Intro to PGT Flashcards
What is PGT, when does this occur and what cells are used?
genetic testing on biopsied cells from embryos
cells are biopsied usually from a day 5-7 embryo at blastocyst stage (possibility to day 3 but moved away from this method)
- trophectoderm cells (become placenta cells later on stay away from ICM which would develop into fetus
What happens to the embryo if it was genetically tested and was fine?
frozen
(when doing day 3 biopsies could have a fresh ET)
Preimplantation genetic screening (PGS) is now referred to as?
Preimplantation genetic testing for aneuploidies (PGT-A)
preimplantation genetic diagnosis (PGD) for monogenic/single gene disorders are now known as?
preimplantation genetic testing monogenic/single gene disorders (PGT-M)
preimplantation genetic diagnosis (PGD) chromosomal structural rearrangements are now known as?
preimplantation geneting testing for chromosomal structural rearrangements (PGT-SR)
What are the two groups of patients needing PGT?
- infertility patients (may have an underlying genetic condition causing infertility)
- advanced maternal age (above 38 years
- recurrent miscarriage (3 +)
- recurrent implantation failure (over 10 embryos transferred without successful pregnancy)
- patients with known genetic risk (usually are fertile but want to avoid heritable disease)
- chromosomal translocations and other structural rearrangements (PGT-SR used)
- sex selections (haemophilia, autism)
- monogenic diseases (PGT-M)
Before genetic testing occurs a biopsy is taken from the trophectoderm cell of a blastocyst. What needs to happen to these cells before testing is completed?
- cells placed in a PCR tube to undergo cell lysis
- samples gent sent to genetics lab where DNA amplification occurs ( not many cells collected need to increase DNA amount)
happens with every sample regardless of which genetic test is being done
How and why is PGT-A (looking for aneuploidies) done and what are they looking for?
karyotyping looks at whole chromosome
aneuploidy is the wrong number of chromosomes:
- most are lethal
- very common (50-70% of early embryos)
- about 60% of spontaneously miscarried pregnancies are aneuploid
- monosmomies usually dont even have the ability to implant
- non lethal: 21, 18, 13, sex chromosome trisomies
what is the relationship between chromosome abnormality and maternal age?
abnormalities are common in oocytes and increases with advancing maternal age (after 30-35 years increases more rapidly)
- same occurs in embryos
this correlates to increased miscarriage rates and decreases in live births
How do aneuploidies occur ?
- nondisjunction in meiosis 1
- homologous pairs do not seperate correctly
- results at end the of meiosis 2 with: 2 with 24 chromosomes and 2 with 22 chromosomes (all are incorrect haploid) - nondisjunction during meiosis 2
- sister chromatids do not seperate correctly
- results in 2 normal 23, one 22 and one 24 (2 incorrect haploid numbers - some aneuploidies occur later after fertilisation due to incorrect cell division (post mitotic error)
What occurs during fertilisation of an egg cell with n+1 and a normal sperm cell?
zygote has a normal pairs then a trisomy (2n+1)
What does PGT-SR test for?
chromosomal structural rearrangements
looking at segments of chromosomes
chromosomal translocations are the most common structural abnormalities. What are these?
caused by rearrangements of parts between non homologous chromosomes (not the same number)
types:
reciprocal translocation
- pieces of two different chromosomes break and they swap places
robertsonian
- can only occur in acrocentric chromosomes (13,14,15,21,22)
- long q arm and short p arm (cap)
- two q arms join together create one long chromosome
- would be 45 XY not 46 as it looks like a chromosome is missing
Reciprocal translocations are the most common chromosome rearrangements in the population (1 in 500). Why is this?
Usually they do not know they are carries of reciprocal translocations
carriers are balanced (they do not gain or lose genetic material)
How do people usually find out if they are a carrier of a reciprocal translocation?
usually based on a family history of:
- infertility
- recurrent miscarriage
- pregnancies with congenital abnormalities
people will find out their a carrier when this is occurring
If carriers of balanced reciprocal translocations why do they have issues with fertility?
even though it does not affect carriers health it can cause chromosomal abnormalities in their gametes manifesting as:
- infertility
- recurrent pregnancy loss
- offspring with intellectual and physical abnormalities
it will also depend on which chromosomes are involved and the size of the segments of chromosomes which were translocated
How are gametes affected by reciprocal translocations?
During metaphase meiosis 1 when homologous chromosomes pair up and line on the equator the translocation does not allow for pairing to occurring
So they form a quadrivalent or tetravalent structure (instead of 1 paring with 1 the translocation piece of chromosome with pair up with its original owner causing there to be 1 and 2 pared together)
when segregation occurs these piece then go anywhere as they have not aligned properly
can either result in:
- balanced cell (end up with the translocation
- normal ( happen to get the parts unaffected by the translocation)
- unbalanced (extra or missing pieces of DNA)
Other than translocations what is another type of chromosomal structural arrangements?
Inversions
- paracentric (breaks, flips and reinserts)
— gamete from this cell will end up with this chromosome having two or no centromeres and the gamete would no be viable) - pericentric (breaks in chromosome including centromere, flips and reinserts)
— some gametes will be unbalanced and have duplication of short arm with deletion of long arm (areas outside of inversion only) or vice versa with long arm being duplicated
For translocation carriers why should patients be counselled?
only a minority of embryos will be suitable for transfer due to the result of meiosis only having 2 ways that could create a good embryo (only unbalanced embryos not used)
In many cycles there will be no embryos to transfer
For a translocation carrier if a balanced embryo is identified can this be used for ART?
yes pregnancy rate is good with balanced embryos and miscarriage rate is low
What technique can be used for both PGT-A and PGT-SR?
Next Gen sequencing (NGS)
What is NGS
massive parallel sequencing of millions of DNA fragments
multiple samples can be analysed simultaneously
technology used for sample prep is embryo map by vitrolife and for sequencing we use machines called Miseq by Illumina
What are the steps of NGS from embryo biopsy
- biopsy
- lysed in PCR tubes
- during DNA amplification barcodes attached to segments which will help identify samples/ DNA fragments (template preperation)
- prepare sample library
- sequence libraries on machine
- data analysis
How to read NGS data
copy number on left axis
chromosome number x axis
normal copy number = 2
above 2 = +1
below 2 = -1
sex chromosomes
XX = 2 X 0 on Y
XY = 1 X and 1Y
What is chromosomal mosaicism in embryos?
different cells within one embryo have different chromosome make up
some are diploid some are aneuploid
What would happen if you were to biopsy an embryo with mosaicism and run the DNA through NGS?
they would show an intermediate copy number (e.g. 2.5)
this would indicate the biopsies embryo may be at risk of having mosaicism
What are the recommendations for the laboratory when regarding mosaicism embryos?
- for reliable detection 5 cells should be biopsied with little cell damage
- cut off point for mosaicism is above 20% (green dots do not always align nicely due to background noise of the test
- every lab should fo their own internal validation i to determine which level of mosaciasm should be reported wither its 30% or more than 50%
What are the recommendations for the clinician regarding an embryo with mosaicism?
to transfer these embryos patients need to be genetically counselled (we are still learning also so can be hard to predict outcomes of mosaicism)
appropriate monitoring and prenatal diagnosis of any resulting pregnancy
What guidelines were initially recommended/ suggested when it came to transferring mosaic embryos?
monosomy preferred over trisomy
mosaic trisomy depends on chromosomes involved
- aneuploidy with low risk chromosomes first
- less priority is chromosomes involved in UPD, intrauterine growth retardation, capable of liveborn viability
Currently what is known about transferring mosaic embryos
many result in normal pregnancies and it is not dangerous but each clinic does its own thing and it is also up to patient preference
Can the morphology of the embryo show signs of aneuploidy?
no
PGT is a selection tool. what does this mean?
cannot repair embryos but gives you the highest chance of implantation
PGT-M test for single gene disorders looking at single nucleotides of DNA. What are the different types of inheritance of these genes?
autosomal (non sex chromosomes)
- dominant
- recessive
X linked inhertience
- dominant (father affected all daughters affected)
- recessive (mother affected all daughters are carriers son may be affected)
What is karyomapping ?
uses linked markers (SNP’s) on a bead array to track known gene mutations
use linkage to look for SNP’s around known gene mutations
- needs reference DNA (known status) used to establish phase (assigns a reference affected or unaffected for that particular haplotype
determines inheritance from parental/grandparental haplo blocks (inherited chromosomal segments)
The genotype of each embryo is lined up against the reference to establish a similarity or difference
How are DNA samples obtained for karyomapping?
DNA samples taken from:
- couple
- 1-2 family members of known genetic status
—- test is done by linkage need knowns to link against
What are SNP’s
single nucleotide polymorphisms
one base changed in a gene
What are the advantaged to karyotyping?
much quicker workup and treatment
more information about other chromosomes
suitable for most couple with a single gene disorders
