ART + Inhibin and Related Proteins

Question 1

Describe the IVF cycle?

Answer 1

Day 0: oocyte retrieval- oocytes are retrieved from follicular fluid which is aspirated- the number of follicles does not equal the number of oocytes retrieved (the average number is 8-10)

3-6 hours later: insemination via standard inseminaiton (with sperm that is motile) or ICSI

Day 1: Fertilisation occurs (only mature oocytes in metaphase II can do this)

Day 1-5: Embryos are grown in culture for 2-5 days

Day 5: The embryos have developed into blastocysts with approx 60 cells and differentiated into a ICM and TE
- These embryos are frozen or implanted via a transfer catheter

Question 2

What are the main techniques for sperm retrieval?

Answer 2

1. Ejaculation
2. Testicular biopsy (if there is obstructive azoospermia due to vasectomy or non-obstructive azoospermia due to issues such as Kleinfelters

• Sperm is then sorted into motile and non-motile/abnormal sperm
• The motile sperm are used for insemination

Question 3

When is ICSI used?

Answer 3

• When sperm quality is suboptimal
• Can help if sperm-egg interaction fails due to lack of receptors etc.
• Involves direct injection of sperm into cytoplasm of oocyte

Question 4

Describe the stages of embryo development critical in IVF:

Answer 4

Day 2: 2-4 cell stage
Day 3: 6-8 cell stage (the switch from maternal to embryonic genome control has occurred)
Day 4: Morula
Day 5: Blastocyst (60 cells and ICM and TE)

Question 5

What issues occur during days 2-3 of embryonic development?

Answer 5

1. Multinucleation:
   - More than one nucleus evident in any individual cell on either day 2 or 3
   - Multinucleation leads to decreased implantation, increased risk of chromosomal abnormalities and an increased risk of spontaneous abortion
2. Embryo fragmentation:
   - Fragments of cytoplasm break off during cell division
   - A small degree of fragmentation has no impact on implantation fertilisation but extensive fragmentation is negatively correlated with implantation
3. Embryo Arrest:
   - 10-15% of IVF embryos permanently arrest in mitosis at the 2-4 cell stage
   - 70% of these arrested embryos display gross chromosomal abnormalities
   - Embryo arrest can be due to a failure to activate the embryonic genome

Question 6

What role do oocyte mitochondria play in fertility?

Answer 6

• There is a correlation between low mtDNA copy number and the ability of an oocyte to be fertilised (the lower the mtDNA copy number- the better the embryos implantation potential)
• The ratio of mtDNA/nDNA was classified as the mitochondrial score (Ms) and used as an indicator of the mitochondrial copy number per cell
• Mitochondrial dysfunction is often associated with substantial mitochondrial hyperproliferation; therefore, the pathogenic consequence of “mitochondrial distress’’ is also a marked increase in mitochondrial proliferation
• Suggests an increase in mtDNA copy number in early embryos would be indicative of metabolic stress and that the embryo is trying to compensate for this stress by increasing its mtDNA
• So the lower the Ms, the more viable the oocyte is

Question 7

Where are BMP15 and GDF9 expressed?

• What is their role?

Answer 7

• BMP15 and GDF9 are secreted by the oocyte throughout follicular development
• They act on the surrounding granulosa cells
• In early primary follicles they increase proliferation of granulosa cells and in later follicle development they control the differentiation of granulosa cells (into the cumulus phenotype)
• BMP15 activates pSMAD 1/5
• GDF9 activates pSMAD 2/3

Question 8

What are the species differences in BMP15 and GDF9?

Answer 8

BMP15:

• Has evolved in rodents to have minimized expression and activity
• Has evolved in primates to maximise expression and activity

GDF9:

• Rodent GDF9 is produced in a highly active form
• Human GDF9 is produced in a latent form (as it binds very strongly to its pro-domain so will not bind well to receptor)
• Although human GDF9 is not produced in an active form it acts in synergy with BMP15 produced and becomes active

Question 9

What is cumulin?

Answer 9

• BMP15 and GDF9 can dissociate and reform as a heterodimer which is cumulin
• This is especially apparant in species such as humans where GDF9 is synthesised in a latent form
• Species that produce cumulin rather than active GDF9 appear to have lower fecundity
• Cumulin is being developed as part of a fertilty treatment known as enhanced IVM