Download Non-invasive pre-implantation genetic diagnosis of X

Medical Hypotheses 83 (2014) 506–508
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Medical Hypotheses
journal homepage: www.elsevier.com/locate/mehy
Non-invasive pre-implantation genetic diagnosis of X-linked disorders
Said Assou a,b, Ounissa Aït-Ahmed a,b, Safia El Messaoudi c, Alain R. Thierry c, Samir Hamamah a,b,d,⇑
a
Université Montpellier 1, UFR de Médecine, Montpellier, France
CHU Montpellier, Institute for Regenerative Medicine and Biotherapy, Hôpital Saint-Eloi, INSERM U1040, Montpellier, France
c
Institut de Recherche en Cancérologie de Montpellier, INSERM U896, Montpellier, France
d
ART-PGD Department, CHU Montpellier, Hôpital Arnaud de Villeneuve, Montpellier, France
b
a r t i c l e
i n f o
Article history:
Received 7 February 2014
Accepted 18 August 2014
a b s t r a c t
Pre-implantation genetic diagnosis (PGD) is a powerful clinical tool to identify embryos with or at risk of
specific genetic diseases before implantation in utero after in vitro fertilization (IVF). PGD is performed on
embryo biopsies that are obtained by aspiration of one or two cells from pre-implantation embryos at day
3 or day 5/6 of culture. However this is a traumatic method that cannot be avoided because non-invasive
procedures to assess the genetic status of pre-implantation embryos are not available yet. We hypothesize that cell-free nucleic acids, which are released by embryos in the culture medium during the IVF
procedure, could be used for genetic screening. To test our hypothesis we will focus first on X-linked disorders because these single-gene diseases due to the presence of defective genes on the X chromosome
are dominant in males. Therefore the objective here is to discriminate between female (XX) and
male (XY) embryos by detecting Y chromosome-specific sequences in cell-free nucleic acids. Using culture medium from embryos we are able to discriminate between male and female embryos. This opens
new avenues for the development of a non-invasive PGD method.
Ó 2014 Elsevier Ltd. All rights reserved.
Background
In vitro fertilization (IVF) combined with pre-implantation
genetic diagnosis (PGD) has been used successfully for increasing
the chances of having a healthy baby [1]. However, the PGD procedure involves an embryo biopsy at day 3 or day 5/6 post-fertilization by aspiration of one or two embryo cells for genetic testing.
The possible negative effects of embryo biopsy on the future development of the baby are still debated 24 years after the first birth
following PGD-based embryo selection. Embryo biopsy is not only
invasive but it is also an expensive procedure; thus, PGD centers
are trying to develop safer and cheaper non-invasive options [2]
for PGD. The discovery of cell-free nucleic acids has brought new
possibilities for non-invasive diagnosis in various medical fields,
including reproductive medicine [3,4]. Indeed, cell-free nucleic
acids such as genomic DNA (gDNA) have been detected in fluid
samples from the blastocoel (the fluid-filled cavity of blastocysts)
of day 5 human embryos [5]. This discovery opens the possibility
of using gDNA in blastocoel fluid samples to develop a new PGD
⇑ Corresponding author at: ART/PGD Department, Hôpital Arnaud de Villeneuve,
Montpellier 34295, France. Tel.: +33 4 67 33 64 04; fax: +33 4 67 33 62 90.
E-mail address: [email protected] (S. Hamamah).
http://dx.doi.org/10.1016/j.mehy.2014.08.019
0306-9877/Ó 2014 Elsevier Ltd. All rights reserved.
approach. However, harvesting blastocoel fluid for DNA analysis
is still a rather invasive approach for the embryos.
Hypothesis
We hypothesize that biomarkers to be used for PGD purposes
may be identified in cell-free nucleic acids released in the culture
medium by embryos during IVF procedures (Fig. 1).
Evaluation of the hypothesis
First of all, we need to determine whether (i) cell-free DNA
(cfDNA) is present and can be quantified in samples of culture
medium of day 3 embryos and day 5/6 blastocysts prior to transfer;
and whether (ii) such cfDNA can be used to detect embryo-specific
sequences. As a proof of concept, we first target genes that are specific for the Y chromosome in order to determine the embryo sex.
To this aim, culture medium samples were amplified using a quantitative polymerase chain reaction (PCR)-based method that we
developed to analyze circulating cfDNA [6]. In line with recent
work showing the presence of both nuclear and mitochondrial
DNA in embryo culture medium samples [7], we confirmed that
significant amount of cfDNA can be detected in all tested culture
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S. Assou et al. / Medical Hypotheses 83 (2014) 506–508
Hypothesis
Embryo
CfDNA
Culture
medium
Published data
Blastocyst
Culture
medium
CfDNA
Cell free DNA in culture
medium
Quantitative
PCR
Cleavage-stage embryo
blastomere biopsy
Blastocyst,
trophectoderm biopsy
Cells (blastomere, trophectoderm)
Sequencing
Non-invasive approach
PCR-based
methods
24-chromosome
SNP array
Comparative
genomic
hybridization
Invasive approach
Fig. 1. Non-invasive PGD and classical PGD based on embryo biopsy. In conventional PGD, material is obtained by microsurgical removal of blastomeric or of trophectodermal
cells. The genetic status of the embryo can then be determined with various methods, such as quantitative polymerase chain reaction (Q-PCR), sequencing, 24-chromosome
single-nucleotide-polymorphism (SNP) arrays and comparative genomic hybridization (CGH) arrays. We hypothesize that cell-free nucleic acids that cross the embryo barrier
can be detected in embryo culture medium samples, thus providing an alternative to the existing invasive tests for the identification of embryos with genetic disorders.
medium samples. Currently the limit of detection of our test is
1.5 ng/ml cfDNA per sample, which represents a minimum of
two genome equivalents (GE). Up to 27 ng/ml of cfDNA (or 36
GE) was measured in samples from day 5/6. We then investigated
the possibility of using the cfDNA present in different drops of
embryo culture medium to sex embryos by amplifying TSPY1, the
Y chromosome specific gene. The ALU repeated sequence was used
as a control (Fig. 2). As expected, TSPY1 sequence was detected only
in some samples (D1) but not in others (D2). We show unambiguously that the difference relies only on sex difference as TSPY is
detected in cells that XY (human foreskin fibroblasts) but not in
XX cells (human cumulus cells from the mother). In contrast ALU
sequences are detected in all the samples regardless of their origin.
These findings confirm that (i) cfDNA is present in the culture medium of pre-implantation embryos and (ii) that it can be used to
assess the sex of the embryo.
sex-linked diseases is within reach. Its extension to the detection
of autosomal dominant disorders, such as myotonic dystrophy
and Huntington’s disease [11], should be straightforward. Finally,
thanks to the development of single-molecule droplet-based PCR
and next generation genome sequencing to analyze trace amounts
of cfDNA in embryo culture medium, the method should be applicable to any (dominant or recessive) genetic disease.
Important questions must be addressed before the clinical
application of such a procedure: (i) for how long (3 or 5/6 days)
embryos should be cultured before the culture medium can be
used for PGD; (ii) The minimum volume of medium required for
robust diagnosis; (iii) the required quality of cell-free DNA; (iv)
whether the results may be obtained the very same day of sample
collection; (v) how to discriminate between maternal DNA (from
the surrounding CCs) and embryo DNA; (vi) the extent of contamination of culture medium by cells that contain full length genomic
DNA.
Discussion
Implications of the hypothesis
As a proof of concept, non-invasive PGD may be first performed
for X-linked disorders, which are a major issue for males. Prior
embryo sexing will facilitate the development of genetic testing.
SRY (Sex determining Region Y) and other Y chromosome specific
genes, such as TSPY1, TBC1D3, RPS4Y1 (Ribosomal protein S4,
Y-linked 1), DDX3Y (DEAD (Asp-Glu-Ala-Asp) box polypeptide 3,
Y-linked) and EIF1AY (Eukaryotic translation initiation factor 1A,
Y-linked) may be used to determine the embryo sex by PCR amplification. The presence of deleterious alleles for the most common
and serious X-linked disorders, such as Duchenne muscular dystrophy [8] and hemophilia [9,10], will then be tested only in XY
embryos. We strongly believe that this methodology for PGD of
Despite these challenging questions, we can reasonably predict
that the non-invasive diagnosis of genetic disorders using cell-free
nucleic acids, including cfDNA released by human embryos in the
culture medium, will become a reality in the near future. This prediction is supported by our better understanding of the biology of
cell-free nucleic acids and the technological advances in their
detection and analysis [12,13]. As culture medium is easily accessible and can be sampled at different intervals, it is an ideal material for assessing the embryo quality and for genetic testing to
improve the selection of healthy embryos before implantation or
preservation.
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S. Assou et al. / Medical Hypotheses 83 (2014) 506–508
A
B
TSPY
Fluorescence
Fluorescence
ALU
XY
D1
XY
XX
D1
D2
XX
D2
C
Cycles
Cycles
Fluorescence
TSPY
330 pg
D1
D3
110 pg
11 pg
XX
D2
Cycles
Fig. 2. Detection of the TSPY1 gene in embryo culture medium samples. (A) TSPY1 expression was assessed in two different culture medium samples (D1 and D2) by dropletbased PCR amplification. Only sample D1 was TSPY1-positive. DNA from human foreskin fibroblasts (XY) was used as positive control for Y chromosome assessment. DNA
from maternal cumulus cell (XX) was used as a negative control. (B) Human ALU repeats were amplified in all samples used in (A) (XY, D1, D2 and XX). (C) The sensitivity of
TSPY1 sequence amplification was evaluated using DNA from human foreskin fibroblasts (XY) at three concentrations (11 pg, 110 pg and 330 pg). The TSPY1 signal for D1 and
D3 (another culture medium sample) was between the signals obtained with 11 pg and 110 pg of DNA from human foreskin fibroblasts. QPCR experiments were performed
on Roche LC480. Fluorescence was acquired at each cycle and plotted against the cycle number. The increasing amount of the measured fluorescence is proportional to the
amount of PCR product generated during the reaction.
Conflict of interest statement
The authors have no personal or financial conflicts of interest
pertaining to this manuscript.
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