U.S. patent application number 17/472848 was filed with the patent office on 2022-04-07 for methods for determining the quality of an embryo.
The applicant listed for this patent is CENTRE HOSPITALIER UNIVERSITAIRE DE MONTPELLIER, INSERM (INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE), INSTITUT REGIONAL DU CANCER DE MONTPELLIER, UNIVERSITE DE MONTPELLIER. Invention is credited to Said ASSOU, Safia EL MESSAOUDI, Samir HAMAMAH, Alain THIERRY.
Application Number | 20220106643 17/472848 |
Document ID | / |
Family ID | |
Filed Date | 2022-04-07 |
![](/patent/app/20220106643/US20220106643A1-20220407-D00001.png)
![](/patent/app/20220106643/US20220106643A1-20220407-D00002.png)
![](/patent/app/20220106643/US20220106643A1-20220407-D00003.png)
![](/patent/app/20220106643/US20220106643A1-20220407-D00004.png)
![](/patent/app/20220106643/US20220106643A1-20220407-D00005.png)
![](/patent/app/20220106643/US20220106643A1-20220407-D00006.png)
![](/patent/app/20220106643/US20220106643A1-20220407-D00007.png)
United States Patent
Application |
20220106643 |
Kind Code |
A1 |
HAMAMAH; Samir ; et
al. |
April 7, 2022 |
METHODS FOR DETERMINING THE QUALITY OF AN EMBRYO
Abstract
The present invention relates generally to the fields of
reproductive medicine. More specifically, the present invention
relates to in vitro non invasive methods and kits for determining
the quality of an embryo by determining the level of the cell free
nucleic acids and/or determining the presence and/or expression
level of at least one specific nucleic acid sequence in the nucleic
acid extraction.
Inventors: |
HAMAMAH; Samir;
(Montpellier, FR) ; EL MESSAOUDI; Safia;
(Montpellier, FR) ; THIERRY; Alain; (Montpellier,
FR) ; ASSOU; Said; (Montpellier, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INSERM (INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE
MEDICALE)
UNIVERSITE DE MONTPELLIER
CENTRE HOSPITALIER UNIVERSITAIRE DE MONTPELLIER
INSTITUT REGIONAL DU CANCER DE MONTPELLIER |
Paris
Montpellier
Montpellier Cedex 05
Montpellier |
|
FR
FR
FR
FR |
|
|
Appl. No.: |
17/472848 |
Filed: |
September 13, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16135293 |
Sep 19, 2018 |
11149314 |
|
|
17472848 |
|
|
|
|
14898591 |
Dec 15, 2015 |
|
|
|
PCT/EP2014/062895 |
Jun 13, 2014 |
|
|
|
16135293 |
|
|
|
|
International
Class: |
C12Q 1/6883 20060101
C12Q001/6883 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 18, 2013 |
EP |
13305820.6 |
Claims
1. A method for selecting a human embryo and subsequently
implanting said embryo in a female undergoing in vitro
fertilization, said method comprising the steps consisting of: i)
providing a sample of the culture medium where the embryo is grown;
ii) extracting the cell free nucleic acids from the sample; iii)
detecting the presence of at least one specific nucleic acid
sequence in the nucleic acid extraction; iv) selecting the embryo
in view of the results obtained in step iii); and v) implanting the
embryo selected in step iv) in said female.
2. The method according to claim 1, wherein step iii) comprises
detecting the presence of at least one genetic alteration or
genetic abnormality in the nucleic extraction.
3. The method according to claim 1, wherein the step of detecting
the presence of at least one specific nucleic acid sequence is
performed by PCR or quantitative PCR.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to the fields of
reproductive medicine. More specifically, the present invention
relates to methods and kits for determining the quality of an
embryo.
BACKGROUND OF THE INVENTION
[0002] Currently, there is no reliable commercially available
genetic or non-genetic procedure for determining the quality of an
embryo during assisted reproductive technology (ART). Notably an
essential issue remains to determine whether an embryo is capable
of yielding viable offspring when transferred to an appropriate
uterine environment. Another important issue is to determine the
genetic profiling of an embryo that will render the development of
the foetus and even after of the child viable.
[0003] The selection of embryos with higher implantation potential
is one of the major challenges in assisted reproductive technology
(ART). Initially, multiple-embryo transfer (MET) was used to
maximize pregnancy rates. However, improved embryo quality and
raising multiple pregnancy rates have resulted in the decrease in
the number of embryos for replacement. Therefore, selection of the
`best` embryo has become crucial, particularly with elective single
embryo transfer (SET) being strongly recommended. There is
therefore a need to develop new objective approaches for embryo
selection. The classical methods to select healthy embryos under
IVF and ICSI conditions are based on subjective morphological
criteria such as fragmentation degree and the presence of
multi-nucleation, the number and size of blastomeres, early
embryonic cleavage (Ebner et al., 2003; Fenwick et al., 2002).
However, most studies suggest that embryos with proper
morphological appearance alone are not sufficient to predict a
successful implantation. Considering the limitation of morphologic
evaluation and cytogenetic screening methods, there is now a
movement toward more sophisticated, high-performance technologies
and the emerging `omics` science, such as transcriptomics and
metabolomics. These approaches focus on a variety of bodily cells
as well as embryonic culture media. An indirect and attractive
approach for predicting embryo and pregnancy outcomes has been
reported by our team using transcriptomic data of cumulus cells
(CCs) gene expression (Assou et al., 2011; Assou et al., 2008). We
observed that there was no relationship between embryo
morphological aspects and the CC gene expression profile (Assou et
al., 2010). Other studies reported that metabolomic profile of
spent culture media by Raman or near-infrared (NIR) spectroscopy
correlates with reproductive potential of individual embryos (Seli
et al., 2007; Vergouw et al., 2008). They showed also that
metabolomic profiling of culture media from embryos was independent
of morphology.
[0004] Another major cause of reduced implantation rate is poor
genetic quality of the implanted embryo. For example, most
embryonic wastage and loss are caused by aneuploidies (chromosome
number abnormalities) that are lethal and occur in approximately
60% of all spontaneous abortions and still births. Other genetic
abnormalities include chromosomal aneuploidy, amplification,
translocation, insertion/deletion, inversion, short tandem repeat
polymorphisms, microsatellite polymorphisms, single nucleotide
polymorphisms (SNPs), and other structural abnormalities. Genetic
abnormalities can cause many phenotypic diseases and some are even
lethal. If genetic abnormalities occur in embryos, many types of
prenatal conditions and congenital diseases are likely to develop.
Screening these abnormalities by preimplantation genetic diagnosis
(PGD) is very important to ensure a structurally normal embryo
selection and viable implantation. However, current methods are
invasive may cause prejudice to the embryo.
[0005] It was reported that cell free DNA may be detected in
biological fluids such as blood, ascite, urine, amniotic fluid,
feces, saliva or cerebrospinal fluids. Various nucleic acids such
as DNA, RNA, miRNA were indeed isolated and detected in cell free
form. cfDNA was found detectable amount in healthy subjects as well
as, in greater amount, in some pathological disorders (cancer,
myocardial infarction, autoimmune disease, sepsis, trauma, . . . )
or specific physiological state (intense effort, . . . ). The
mechanisms of release of cfDNA are very poorly known, but it has
been suggested that necrosis, apoptosis, phagocytosis or active
release might be implicated. CfDNA analysis is an area of active
investigation in the diagnostic field especially in two areas is
subjected to high scrutiny at this time. However, detection of
cfDNA has not yet been investigated for determining the quality of
an embryo.
SUMMARY OF THE INVENTION
[0006] The present invention is based on the dramatic discovery of
the presence of amounts of cell free nucleic acids in the culture
medium where the embryo is grown under in vitro fertilization
conditions. The inventors demonstrate that the level of said cell
free nucleic acids in the culture medium is informative about the
ability of the embryo to give rise to a pregnancy. Moreover, the
inventors demonstrate that the analysis of said cell free nucleic
acids make the detection and expression of a specific sequence gene
expression possible and pave the way for the development of a
non-invasive method for the genetic profiling of an embryo.
DETAILED DESCRIPTION OF THE INVENTION
[0007] The present invention relates to an in vitro non invasive
method for determining the quality of an embryo comprising the
steps consisting of i) providing a sample of the culture medium
where the embryo is grown, ii) extracting the cell free nucleic
acids from the sample and iii) determining the level of the cell
free nucleic acids in the nucleic acid extraction and/or
determining the presence and/or expression level of at least one
specific nucleic acid sequence in the nucleic acid extraction.
[0008] As used herein the term "embryo" has its general meaning in
the art and refers to a fertilized oocyte or zygote. The term
"embryo" also refers to cells in all stages of development from a
fertilized oocyte or zygote up to the 5 or 6 days (blastocyst
stage). Said fertilization may intervene under a classical in vitro
fertilization (cIVF) conditions or under an intracytoplasmic sperm
injection (ICSI) procedure. Examples of embryos that may be
assessed by the methods of the invention include 1-cell embryos
(also referred to as zygotes), 2-cells embryo, 3-cells embryo,
4-cells embryo, 5-cells embryo, 6-cells embryo, 8-cells embryo,
etc. typically up to and including 16-cells embryo, any of which
may be derived by any convenient manner, e.g. from an oocyte that
has matured in vivo or from an oocyte that has matured in vitro. As
used herein, the term "blastocyst" refers to the structure formed
in the early embryogenesis of mammals, after the formation of the
morula. It possesses an inner cell mass (ICM), or embryoblast,
which subsequently forms the embryo, and an outer layer of cells,
or trophoblast, which later forms the placenta. The trophoblast
surrounds the inner cell mass and a fluid-filled blastocyst cavity
known as the blastocoele. The human blastocyst comprises 70-100
cells. Blastocyst formation begins at day 5/6 after fertilization
in humans.
[0009] According to the invention, the oocyte may result from a
natural cycle, a modified natural cycle or a stimulated cycle for
cIVF or ICSI. The term "natural cycle" refers to the natural cycle
by which the female or woman produces an oocyte. The term "modified
natural cycle" refers to the process by which, the female or woman
produces an oocyte or two under a mild ovarian stimulation with
GnRH antagonists associated with recombinant FSH or hMG. The term
"stimulated cycle" refers to the process by which a female or a
woman produces one or more oocytes under stimulation with GnRH
agonists or antagonists associated with recombinant FSH or hMG.
[0010] The term "classical in vitro fertilization" or "cIVF" refers
to a process by which oocytes are fertilised by sperm outside of
the body, in vitro. IVF is a major treatment in infertility when in
vivo conception has failed. The term "intracytoplasmic sperm
injection" or "ICSI" refers to an in vitro fertilization procedure
in which a single sperm is injected directly into an oocyte. This
procedure is most commonly used to overcome male infertility
factors, although it may also be used where oocytes cannot easily
be penetrated by sperm, and occasionally as a method of in vitro
fertilization, especially that associated with sperm donation.
[0011] By "determining the quality of an embryo" it is meant that
the method of the invention aims at determining whether an embryo
is competent and/or bears a genetic abnormality or a specific
sequence in the context of in vitro fertilization. The method of
the invention allows the assessment of the ability of an embryo to
perform successfully either or both in terms of conferring a high
pregnancy rate and/or resulting in a healthy person. Accordingly
the method of the invention is able to combine pre-implantation
genetic testing and selection of the best embryo that is able to
give rise to pregnancy.
[0012] The term "competent embryo" refers to an embryo with a high
implantation rate leading to pregnancy. The term "high implantation
rate" means the potential of the embryo when transferred in uterus,
to be implanted in the uterine environment and to give rise to a
viable fetus, which in turn develops into a viable offspring absent
of a procedure or event that terminates said pregnancy.
[0013] As used herein the term "genetic abnormality" refers to any
event that can exist in the genome of an individual (i.e. an
embryo) that can give rise to cause a phenotypic disease and
lethality. Genetic abnormalities include but are not limited to
aneuploidy, translocation, gene/locus amplification, insertions,
deletions, reversions, short tandem repeat (STR) polymorphisms,
microsatellite polymorphisms, single nucleotide polymorphisms
(SNPs), single genetic mutations responsible for inherited
diseases, or a combination thereof. In particular, any genetically
transmissible disease may be detected according to the present
method. For example genetic alteration can include known
alterations in one or more of the genes: CFTR, Factor VIII (F8
gene), beta globin, hemachromatosis, G6PD, neurofibromatosis,
GAPDH, beta amyloid, and pyruvate kinase. The sequences and common
mutations (e.g., single nucleotide polymorphisms or SNPs) of the
genes are known. Other genetic abnormalities may be detected, such
as those involving a sequence which is deleted in a human
chromosome, is moved in a translocation or inversion, or is
duplicated in a chromosome duplication, wherein said sequence is
characterized in a known genetic disorder in the fetal genetic
material. For example chromosome aneuploidy, such as Down syndrome
(or trisomy 21), Edwards syndrome (trisomy 18), Patau syndrome
(trisomy 13), Turner Syndrome (45X0) Klinefelter's syndrome (a male
with 2.times. chromosomes), Prader-Willi syndrome, and DiGeorge
syndrome. A listing of known genetic abnormalities may be found in
the OMIM database (http://omim.org/).
[0014] The method of the invention is applicable preferably to
women but in theory may be applicable to other mammals (e.g.,
primates, dogs, cats, pigs, cows, mouse . . . ).
[0015] As used herein the term "nucleic acid" has its general
meaning in the art and refers to refers to a coding or non coding
nucleic sequence. Nucleic acids include DNA (deoxyribonucleic acid)
and RNA (ribonucleic acid). Example of nucleic acid thus include
but are not limited to DNA, mRNA, tRNA, rRNA, tmRNA, miRNA, piRNA,
snoRNA, and snRNA. According to the invention, the nucleic acid may
originate form the nucleus of the embryo or for the mitochondrial
compartment of the embryo. By "cell free nucleic acid" it is meant
that the nucleic acid is released by the embryo and present in the
culture medium wherein the embryo is grown after in vitro
fertilization or intracytoplasmic sperm injection (ICSI).
[0016] In a particular embodiment, sample is prepared when the
embryo has reached the balstocyst stage corresponding to day 5 or 6
of the embryo development. Any methods well known in the art may be
used for preparing a sample of the culture medium where the embryo
was grown after in vitro fertilization or intracytoplasmic sperm
injection (ICSI). One essential feature of the invention is that
the embryo remains viable during preparation of the sample. No
lytic enzyme or chemical reagents-based lysis solution are used to
maintain the integrity of the embryo. The method of the invention
is a perfect non-invasive method and only relies to the fact that
an embryo is capable to release nucleic acids in the culture medium
by a mechanism not yet determined.
[0017] Any methods well known in the art may be used by the skilled
artisan in the art for extracting the free cell nucleic acid from
the prepared sample. For example, the method described in the
example may be used.
[0018] In a particular embodiment the method of the invention
comprises the steps consisting of i) determining the level of the
nucleic acid in the nucleic acid extraction, ii) comparing the
level determined at step i) with a reference value, and iii)
concluding that the embryo is competent when the level determined
at step i) is lower than the reference value.
[0019] Determination of the level of the nucleic acid can be
performed by a variety of techniques well known in the art. In a
particular embodiment, quantitative PCR may be performed for
determining the level of DNA such as described in El Messaoudi et
al., 2013; Mouliere et al., 2013; Thierry et al., 2013 and
WO2012/028746. In particular, the determination of the level of the
nucleic acid may be performed as described in the example.
[0020] In a particular embodiment, the reference value consists in
the level of the nucleic acids determined in an embryo culture
medium at day 3 of embryo development. Accordingly, the decrease of
the level between day 3 of embryo development and day 5 or 6
(blastocyst stage) indicates that the embryo is competent
[0021] In a particular embodiment, the reference value is a
threshold value or a cut-off value that can be determined
experimentally, empirically, or theoretically. A threshold value
can also be arbitrarily selected based upon the existing
experimental and/or clinical conditions, as would be recognized by
a person of ordinary skilled in the art. The threshold value has to
be determined in order to obtain the optimal sensitivity and
specificity according to the function of the test and the
benefit/risk balance (clinical consequences of false positive and
false negative). Typically, the optimal sensitivity and specificity
(and so the threshold value) can be determined using a Receiver
Operating Characteristic (ROC) curve based on experimental data.
Preferably, the person skilled in the art may compare the nucleic
acid levels (obtained according to the method of the invention)
with a defined threshold value. In one embodiment of the present
invention, the threshold value is derived from the nucleic acid
levels (or ratio, or score) determined in an embryo culture mediums
derived from one or more patients undergoing IVF or ISCI.
Furthermore, retrospective measurement of the nucleic acid levels
(or ratio, or scores) in properly banked historical embryo culture
mediums of patients undergoing IVF or ISCI may be used in
establishing these threshold values.
[0022] In a particular embodiment the method of the invention
comprises the steps consisting of i) detecting at least one
mutation in the nucleic extraction, and ii) concluding that the
embryo bears a genetic abnormality when the mutation is
detected.
[0023] Typical techniques for detecting a mutation in a nucleic
acid in particular DNA or mRNA include but are not limited
restriction fragment length polymorphism, hybridisation techniques,
sequencing, exonuclease resistance, microsequencing, solid phase
extension using ddNTPs, extension in solution using ddNTPs,
oligonucleotide assays, methods for detecting single nucleotide
polymorphism such as dynamic allele-specific hybridisation,
ligation chain reaction, mini-sequencing, DNA "chips",
allele-specific oligonucleotide hybridisation with single or
dual-labelled probes merged with PCR or with molecular beacons, and
others.
[0024] Typically, mutations are detected after amplification. For
instance, the isolated RNA may be subjected to coupled reverse
transcription and amplification, such as reverse transcription and
amplification by polymerase chain reaction (RT-PCR), using specific
oligonucleotide primers that are specific for a mutated site or
that enable amplification of a region containing the mutated site.
According to a first alternative, conditions for primer annealing
may be chosen to ensure specific reverse transcription (where
appropriate) and amplification; so that the appearance of an
amplification product be a diagnostic of the presence of a
particular mutation. Otherwise, RNA may be reverse-transcribed and
amplified, or DNA may be amplified, after which a mutated site may
be detected in the amplified sequence by hybridization with a
suitable probe or by direct sequencing, or any other appropriate
method known in the art. For instance, a cDNA obtained from RNA may
be cloned and sequenced to identify a mutation.
[0025] In particular sequencing represents an ideal technique that
can be used in the context of the present invention. The one
skilled in the art is familiar with several methods for sequencing
of polynucleotides. These include, but are not limited to, Sanger
sequencing (also referred to as dideoxy sequencing) and various
sequencing-by-synthesis (SBS) methods as reviewed by Metzger
(Metzger ML 2005, Genome Research 1767), sequencing by
hybridization, by ligation (for example, WO 2005/021786), by
degradation (for example, U.S. Pat. Nos. 5,622,824 and 6,140,053),
nanopore sequencing. Preferably in a multiplex assay deep
sequencing is preferred. The term "deep sequencing" refers to a
method of sequencing a plurality of nucleic acids in parallel. See
e.g., Bentley et al, Nature 2008, 456:53-59. The leading
commercially available platforms produced by Roche/454 (Margulies
et al., 2005a), Illumina/Solexa (Bentley et al., 2008), Life/APG
(SOLiD) (McKernan et al., 2009) and Pacific Biosciences (Eid et
al., 2009) may be used for deep sequencing. For example, in the 454
method, the DNA to be sequenced is either fractionated and supplied
with adaptors or segments of DNA can be PCR-amplified using primers
containing the adaptors. The adaptors are nucleotide 25-mers
required for binding to the DNA Capture Beads and for annealing the
emulsion PCR Amplification Primers and the Sequencing Primer. The
DNA fragments are made single stranded and are attached to DNA
capture beads in a manner that allows only one DNA fragment to be
attached to one bead. Next, the DNA containing beads are emulsified
in a water-in-oil mixture resulting in microreactors containing
just one bead. Within the microreactor, the fragment is
PCR-amplified, resulting in a copy number of several million per
bead. After PCR, the emulsion is broken and the beads are loaded
onto a pico titer plate. Each well of the pico-titer plate can
contain only one bead. Sequencing enzymes are added to the wells
and nucleotides are flowed across the wells in a fixed order. The
incorporation of a nucleotide results in the release of a
pyrophosphate, which catalyzes a reaction leading to a
chemiluminescent signal. This signal is recorded by a CCD camera
and a software is used to translate the signals into a DNA
sequence. In the Illumina method (Bentley (2008)), single stranded,
adaptor-supplied fragments are attached to an optically transparent
surface and subjected to "bridge amplification". This procedure
results in several million clusters, each containing copies of a
unique DNA fragment. DNA polymerase, primers and four labeled
reversible terminator nucleotides are added and the surface is
imaged by laser fluorescence to determine the location and nature
of the labels. Protecting groups are then removed and the process
is repeated for several cycles. The SOLiD process (Shendure (2005))
is similar to 454 sequencing, DNA fragments are amplified on the
surface of beads. Sequencing involves cycles of ligation and
detection of labeled probes. Several other techniques for
high-throughput sequencing are currently being developed. Examples
of such are The Helicos system (Harris (2008)), Complete Genomics
(Drmanac (2010)) and Pacific Biosciences (Lundquist (2008)). As
this is an extremely rapidly developing technical field, the
applicability to the present invention of high throughput
sequencing methods will be obvious to a person skilled in the
art.
[0026] In a particular embodiment the method of the invention
comprises the steps consisting of i) determining the level of at
least one specific nucleic acid sequence, ii) comparing the level
determined at step i) with a reference value and iii) concluding
that the embryo bears a genetic abnormality when the level
determined at step i) is different from the reference value (i.e.
lower or higher depending on the nucleic acid looked).
[0027] Determining the expression level of a nucleic acid (in
particular a gene, miRNA, snRNA, and snoRNA) may be assessed by any
of a wide variety of well-known methods. Typically the prepared
nucleic acid can be used in hybridization or amplification assays
that include, but are not limited to, Southern or Northern
analyses, polymerase chain reaction analyses, such as quantitative
PCR (TaqMan), and probes arrays such as GeneChip.TM. DNA Arrays
(AFF YMETRIX). Advantageously, the analysis of the expression level
of a nucleic acid involves the process of nucleic acid
amplification, e. g., by RT-PCR (the experimental embodiment set
forth in U.S. Pat. No. 4,683,202), ligase chain reaction (BARANY,
Proc. Natl. Acad. Sci. USA, vol. 88, p: 189-193, 1991), self
sustained sequence replication (GUATELLI et al., Proc. Natl. Acad.
Sci. USA, vol. 57, p: 1874-1878, 1990), transcriptional
amplification system (KWOH et al., 1989, Proc. Natl. Acad. Sci.
USA, vol. 86, p: 1173-1177, 1989), Q-Beta Replicase (LIZARDI et
al., Biol. Technology, vol. 6, p: 1197, 1988), rolling circle
replication (U.S. Pat. No. 5,854,033) or any other nucleic acid
amplification method, followed by the detection of the amplified
molecules using techniques well known to those of skill in the art.
Real-time quantitative or semi-quantitative RT-PCR is preferred. In
a particular embodiment, the determination comprises hybridizing
the sample with selective reagents such as probes or primers and
thereby detecting the presence, or measuring the amount of the
nucleic acid. Hybridization may be performed by any suitable
device, such as a plate, microtiter dish, test tube, well, glass,
column, and so forth. Nucleic acids exhibiting sequence
complementarity or homology to the nucleic acid of interest herein
find utility as hybridization probes or amplification primers. It
is understood that such nucleic acids need not be identical, but
are typically at least about 80% identical to the homologous region
of comparable size, more preferably 85% identical and even more
preferably 90-95% identical. In certain embodiments, it will be
advantageous to use nucleic acids in combination with appropriate
means, such as a detectable label, for detecting hybridization. A
wide variety of appropriate indicators are known in the art
including, fluorescent, radioactive, enzymatic or other ligands
(e.g. avidin/biotin). The probes and primers are "specific" to the
nucleic acid they hybridize to, i.e. they preferably hybridize
under high stringency hybridization conditions (corresponding to
the highest melting temperature --Tm--, e.g., 50% formamide,
5.times. or 6.times.SCC. 1.times.SCC is a 0.15 M NaCl, 0.015 M
Na-citrate). Many quantification assays are commercially available
from Qiagen (S.A. Courtaboeuf, France) or Applied Biosystems
(Foster City, USA). Expression level of the nucleic acid may be
expressed as absolute expression profile or normalized expression
profile. Typically, expression profiles are normalized by
correcting the absolute expression profile of the nucleic acid of
interest by comparing its expression to the expression of a nucleic
acid that is not a relevant, e.g., a housekeeping mRNA that is
constitutively expressed. Suitable mRNA for normalization include
housekeeping mRNAs such as the U6, U24, U48 and S18. This
normalization allows the comparison of the expression profile in
one sample, e.g., a patient sample, to another sample, or between
samples from different sources.
[0028] Probe and or primers are typically labelled with a
detectable molecule or substance, such as a fluorescent molecule, a
radioactive molecule or any others labels known in the art. Labels
are known in the art that generally provide (either directly or
indirectly) a signal. The term "labelled" is intended to encompass
direct labelling of the probe and primers by coupling (i.e.,
physically linking) a detectable substance as well as indirect
labeling by reactivity with another reagent that is directly
labeled. Examples of detectable substances include but are not
limited to radioactive agents or a fluorophore (e.g. fluorescein
isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine
(Cy5)).
[0029] The reference value may be determined as above described and
will depend on the nucleic acid for which the determination of the
expression level is required for concluding that the embryo bears a
genetic abnormality or a nucleic acid specific sequence.
[0030] The method of the invention is particularly suitable for
reaching a clinical decision. As used herein the term "clinical
decision" refers to any decision to take or not take an action that
has an outcome that affects the health or survival of the embryo.
In particular, in the context of the invention, a clinical decision
refers to a decision to implant or not the embryo of in the uterus
of the patient. A clinical decision may also refer to a decision to
conduct further testing, to take actions to mitigate an undesirable
phenotype, or to take actions to prepare for the birth of a child
with abnormalities. In particular the method as above described
will thus help embryologist to avoid the transfer in uterus of
embryos with a poor potential for pregnancy outcome. The method as
above described is also particularly suitable for avoiding multiple
pregnancies by selecting the competent embryo able to lead to an
implantation and a pregnancy and therefore fewer embryos could be
transferred at each cycle, resulting in a decreased incidence of
multiple pregnancies.
[0031] The methods of the invention are particularly suitable for
enhancing the pregnancy outcome of a child with a minimum of risk
of having a genetic abnormality. Accordingly the invention also
relates to a method for enhancing the pregnancy outcome of a
patient comprising the steps consisting of i) providing a plurality
of embryos, ii) determining the quality of the embryo by the method
according to the invention and iii) selecting the most competent
embryo with the minimum risk of bearing a genetic abnormality, and
iv) implanting the embryo selected at step iii) in the uterus of
said patient.
[0032] The invention also relates to a kit for performing the
methods as above described, wherein said kit comprises means for
determining the level of the cell free nucleic and/or means for
determining the expression level of at least one specific nucleic
acid and/or means for detecting at least one mutation, one SNP or a
specific sequence in the nucleic acid extraction. Typically, the
kits include probes, primers macroarrays or microarrays as above
described. For example, the kit may comprise a set of probes as
above defined, and that may be pre-labelled. Alternatively, probes
may be unlabelled and the ingredients for labelling may be included
in the kit in separate containers. The kit may further comprise
hybridization reagents or other suitably packaged reagents and
materials needed for the particular hybridization protocol,
including solid-phase matrices, if applicable, and standards.
Alternatively the kit of the invention may comprise amplification
primers (e.g. stem-loop primers) that may be pre-labelled or may
contain an affinity purification or attachment moiety. The kit may
further comprise amplification reagents and also other suitably
packaged reagents and materials needed for the particular
amplification protocol.
[0033] The invention will be further illustrated by the following
figures and examples. However, these examples and figures should
not be interpreted in any way as limiting the scope of the present
invention.
FIGURES
[0034] FIG. 1: cfDNA concentrations in culture medium of several
embryos from two patients at Day3.
[0035] FIG. 2: cfDNA concentrations in culture medium of several
embryos from 9 patients at Day5/6.
[0036] FIG. 3: cfDNA concentrations in culture medium of several
embryos from two patients at Day3 and Day5. Dark histogram, D5/6
concentration. Clear histogram, D3 concentration.
[0037] FIG. 4A: Difference of the cfDNA concentrations between Day3
and Day5/6 respective to embryo grade. Serie 1 histograms, D3-D5/6
concentration (ng/mL cfDNA); serie 2 histograms, ranking respective
to growing grade (1-10). CfDNA concentration values obtained in the
serie of the embryo of the HSC patient.
[0038] FIG. 4B: cfDNA concentrations respective to embryo grade.
Serie 1 histograms, Day5/6 concentration (ng/mL cfDNA); serie 2
histograms, ranking respective to growing grade (1 to 10). CfDNA
concentration values obtained in the serie of the embryo of the HSC
patient.
[0039] FIG. 5: Relationship between cfDNA in culture media and
pregnancy outcome. Histograms comparing the mean cfDNA quantity in
embryo culture media at day 5/6 issued from positive pregnancy
patients and negative pregnancy patients.
[0040] FIGS. 6A and 6B: Histograms show the microarray signal
values for genes in ovary, testes, MII oocytes, day 3 embryos, day
5/6 blastocysts, trophectoderm and endometrium samples. Microarray
data from MII oocytes, day 3 embryos, day 5/6 blastocysts,
trophectoderm and endometrium samples were obtained from our team
and those for ovary and testes samples were obtained from the Gene
Expression Omnibus (GEO) through the provisional accession numbers
(GPL570).
EXAMPLE
[0041] Material & Methods
[0042] IVF Procedure
[0043] The women underwent a gonadotropin-releasing hormone (Gn-RH)
long or antagonist protocols treatment, which was followed by
ovarian stimulation by hMG (human menopausal gonadotropin) or
recombinant follicle-stimulating hormone (FSH). When at least three
follicles reached a mean diameter of 17 mm under transvaginal
ultrasound examination, 5000 IU hCG was administrated. Then, 36 h
later, the oocytes were retrieved by ultrasound-guided
trans-vaginal puncture. Conventional IVF or ICSI was use as
indicated. Fertilization was confirmed 16 to 20 h after oocyte
insemination or microinjection by the presence of two distinct
pronuclei under the inverted microscope+two polar bodies. The
zygotes were then placed individually into fresh 30 .mu.l droplets
of culture medium (G1.5, Vitolife, Sweden) covered with mineral oil
and maintained in a tri-gas incubator, which provide a 5% oxygen
environment. All embryos were cultured in individual droplets at
all times. The embryos were placed into extended culture media and
continued until day 5. G2.5 medium (Vitolife, Sweden) was used for
extended culture.
[0044] Quantification of the cfDNA in Culture Media of the
Invention
[0045] Embryo Culture Media Sampling After the removal of the
embryos, the culture media were placed individually into labeled
cryovials and then labeled again with a randomly assigned accession
number. The collected specimens were immediately frozen and stored
at -80.degree. C. A control sample incubated under the same
conditions without an embryo was also collected. Up to 50 .mu.L may
be sampled from the culture media.
[0046] cfDNA Extraction
[0047] For Day 3 or Day5/6 samples, initial volume of 30 .mu.L was
completed to 200 .mu.L with 170 .mu.L of PBS 1.times.. For D5
samples, initial volume of 10 .mu.L was completed to 200 .mu.L with
190 .mu.L of PBS 1.times.. Subsequently, samples were either
immediately handled for DNA extraction. CcfDNA was extracted from
200 .mu.L of the sample using the QIAmp DNA Mini Blood Kit (Qiagen,
Hilden, Germany) according to the "Blood and body fluid protocol."
DNA samples were kept at -20.degree. C. until use.
[0048] cfDNA Quantification by Q-PCR
[0049] The methodology and the data description were carried out
according to the MIQE guidelines.q-PCR amplifications were carried
out at least in duplicate in a 25-.mu.l reaction volume on a
CFX96.TM. real-time PCR detection system using the CFX Manager.TM.
software (Bio-Rad, Hercules, Calif.). Each PCR mixture was composed
of 12.5 .mu.l of PCR mix (Bio-Rad Supermix SYBR Green), 2.5 .mu.l
of each amplification primer (0.3 pmol/.mu.l), 2.5 .mu.l of
PCR-analyzed water, and 5 .mu.l of DNA extract. Thermal cycling
consisted of three repeated steps: a 3-minute hot-start polymerase
activation-denaturation step at 95.degree. C. followed by 40
repeated cycles at 95.degree. C. for 10 seconds and then at
60.degree. C. for 30 seconds. Melting curves were obtained by
increasing the temperature from 55 to 90.degree. C. with a plate
reading every 0.2.degree. C. Serial dilutions of genomic DNA from
human placenta cells (Sigma, Munich, Germany) were used as standard
for quantification and their concentration and quality was assessed
using a Qubit.RTM. 2.0 Fluorometer (Life Technologies). Every Q-PCR
run comprised routine quality negative and positive controls. Each
sample was analyzed in triplicate and each assay was repeated at
least once. The cfDNA concentrations obtained were normalized to
the precise concentration using the standard curve. The coefficient
of variation of the concentration value due to cfDNA extraction and
Q-PCR analysis was calculated as 24% from two experiments (n=12).
Quantification of cfDNA in samples and realization of the standard
curve were performed by using the primer systems described in Table
1 (KRAS B1 inv k Sense: SEQ ID NO: 1; and KRAS B2 inv k Antisense:
SEQ ID NO:2). Concentration value determined by the test exhibit a
coefficient of variation of 24%.
TABLE-US-00001 TABLE 1 Intplex primers used for cfDNA
quantification. Intplex Direction Primer Sequence Tm Amplicon
Species Gene Name 5'-3' (.degree. C.) Size (bp) Human Kras KRAS B1
CCTTGGGTTT 54.0 67 Inv k CAAGTTATATG Sense Human Kras KRAS B2
CCCTGACATA 59.4 Inv k CTCCCAAGGA Antisense
[0050] Primer Design
[0051] The sequences and characteristics of the selected primers
are presented in Table 1. The primers were designed using the
Primer 3 software and all sequences were checked for self-molecular
or intermolecular annealing with nucleic acid folding software
(mfold and oligoAnalyzer 1.2). We performed local alignment
analyses with the BLAST program to confirm the specificity of the
designed primers. Oligonucleotides were synthesized and purified on
high performance liquid chromatography (HPLC) by Eurofins
(Ebersberg, Germany) and quality control of the oligonucleotides
was performed by matrix-assisted laser desorption ionization-time
of flight (MALDI-TOF).
[0052] The Q-PCR system was designed to be able to quantify a
sequence present in two copies of the human genome. It enables the
highly specific and sensitive quantification of this sequence in
one allele. Higher specificity is obtained by using Allele Specific
with Blocker PCR with using the same primers (Mouliere, 2011). This
method allows for distinguishing two sequences having only one
nucleotide difference with a 0.005 mutant/WT ratio. Thus detecting
and quantifying a specific sequence may correspond either at
distinguishing a WT sequence versus sequences with a few
nucleotides difference up to only one nucleotide difference such as
in sequences with a point mutation or a SNP. Therefore the
demonstration of the quantification of cfDNA in embryo culture
medium as shown here, show the potential of this method to detect
the presence of a single nucleotide mutation, SNP, or other genetic
alterations.
[0053] Higher concentration values may be obtained when targeting
repeated sequences in the nuclear genome such as the lyne sequence,
or mitochondrial sequences.
[0054] Results
[0055] Detection of cfDNA in Embryo Culture Media
[0056] The targeted sequence has 2 copies per genome of the nucleus
of diploid cell. CfDNA could significantly be detected in embryo
culture media at D3 or D5/6 (FIG. 1). The test can detect down to
1.5 ng/ml medium and as such a minimum of 2 GE copy were found in
culture medium. Up to of 27 ng/ml cfDNA or 36 GE was observed in
D5/6 culture medium (FIG. 2). Note, those number may be relevant to
the embryo development. As such this data reveals the possibility
of the detection of the presence of a specific DNA sequence (at the
most 2 copies per diploid cell) and therefore the potential
presence of homozygous or heterozygous genetic or epigenetic
alteration. CfDNA could significantly be detected in all samples
and for each patient (FIG. 2).
[0057] There are significant (1 Log) intra and inter variation
between samples buttressing the notion that dynamics of measurement
enables comparison between samples (FIG. 3).
[0058] Relationship Between cfDNA and In Vitro Embryo Outcome
[0059] The relation between cfDNA content of embryo culture medium
and in vitro embryo development was also investigated:
[0060] cfDNA conc. determined at D3 and D5/6 could be compared in
culture media of eleven embryos. As shown in (FIG. 4A), the values
of the difference of the cfDNA concentration between D3 and D5/6
are growing respectively with the good embryo development as
evaluated by morphological criteria. As presented in (FIG. 4B)
cfDNA conc. values are inversely proportional to the good embryo
development. Thus, both D5/6 cfDNA conc. and D3-D5/6 conc. decrease
appear as a marker of in vitro embryo development.
[0061] cfDNA isolated from embryo culture media that developed into
good quality 8-cells embryo at day 3 and leading to blastocyst
stage at day 5/6 were selected and divided in three groups: i)
cfDNA from embryo at day 3 that developed into good blastocyst
quality at day 5/6 (grade 4AA, 4AB or 4BA, 5AA, 5AB or 5BA) and
leading to pregnancy, (ii) cfDNA from embryo at day 3 developed
into intermediary blastocyst quality at day 5/6 (grade 4BB or 5
BB), (iii) cfDNA from embryo at day 3 developed into bad blastocyst
quality at day 5/6 (grade 4CC or 5CC) (see table 2 for patient
HSC). The quantity of cfDNA in culture medium from embryo at day 3
that developed into good blastocyst quality at day 5/6 (grade 4AA,
4AB, 4BA) and leading to pregnancy was 22.16 ng/ml and 2.75 ng/ml
at day 3 and day 5/6 respectively (88%, decrease). The variation in
the cfDNA value between day 3 and day 5/6 decreased to 7.55 ng/ml
and 1.80 ng/ml (76%, decrease) in the intermediary grade and to
6.46 ng/ml (day 3) and 3.78 (day 5/6) (41%, decrease) in the no
good blastocyst grade (Table 3). Interestingly, this variation is
very low in the lyzed embryo 8.36 ng/ml (day 3) and 5.57 (day 5/6)
(33%, decrease). Additionally, cfDNA quantities in embryo culture
media at day 5/6 were evaluated according to patient outcome. We
show that cfDNA were greater in embryo culture media at day 5/6
from no-pregnant patients than that of pregnant patients (FIG.
5).
[0062] cfDNA in Embryo Culture Medium could be Used to Detect Male
Embryos
[0063] Genes such as TSPY1 (Testis specific protein, Y-linked 1)
and RPS4Y1 (Ribosomal protein S4, Y-linked 1) could be used to
revealed embryo sex. This opens up an appropriate strategy for
screening embryos from couples known to be at risk for an X-linked
disease. The high-density oligonucleotide Affymetrix HG-U133P
microarray chips were used to investigate the expression of TSPY1
and RPS4Y1 in XX and XY samples. Our results reveal that TSPY1 and
RPS4Y1 may prove valuable as biomarkers of embryo sex determination
by amplifying the multicops of these genes (cfDNA) in the embryo
culture medium (FIGS. 6A and 6B). The methods may be applicable to
other genes localized on the chromosome Y: DDX3Y (DEAD
(Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked), EIF1AY (Eukaryotic
translation) and Y chromosome gene (SRY).
TABLE-US-00002 TABLE 2 ICSI outcome of a patient (HSC) in relation
to cfDNA detected in embryo culture media. Data generated from each
mature MII oocyte (fertilization, embryo cleavage and blastocyst
development) were recorded by an embryologist according to the
morphological criteria of Gardner and Schoolcraft 1999. Day 2 Day 3
Patient Day 1 Blastomere Blastomere % of Blastomere Blastomere
(HSC) Fertilization number uniformity fragmentatio number
uniformity No2 Unfertilized No3 fertilized 4bl Equal. Homo. 10% 7bl
Equal. Homo. No4 fertilized 2PB 2PB No5 Unfertilized No6 fertilized
4bl Unequal. .+-.Homo. 30% 8bl Unequal. Homo. No7 fertilized 4bl
Equal. Homo. 10% 8bl Equal. Homo. No8 fertilized 4bl Equal.
.+-.Homo. 5% 8bl Equal. Homo. No9 Unfertilized No10 fertilized 4bl
Equal. Homo. 10% 8bl Equal. Homo. No11 Unfertilized No12 fertilized
4bl Equal. Homo. 10% 8bl Equal. Heter. No13 Unfertilized No14
fertilized 4bl Equal. Homo. 15% 8bl Equal. Homo. No15 fertilized
4bl Unequal. Homo. 15% 6bl Unequal. Heter. No16 Unfertilized No17
fertilized 4bl Unequal. .+-.Homo. 20% 10bl Unequal. .+-.Homo. No18
fertilized 4bl Unequal. .+-.Homo. 15% 9bl Unequal. Heter. No19
Unfertilized No20 fertilized 5bl Unequal. .+-.Homo. 10% 8bl
.+-.Equal. Homo. No21 fertilized 3bl Equal. Homo. 30% 8bl Unequal.
Homo. No22 fertilized 4bl Equal. Homo. 20% 8bl .+-.Equal. Homo.
No23 fertilized 4bl Equal. Homo. 10% 8bl .+-.Equal. .+-.Homo. No24
Unfertilized Drop Day 3 Drop Day 5 Day 3 Concentration
Concentration Patient % of Day 5 Day 6 CfDNA CfDNA (HSC)
fragmentatio Grade Grade Pregnancy (ng/ml) (ng/ml) No2 13.871 No3
15% 3CC 6.46 3.787 No4 2.938 No5 4.73 No6 40% Lyzed 8.365 5.572 No7
30% 5BB 12.725 (Frezing) No8 15% STOP 12.137 16.668 No9 4.255 No10
15% B1 other 12.653 2.435 No11 33.647 No12 10% 4AB 13.542 3.81
(Frezing) No13 0.777 No14 15% B1+ 5BB 7.55 1.804 (Frezing) No15 30%
B1 4BC 11.167 3.955 (Stop) No16 6.923 No17 20% 3CC 5CC 20.515
(Stop) No18 30% 4AC 5BA 12.07 N/A (Frezing) No19 20.968 No20 15% B1
5BB 75.392 2.28 (Frezing) No21 30% B1 5CC 14.219 2.81 (Stop) No22
25% 3BB 3BB 14.033 N/A (Frezing) No23 20% AA Positive 22.158 2.755
(Transfer) No24 8.41
TABLE-US-00003 TABLE 3 Relationship between cfDNA in culture media
and in vitro embryo development. Three grades of blastocysts at day
5/6 (good (AA), intermediary (BB) or bad quality (CC)) were
obtained from good 8-cell embryos at day 3. The results indicate
that the cfDNA variation in culture media between day 3 and day 5/6
is different according to blastocyst grade. Day 2 Day 3 Patient Day
1 Blastomere Blastomere % of Blastomere Blastomere (HSC)
Fertilization number uniformity fragmentation number uniformity
No23 fertilized 4bl Equal. Homo. 10% 8bl .+-.Equal. .+-.Homo. No14
fertilized 4bl Equal. Homo. 15% 8bl Equal. Homo. No3 fertilized 4bl
Equal. Homo. 10% 7bl Equal. Homo. Drop Day 3 Drop Day 5 Day 3
Concentration Concentration Patient % of Day5 Day 6 CfDNA CfDNA
(HSC) fragmentation Grade Grade Pregnancy (ng/ml) (ng/ml)) No23 20%
5AA Positive 22.168 2.755 (Transfer) No14 15% B1+ 5BB 7.55 1.804
(Frezing) No3 15% 3CC 6.46 3.787
REFERENCES
[0064] Throughout this application, various references describe the
state of the art to which this invention pertains. The disclosures
of these references are hereby incorporated by reference into the
present disclosure. [0065] Antonatos, D., Patsilinakos, S.,
Spanodimos, S., Korkonikitas, P. and Tsigas, D. (2006). Cell-free
DNA levels as a prognostic marker in acute myocardial infarction.
Ann N Y Acad Sci 1075, 278-81. [0066] Arnalich, F., Maldifassi, M.
C., Ciria, E., Quesada, A., Codoceo, R., Herruzo, R.,
Garcia-Cerrada, C., Montoya, F., Vazquez, J. J., Lopez-Collazo, E.
et al. (2010). Association of cell-free plasma DNA with
perioperative mortality in patients with suspected acute mesenteric
ischemia. Clin Chim Acta 411, 1269-74. [0067] Assou, S., Boumela,
I., Haouzi, D., Anahory, T., Dechaud, H., De Vos, J. and Hamamah,
S. (2011). Dynamic changes in gene expression during human early
embryo development: from fundamental aspects to clinical
applications. Hum Reprod Update 17, 272-90. [0068] Assou, S.,
Haouzi, D., De Vos, J. and Hamamah, S. (2010). Human cumulus cells
as biomarkers for embryo and pregnancy outcomes. Mol Hum Reprod 16,
531-8. [0069] Assou, S., Haouzi, D., Mahmoud, K., Aouacheria, A.,
Guillemin, Y., Pantesco, V., Reme, T., Dechaud, H., De Vos, J. and
Hamamah, S. (2008). A non-invasive test for assessing embryo
potential by gene expression profiles of human cumulus cells: a
proof of concept study. Mol Hum Reprod 14, 711-9. [0070]
Czamanski-Cohen, J., Sarid, O., Cwikel, J., Lunenfeld, E.,
Douvdevani, A., Levitas, E. and Har-Vardi, I. (2013). Increased
plasma cell-free DNA is associated with low pregnancy rates among
women undergoing IVF-embryo transfer. Reprod Biomed Online 26,
36-41. [0071] Destouni, A., Vrettou, C., Antonatos, D., Chouliaras,
G., Traeger-Synodinos, J., Patsilinakos, S., Kitsiou-Tzeli, S.,
Tsigas, D. and Kanavakis, E. (2009). Cell-free DNA levels in acute
myocardial infarction patients during hospitalization. Acta Cardiol
64, 51-7. [0072] Ebner, T., Moser, M., Sommergruber, M.,
Gaiswinkler, U., Wiesinger, R., Puchner, M. and Tews, G. (2003).
Presence, but not type or degree of extension, of a cytoplasmic
halo has a significant influence on preimplantation development and
implantation behaviour. Hum Reprod 18, 2406-12. [0073] European
patent PCT No EP2011/065333 AR Thierry and F. Molina, Analytical
methods for cell free nucleic acids and application, 5th of Sep.
2011. [0074] Fenwick, J., Platteau, P., Murdoch, A. P. and Herbert,
M. (2002). Time from insemination to first cleavage predicts
developmental competence of human preimplantation embryos in vitro.
Hum Reprod 17, 407-12. [0075] Goldshtein H, Hausmann M J,
Douvdevani A. (2009). A rapid direct fluorescent assay for
cell-free DNA quantification in biological fluids. Ann Clin
Biochem. 46(Pt 6):488-94. [0076] Lazar, L., Rigo, J., Jr., Nagy,
B., Balogh, K., Mako, V., Cervenak, L., Mezes, M., Prohaszka, Z.
and Molvarec, A. (2009). Relationship of circulating cell-free DNA
levels to cell-free fetal DNA levels, clinical characteristics and
laboratory parameters in preeclampsia. BMC Med Genet 10, 120.
[0077] Li C N, Hsu H L, Wu T L, Tsao K C, Sun C F, Wu J T. (2003).
Cell-free DNA is released from tumor cells upon cell death: a study
of tissue cultures of tumor cell lines. J Clin Lab
Anal.17(4):103-7. [0078] Mouliere, F., Robert B, Arnau Peyrotte E,
Del Rio M, Ychou M, Molina F, Gongora C, Thierry A R. (2011). High
Fragmentation Characterizes Tumour-Derived Circulating DNA. Plos
One 6. [0079] Mussolin, L., Burnelli, R., Pillon, M., Carraro, E.,
Farruggia, P., Todesco, A., Mascarin, M. and Rosolen, A. (2013).
Plasma cell-free DNA in paediatric lymphomas. J Cancer 4, 323-9.
[0080] Schwarzenbach, H., Hoon, D. S. B. & Pantel, K. (2011).
Cell-free nucleic acids as biomarkers in cancer patients. Nature
Reviews Cancer 11, 426-437. [0081] Seli, E., Sakkas, D., Scott, R.,
Kwok, S. C., Rosendahl, S. M. and Burns, D. H. (2007). Noninvasive
metabolomic profiling of embryo culture media using Raman and
near-infrared spectroscopy correlates with reproductive potential
of embryos in women undergoing in vitro fertilization. Fertil
Steril 88, 1350-7. [0082] Thierry, A. R., Mouliere F, Gongora C,
Ollier J, Robert B, Ychou M, Del Rio M, Molina F.(2010). Origin and
quantification of circulating DNA in mice with human colorectal
cancer xenografts. Nucleic Acids Res 38, 6159-6175. [0083] Vergouw,
C. G., Botros, L. L., Roos, P., Lens, J. W., Schats, R., Hompes, P.
G., Burns, D. H. and Lambalk, C. B. (2008). Metabolomic profiling
by near-infrared spectroscopy as a tool to assess embryo viability:
a novel, non-invasive method for embryo selection. Hum Reprod 23,
1499-504. [0084] Wroclawski, M. L., Serpa-Neto, A., Fonseca, F. L.,
Castro-Neves-Neto, O., Pompeo, A. S., Machado, M. T., Pompeo, A. C.
and Del Giglio, A. (2013). Cell-free plasma DNA as biochemical
biomarker for the diagnosis and follow-up of prostate cancer
patients. Tumour Biol. May 29. [0085] Yakimovich A, Gumpert H,
Burckhardt C J, Lutschg VA, Jurgeit A, Sbalzarini I F, Greber U F.
(2012). Cell-free transmission of human adenovirus by passive mass
transfer in cell culture simulated in a computer model. J Virol.
September; 86(18).
Sequence CWU 1
1
2121DNAHomo sapiens 1ccttgggttt caagttatat g 21220DNAHomo sapiens
2ccctgacata ctcccaagga 20
* * * * *
References