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At the Forefront in PGD
PGD aneuploidy by CGH arrays (CGHa)
PGS-CGHa fundamentals
Aneuploidy is a very frequent phenomenon in preimplantation embryos. It is forecast that up to 70% of the IVF embryos are aneuploids.
The PGS-CGHa allows us to detect aneuploidy studding the 24 chromosomes in cleavage stage embryos and blastocyst.
What is an array of DNA?
An array of DNA (Chip or microchip of DNA) is a solid surface with
DNA sequences added in a micro-points known as spots.
We analyzed
gene regions
covering the
entire
chromosome
Cromosome
Sequences of DNA
What are the arrays of CGH or CGHa?
The CGHa are based on the compared genome hybridization. This
technique compares the genome of the sample with a reference
genome (“normal”), in order to detect gains or losses of genetic
material.
How the CGHa work in PGS?
In the array there are fixed regions of DNA representing of all the
chromosomes. On the array we add DNA of the blastomere or
trophectoderm, with a normal DNA. These two DNAs compete among
themselves to join the regions attached to the support. The two DNAs
are marked with fluorescent molecules. Each DNA emits a different
color (red and green). A laser system “reads” each of the points
(spots) where the concrete genetic regions of each chromosome are.
The Software interprets the fluorescents signs and it allows us to
determine if the DNA from the blastomere o trophectoderm, has gains
or losses of chromosomal material with respect to the normal pattern.
ó
Genomic
Amplification
Aneuploid embryo, with different minosomies:
-7,-8,-16,-20
Marking
«normal»
DNA
Competitive
Hybridization
Euploid embryo (normal)
Advantages of the PGS-CGHa over the PGS-FISH

We get information from all the chromosomes

Genetic regions distributed along the full
chromosome are studied

The subjectivity of FISH in the fixation of the
nuclei and the interpretation of the results is
eliminated

Up to 50% more of aneuploidy and 20% more
of anormal embryos is detected
Limitations of the CGHa

Haploid and polyploidy homogenic embryos
(69XXX, 92XXXX, 92XXYY) are not detected.
They represent 0.2% of total embryos.

Deletions/duplications of a very small size
(<1MB) are not detected.
Reading
Total duration of: 30 h
6/10/2011 v2.0
At the Forefront in PGD
Combined Molecular PGD:
PGD of monogenic disorders + aneuploidy (24 chromosomes)
PGD of monogenic disorders + translocation/inversion + aneuploidy (24 chromosomes)
This technology combines the techniques of whole genome amplification, the PCR and the microrrays of CGH. It
allows us to perform PGD of a single-gene disorder, and the screening of aneuploidies for all the chromosomes in the
same cell simultaneously. In addition, we can detect normal/balanced embryos generated from carriers of balanced
translocations/inversions.
The process
Fundamentals of molecular PGD combined with
aneupolidy screening (24 chromosomes)
Combined molecular PGD is beging with the
amplification of the complete genome of blastomeres
or trophectoderm. One part of the amplified DNA is
processed by fluorescent PCR, obtaining the
diagnostic of the disease in 24h. Simultaniously, an
aliquot part of the amplified DNA, is hybridized in a
CGH microarray for the study of aneuploidy of the 24
chromosomes.
Finally, in 36 hours it is obtained the complete result
of the two analyses. The final report includes the
results of each embryo individually in relation to the
monogenic disease and aneuploidy.
PGD of monogenic disorders by PCR does not allow
to obtain information of the number of chromosomes
of the analyzed embryo. Thus, PGD of monogenic
diseases not ensure that the embryo will be euploid.
On the other hand, it is well known that the aneuploidy
is a common phenomenon in primplantation embryos,
involving monosomies and trisomies of the 22
autosomes and sex chromosomes.
In conclusion, combined molecular PGD allows the
transfer of healthy embryos of the monogenic disorder
and normal respect to number of chromosomes.
Option 1. Blastomere biopsy (D+3)
Option 2. Trophectodrm biopsy (D+5)
Part of the DNA that is
studied by PCR to diagnose
the monogenic disease
Blastomere or
trophectoderm
Whole genome
amplification
36 h
Joint results report
Part of the DNA is
studied byCGHa
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At the Forefront in PGD
Combined Chromosomal PGD:
Simultaneous PGD or transloction/inversión+aneuplidy (24 chromosomes)
Fundamentals of combined chromosomal PGD
Couples with one member carrying a balanced chromosomal
rearrangement (translocation or inversion) have an increased risk of
generating abnormal embryos as a result of segregation of the
balanced abnormality. This causes, recurrent abortions and, in
many cases, infertility. PGD using FISH techniques allows detect
altered embryos (unbalanced) for a specific chromosomal
rearrangement. However, the main limitation is that it does not
provide information of the rest of chromosomes. Combined
chromosomal PGD is based on CGH arrays technology. It allows to
identify the altered embryos (unbalanced) in relation to the
translocation/inversion and it also allows us to study aneuploidy for
24 chromosomes, simultaniously and in the same cell. The
information of the non involved chromosomes in the rearrangements
is important for two reasons:
1. Aneuploidy is a very frequent phenomenon in embryos at
preinplantation stages. It is estimated that up to 70% of
the embryos generated by IVF are aneuploid.
2. The interchromosomal effect: There are chromosomes
not involved in the translocations, that are affected in the
offspring.
We analyzed
gene regions
covering the
entire
chromosome
Cromosome
Normal embryo/balanced
Unbalanced embryo from a patient with cariotipe:
46,XX,t(12;14)(q24,q22)
Option 1. Blastomere Biopsy (D+3)
Option 2. Trophectoderm biopsy (D+5)
Blastomere or
trophectoderm
Genome
Amplification
Marking
«normal»
DNA
30 h
Competitive
hybridization
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