Lecture 7 - ART

Question 1

Define infertility

Answer 1

Failing to get pregnant after 2 years of unprotected sex

Question 2

What is ART?

Answer 2

Bringing sperm and oocyte together to increase chance of fertilisation and pregnancy

Question 3

What is the protocol for IVF?

Answer 3

1. Superovulation and semen collection
2. Insemination
3. Embryo culture and transfer
4. Luteal support

Question 4

What is superovulation?

Answer 4

Stimulation of ovaries to produce multiple follicles. Suppress GnRH to suppress normal cycle and then high dose of LH and FSH to increase follicle number

Question 5

How do they assess the viability of sperm?

Answer 5

Mobility and morphology, fluoro staining, membrane integrity (green/red), FITC to stain for acrosome, flow cytometry and FACs

Question 6

What is the success rate of IVF?

Answer 6

25% - still not great but it is the accepted protocol now

Question 7

What is ICSI?

Answer 7

Intracytoplasmic sperm injection. Sperm injected directly into the oocyte. Can be done with immature sperm but there is a subjective bias and you can’t be sure that the sperm is OK

Question 8

How is pre-implantation genetic diagnosis carried out?

Answer 8

1. Routine IVF
2. In vitro embryo maturation - 2/3 days
3. Zona drilling and removal of 1-2 blastomeres
4. Cells screened for marker of genetic conditions
   5 Embryo transfer of unaffected embryos

Question 9

How is mitochondrial donation carried out?

Answer 9

1. Nucleus removed from oocyte with damaged mito DNA
2. Remove nuc from donor oocyte with healthy mito DNA
3. Insert nuc into healthy oocyte
4. Fertilise and implant

Question 10

What is the benefit of mito donation?

Answer 10

Mito DNA mutations alters how a cell uses energy and often leads to life threatening conditions

Question 11

How is gene editing carried out?

Answer 11

CRISPR/Cas9 used to identify, remove and replace faulty genes

Question 12

What are some things gene editing can be used for?

Answer 12

Removal of mutations that cause developmental problems. Removal/prevent spread of disease. Promote beneficial traits. Remove viruses (pig TP)

Question 13

How does CRISPR/Cas9 work?

Answer 13

1. CRISPR RNA seq generated to reflect seq of target gene
2. CRISPR/Cas9 complex injected into embryo/cell
3. Target sequence snipped out of DNA to disable gene
4. Synthetic DNA can be spliced at the cut site to introduce a desired trait