Assisted Reproductive Technologies Flashcards
What is infertility?
“failing to get pregnant after 2 years of unprotected sex”
Decreasing
Affects 1 in 6 couples
Cause of infertility spilt equally between sexes
What is the objective of ART?
Bring sperm and oocyte close together to increase chance of fertilisation and achieve pregnancy
What are some forms of ART?
In Vitro Fertilisation (IVF) Intra Cytoplasmic Sperm Injection (ICSI) Preimplantation genetic diagnosis Mitochondrial donation Gene editing Stem cell transplantation
What are reasons for using ART?
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
How is IVF - embryo transfer achieved?
Super-ovulation Semen collection Insemination Embryo culture Embryo transfer Luteal support - stimulate ovaries to produce multiple follicles - manipulate hypothalamic - pituitary - gonadal hormone axis
What is super-ovulation?
← hypothalamus
↓ ↓ GnRH
← pituitary gland →
↓ ↓ ↓ LH/FSH
→ ovary ←
GnRH = suppression of normal cycle using GnRH agonist
LH/FSH = high dose increases follicle number
Is there a cancer risk with super ovulation?
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
How is oocyte collection performed?
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
What is fertilisation and in-vitro maturation?
Sperm collected
Oocytes fertilised in vitro to 4-8 cell stage
Applied to animal systems
What is large offspring syndrome?
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
What is embryo transfer?
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
What is the success rate of IVF?
Approx. 25%
- age
- sperm quality
- response to super-ovulation
What is ICSI?
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
How are the optimum sperm for ICSI selected?
Morphology reflects genetic viability - so sperm with the highest % normal karyotype are selected
What is Pre-implantation genetic diagnosis?
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??
How is PGD performed?
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
What is mitochondrial donation?
Healthy children can be born to a mother who carries faulty mitochondrial DNA
- Remove nucleus from oocyte harvested from a patient with defective mitochondrial DNA
- Remove and discard the nucleus from a donor’s oocyte with healthy mitochondria
- Insert patients nucleus into donor oocyte (healthy mitochondria)
- Fertilise oocyte with sperm (IVF/ICSI)
- Implant embryo into patient
What is gene editing in humans?
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
What are implications of embryo editing?
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
How is CRISPR - Cas9 used in the lab?
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
How is gene editing used for organ transplant?
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
How are mosquitoes genetically manipulated?
CRISPR used to make offspring sterile
- gene edit has fluroescent protein to confirm success
How is stem cell transplantation used?
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
What are potential benefits of introduction of SSCs to a hosts testis?
Infertility treatments Breeding from an animal after its death Preserve endangered species Rapid generation of transgenic animals Re-introduce fertility in cancer survivors
What is germ cell transplantation?
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
