Assisted Reproductive Technologies

Question 1

What is infertility?

Answer 1

“failing to get pregnant after 2 years of unprotected sex”
Decreasing
Affects 1 in 6 couples
Cause of infertility spilt equally between sexes

Question 2

What is the objective of ART?

Answer 2

Bring sperm and oocyte close together to increase chance of fertilisation and achieve pregnancy

Question 3

What are some forms of ART?

Answer 3

In Vitro Fertilisation (IVF)
Intra Cytoplasmic Sperm Injection (ICSI)
Preimplantation genetic diagnosis 
Mitochondrial donation 
Gene editing 
Stem cell transplantation

Question 4

What are reasons for using ART?

Answer 4

Infertility 
- female factor 
Absence of a partner 
- Lesbian, gay, trans 
- single woman
- death of a spouse 
Genetic engineering 
- eliminate hereditary disorders 
- sex selection 
- promote beneficial traits 
- generate transplant tissue 
- produce engineered species

Question 5

How is IVF - embryo transfer achieved?

Answer 5

Super-ovulation 
Semen collection
Insemination 
Embryo culture 
Embryo transfer 
Luteal support 
- stimulate ovaries to produce multiple follicles 
- manipulate hypothalamic - pituitary - gonadal hormone axis

Question 6

What is super-ovulation?

Answer 6

← hypothalamus
↓ ↓ GnRH
← pituitary gland →
↓ ↓ ↓ LH/FSH
→ ovary ←
GnRH = suppression of normal cycle using GnRH agonist
LH/FSH = high dose increases follicle number

Question 7

Is there a cancer risk with super ovulation?

Answer 7

Stimulates multiple follicles
Mild + severe side effects - ovarian hyper-stimulation syndrome, acute inflammatory condition can be fatal
IVF drugs have been linked to ovarian cancer, 1/3 more likely - study suggests the drugs aren’t to blame and that infertile women are pre-disposed to develop cancer

Question 8

How is oocyte collection performed?

Answer 8

Monitor follicular development by trans-vaginal ultrasound
2+ follicles @ 16-18mm diameter
10,000IV hCG (LH like action) induces oocyte maturation
Oocyte aspirated by ultrasound guided catheter

Question 9

What is fertilisation and in-vitro maturation?

Answer 9

Sperm collected
Oocytes fertilised in vitro to 4-8 cell stage
Applied to animal systems

Question 10

What is large offspring syndrome?

Answer 10

Bovine embryos exposed to human maturation media prior to blastocyst stage = large offspring syndrome
- often fatal for mother and calf
- organ defects in surviving calf
also observed in cloning

Question 11

What is embryo transfer?

Answer 11

Embryos transferred to uterus 2-5 days after IVF (4-8 cell stage)
2 embryos transferred
Trans-abdominal ultrasound guides catheter through the cervix into uterus
Cryopreserve excess embryos - permission from both parents to use embryos in future

Question 12

What is the success rate of IVF?

Answer 12

Approx. 25%

• age
• sperm quality
• response to super-ovulation

Question 13

What is ICSI?

Answer 13

Modified IVF-ET technique 
Sperm is directly injected into oocyte 
Higher success rate 
Embryo cleavage 24 hours after injection 
Used if sperm are unable to fertilise:
- abnormal motility and/or morphology 
- damaged acrosome 
Used with immature sperm 
- post meiosis (haploid)
- pre-spermatogenesis
- pre-epididymal modification

Question 14

How are the optimum sperm for ICSI selected?

Answer 14

Morphology reflects genetic viability - so sperm with the highest % normal karyotype are selected

Question 15

What is Pre-implantation genetic diagnosis?

Answer 15

Screen embryos for genetic diseases e.g. Huntington’s disease, cystic fibrosis, sickle cell anaemia
Prevents inheritable conditions being passed onto offspring
Human Leukocyte Antigen (HLA) typing
- how closely donor tissues match a recipient
Designer babies??

Question 16

How is PGD performed?

Answer 16

Routine IVF treatment
Zona drilling
Remove 1-2 cells, no detrimental effect on embryo
Screen for genetic conditions
Unaffected embryos transferred
Cryopreservation of unaffected embryos, can donate embryos for free treatment
Affected embryos allowed to perish

Question 17

What is mitochondrial donation?

Answer 17

Healthy children can be born to a mother who carries faulty mitochondrial DNA

1. Remove nucleus from oocyte harvested from a patient with defective mitochondrial DNA
2. Remove and discard the nucleus from a donor’s oocyte with healthy mitochondria
3. Insert patients nucleus into donor oocyte (healthy mitochondria)
4. Fertilise oocyte with sperm (IVF/ICSI)
5. Implant embryo into patient

Question 18

What is gene editing in humans?

Answer 18

Form of genetic engineering, CRISPR - Cas9 technology
Enables identification, removal and replacement of “faulty” genes
Fertility benefits = understand genes involved in human embryo development + improve embryo development after IVF

Question 19

What are implications of embryo editing?

Answer 19

Limited use in differentiated cells, specific cells must be targeted, each individual must be treated
CRISPR-Cas9 can edit the human germ line
- embryo or gametes
- primate embryo editing routine
Edited information can be inherited by the parents children/grandchildren
- correct genetic flaw
- enhance a desired trait
Implications of editing the germ line = serious and unpredictable

Question 20

How is CRISPR - Cas9 used in the lab?

Answer 20

Disable/insert DNA into an embryo/other cell
CRISPR RNA sequence generated to reflect the sequence of a target gene, Cas 9 restriction enzyme
CRISPR-Cas9 complex introduced into embryo/cell
- target sequence is stripped out of the cellular DNA to disable the gene and/or synthetic DNA can be spliced at the cut site to introduce a desired trait

Question 21

How is gene editing used for organ transplant?

Answer 21

Pig organs are a similar size to humans
Unable to transplant organs as genome contains viruses - Porcine Endogenous Retroviruses (PERV) capable of infecting human cells
CRISPR-Cas9 used to remove 62 PERV genes from pig kidney and lung cells
- mix CRISPR kidney cells and human cells - no infection occurs

Question 22

How are mosquitoes genetically manipulated?

Answer 22

CRISPR used to make offspring sterile

- gene edit has fluroescent protein to confirm success

Question 23

How is stem cell transplantation used?

Answer 23

Introduction of spermatogonial sperm cells into a host testis if the host does not make his own sperm or might ejaculate a mix of two males sperm

Question 24

What are potential benefits of introduction of SSCs to a hosts testis?

Answer 24

Infertility treatments 
Breeding from an animal after its death 
Preserve endangered species 
Rapid generation of transgenic animals 
Re-introduce fertility in cancer survivors

Question 25

What is germ cell transplantation?

Answer 25

Transplant stem cells into the testis via the rete testis or the vas deferens
- restoration of spermatogenesis takes several weeks
Ocelot a good candidate for SSC transplantation - single offspring, every other year, slow growth, vulnerable to population loss
Ocelot SSC’s colonise domestic cat testis & produce ocelot sperm
- 2 weeks sperm in seminiferous tubules
- 13 weeks sperm in epididymis