20.05.11 PGD - genetics diagnosis and screening

Question 1

What is PGD

Answer 1

• 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

Question 2

What 3 stages of embryonic development can PGD be performed

Answer 2

1. Biopsy of first polar body (prior to conception) and second polar body (after fertilisation)
2. Day 3 cleavage stage (5-8 cell embryonic stage), biopsy of 1-2 blastomeres.
3. Trophectoderm biopsy performed on day 5-6, where embryos have ~120 cells. 5-10 cells are removed. Advantageous to test more cells.

Question 3

Applications of PGD

Answer 3

• Monogenic disease (HD, CF, DMD, FRAX)

- Carriers of certain Robertsonian translocations, reciprocal translocations and inversions (>5Mb)

Question 4

What is PGT

Answer 4

• 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)

Question 5

What two types of PGT are there

Answer 5

• Diagnosis (PGD)

- Screening (PGS)

Question 6

What is PGS

Answer 6

• 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.

Question 7

NHS criteria for commission of 3 cycles of PGD to couples

Answer 7

• 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.

Question 8

What is the HFEA

Answer 8

• Human Fertilisation & Embryology Authority

- UK independent regulator of fertility treatment and research using human embryos

Question 9

What examples are excluded from NHS policy for commissioning PGT

Answer 9

• 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

Question 10

Steps of PGD

Answer 10

1. Culture and IVF
2. Biopsy of cells
3. Genetic tests

Question 11

Review of culture and IVF step of PGD

Answer 11

• 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

Question 12

What are the pros and cons to testing polar bodies

Answer 12

• 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)

Question 13

Review of testing blastomere

Answer 13

• 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)

Question 14

Review of testing blastocyst

Answer 14

• 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.

Question 15

What is bastocentesis

Answer 15

-New procedure where fluid removed from blastocoelic cavity of day 5 blastocyst as an alternative source of embryonic DNA.

Question 16

Future of PGS

Answer 16

-Non-invasive PGS

Question 17

What is Non-invasive PGS

Answer 17

• 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

Question 18

Issues with PGD PCR

Answer 18

-Prone to contamination from extra-embryonic material leading to allele dropout. Could result in the transfer of an affected embryo.

Question 19

Review of PGD for mtDNA mutations

Answer 19

• 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.

Question 20

What is PGH

Answer 20

• Preimplantation genetic haplotyping

- Screening embryos conceived by IVF for presence of familial disease haplotypes rather than specific mutations

Question 21

What are the benefits of PGH

Answer 21

• 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

Question 22

What is multiple displacement amplification (MDA)

Answer 22

• Utilises bacteriophage Φ29 polymerase for efficient whole genome amplification.
• Generates large amounts of DNA from small samples.

Question 23

Main limitation of PGD

Answer 23

1. Misdiagnosis and adverse outcomes
   - Confusion of embryo and cell number, transfer of wrong embryo, maternal/paternal contamination causing ADO, probe/primer failure, chromosome mosaicism.
2. 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

Question 24

Ethical considerations of PGD

Answer 24

• 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.