20.05.11 PGD - genetics diagnosis and screening Flashcards
1
Q
What is PGD
A
- Preimplantation genetic diagnosis.
- Genetic profiling of embryos prior to implantation
- Testing cells from embryos obtained by IVF
- prevents transmission of disease-causing genetic mutations, where parents are affected or carriers of a known genetic abnormality
2
Q
What 3 stages of embryonic development can PGD be performed
A
- Biopsy of first polar body (prior to conception) and second polar body (after fertilisation)
- Day 3 cleavage stage (5-8 cell embryonic stage), biopsy of 1-2 blastomeres.
- Trophectoderm biopsy performed on day 5-6, where embryos have ~120 cells. 5-10 cells are removed. Advantageous to test more cells.
3
Q
Applications of PGD
A
- Monogenic disease (HD, CF, DMD, FRAX)
- Carriers of certain Robertsonian translocations, reciprocal translocations and inversions (>5Mb)
4
Q
What is PGT
A
- Preimplantation genetic testing
- Practice of obtaining a cellular biopsy sample and evaluating genetic composition to determine which embryos will be optimal for subsequent uterine transfer.
- Can be from developing oocyte (prior to fertilisation= 1st polar body), zygote (1 or 2 polar body), embryo (prior to implantation)
5
Q
What two types of PGT are there
A
- Diagnosis (PGD)
- Screening (PGS)
6
Q
What is PGS
A
- Identifying optimal embryos for uterine transfer in an IVF cycle.
- Parents are assumed normal and testing for aneuploidy is carried out to select for a euploid embryo
- Improved pregnancy rates by selecting euploid embryos for transfer
- Current use is controversial as several randomised trial shave shown no clinical benefit. Not available in NHS.
7
Q
NHS criteria for commission of 3 cycles of PGD to couples
A
- Couple at risk of having a child with a serious genetic condition
- Couple should be referred by Clinical Genetics service and have received genetic counselling
- Risk of having an affected pregnancy should be higher than 10%
- Female should be under 40yrs and BMI >19 and <30
- Non smokers
- No living unaffected child from current relationship
- HFEA (Human Fertilisation & Embryology Authority) must have licensed the indication for PGD
- Test must be included on a list of UKGTN approved tests.
8
Q
What is the HFEA
A
- Human Fertilisation & Embryology Authority
- UK independent regulator of fertility treatment and research using human embryos
9
Q
What examples are excluded from NHS policy for commissioning PGT
A
- Non-medical gender selection (i.e. for family balancing)
- PGD to address infertility or to prevent miscarriages of unknown etiology
- PGS, screening embryos for chromosomal abnormalities
10
Q
Steps of PGD
A
- Culture and IVF
- Biopsy of cells
- Genetic tests
11
Q
Review of culture and IVF step of PGD
A
- Ovarian stimulation and oocyte removal
- ICSI (Intracytoplasmic sperm injection) or IMSI (intracytoplasmic morphologically selected sperm injection)
- Number of embryos to be transferred is agreed by doctor and patients. Risk with multiple pregnancies
12
Q
What are the pros and cons to testing polar bodies
A
- Pro: Does not compromise the functional part of embryo
- Con: does not take into account paternal contribution, needs analysis of each polar body therefore generates unnecessary work (some oocytes won’t be fertilised and some zygotes won’t reach blastocyst stage)
13
Q
Review of testing blastomere
A
- 1-2 blastomeres are removed from day 3 cleavage-stage embryos
- 60% of embryos at cleavage-stage exhibit mosaicism, where at least one cell has a different ploidy (many will self-correct by blastocyst stage)
14
Q
Review of testing blastocyst
A
- 5-10 cells are removed
- Means more material to test
- Up to 50% of embryos survive to day 5-6 stage.
- Although fewer embryos survive to this stage, those that are tested are more robust and more likely to be implanted.
15
Q
What is bastocentesis
A
-New procedure where fluid removed from blastocoelic cavity of day 5 blastocyst as an alternative source of embryonic DNA.
16
Q
Future of PGS
A
-Non-invasive PGS
17
Q
What is Non-invasive PGS
A
- Embryonic DNA from blastocyst culture conditioned medium (BCCM) is combined with blastocoel fluid
- Pros: less technically challenging, less invasive, more cost effective, BF DNA is a good representation of blastocyst chromosomal composition
18
Q
Issues with PGD PCR
A
-Prone to contamination from extra-embryonic material leading to allele dropout. Could result in the transfer of an affected embryo.
19
Q
Review of PGD for mtDNA mutations
A
- mtDNA is transmitted mother to child
- Chance of disease expression depends on proportion of mutated mtDNA present in their tissues (heteroplasmy)
- PGD can select embryos with lower levels of mtDNA mutations
- Less prone to allele dropout due to high copy number of mtDNA
- PGD not suitable where there is a high proportion of abnormal mtDNA or homoplasmic. Due to random segregation of mitochondria and bottle neck effects, could result in a baby with a high proportion of mutated mtDNA in critical tissues
- Can be overcome by using donated oocytes, although child isn’t genetically related to mother.
- Maternal spindle transfer and pronuclear transfer is available since 2015, where transmission of both parents’ nuclear DNA into a donor oocyte.
20
Q
What is PGH
A
- Preimplantation genetic haplotyping
- Screening embryos conceived by IVF for presence of familial disease haplotypes rather than specific mutations
21
Q
What are the benefits of PGH
A
- Same test can be used for all families, even if there is heterogeneity in pathogenic mutations. Less labour intensive.
- Not limited to common mutations
- Where disease causing variant hasn’t been identified.
- When disease causing change is not amenable to PCR amplification
22
Q
What is multiple displacement amplification (MDA)
A
- Utilises bacteriophage Φ29 polymerase for efficient whole genome amplification.
- Generates large amounts of DNA from small samples.
23
Q
Main limitation of PGD
A
- Misdiagnosis and adverse outcomes
- Confusion of embryo and cell number, transfer of wrong embryo, maternal/paternal contamination causing ADO, probe/primer failure, chromosome mosaicism. - Methylation/ epigenetics
- Children born by assisted reproductive technology (ART) are at a higher risk of imprinting disorders (10x risk of BWS)
- ART is thought to interfere with restoration of epigenome during epigenetic reprogramming
24
Q
Ethical considerations of PGD
A
- Selective transfer over selective abortion, as there is the belief that a fetus has a higher moral value than an embryo.
- Designer babies
- When is it acceptable to utilise PGD (severity of disease, treatability, age of onset, penetrance). Testing for late onset conditions is controversial.
