embryo is approx. 40 micrometers in diameter

Question 11

What are the two types of cryoprotectants and how do they work?

Answer 11

permeating:
- small molecules can diffuse across membranes
- form hydrogen bonds with water to prevent or slow intra/extra cellular ice formation
- protect cell from solution effects by diluting the electrolytes which can be toxic to the cell
- ex: propylene glycol, glycerol, DMSO

non-permeating:
- larger molecules remain extracellular
- slow or prevent extracellular ice formation
- control dehydration
- often used in combo with permeating
- ex: sucrose (disaccharide)

Question 12

As cells cool, the extracellular fluid becomes —– concentrated

Answer 12

more

Question 13

What is the typical post-thaw motility of frozen sperm? Why? What does this mean overall?

Answer 13

20-65% post thaw motility

decreased integrity of membranes (plasma m., acrosomal m., intracellular compartment m.)

altered energy metabolism and synthesis

overall, fresh sperm is better than frozen

Question 14

What is the thaw survival rate for frozen embryos?
How do pregnancy rates compare for fresh vs frozen embryos?

Answer 14

thaw: day 5 blastocysts >90% survival

pregnancy: greater for frozen embryos than fresh embryos

Question 15

What damage can happen to oocytes during freezing?

Answer 15

meiotic spindle -
- highly sensitive to temperature
- microtubule and microfilament damage may lead to abnormal division of chromosomes
- spindle reforms after thaw and may reform in an abnormal way

zona pellucida -
- hardening through premature cortical granule release
- cortical reaction normally occurs after fertilization to prevent polyspermy
- frozen oocytes require ICSI

Question 16

What is vitrification? What does it do?

Answer 16

vitrification - water creates glassy, amorphous solid free from crystalline structure

ultra rapid freezing provess (1,000-10,000 C/min) is so fast it literally allows no time for ordered ice crystals to form

Question 17

What are the some of the paths for preserving fertility in cancer patients?

Answer 17

ovarian stimulation and oocyte retrieval:
either IVF (if they have a partner) then freeze embryos for future embryo transfer
OR
freeze oocytes for future IVF (with ICSI) and embryo transfer

surgical removal of the ovary:
either freeze whole ovary (for later transplantation and natural conception) OR
ovarian tissue prep, thaw when needed, transplantation…

auto-heterotropic transplantation, IVF, and ET

auto-orthotopic transplantation, IVF and ET or natural conception

xeno transplantation, IVF and ET

Question 18

What are autotransplantation, allotransplantation, and xenotransplantaiton?

Answer 18

autotransplantation: transplant tissue back into same individual

allotransplantation: transplant tissue into same species but a different individual

xenotransplantaiton:
transplant tissue from one species to another

Question 19

What are orthotopic and heterotopic transplantation?

Answer 19

orthotopic:
transplant tissue back into its natural position (allows for possibility of natural conception, but longevity of transplant is uncertain)

heterotopic:
transplant tissue so it is not in its normal position
(spontaneous conception is not possible)