U.S. patent application number 12/974781 was filed with the patent office on 2011-10-06 for identification of genes or polypeptides the expression of which correlates to fertility, ovarian function and/or fetal/newborn viability.
Invention is credited to Jose Cibelli, Javier Crosby, Guilherme Jordao De Rosa, Emilio Fernandez, Arif Kocabas.
Application Number | 20110244464 12/974781 |
Document ID | / |
Family ID | 38779167 |
Filed Date | 2011-10-06 |
United States Patent
Application |
20110244464 |
Kind Code |
A1 |
Cibelli; Jose ; et
al. |
October 6, 2011 |
IDENTIFICATION OF GENES OR POLYPEPTIDES THE EXPRESSION OF WHICH
CORRELATES TO FERTILITY, OVARIAN FUNCTION AND/OR FETAL/NEWBORN
VIABILITY
Abstract
A genetic means of determining whether a female subject produces
"pregnancy competent" oocytes is provided. The means comprises
detecting the level of expression of one or more genes that are
expressed at characteristic levels (upregulated or downregulated)
in cumulus cells derived from pregnancy competent oocytes. This
characteristic gene expression level, or pattern referred to herein
as the "pregnancy signature", also can be used to identify subjects
with underlying conditions that impair or prevent the development
of a viable pregnancy, e.g., pre-menopausal condition, other
hormonal dysfunction, ovarian dysfunction, ovarian cyst, cancer or
other cell proliferation disorder, autoimmune disease and the like.
Microarrays containing "pregnancy signature" genes or corresponding
polypeptides provide another preferred aspect of the invention.
Still further, the subject invention can be used to derive animal
models, e.g., non-human primate animal models, for the evaluation
of the efficacy of putative female fertility treatments.
Inventors: |
Cibelli; Jose; (East
Lansing, MI) ; Crosby; Javier; (Santiago, CL)
; Fernandez; Emilio; (Santiago, CL) ; Kocabas;
Arif; (East Lansing, MI) ; De Rosa; Guilherme
Jordao; (Middleton, WI) |
Family ID: |
38779167 |
Appl. No.: |
12/974781 |
Filed: |
December 21, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11584580 |
Oct 23, 2006 |
7858308 |
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12974781 |
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11437797 |
May 22, 2006 |
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11584580 |
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11091883 |
Mar 29, 2005 |
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11437797 |
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60556875 |
Mar 29, 2004 |
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Current U.S.
Class: |
435/6.12 ;
435/6.1 |
Current CPC
Class: |
C12Q 1/6883 20130101;
C12Q 2600/158 20130101 |
Class at
Publication: |
435/6.12 ;
435/6.1 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1-45. (canceled)
46. A method of identifying oocytes that are capable of giving rise
to a viable pregnancy when fertilized comprising the following
steps: (i) obtaining at least one cumulus cell associated with an
oocyte; (ii) assaying the expression of at least 5 genes selected
from the genes in Table 1, wherein at least 2 of said genes are
selected HAS2, PTX3, DHFR, ZNF93, DUSP12, STK35, WTAP, AQP3 by said
at least one oocyte associated cell, the expression of which
correlates to the capability of an oocyte associated with said cell
to yield a viable pregnancy upon fertilization and transferral into
a suitable uterine environment; and (iii) identifying, based on the
level of expression of said at least one gene, whether said oocytes
is potentially capable of yielding a viable pregnancy upon
fertilization and transferral into a suitable uterine
environment.
47. The method of claim 46, wherein said oocyte is a mammalian
oocyte.
48. The method of claim 47, wherein said oocytes is a human
oocyte.
49. The method of claim 47, wherein said oocyte is a non-human
primate oocyte.
50. The method of claim 46, wherein the expression of at least 10
genes, the expression of which correlates to the capability of an
oocyte to potentially yield a viable pregnancy are identified.
51. The method of claim 46 wherein said at least one gene
additionally includes at least one gene selected from the group
consisting of PRMT5, PTG2, ACTB, or a variant thereof possessing at
least 95% sequence identity to one of the sequences contained in
FIG. 19.
52. The method of claim 46, wherein the expression of at least 15
genes, the expression of which correlates to the capability of an
oocyte to potentially yield a viable pregnancy are measured.
53. The method of claim 46, wherein the expression of at least 20
genes, the expression of which correlates to the capability of an
oocyte to potentially yield a viable pregnancy are identified.
54. The method of claim 53, wherein the expression of at least 20
to 50 genes, the expression of which correlates to the capability
of an oocyte to potentially yield a viable pregnancy are
identified.
55. The method of claim 54, wherein the expression of at least 50
to 100 genes, the expression of which correlates to the capability
of an oocyte to potentially yield a viable pregnancy are
identified.
56. The method of claim 46 wherein the method of assaying gene
expression uses a method that monitors differential gene
expression.
57. The method of claim 56 wherein said method comprises indexing
differential display reverse transcriptase polymerase chain
reaction (DDRT-PCR).
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation in part of U.S. Ser. No.
11/ 437,797 filed on May 22, 2006, which is in turn a
continuation-in-part of U.S. Ser. No. 11/091,883 filed on Mar. 29,
2005. This application further claims the benefit of provisional
application No. 60/556,875 filed Mar. 29, 2004. All of these
applications are incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The present invention provides genetic methods that provide
for the identification of "pregnancy competent" oocytes, i.e.,
oocytes that when fertilized and transferred to a suitable uterine
environment are capable of yielding a viable pregnancy. The present
invention further provides genetic methods of identifying female
subjects, preferably human females having impaired fertility
function, e.g., as a result of impaired ovarian function, e.g., as
a result of age (menopause) or an underlying disease condition or
therapy.
[0003] Also, the invention provides methods of evaluating the
efficacy of a putative fertility treatment based on its effect on
the expression of specific genes.
[0004] Further, the invention identifies genes which are
differentially expressed by cumulus cells that correlate to the
pregnancy potential of oocytes that are associated therewith.
[0005] Further, the present invention provides an improved mRNA
amplification protocol that is especially suited for gene
expression profiling of biological samples of small quantity, such
as cumulus or stem cell containing cell samples.
BACKGROUND OF THE INVENTION
[0006] Currently, there is no available genetic procedures for
identifying whether a female subject produces oocytes that are
"pregnancy competent", i.e., oocytes which when fertilized by
natural or artificial means are capable of giving rise to embryos
that in turn are capable of yielding viable offspring when
transferred to an appropriate uterine environment. Rather,
conventional fertility assessment methods assess fertility e.g.,
based on hormonal levels, visual inspection of numbers and quality
of oocytes, surgical or non-invasive (MRI) inspection of the female
reproduction system organs, and the like. Often, when a woman has a
problem in producing a viable pregnancy after a prolonged duration,
e.g., more than a year, the diagnosis may be an "unexplained"
fertility problem and the woman advised to simply keep trying or to
seek other options, e.g., adoption or surrogacy. Therefore,
providing alternative and more predictive methods for identifying
women with fertility problems would be highly desirable. Likewise,
novel and improved methods for treating fertility problems would be
highly desirable.
[0007] Still further, the identification of women with fertility
problems, preferably earlier on than by current methods is
desirable, as fertility problems may correlate to other health
issues that preclude pregnancy, e.g., cancer, menopausal condition,
hormonal dysfunction, ovarian cyst, or other underlying disease or
health related problems.
BRIEF DESCRIPTION AND OBJECTS OF THE INVENTION
[0008] It is an object of the invention to provide a novel and
improved method of detecting infertility problems and the genetic
basis thereof.
[0009] It is a more specific object of the invention to provide a
novel method of detecting female fertility or infertility which
method comprises evaluating the capability of oocytes produced by
said female to potentially give rise to a viable pregnancy upon
fertilization and transferral into a suitable uterine environment,
wherein said method involves detecting the levels of expression of
specific ("pregnancy signature") genes or polypeptides encoded
thereby by cells that are oocye-associated, e,g., cumulus
cells.
[0010] It is another specific object of the invention to provide a
method of evaluating whether a subject produces oocytes capable of
giving rise to a viable pregnancy comprising:
[0011] (i) measuring the expression levels of genes in a
oocyto-associated cell, e.g., a cumulus cell, wherein said genes
are expressed or not expressed at characteristic levels ("pregnancy
signature") in cells associated with oocytes capable of yielding a
viable pregnancy; and
[0012] (ii) detecting the "pregnancy potential" of said oocytes
based on the level of similarity of said gene expression pattern to
said "pregnancy signature".
[0013] It is another specific object of the invention to identify a
female subject putatively having a condition that inhibits or
prevents pregnancy by detecting whether said subject produces
oocytes associated with cells, e.g., cumulus cells, which do not
express one or more genes in a manner characteristic of "pregnancy
competent" oocytes; wherein said method comprises detecting the
expression of said one or more "pregnancy signature" genes in at
least one cell associated with an oocyte isolated from said female
subject; and thereby identifying the subject as potentially having
a health problem which prevents or precludes fertility based on an
abnormal expression pattern of at least one of said "pregnancy
signature" genes.
[0014] It is another object of the invention to provide a method of
evaluating the efficacy of a female fertility treatment which
comprises:
[0015] (i) treating a female subject putatively having a problem
that prevents or inhibits her from having a "viable pregnancy"
and
[0016] (ii) isolating at least one oocyte from said female subject
and cells associated therewith after said fertility treatment;
[0017] (iii) isolating at least one cell associated with said
isolated oocyte, preferably a cumulus cell, and detecting the level
of expression of at least one gene that is expressed at a
characteristic level of expression in "pregnancy competent"
oocytes; and
[0018] (iv) determining the putative efficacy of said fertility
treatment based on whether said gene is expressed at a level
characteristic of "pregnancy competent" oocytes as a result of
treatment.
[0019] It is another object of the invention to provide animal
models for evaluating the efficacy of putative fertility treatments
comprising identifying genes which are expressed at characteristic
levels in cumulus cells associated with pregnancy competent oocytes
of a non-human animal, e.g., a non-human primate; and assessing the
efficacy of a putative fertility treatment in said non-human animal
based on its effect on said gene expression levels, i.e., whether
said treatment results in said gene expression levels better
mimicking gene expression levels observed in cumulus cells
associated with pregnancy competent oocytes, ("pregnancy
signature").
[0020] It is another object of the invention to identify specific
human genes that are differentially expressed by cumulus cells and
other oocyte-associated cells and to assay the expression of one or
more of such specific genes by cumulus or other oocyte-associated
cells as an indicator of fertility and ovarian function.
[0021] It is another object of the invention to provide a novel
mRNA amplication protocol especially suited for biological samples
of small quantity that combines the use of specific primers, i.e.,
SMART II oligonucleotide (Clontech, CA) and T7-oloigo(dT)24V
promoter primers (Ambion, Tex.).
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIGS. 1A-1C and 1D-1I depict schematically a genetic
fertility testing method according to the invention. FIG. 1A shows
a freshly ovulated egg containing a polar body, zona pellucida and
cumulus cells. FIG. 1B shows the fertilization and transferral of
this egg into a uterine environment. FIG. 1C shows the recovery of
cumulus cells from the oocytes shown in 1A which are to be used for
genetic testing. FIG. 1D-1I show the isolation of RNAs from said
cumulus cells, microarray analysis of said RNAs, validation of 100
genes by real time RT-PCR, correlation of the levels of expression
of said genes (upregulated or downregulated) to the ability of an
oocyte to give rise to a viable pregnancy, and the use of this gene
expression profile to identify a set of genes, the expression of
which correlates to the capability of an oocyte to yield a viable
pregnancy ("pregnancy signature")
[0023] FIG. 2 contains a flow chart of the CRL amplification
protocol used in the present invention.
[0024] FIG. 3 contains a digital RNA gel-like image of total RNA
sample isolated from 8 mature oocytes run 3 times.
[0025] FIG. 4 contains RT-PCR verification of GeneChip array
results for samples as described infra.
[0026] FIG. 5 contains selected overrepresented GO biological
processes in oocytes identified by EASE.
[0027] FIG. 6 contains Venn diagrams depicting intersection of
differentially expressed genes in human and mouse oocytes and
ESCs.
[0028] FIG. 7 contains primers and sequences used to validate
RT-PCR microarray experimental results.
[0029] FIG. 8 contains selected representations of genes common to
human and mouseoocytes with homologs functionally characterized in
the mouse oocye (16 genes out of top 100 genes).
[0030] FIG. 9 contains 66 unique genes in common between human
oocytes, mouse oocytes, hESCs, and mESCs.
[0031] FIG. 10 contains genes differentially expressed in human
oocytes corresponding to TGF-beta signaling pathway.
[0032] FIG. 11 contains gene that are differentially expressed in
human and mouse oocytes that are in the estrogen receptor signaling
pathway.
[0033] FIG. 12 contains progeny distribution of 27 analyzed in
vitro fertilization (IVF) patients.
[0034] FIG. 13 contains a detailed flow chart describing the steps
of the invention starting from the IVF clinic to the gene
expression results. Panel A illustrates a sample collection from
freshly ovulated mature cumulus-oocyte complex in vivo. Panel B
shows quality and quantity checking of total RNA from 4 different
cumulus cell lysates.
[0035] FIG. 14 depicts schematically the experimental design of the
protocol in more detail than FIG. 1.
[0036] FIG. 15 contains a summary of CRL RNA amplification protocol
described herein. Panel B shows the means of validating amplication
fidelity.
[0037] FIG. 16 shows clustering of samples using genes that are
called present on 4 pregnant and 4 non-pregnant donor samples (442
probe sets).
[0038] FIG. 17 contains experimental patterns of 3 candidate
pregnancy markers in cumulus cells that resulted in pregnancy
versus no pregnancy.
[0039] FIG. 18 contains a Table containing the top 10 functional
categories overreported in differentially expressed genes between
cumulus cells coming from oocytes that produce progeny and those
that fail to produce progeny. Gene orthology biological properties
and molecular function as detected by EASE when oocyte expressed
genes were compared to the genes represented in the GeneChip
array.
[0040] FIG. 19 contains the sequences of three differentially
expressed genes in FIG. 17 that are expressed on average at
substantially elevated amounts in cumulus cells derived from
occytes that give rise to progeny versus those which do not.
DETAILED DESCRIPTION OF THE INVENTION
[0041] Prior to discussing the invention in more detail, the
following definitions are provided. Otherwise all words and phrases
in this application are to be construed by their ordinary meaning,
as they would be interpreted by an ordinary skilled artisan within
the context of the invention.
[0042] "Pregnancy-competent oocytes": refers to a female gamete or
egg that when fertilized by natural or artificial means is capable
of yielding a viable pregnancy when it is comprised in a suitable
uterine environment.
[0043] "Viable-pregnancy": refers to the development of a
fertilized oocyte when contained in a suitable uterine environment
and its development into a viable fetus, which in turn develops
into a viable offspring absent a procedure or event that terminates
said pregnancy.
[0044] "Cumulus cell" refers to a cell comprised in a mass of cells
that surrounds an oocyte. This is an example of an "oocyte
associated cell". These cells are believed to be involved in
providing an oocyte some of its nutritional and or other
requirements that are necessary to yield an oocyte which upon
fertilization is "pregnancy competent".
[0045] "Differential gene expression" refer to genes the expression
of which varies within a tissue of interest; herein preferably a
cell from an oocyte, e.g., a cumulus cell.
[0046] "Real Time RT-PCR": refers to a method or device used
therein that allows for the simultaneous amplification and
quantification of specific RNA transcripts in a sample.
[0047] "Microarray analysis": refers to the quantification of the
expression levels of specific genes in a particular sample, e.g.,
tissue or cell sample.
[0048] "Pregnancy signature": refers to a phrase coined by the
inventors which refers to the characteristics levels of expression
of a set of one or more genes, preferably at least 5, more
preferably at least 10 to 20 genes, and still more preferably, at
least 50 to 100 genes, that are expressed at characteristic levels
in oocyte cells, preferably cumulus cells, that surround "pregnancy
competent" oocytes. This is intended to encompass the level at
which the gene is expressed and the distribution of gene expression
within cells analyzed.
[0049] "Pregnancy signature gene": refers to a gene which is
expressed at characteristic levels by a cell, e.g., cumulus cell,
on a "pregnancy competent" oocyte.
[0050] "IVF": refers to in vitro fertilization.
[0051] "Zona pellucida" refers to the outermost region of an
oocyte.
[0052] "Method for detecting differential expressed genes"
encompasses any known method for evaluating differential gene
expression. Examples include indexing differential display reverse
transcription polymorase chain reaction (DDRT-PCR; Mahadeva et al,
1998, J. Mol. Biol. 284:1391-1318; WO 94/01582; subtractive mRNA
hybridization (See Advanced Mol. Biol.; R. M. Twyman (1999) Bios
Scientific Publishers, Oxford, p. 334, the use of nucleic acid
arrays or microarrays (see Nature Genetics, 1999, vol. 21, Suppl.
1061) and the serial analysis of gene expression. (SAGE) See e.g.,
Valculesev et al, Science (1995) 270:484-487) and real time PCR
(RT-PCR). For example, differential levels of a transcribed gene in
an oocyte cell can be detected by use of Northern blotting, and/or
RT-PCR.
[0053] CRL amplification protocol refers to the novel total RNA
amplification protocol depicted schematically in FIG. 2 that
combines template-switching PCR and T7 based amplification methods.
As noted above, this protocol is well suited for samples wherein
only a few cells or limited total RNA is available.
[0054] Preferably, the "pregnancy signature" genes will be detected
by hybridization of RNA or DNA to DNA chips, e.g., filter arrays
comprising cDNA sequences or glass chips containing cDNA or in situ
synthesized oligonucleotide sequences. Filtered arrays are
typically better for high and medium abundance genes DNA chips can
detect low abundance genes. In the exemplary embodiment the sample
may be probed with Affymetrix GeneChips comprising genes from the
human genome or a subset thereof.
[0055] Alternatively, polypeptide arrays comprising the
polypeptides encoded by pregnancy signature genes or antibodies
that bind thereto may be produced and used for detection and
diagnosis.
[0056] "EASE" is a gene ontology protocol that from a list of genes
forms subgroups based on functional categories assigned to each
gene based on the probability of seeing the number of subgroup
genes within a category given the frequency of genes from that
category appearing on the microarray.
[0057] As noted above, the present invention preferably provides a
novel method of detecting whether a female subject, human or
non-human, produces "pregnancy competent" oocytes. The method
involves detecting the levels of expression of one or more genes
that are expressed or not expressed at characteristic levels by
cumulus cells associated with (surrounding) oocytes that are
"pregnancy competent", i.e., which when fertilized by natural or
artificial means (IVF), and transferred into a suitable uterine
environment are capable of yielding a viable pregnancy, i.e.,
embryo that develops into a viable fetus and eventually an
offspring unless the pregnancy is terminated by some event or
procedure, e.g., a surgical or hormonal intervention.
[0058] The invention further provides a novel and improved means
for amplying the total RNA from a particular cell sample that
combines template-switching PCR and T7-based amplification methods
(referred to herein as CRL amplification protocol). While this
method is preferably used for assaying gene expression by oocyte,
cumulus, or ES total RNA samples it is applicable for any cell
sample, preferably a cell sample wherein amplifiable RNA is only
available in small quantity.
[0059] The invention further provides transcriptome data obtained
from oocyte, cumulus, or ES cells that identifies genes which are
differentially expressed therein.
[0060] The invention in particular identifies 1626 genes that are
differentially expressed by human ES cells.
[0061] The invention further identifies 5331 transcripts
upregulated and 7074 transcripts down-regulated in human oocyte
sample. Upregulated genes include FIGLA, STELLA, VASA, DAZL, GDF9,
ZP1, ZP2, MOS, OCT4, NPM2, and H1FOO.
[0062] The invention further compares transcriptomes from human and
mouse oocytes and identifies 1587 genes common (differentially
expressed) to both.
[0063] The invention further compares the transcriptomes of oocytes
and ES cella and identifies 388 (human) and 591 (mouse)genes
differentially expressed in both as well as a set of 66 genes that
are preferentially differentially expressed in each of human and
mouse ESCs and oocytes.
[0064] In particular the invention provides a comprehensive
expression baseline of gene transcripts present in in vivo matured
metaphase II human oocytes.
[0065] In preferred embodiments, the inventive methods will be used
to identify women subjects who produce or do not produce pregnancy
competent oocytes based on the levels of expression of a set of
differentially expressed genes. However, the inventive methods are
applicable to non-human animals as well, e.g., other mammals,
avians, amphibians, reptiles, et al. For example, the subject
invention may be used to derive animal models for the study of
putative female fertility treatments.
[0066] Additionally, the present invention may be used to identify
female subjects who have an abnormality that precludes or inhibits
their ability to produce pregnancy competent oocytes, e.g., ovarian
dysfunction, ovarian cyst, pre-menopausal or menopausal condition,
cancer, autoimmune disorder, hormonal dysfunction, cell
proliferation disorder, or another health condition that inhibits
or precludes the development of pregnancy competent oocytes.
[0067] For example, subjects who do not express specific pregnancy
signature genes at characteristic expression levels will be
screened to assess whether they have an underlying health condition
that precludes them from producing pregnancy competent oocytes.
Particularly, such subjects will be screened to assess whether they
are exhibiting signs of menopause, whether they have a cancer,
autoimmune disease or ovarian abnormality, e.g., ovarian cyst, or
whether they have another health condition, e.g., hormonal
disorder, allergic disorder, etc., that may preclude the
development of "pregnancy competent" oocytes.
[0068] Additionally, the subject methods may be used to assess the
efficacy of putative female fertility treatments in humans or
non-human female subjects. Essentially, such methods will comprise
treating a female subject, preferably a woman, with a putative
fertility enhancing treatment, isolating at least one oocyte and
associated surrounding cells from said woman after treatment,
optionally further isolating at least one oocyte and associated
surrounding cells prior to treatment, isolating at one cumulus cell
from each of said isolated oocytes; detecting the levels of
expression of at least one gene that is expressed or not expressed
at characteristic levels by cumulus cells that are associated with
(surround) pregnancy competent oocytes; and assessing the efficacy
of said putative fertility treatment based on whether it results in
cumulus cells that express at least one pregnancy signature gene at
levels more characteristic of cumulus cells that surround pregnancy
competent oocytes (than without treatment). As noted, while female
human subjects are preferred, the subject methods may be used to
assess the efficacy of putative fertility treatments in non-human
female animals, e.g., female non-human primates or other suitable
animal models for the evaluation of putative human fertility
treatments.
[0069] Still further, the present invention may be used to enhance
the efficacy of in vitro or in vivo fertility treatments.
Particularly, oocytes that are found to be "pregnancy incompetent",
or are immature, may be cultured in a medium containing one or more
gene products that are encoded by genes identified as being
"pregnancy signature" genes, e.g., hormones, growth factors,
differentiation factors, and the like, prior to, during, or after
in vivo, or in vitro fertilization. Essentially, the presence of
these gene products should supplement for a deficiency in
nutritional gene products that are ordinarily expressed by cumulus
cells that surround "pregnancy competent" oocytes, and which
normally nurture oocytes and thereby facilitate the capability of
these oocytes to yield viable pregnancies upon fertilization.
[0070] Alternatively, one or more gene products encoded by said
pregnancy signature genes may be administered to a subject who is
discovered not to produce pregnancy competent oocytes according to
the methods of the invention. Such administration may be
parenteral, e.g., by intravenous, intramuscular, subcutaneous
injection or by oral or transdermal administration. Alternatively,
these gene products may be administered locally to a target site,
e.g., a female ovarian or uterine environment. For example, a
female subject may have her uterus or ovary implanted with a drug
delivery device that provides for the sustained delivery of one or
more gene products encoded by "pregnancy signature" genes.
[0071] Also, the novel CRL amplication protocol of the invention
may be used to identify differentially expressed genes from any
cell sample, preferably those only available in limited numbers
such as e.g., samples used in forensic analysis, pathological
samples such as cancer cells, especially cancer stem cells, cell
samples suspected of containing an unknown pathogen, cell samples
obtained from cells undergoing specific cellular processes such as
differentiation, apoptosis, angiogenesis, and the like. This
protocol has been found to faithfully and consistently amplify
small amounts of RNA to quantities required for microarray
analysis.
[0072] Thus, in general, the present invention involves the
identification and characterization, in terms of gene identity and
relative abundance, of genes that are expressed by desired cells,
e.g., cumulus cells derived from an egg, preferably human egg, at
the time of ovulation, preferably cumulus cells, the expression
levels of which correlate to the capability of said egg to give
rise to a viable pregnancy upon natural or artificial fertilization
and transferal to a suitable uterine environment. Also, the
invention identifies a set of genes differentially expressed by
human or murine ESCs and metaphase II oocytes.
[0073] In one preferred embodiment, of the invention at least 50 to
100 genes that are significantly upregulated or downregulated, by
cumulus cells that correlate to the "pregnancy competency" of an
oocyte from which said cumulus cells are associated with will be
chosen and monitored in the inventive genetic testing methods.
[0074] However, while the invention preferably will select at least
50-100 genes from each of said categories, it is anticipated that
the inventive methods alternatively may be practiced by monitoring
the expression levels of fewer numbers of cumulus cell expressed
genes, wherein said genes are similarly selected to be those which
correlate to cumulus cells associated with "pregnancy competent"
oocytes, i.e., those that are capable of yielding viable
pregnancies.
[0075] According to the invention, gene expression levels will
preferably be detected by the novel CRL amplification protocol
provided herein. However other known methods, preferably real time
detection methods such as mentioned above may be used to detect and
quantify gene expression. Methods for detecting relative gene
expression levels are known in the art and well within the purview
of the ordinary skilled artisan.
[0076] As noted supra, this invention further provides a novel mRNA
amplication protocol that is well suited for small cell samples
such as those containing only a few or even a single cumulus cell
or oocyte or ESC or other desired cells. This amplification
protocol is well suited as well for forensic applications where
only a minute nucleic acid sample may be available. Also, this
technique is useful wherein only a few cells may be isolated from
an individual such as adult stem cells, cancer stem cells, other
differentiation specific cells, olfactory cells, taste cells, and
the like. The present protocol will be useful in the biomedical
field such as by medical and veterinary pathologists, e.g. in
coordination with Laser-assisted Microdissection of tissues.
Particularly, such applications may include cancer-related
applications, research and disease diagnosis.
[0077] Previously, in order to generate a biotin-labeled antisense
aRNA target for GeneChip experiments from limited amount of RNA
samples, this entailed the use of commercial kits e.g., those
available from a few venders (such as Ambion TX, and Arcturus, CA).
All of these kits use the same approach based on the Eberwine T7
amplification method (See Eberwine Biotechniques 20:584-591
(1996)).
[0078] By contrast, the present invention provides an improvement
thereover that faithfully and consistently amplifies small amounts
of RNA to quantities required to perform microarray experiments.
The CRL amplification protocol disclosed herein provides a
practical approach to facilitate the analysis of gene expression in
samples of small quantity while maintaining the relative gene
expression profile throughout reactions (Kocabas et al.,
"Transcriptome Analysis of the Human Ooocyte" In Press, 2006).
[0079] This amplification protocols achieves at least the following
advantages versus available protocols:
[0080] (i) global mRNA amplification is possible for a limited
number of cells, tissues and micro-dissected biopsy using other
(non-CRL) PCR amplification method;
[0081] (ii) the protocol is comprised of simple laboratory
manipulations;
[0082] (iii) the simplicity of the protocol contributes to a high
level of reproducibility from experiment to experiment; and
[0083] (iv) the protocol time is shorter than other methods, in
particular when multiple rounds are performed.
[0084] Based on these advantages this methodology is well suited
for use in the present differential gene expression based-assays
which detect genes the expression of which correlates to oocytes or
embryonic stem cells as well as other applications wherein the
detection of expressed genes in a sample is desired.
[0085] Essentially, the CRL protocol is depicted in FIG. 2 and
comprises (i) first strand cDNA synthesis coupled with dC tailing
by MMLV RT (Rnase H-); (ii) template switching and chain extension
by RT; (iii) amplification of cDNA a requisite number of cycles,
typically 5 to 50, more preferably around 10-20, more preferably
around 15 cycles by LD-PCR using SMART primers; (iv) and production
of double stranded DNAs by in vitro transcription using T7 RNA
polymerase. As noted this methodology is applicable for
amplification of mRNAs in any sample, but preferably is used in
amplifying mRNAs from relatively small cell samples such as samples
containing a few number of cumulus cells, oocytes, or stem
cells.
[0086] In the inventive pregnancy signature gene detection methods,
cumulus cells will be isolated from oocytes of different female
subjects, the oocytes fertilized by known IVF procedures, and the
cumulus cells of the corresponding isolated oocytes being subjected
to gene expression analysis, i.e., by isolation of total RNA
therefrom, amplification of said total RNA, quantification of the
relative gene expression levels of said RNAs by microarray analysis
and RT-PCR, and the identification of genes, the expression of
which correlates to oocytes that give rise to a viable
pregnancy.
[0087] To effect such identification, as a separate step, the
status of embryos fertilized with oocytes derived from each of said
cumulus cell samples will be monitored and pregnancy data recorded.
Particularly, the relative birth rate and the health status of the
newborn for each oocyte will be recorded and the gene expression
levels of cumulus cells associated with each oocyte assessed as a
function of pregnancy rate, newborn health, among other parameters,
e.g., gender. Based on these results, a set of genes the expression
of which correlates to pregnancy/health outcome or gender will be
identified. ("pregnancy signature")
[0088] This set of genes, and the corresponding expression levels
is referred to herein as the "pregnancy signature" because these
gene expression levels correlate to the development of a viable
pregnancy and ultimately the production of a healthy newborn. While
this "pregnancy signature" may comprise as many as 50, 100 or even
200 genes, it is anticipated that a fewer number of genes, e.g., on
the order of 20 or less genes, may be sufficient to develop a
suitable "pregnancy signature".
[0089] The genes which constitute the "pregnancy signature" may
include genes which encode gene products that are involved in the
nutritional and developmental requirements of the oocyte, i.e.,
maturation and development, and the potential of the oocyte to be
capable of yielding a viable pregnancy. These gene products may
include growth factors, hormones, transcription factors,
differentiation promoting agents, and the like. After the
"pregnancy signature" is obtained, the corresponding genes are
sequenced, the DNA sequences are then used to deduce the identify
the corresponding polypeptide sequences, and these sequences then
compared to databases of available human or other gene sequences to
identify the identity of the gene products that correlate to the
ability of an oocyte to yield a viable pregnancy. Genes which are
differentially expressed by human oocytes are identified infra and
include such pregnancy signature genes. Further statistical
analysis of the relative levels of expression of these genes, or
subsets of such genes, will identify preferred subsets of these
genes that constitute a "pregnancy signature" of a viable oocyte,
i.e., one that is pregnancy competent. Some genes found to be
differentially expressed in cumulus cells are contained in SEQ ID
NO's 1-513 infra. Additionally, FIG. 19 contains the sequences of
three genes which are differentially expressed and expressed at
different levels in human cumulus cells associated with oocytes
that give rise to viable pregnancies versus cumulus cells
associated with oocytes that do not give rise to viable
pregnancies. These three genes are the human arginine
methyltransferase gene (PRMT5) and its transcript variants and
other allelic variants, the human gene identified in FIG. 19 as
Clone IMAGE 5299642 and contained in deposited NCBI Accession
Sequence BC041913; ACTB, and human BTG family member, member 2,
BTG2, and contained in deposited NCBI Accession Sequence
NM.sub.--006763. The results of the gene expression experiments
contained in Example 3 suggest that these 3 genes are all expressed
at detectably higher amounts in cumulus cells that are associated
with oocytes that give rise to viable pregnancies versus those that
do not. While the expression results are qualitative and have not
yet been quantified the qualitative results would reasonably
suggest that these genes are all expressed at levels which are
least 2-3 fold greater in cumulus cells associated with oocytes
that give rise to viable pregnancies, and likely up to 5-10-fold
greater. Therefore, detecting the expression of these genes by
cumulus cells derived from different donor ooytes, e.g., those to
be used in an IVF procedure may be used as one means of predicting
the pregnancy potential of the oocytes associated therewith, i.e.,
cumulus cells with higher levels of expression of these genes are
more likely to be associated with oocytes useful in IVF procedures.
Therefore, these genes may be used as part of the pregnancy
signature set of genes the expression of which is assayed in order
to assess the pregnancy potential of oocytes from a donor, e.g., a
patient who is contemplating being an IVF donor. Also, these gene
expression procedures may be used to assess the pregnancy potential
of oocytes in an individual who is undergoing fertility treatments,
an individual who is near menopause (perimenopause), or an
individual who has or had a disease or condition or treatment that
potentially would impact the viability and quality of her oocytes
such as radiation or chemotherapy.
[0090] As noted previously, these polypeptide gene products which
are found to be deficient in pregnancy incompetent oocytes may be
added to in vitro culture media containing oocytes in order to
enhance their pregnancy competency or alternatively may be
administered in vivo as part of a fertility treatment regimen.
EXAMPLE 1
Exemplification of CRL Amplification Protocol of the Invention with
Oocyte and ES Cell Samples
[0091] Oocte Collection, Total RNA Extraction and Reference RNA
[0092] Human oocytes were obtained from 3 patients undergoing an
assisted reproductive treatment (ART) at the unit of Reproductive
Medicine at Clinica Las Condes, Santiago Chile. The selection
criteria for the donors was a) less than 35 years old, (b)
reproductively healthy with regular ovulatory cycles; (c) male
factor as the only cause of infertility, (d) considerable number of
developing follicles that assured spared oocytes. The experimental
protocol was reviewed and approved by a local independent Ethics
Review Board. All donors signed informed consent. At the time of
this application filing, all three donors had already conceived,
two of them got pregnant during the ART cycle in which the samples
were collected, and the third one got pregnant following a
spontaneous cycle with artificial insemination using donated sperm.
Ovarian stimulation and oocyte retrieval and isolation were
performed as described herein.
[0093] Three groups of 10 oocytes were used. Total RNA was isolated
following the guanidium thiocyanate method (28) using the PicoPure
RNA isolation kit(Arcturus, CA) following manufacturer's
instructions except only 6.6 micromolar elution buffer was used and
the elution was repeated at least 3 times using the first eluate.
All RNA samples within the purification column were treated with
the Rnase-Free Dnase (Qiagen, CA). Extracted RNA was stored at -80
degrees C. until used as a template for cDNA synthesis. The quality
and quantity of extracted total RNA from 8 matured oocytes
(independent from the 30 oocytes used in this experimental
study(was evaluated using the Agilent 2100 bioanalyzer (Agilent
Technologies, CA). Each mature oocyte was found to have about 330
pg total RNA when the Arcturus' RNA isolation kit was used. Quality
of RNA was intact as shown in FIG. 3. Reference RNA (100
micrograms) was prepared by mixing 10 micrograms total RNA from
each of 10 different normal human tissues including skeletal
muscle, kidney, lung, colon, liver, spleen, breast, brain, heart
and stomach (Ambion, TX).
[0094] RNA Amplification for GeneChip Analysis (FIG. 4a)
[0095] First-strand cDNA synthesis: the following reagents were
added to each of 0.5 ml Rnase-free tube: 5 micromolar total RNA (3
ng for the reference and 5 microliters, about 3 ng, for the oocyte
samples) and 300 ng of an anchored T7-Oligo(dT)24 V promoter primer
(Ambion TX). The reaction tubes were incubated in preheated PCR
machine at 70 degrees C. for 2 min and transferred to ice. After
denaturation, the following reagents were added to each tube: 1.4
microliters of SMART II A oligonucleotide
(5'-AAGCAGTGGTATCAACGCAGAGTACGCGrGrGr-3'') (Clonetech, CA), 4
microliters of 5.times. first-strand buffer, 2 microliters of 20 mm
DTT, 0.6 microliters of 5 mg/ml T4 Gene 32 Protein (Roche, IN), 2
microliters of 10 mM dNTPs, 20 U Rnase inhibitor (Ambion, TX) and 1
microliter PowerScript Reverse Trasnscriptase (Clontech, CA). After
gentle mixing, reaction tubes were incubated at 42 degrees C. for
60 minutes in a hot-lid thermal cycler. The reaction was terminated
by heating at 70 degrees C. for 15 minutes and purified by
NucleoSpin Extraction Kit (Clontech, CA).
[0096] Double-stranded cDNA synthesis by Long-distance (LD)-PCR,
cDNA purification: PCR Advantage 2 mix (9 microliters) was prepared
as follows: 5 microliters of 10.times.PCR Advantage buffer
(Clontech, CA), 1 microliter of 10 mM dNTPs, 100 ng 5' SMART upper
primer (5'-AAGCAGTGGTATCAACGCAGAGTA-3'), 100 ng 3' SMART lower
primer (5'-CGGTAATACGACTCACTATAGGGAGAA-3'), and 1 microliter of
Polymerase Mix Advantage 2 (clontech, CA). This mix was added to 41
microliters of the first-strand cFDNA synthesis product, and
thermal cycling was carried out in the following conditions: 95
degrees C. for 1 minute, followed by 15 cycles, each consisting of
denaturation at 94 degrees C. for 30 sec, annealing at 62 degrees
for 30 sec, and extension at 68 degrees C. for 10 min. The cDNA was
purified by NucleoSpin Extraction Kit following the manufacturer's
instructions.
[0097] In vitro transcription (IVT), biotin labeled aRNA
purification and aRNA fragmentation is described herein.
[0098] Microarray Analysis: Transcription profile of each sample
was probed using Affymetrix Human Genome U133 Plus 2.0 GeneChips.
The raw data obtained after scanning the arrays was analyzed by
dChip (29). A smoothing spline normalization method was applied
prior to obtaining model-based gene expression indices, a.k.a.
signal values. There were no outliers identified by dChip so all
samples were carried on for subsequent analysis.
[0099] Pathway analysis was performed using Ingenuity Software
Knowledge Base (Redwood City, Calif.) which is a manually created
database of previously published findings on mammalian biology from
the public literature. We used the network analysis using the
knowledge base to identify interactions of input genes within the
context of known biological pathways.
[0100] Gene ontology (GO) was performed using EASE. Given a list of
genes, EASE forms subgroups based on the functional categories
assigned to each gene. EASE assigns a significance level (EASE
score) to the functional category based on the probability of
seeing the number of subgroup genes within a category given the
frequency of genes fro that category appearing on that microarray
(30)
[0101] Comparison with External Data Sets
[0102] MouseMII oocyte transcriptome data was obtained from Su et
al., who used custom designed Affymetrix chips to obtain gene
expression profiles of oocytes and 60 other mouse tissue types.
(31) Using their expression database we identified 3,617
differentially upregulated transcripts in the mouse oocyte by using
the median expression value of the remaining 60 samples as the
baseline (Supplementary dataset 1, not shown). We selected
transcripts with an expression value in oocyte samples that are
2-fold higher than the base-line.
[0103] Human embryonic stem cell (hESC) data was derived from the
work of Sato et al. who profiled human stem cells and their
differentiated counterparts using Affymetrix HG-U133A representing
around 2200 transcripts. (32)
[0104] We analyzed raw data using dChip and identified 1,626 hESC
genes by selecting transcripts significantly upregulated in human
stem cells compared to their differentiated counterparts
(Supplementary dataset 2, not shown).
[0105] Finally for mouse ES cells we used a list of 1,687
differentially upregulated mouse ES genes published by Fortunel et
al (33) which were identified by comparing mouse ES cells to
differentiated cells using Affymetrix MG-U74Av2 chips representing
around 1200 transcripts (Supplementary dataset 3, not shown). We
used Affymetrix NetAfffx tool for mapping genes across organisms
and platforms used in the respective studies.
RESULTS AND CONCLUSIONS
[0106] Validation of amplification Fidelity (Amplified vs.
non-amplified RNA)
[0107] A critical step in the analysis of gene expression on small
samples is the faithful amplification of mRNA molecules present in
the sample. We have designed a PCR based amplification system using
the combination of SMART UII A oligonucleotide (Clontech, CA) and
T7-Oligo (dT) promoter primers ("CRL amplification protocol").
(FIG. 3a) We have isolated total RNA from a human cell line and 20,
3 and 1.5 ng input total RNA was amplified using CRL amplification
protocol. For each experiment, 15 micrograms of fragmented aRNA was
hybridized to a single Affymetrix Human Genome U133 Plus 2.0 array.
Non-amplified RNA from the original sample (12 micrograms) was run
in parallel by using the MessageAmp II aRNA Kit (Ambion, TX). Gene
expression results from both amplified vs non-amplified RNA samples
were compared and the correlation coefficients were found to be
0.94. (FIGS. 3b), 0.03, and 0.91 respectively for 20 ng, 3 ng, and
1.5 ng of total input RNA respectively. CRL amplification protocol
was repeated two times with 20 ng initial total RNA from the same
cell type and the correlation between the two experiments was 0.99,
These results show that the subject RNA amplification strategy
faithfully and consistently amplifies even small amounts of RNA to
quantities required to perform microarray experiments. The CRL
amplification protocol provides a practical approach to facilitate
the analysis of gene expression in samples of small quantity while
maintaining the relative gene expression profile throughout
reactions.
[0108] Differentially Upregulated Genes in the Human Oocyte
[0109] We generated a databases of the human oocyte transcriptome
by comparing the transcripts in the oocyte and the reference
samples which contain mRNA from several somatic tissues. A complete
list of up and down regulated genes, functional comparative and
correlation analysis is provided (see Supplementary dataset 4).
Compared to reference samples there were 5,331 transcripts
significantly up-regulated and 7,074 transcripts significantly
down-regulated in the oocyte. Genes up-regulated in oocyte samples
included most of the well-known germ cell specific genes, such as
FLGLA, STELLA, VASA, DAZL, GDF9, ZP1, ZP2, MOS, OCT4, NPM2, and
H1F00. (FIG. 4), Our analysis also confirms the presence of
pathways previously described in the mouse, in particular the
TGF-beta pathway (FIG. 5)
[0110] Validation of microarray data
[0111] A selected list of genes known to be expressed in the oocyte
was used to validate the microarray results by TR-PCR (FIG. 4).
These genes were found to be present in the oocyte sample and
absent in the reference RNA.
[0112] Functional Annotation of Genes Over-Expressed in the Human
Oocyte
[0113] To examine the biological processes performed by the oocyte,
we implemented EASE (36), contrasting the genes over-expressed in
the oocyte with all the genes present in the Affymetrix chip (Table
1). One of the top over-represented categories found in oocytes was
related to RNA metabolism. This is in agreement with the fact that
oocytes store RNA to support the events of fertilization and early
embryonic development until the embryonic gene is activated.
(34,35,36) DNA metabolism and chromatin modification were also
over-represented categories, in agreement with the need of the
oocyte to remodel the sperm chromatin upon fertilization.
[0114] Cell cycle related categories were the most
over-represented. Many genes known to be involved in the regulation
of the meiotic cell cycle Many genes known to be involved in the
regulation of the meiotic cell cycle were detected (MOS, AKT2,
CDC25, and PLK1) (37) Detection of gametogenesis and reproduction
as over-represented categories further suggests the accuracy of
this transcriptional profiling. Protein kinases and phosphatases
denoted another functional category over-represented in oocytes.
Many of the cell cycle regulatory genes (AURKB, CDC25, DCD7, PLK1,
CDC23 and plk3) and some receptors of the TGF superfamily (ACVR1,
ACVR2B, and BMPR1A and 1B) were in this category.
[0115] An important category that is highly represented in the
oocyte was related to nucleic acid metabolism and regulation of
transcription. Although transcriptionally silent at the MII stage,
the oocyte is very active in transcription and translation during
its growth phase and must be prepared to initiate transcription at
the time of embryonic genome activation, 4- to 8-cell stage in
human (38). Many of the genes in this category represent
Zinc0-finger proteins that are not yet fully characterized,
providing an opportunity to discover new transcriptional regulatory
networks that operate during embryonic genome activation.
[0116] We also found that chromatin remodeling genes are well
represented in the human oocyte. Genes in this category expressed
in the human oocyte were: DNA methyltransferases (DNMT1, DNMT3a,
and DNMT3B), histone acetyltransferases (NCOA1,and 3, SRCAP, GCN5L2
and TADA2L), histone deacetyltransferases (HDAC3 HDAC9, SIRT7),
methyl-CpG-binding proteins (MBD2 and MBD4), histone
methyltransferases (EHMT1 and SET8), ATP-dependent remodeling
complexes (SMARCA1, SMARCA5, SMARCAD1, SMARCC2, SMARCD!) and other
chromatin modifying genes (ESR1, NCOA6, HMGB3, HMGN1 and
HMGA1).
[0117] These GO results validate our transcriptome analysis when
compared with candidate gene analysis already reported in other
species but more importantly, shed new light into a large number of
biological processes that take place in the human oocyte.
[0118] Intersection Between Human Oocyte and Mouse Oocyte
Transcriptome
[0119] Mouse has been the best model for genetic studies and
several groups have already reported the transcriptome analysis of
mouse oocytes.(39) In an effort to find differences and
similarities between the human and mouse oocyte, we compared our
human oocyte transcriptome results with that of mouse oocyte
transcriptome derived from, data of Su et al. (35) The intersection
of the two transcriptomes yielded a set of 1587 genes to be common
in both mouse and human oocytes, indicating genes of conserved
function in mammalian oocytes (FIG. 6a, Supplementary dataset 5).
Table S2 shows the functional characterization of 16 of the top 100
intersected genes which have functions described in mouse oocytes.
Many of these genes relate to oocyte maturation, from the first
meiotic division to MII arrest, encompassing various controls of
cell cycle checkpoints and cellular machinery for DNA segregation
and cell division. Using the Ingenuity software to analyze the
intersection of these two datasets we found that the estrogen
receptor (ER) signaling pathway is represented in human and mouse
oocytes (FIG. 6) Genes significantly upregulated in this pathway
were CTBP2, ESR1, GTFH1, GTF2H2, MAP2K1, NCOA1, NCOA3, PCQAP, PHB2,
POLR2C, POLR2J, RBM9, TAF3, TAF4, TAF4B, TAF5, TAF6, TAF12, and
TBP. Recent studies in knockout models for aromatase have shown
that estrogen is not required for the generation of preimplantation
embryos. (40) However, our study suggests that in the oocyte some
genes associated with the ER pathways are transcribed, perhaps in
response to hormonal stimulation during folliculogenesis and oocyte
maturation. Like with the EGF pathway, it remains to be determined
whether the ER pathway has a role during preimplantation
development in human embryos.
[0120] Considering the high degree of similarity
EXAMPLE 2
Description
[0121] Phase I: At the clinic, embryologists will remove the
cumulus cells of two eggs and fertilize them. Embryos will be
transferred to the uterus of a woman and cumulus cells sent to the
laboratory for analysis. Once the cells arrive to the laboratory,
RNA will be isolated and microarray analysis performed using
Affymetrix platform. Pregnancy tests will be done by ultrasound on
day 30 and embryonic sacs counted. There will be three kinds of
outcomes. 1) 0 sacs; 2) 1 sac and 3) 2 sacs. A minimum of 30
volunteer women will participate during this phase. Ten with no
sacs, ten with one sac and ten with 2 sacs. Pregnancy data will be
correlated with gene expression obtained from the cumulus cells
isolated from those same eggs. One hundred genes that directly
correlate with pregnancy--either by upregulation or
downregulation--will be further analyzed using real time RT-PCR.
The best 20 genes that correlate with pregnancy (positively or
negatively) will be called "pregnancy signature" and used for later
testing at the clinic.
[0122] Phase II: Blind validation of genes in the pregnancy
signature. At the clinic, the embryologist will isolate RNA from
cumulus cells from each oocyte that will be later fertilized. Half
of the RNA will be sent to our laboratory and the rest will be used
for real time RT-PCR analysis to be performed on site. Gene
expression of the "pregnancy signature" will be measured.
Embryologists will transfer embryos without knowing the outcome of
gene expression analysis. One hundred women will be asked to
participate as volunteers in this part of the study. At the time
pregnancy results are obtained, the study will be unmasked and
results from each individual will be correlated with gene
expression analysis. We anticipate that the "pregnancy signature"
put forward in phase 1 will be validated during this phase.
[0123] Alternative strategy: In the event of an unexpected outcome
i.e., the pregnancy signature is not validated; microarray analysis
will be run once more using the RNA provided by the clinic in phase
2. It is anticipated that having 100 more samples will result in
the identification of a clear pattern of gene expression in cumulus
cells from eggs capable (or non-capable) of generating a healthy
pregnancy/baby.
[0124] Using microarray analysis as described above, the genes
identified infra were found to be differentially expressed by
cumulus cells obtained from eggs of women donors. The expression of
those particular genes which correlate to pregnancy (positive or
negative) will establish a "pregnancy signature", i.e., genes the
expression or absence of expression of which correlates to a
positive pregnancy outcome and "infertility signature", i.e.,
specific genes the expression or absence of expression correlate to
fertility problems or abnormalities.
[0125] This is effected preferably by microarray analysis. For
example, comparison of expression between two samples on filter
arrays may be performed by comparing nucleic acids obtained from
normal oocyte cells to those obtained from a donor suspected of
having ovarian dysfunction that renders oocytes pregnancy
incompetent on two duplicate filters or alternatively a single
filter may be used that is stripped and hybridized
sequentially.
[0126] Direct comparison of gene expression in two samples can be
achieved on glass arrays by labeling the two samples with different
flourophores. This technique allows the evolution of repression of
gene expression as well as induction of expression. The two
flouresently-labeled cDNAs are then mixed and hybridized on a
single glass or filter array. Glass arrays have the advantage of
allowing the simultaneous analysis of two samples on the same array
under the same hybridization conditions.
[0127] Gene arrays containing sequences of genes implicated in
pregnancy ("pregnancy signature") will allow high-throughput
screening of individuals for diagnostic purposes or tailor-made
treatments.
[0128] Arrays of polynucleotides, the expression of which
corresponds to, or are complementary to the sequences of genes
identified by the method of the invention therefore provide a
further aspect of the invention. Such an array will include at
least two nucleic acid sequences, preferably at least 10, and more
preferably at least 20, e.g., 50 genes or more that correspond to
the sequence of, or are complementary to genes, the expression of
which (positive or negative) the positive pregnancy outcome in
cells obtained from oocyte donors, e.g., women suspected to have
ovarian dysfunction as a result of disease, age, and the like.
Protein arrays form a further aspect of the invention and will
contain polypeptides encoded by such pregnancy signature genes or
antibodies which bind thereto.
[0129] Recent developments in the field of protein and antibody
arrays allow the simultaneous detection of a large number of
proteins.
EXAMPLE 3
Identification of 3 Pregnancy Signature Genes Which Are
Differentially Expressed by Cumulus Cells and Wherein The
Expression Levels of Which Correlate to Pregnancy Outcome of
Associated Human Oocytes
[0130] There are no prior reports describing correlations between
overall gene expression in cumulus cells and pregnancy
establishment of human embryos. A study essentially as described in
Example 2 was conducted to identify differentially expressed genes
between cumulus cells surrounding competent and noncompetent
oocytes using Affymetrix GeneChip technology. To achieve this goal
cumulus cells were classified according to pregnancy outcome of
their matching oocytes following in vitro fertilization (IVF)
treatments. The cumulus cells from 2 oocytes per patient undergoing
IVF treatment were lysed and the oocytes were fertilized and
transferred to the recipient women, 2 embryos per recipient with
the exception of 2 that received a single embryo. Total RNA
isolated from the cumulus cell lysates was amplified by the novel
amplification protocol described supra that combined
template-switching PCR and T7-based amplification methods. A total
of 52 cumulus cell samples were analyzed from 27 donors (FIG. 12).
A gene-by-gene mixture model analysis was used to compare
microarray measurements of gene expression on cumulus cells between
pregnant and non-pregnant patients. 2,604 differentially expressed
genes were considered as potential discriminant candidates of
competent and noncompetent oocytes.
[0131] A flow chart of the specific procedures used is contained in
FIG. 13 which shows how the quality and quantity of total RNA from
different cumulus cell lysates is verified. The Experimental Design
is also depicted schematically in FIG. 14 and the CRL amplification
protocol and a scatter plot of the obtained results by the CRL
amplification versus another method (Ambion) is contained in FIG.
15.
[0132] As shown in FIG. 16 a comparison of the differential
expression profiles of genes by cumulus cells of oocytes that give
rise to pregnancies from 4 pregnant donor samples versus 4 donor
samples that did not give rise to pregnancy reveals a clustering of
samples using genes that are called present on the 4 pregnancy
donor samples and 4 non-pregnant donor samples (442 probe
sets).
[0133] FIG. 17 contains the expression patterns of 3 candidate
pregnancy marker genes in cumulus cells that produced pregnancy
versus non-pregnancy. Additionally, FIG. 18 contains a Table
containing the top 10 functional gene categories overrepresented in
differentially expressed genes between cumulus cells emanating from
oocytes that produce pregnancies versus those that failed to
produce pregnancies. Gene ontology and biological process and
molecular function as detected by EASE when oocyte expressed genes
were compared to the genes represented in the GeneChip array.
[0134] These results provide the first known comprehensive
expression baseline (transcriptome) for the genes which are
expressed in the human cumulus cells that surround a
pregnancy-competent oocyte. As discussed, these genes may serve as
markers for the quality and pregnancy outcome of the oocyte and can
be used to monitor and optimize the factors that are related to
developmental competence, such as patient specific medical
treatments and medical conditions, hormone treatments, and embryo
culture conditions. In addition, the effective development of
pregnancy markers such as the genes which are contained in FIG. 19
can increase the probability of a healthy pregnancy and viable
birth; reduce the chances of multiple births, physical and
emotional burden, and the costs in treatments and hospital stays.
Additionally, identifying these genes may facilitate an
understanding of the underlying biology and help explain how the
levels of expression of these genes may impact or cause female
infertility.
TABLE-US-00001 Lengthy table referenced here
US20110244464A1-20111006-T00001 Please refer to the end of the
specification for access instructions.
TABLE-US-LTS-00001 LENGTHY TABLES The patent application contains a
lengthy table section. A copy of the table is available in
electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20110244464A1).
An electronic copy of the table will also be available from the
USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20110244464A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20110244464A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References