U.S. patent application number 14/168052 was filed with the patent office on 2014-05-29 for methods for selecting oocytes and competent embryos with high potential for pregnancy outcome.
This patent application is currently assigned to Insitut National de la Sante et de la Recherche Medicale (INSERM). The applicant listed for this patent is Insitut National de la Sante et de la Recherche Medicale (INSERM). Invention is credited to Said Assou, John De Vos, Samir Hamamah.
Application Number | 20140148363 14/168052 |
Document ID | / |
Family ID | 40984880 |
Filed Date | 2014-05-29 |
United States Patent
Application |
20140148363 |
Kind Code |
A1 |
Hamamah; Samir ; et
al. |
May 29, 2014 |
Methods for Selecting Oocytes and Competent Embryos with High
Potential for Pregnancy Outcome
Abstract
The present invention relates to a method for selecting a
competent oocyte or a competent embryo.
Inventors: |
Hamamah; Samir; (Montpellier
Cedex, FR) ; De Vos; John; (Montpellier Cedex,
FR) ; Assou; Said; (Montpellier Cedex, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Insitut National de la Sante et de la Recherche Medicale
(INSERM) |
Paris |
|
FR |
|
|
Assignee: |
Insitut National de la Sante et de
la Recherche Medicale (INSERM)
Paris
FR
|
Family ID: |
40984880 |
Appl. No.: |
14/168052 |
Filed: |
January 30, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13264589 |
Dec 8, 2011 |
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PCT/EP2010/054714 |
Apr 9, 2010 |
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14168052 |
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61175503 |
May 5, 2009 |
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Current U.S.
Class: |
506/16 ;
506/18 |
Current CPC
Class: |
C12Q 2600/156 20130101;
G01N 33/689 20130101; C12Q 2600/158 20130101; C12Q 1/6881
20130101 |
Class at
Publication: |
506/16 ;
506/18 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 17, 2009 |
EP |
09305331.2 |
Claims
1-4. (canceled)
5. A kit, comprising: means for measuring the expression level of
genes in a cumulus cell surrounding an oocyte or an embryo, wherein
said means for measuring are selective reagents selected from the
group consisting of probes, primers, and binding partners which are
specific for each of the following 45 genes: WNT6, LRCH4, PAX8,
CABP4, PDE5A, BCL2L11, PCK1, TCF20, SLAMF6, EPOR, CACNG6, NLRP1,
PECAMI, NOS1, ATF3, KRTAP8, GRIK5, SLC24A3, SLC5A12, SLCA10A2,
SLCO1A2, SLC25A5, MG29, NLGN2, PRKACA, FOSB, SIAT6, LOXL2, PRFI,
ADPRH, APBB3, EGR3, CNR2, IFITM1, PLA2G5, CAMTA1, SOX4, NFIB, NFIC,
RBMSI, G0S2, FAT3, SLC40A1, GPC6 and IGFIR.
6. The kit according to claim 5 further comprising: a non-transient
storage medium containing stored reference values for a control
sample having cumulus cells associated with an oocyte or an embryo
wherein said reference values include expression levels for said
control for each of said 45 genes; and means for comparing each of
said stored reference values to measured values obtained with said
means for measuring for each of said 45 genes.
7. The kit according to claim 5, wherein the selective reagents are
hybridization probes and wherein said hybridization probes are
nucleic acids which are suitable for hybridizing to mRNA of WNT6,
LRCH4, PAX8, CABP4, PDE5A, BCL2L11, PCK1, TCF20, SLAMF6, EPOR,
CACNG6, NLRP1, PECAM1, NOS1, ATF3, KRTAP8, GRIK5, SLC24A3, SLC5A12,
SLCA10A2, SLCO1A2, SLC25A5, MG29, NLGN2, PRKACA, FOSB, SIAT6,
LOXL2, PRF1, ADPRH, APBB3, EGR3, CNR2, IFITMI, PLA2G5, CAMTAI,
SOX4, NFIB, NFIC, RBMS1, G0S2, FAT3, SLC40A1, GPC6 and IGFIR under
high stringency hybridization conditions.
8. The kit according to claim 7 wherein the hybridization probes
are chemically attached to a substrate.
9. The kit according to claim 8 wherein the kit comprises a DNA
chip comprising said hybridization probes chemically attached to a
solid support.
10. The kit according to claim 5, wherein the selective reagents
are amplification primers and wherein said amplification primers
are single stranded nucleic acids from 10 to 25 nucleotides which
are suitable for hybridizing to mRNA of WNT6, LRCH4, PAX8, CABP4,
PDE5A, BCL2L11, PCK1, TCF20, SLAMF6, EPOR, CACNG6, NLRP1, PECAMI,
NOSI, ATF3, KRTAP8, GRIK5, SLC24A3, SLC5A12, SLCA10A2, SLCO1A2,
SLC25A5, MG29, NLGN2, PRKACA, FOSB, SIAM, LOXL2, PRF1, ADPRH,
APBB3, EGR3, CNR2, IFITM1, PLA2G5, CAMTA1, SOX4, NFIB, NFIC, RBMSI,
G0S2, FAT3, SLC40A1, GPC6 and IGFIR under high stringency
hybridization conditions.
11. The kit according to claim 10 wherein the kit comprises
reagents for a quantitative or semi-quantitative RT-PCR.
12. The kit according to claim 5, wherein the selective reagents
are binding partners and wherein said binding partners are capable
of selectively interacting with the proteins WNT6, LRCH4, PAX8,
CABP4, PDE5A, BCL2L11, PCK1, TCF20, SLAMF6, EPOR, CACNG6, NLRP1,
PECAM1, NOS1, ATF3, KRTAP8, GRIK5, SLC24A3, SLC5A12, SLCA10A2,
SLCO1A2, SLC25A5, MG29, NLGN2, PRKACA, FOSB, SIAT6, LOXL2, PRF1,
ADPRH, APBB3, EGR3, CNR2, IFITMI, PLA2G5, CAMTA1, SOX4, NFIB, NFIC,
RBMS1, G0S2, FAT3, SLC40A1, GPC6 and IGFIR.
13. The kit according to claim 12 wherein the binding partner is
antibody.
14. The kit according to claim 13 wherein the kit comprises
reagents for an ELISA.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method for selecting a
competent oocyte or a competent embryo.
BACKGROUND OF THE INVENTION
[0002] In assisted reproductive technology (ART), pregnancy and
birth rates following in vitro fertilization (WF) attempts remain
low. Indeed, 2 out of 3 IVF cycles fail to result in pregnancy
(SART 2004) and more than 8 out of 10 transferred embryos fail to
implant (Kovalevsky and Patrizio, 2005). In addition, more than 50%
of IVF-born babies are from multiple gestations (Reddy et al.,
2007). Preterm deliveries that result from multiple pregnancies
caused by ART are estimated to account for approximately $890
million of U.S. health care costs annually (Bromer and Seli,
2008).
[0003] Subjective morphological parameters are still a primary
criterion to select healthy embryos used for in IVF and ICST
programs. However, such criteria do not truly predict the
competence of an embryo. Many studies have shown that a combination
of several different morphologic criteria leads to more accurate
embryo selection (Balaban and Urman, 2006; La Sala et al., 2008;
Scott et al., 2000). Morphological criteria for embryo selection
are assessed on the day of transfer, and are principally based on
early embryonic cleavage (25-27h post insemination), the number and
size of blastomeres on day two or day three, fragmentation
percentage and the presence of multi-nucleation in the 4 or 8 cell
stage (Fenwick et al., 2002).
[0004] However, a recent study has shown that the selection of
oocytes for insemination does not improve outcome of ART as
compared to the transfer of all available embryos, irrespective of
their quality (La Sala et al., 2008). There is a need to identify
viable embryos with the highest implantation potential to increase
IVF success rates, reduce the number of embryos for fresh
replacement and lower multiple pregnancy rates.
[0005] For all these reasons, several biomarkers for embryo
selection are currently being investigated (Haouzi et al., 2008;
Pearson, 2006). As embryos that result in pregnancy differ in their
metabolomic profiles compared to embryos that do not, some studies
are trying to identify a molecular signature that can be detected
by non-invasive evaluation of the embryo culture medium (Brison et
al., 2004; Gardner et al., 2001; Sakkas and Gardner, 2005; Seli et
al., 2007; Zhu et al., 2007).
[0006] Genomics are. also providing vital knowledge of genetic and
cellular function during embryonic development. (McKenzie et al.,
2004) and (Feuerstein et al., 2007) have reported, that the
expression of several genes in cumulus cells, such as
cyclooxygenase 2 (COX2), was indicative of oocyte and embryo
quality. Gremlin 1 (GREM 1), hyaluronic acid synthase 2 (HAS2),
steroidogenic acute regulatory protein (STAR), stearoyl-coenzyme A
desaturase 1 and 5 (SCDI and 5), amphiregulin (AREG) and pentraxin
3 (PTX3) have also been shown to be positively correlated with
embryo quality (Zhang et al. 2005). More recently, the expression
of glutathione peroxidase 3 (GPX3), chemokine receptor 4 (CXCR4),
cyclin D2 (CCND2) and catenin delta I (CTNND1) in human cumulus
cells have been shown to be inversely correlated with embryo
quality, based on early-cleavage rates during embryonic development
(van Montfoort et al. 2008). But, despite the fact that early
cleavage has been shown to be a reliable biomarker for predicting
pregnancy (Lundin et al., 2001; Van Montfoort et al., 2004; Yang et
al., 2007), gene expression profiles of cumulus cells had not been
studied with respect to pregnancy outcome.
SUMMARY OF THE INVENTION
[0007] The present invention relates to a method for selecting a
competent oocyte, comprising a step of measuring the expression
level of 45 genes in a cumulus cell surrounding said oocyte,
wherein said genes are WNT6, LRCH4, PAX8, CABP4, PDE5A, BCL2L11,
PCK 1, TCF20, SLAMF6, EPOR, CACNG6, NLRP1, PECAM1, NOS1, ATF3,
KRTAP8, GRIK5, SLC24A3, SLC5A12, SLCA10A2, SLCO1A2, SLC25A5, MG29,
NLGN2. PRKACA, FOSB, SIAT6, LOXL2, PRFI, ADPRH. APBB3, EGR3, CNR2,
IFITM1, PLA2G5, CAMTA1, SOX4, NFIB, NFIC, RBMS1, G0S2, FAT3,
SLC40A1, GPC6 and IGFIR.
[0008] The present invention also relates to a method for selecting
a competent embryo, comprising a step of measuring the expression
level of 45 genes in a cumulus cell surrounding the embryo, wherein
said genes are WNT6. LRCH4, PAX8, CABP4, PDE5A, BCL2L11, PCK1,
TCF20, SLAMF6, EPOR, CACNG6, NLRP1, PECAM1, NOS1, ATF3, KRTAP8,
GRIK5, SLC24A3, SLC5A12, SLCA10A2, SLCO1A2. SLC25A5, MG29, NLGN2,
PRKACA, FOSB, SIAT6, LOXL2, PRF1, ADPRH, APBB3, EGR3, CNR2, IFITM1,
PLA2G5, CAMTA1, SOX4, NFIB, NFIC, RBMS1, GOS2, FATS, SLC40A1, GPC6
and IGFIR.
[0009] The present invention also relates to a method for selecting
a competent oocyte or a competent embryo, comprising a step of
measuring in a cumulus cell surrounding said oocyte or said embryo
the expression level of one or more genes selected from the groups
A, B or C. wherein group A consists of PCK1, ADPRH, CABP4, SLAMF6,
CAMTA1, CSPG2, and PRF1; group B consists of FOSB, NLGN2, PDE5A,
PLA2G5, GPC6, and EGR3; and group C consists of NFIB, NFIC, IGFIR,
G0S2, GIRK5 and RBMS1.
[0010] Overexpression of one or more genes selected from group A is
predictive of a competent oocyte or embryo leading to pregnancy.
Overexpression of one or more genes selected from group B is
predictive of a non competent oocyte or embryo, the embryo being
unable to implant. Overexpression of one or more genes selected
from group C is predictive of a non competent oocyte or embryo due
to early embryo arrest.
DETAILED DESCRIPTION OF THE INVENTION
[0011] The inventors have determined as set of genes expressed in
cumulus cells that are biomarkers for embryo potential and
pregnancy outcome. They demonstrated that genes expression profile
of cumulus cells which surrounds oocyte correlated to different
pregnancy outcomes, allowing the identification of a specific
expression signature of embryos developing toward pregnancy. Their
results indicate that analysis of cumulus cells surrounding the
oocyte is a non-invasive approach for embryo selection.
Set of Predictive Genes
[0012] All the genes pertaining to the invention are known per se,
and are listed in the below Tables A and B. Tables A and B present
the set of genes whose combined expression profile has been shown
to be informative for selecting a competent oocyte or for selecting
a competent embryo with a high implantation potential leading to
pregnancy.
TABLE-US-00001 TABLE A set of predictive genes. Gene Gene Symbol
Gene name ID WNT6 wingless-type MMTV integration site family,
member 6 7475 LRCH4 leucine-rich repeats and calponin homology (CH)
domain containing 4034 PAX8 paired box 8 7849 CABP4 calcium binding
protein 4 57010 PDE5A phosphodiesterase 5A, cGMP-specific 8654
BCL2L11 BCL2-like 11 (apoptosis facilitator) 10018 PCK1
phosphoenolpyruvate carboxykinase 1 (soluble) 5105 TCF20
transcription factor 20 (AR1) 6942 SLAMF6 SLAM family member 6
114836 EPOR erythropoietin receptor 2057 CACNG6 calcium channel,
voltage-dependent, gamma subunit 6 59285 NLRP1 NLR family. pyrin
domain containing 1 22861 PECAM1 platelet/endothelial cell adhesion
molecule 5175 NOS1 nitric oxide synthase 1 (neuronal) 4842 ATF3
activating transcription factor 3 467 KRTAP8 keratin associated
protein 8-1 337879 GRIK5 glutamate receptor, ionotropic, kainate 5
2901 SLC24A3 solute carrier family 24 (sodium/potassium/calcium
exchanger), member 3 57419 SLC5A12 solute carrier family 5
(sodium/glucose cotransporter). member 12 159963 SLCA10A2 Solute
carrier family 10 (sodium/bile acid cotransporter family), member 2
6555 SLCO1A2 solute carrier organic anion transporter family,
member 1A2 6579 SLC25A5 solute carrier family 25 (mitochondrial
carrier; adenine nucleotide 292 translocator), member 5 MG29 or
synaptophysin-like 2 284612 SYPL2 NLGN2 neuroligin 2 57555 PRKACA
protein kinase, cAMP-dependent, catalytic, alpha 5566 FOSB FBJ
murine osteosarcoma viral oncogene homolog B 2354 SIAT6 ST3
beta-galactoside alpha-2,3-sialyltransferase 3 6487 LOXL2 lysyl
oxidase-like 2 4017 PRF1 perforin 1 (pore forming protein) 5551
ADPRH ADP-ribosylarginine hydrolase 141 APBB3 amyloid beta (A4)
precursor protein-binding, family B, member 3 10307 EGR3 early
growth response 3 1960 CNR2 cannabinoid receptor 2 (macrophage)
1269 IFITM1 interferon induced transmembrane protein 1 (9-27) 8519
PLA2G5 phospholipase A2, group V 5322 CAMTA1 calmodulin binding
transcription activator 1 23261 SOX4 SRY (sex determining region
Y)-box 4 6659 NFIB nuclear factor I/B 4781 NFIC nuclear factor I/C
(CCAAT-binding transcription factor) 4782 RBMS1 RNA binding motif,
single stranded interacting protein 1 5937 G0S2 G0/G1switch 2 50486
FAT3 FAT tumor suppressor homolog 3 (Drosophila) 120114 SLC40A1
solute carrier family 40 (iron-regulated transporter), member 1
30061 GPC6 glypican 6 10082 IGF1R insulin-like growth factor 1
receptor 3480
[0013] An object of the invention relates to a method for selecting
a competent oocyte, comprising a step of measuring the expression
level of 45 genes in a cumulus cell surrounding said oocyte,
wherein said genes are WNT6, LRCH4, PAX8, CABP4, PDE5A, BCL2L11,
PCK1, TCF20, SLAMF6, EPOR, CACNG6, NLRP1, PECAMI, NOS1, ATF3,
KRTAP8, GRIK5, SLC24A3, SLC5A12, SLCA10A2, SLCO1A2, SLC25A5, MG29,
NLGN2, PRKACA, FOSB, SIAT6, LOXL2, PRF1, ADPRH, APBB3, EGR3, CNR2,
IFITM1, PLA2G5, CAMTA1, SOX4, NFIB, NFIC, RBMS1, G0S2, FAT3,
SLC40A1, GPC6 and IGFIR.
[0014] A used herein the term "competent oocyte" refers to a female
gamete or egg that when fertilized produces a viable embryo with a
high implantation rate leading to pregnancy.
[0015] 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 ore more oocytes under stimulation with GnRH
agonists or antagonists associated withrecombinant FSH or hMG.
[0016] The term "cumulus cell" refers to a cell comprised in a mass
of cells that surrounds an oocyte. These cells are believed to be
involved in providing an oocyte some of its nutritional, energy and
or other requirements that are necessary to yield a viable embryo
upon fertilization.
[0017] The methods of the invention may further comprise a step
consisting of comparing the expression level of the genes in the
sample with a control, wherein detecting differential in the
expression level of the genes between the sample and the control is
indicative whether the oocyte is competent. The control may consist
in sample comprising cumulus cells associated with a competent
oocyte or in a sample comprising cumulus cells associated with an
unfertilized oocyte.
[0018] The methods of the invention are applicable preferably to
women but may be applicable to other mammals (e.g., primates, dogs,
cats, pigs, cows . . . ).
[0019] The methods of the invention are particularly suitable for
assessing the efficacy of an in vitro fertilization treatment.
Accordingly the invention also relates to a method for assessing
the efficacy of a controlled ovarian hyperstimulation (COS)
protocol in a female subject comprising:
[0020] i) providing from said female subject at least one oocyte
with its cumulus cells;
[0021] ii) determining by a method of the invention whether said
oocyte is a competent oocyte.
[0022] Then after such a method, the embryologist may select the
competent oocytes and in vitro fertilized them through a classical
in vitro fertilization (cIVF) protocol or under an intracytoplasmic
sperm injection (ICSI) protocol.
[0023] A further object of the invention relates to a method for
monitoring the efficacy of a controlled ovarian hyperstimulation
(COS) protocol comprising:
[0024] i) isolating from said woman at least one oocyte with its
cumulus cells under natural, modified or stimulated cycles;
[0025] ii) determining by a method of the invention whether said
oocyte is a competent oocyte;
[0026] iii) and monitoring the efficacy of COS treatment based on
whether it results in a competent oocyte.
[0027] The COS treatment may be based on at least one active
ingredient selected from the group consisting of GnRH agonists or
antagonists associated with recombinant FSH or hMG.
[0028] The present invention also relates to a method for selecting
a competent embryo, comprising a step of measuring the expression
level of 45 genes in a cumulus cell surrounding the embryo, wherein
said genes are WNT6, LRCH4, PAX8, CABP4, PDE5A, BCL2L11, PCK1,
TCF20, SLAMF6, EPOR, CACNG6, NLRP1, PECAM1, NOS1, ATF3, KRTAP8,
GRIK5, SLC24A3, SLC5A12. SLCA10A2, SLCO1A2, SLC25A5, MG29, NLGN2,
PRKACA, FOSB, SIAT6, LOXL2, PRFI, ADPRH. APBB3. EGR3, CNR2, IFITM1,
PLA2G5, CAMTA1, SOX4, NF1B, NF1C, RBMS1, G0S2, FAT3, SLC40A1, GPC6
and IGFIR.
[0029] The term "embryo" refers to a fertilized oocyte or zygote.
Said fertilization may intervene under a classical in vitro
fertilization (cIVF) or under an intracytoplasmic sperm injection
(ICSI) protocol.
[0030] 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.
[0031] 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 foetus, which in turn develops into a viable offspring
absent a procedure or event that terminates said pregnancy.
[0032] The methods of the invention may further comprise a step
consisting of comparing the expression level of the genes in the
sample with a control, wherein detecting differential in the
expression level of the genes between the sample and the control is
indicative whether the embryo is competent. The control may consist
in sample comprising cumulus cells associated with an embryo that
gives rise to a viable foetus or in a sample comprising cumulus
cells associated with an embryo that does not give rise to a viable
foetus.
[0033] It is to note that the methods of the invention leads to an
independence from morphological considerations of the embryo. Two
embryos may have the same morphological aspects but by a method of
the invention may present a different implantation rate leading to
pregnancy.
[0034] The methods of the invention are applicable preferably to
women but may he applicable to other mammals (e.g. primates, dogs,
cats, pigs, cows . . . ).
[0035] The present invention also relates to a method for
determining whether an embryo is a competent embryo, comprising a
step consisting in measuring the expression level of 45 genes in a
cumulus cell surrounding the embryo, wherein said genes are WNT6,
LRCH4, PAX8, CABP4, PDE5A, BCL2L11, PCK1, TCF20, SLAMF6, EPOR,
CACNG6, NLRP1, PECAM1, NOS1, ATF3, KRTAP8, GRIK5, SLC24A3, SLC5A12,
SLCA10A2, SLCO1A2, SLC25A5, MG29, NLGN2, PRKACA, FOSB, SIAT6,
LOXL2, PRFI, ADPRH, APBB3, EGR3, CNR2. IFITM1, PLA2G5, CAMTAI,
SOX4, NFIB, NFIC, RBMS1, G0S2, FAT3, SLC40A1, GPC6 and IGFIR.
[0036] The present invention also relates to a method for
determining whether an embryo is a competent embryo,
comprising:
[0037] i) providing an oocyte with its cumulus cells
[0038] ii) in vitro fertilizing said oocyte
[0039] iii) determining whether the embryo that results from step
ii) is competent by determining by a method of the invention
whether said oocyte of step i), is a competent oocyte.
[0040] The present invention also relates to a method for selecting
a competent oocyte or a competent embryo, comprising a step of
measuring in a cumulus cell surrounding said oocyte or said embryo
the expression level of one or more genes selected from the groups
A, B or C, wherein group A consists of PCK1, ADPRH, CABP4, SLAMF6,
CAMTAI, CSPG2. and PRFI; group B consists of FOSB, NLGN2, PDE5A,
PLA2G5, GPC6, and EGR3; and group C consists of NFIB. NFIC, IGFIR,
G0S2, GIRK5 and RBMS 1.
[0041] Overexpression of one or more genes selected from group A is
predictive of a competent oocyte or embryo leading to pregnancy.
Overexpression of one or more genes selected from group B is
predictive of a non competent oocyte or embryo, the embryo being
unable to implant. Overexpression of one or more genes selected
from group C is predictive of a non competent oocyte or embryo due
to early embryo arrest. Said one or more genes may be selected for
example from group A alone, group B alone or group C alone.
Typically, 1, 2, 3, 4, 5, 6 or 7 genes may be selected from group
A. Typically, 1, 2, 3, 4, 5, or 6 genes may be selected from group
B. Typically, 1, 2, 3, 4, 5, or 6 genes may be selected from group
C. Alternatively, said genes may be selected for example from
groups A and B, from groups A and C, from groups B and C, or from
groups A, B and C. Typically, 1, 2, 3, 4, 5, 6 or 7 genes may be
selected from group A, and 0, 1, 2, 3, 4, 5, or 6 genes may be
selected from group B and 0, 1, 2, 3, 4, 5, or 6 genes may be
selected from group C. Typically, 0, 1, 2, 3, 4, 5, 6 or 7 genes
may be selected from group A, and 1, 2, 3, 4, 5, or 6 genes may be
selected from group B and 0, 1, 2, 3, 4. 5, or 6 genes may be
selected from group C. Typically, 0, 1, 2, 3, 4, 5, 6 or 7 genes
may be selected from group A, and 0, 1, 2, 3, 4, 5, or 6 genes may
be selected from group B and 1, 2, 3, 4, 5, or 6 genes may be
selected from group C.
[0042] The methods of the invention are particularly suitable for
enhancing the pregnancy outcome of a female. Accordingly the
invention also relates to a method for enhancing the pregnancy
outcome of a female comprising:
[0043] i) selecting a competent embryo by performing a method of
the invention
[0044] iii) implanting the embryo selected at step i) in the uterus
of said female.
[0045] The method as above described will thus help embryologist to
avoid the transfer in uterus of embryos with a poor potential for
pregnancy out come.
[0046] 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.
[0047] In all above cases, the methods described the relationship
between genes expression profile of cumulus cells and embryo and
pregnancy outcomes.
[0048] Methods for Determining the Expression Level of the Genes of
the Invention:
[0049] Determination of the expression level of the genes as above
described in Tables A and B can be performed by a variety of
techniques. Generally, the expression level as determined is a
relative expression level.
[0050] More preferably, the determination comprises contacting the
sample with selective reagents such as probes, primers or ligands,
and thereby detecting the presence, or measuring the amount, of
polypeptide or nucleic acids of interest originally in the sample.
Contacting may be performed in any suitable device, such as a
plate, microtiter dish, test tube, well, glass, column. and so
forth. In specific embodiments, the contacting is performed on a
substrate coated with the reagent, such as a nucleic acid array or
a specific ligand array. The substrate may be a solid or semi-solid
substrate such as any suitable support comprising glass, plastic,
nylon, paper, metal, polymers and the like. The substrate may be of
various forms and sizes, such as a slide, a membrane, a bead, a
column, a gel, etc. The contacting may be made under any condition
suitable for a detectable complex, such as a nucleic acid hybrid or
an antibody-antigen complex, to be formed between the reagent and
the nucleic acids or polypeptides of the sample.
[0051] In a preferred embodiment, the expression level may be
determined by determining the quantity of mRNA.
[0052] Methods for determining the quantity of mRNA are well known
in the art. For example the nucleic acid contained in the samples
(e.g., cell or tissue prepared from the patient) is first extracted
according to standard methods. for example using lytic enzymes or
chemical solutions or extracted by nucleic-acid-binding resins
following the manufacturer's instructions. The extracted mRNA is
then detected by hybridization (e. g., Northern blot analysis)
and/or amplification (e.g., RT-PCR). Preferably quantitative or
semi-quantitative RT-PCR is preferred. Real-time quantitative or
semi-quantitative RT-PCR is particularly advantageous.
[0053] Other methods of Amplification include ligase chain reaction
(LCR), transcription-mediated amplification (TMA), strand
displacement amplification (SDA) and nucleic acid sequence based
amplification (NASBA).
[0054] Nucleic acids having at least 10 nucleotides and exhibiting
sequence complementarity or homology to the mRNA 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).
[0055] Probes typically comprise single-stranded nucleic acids of
between 10 to 1000 nucleotides in length, for instance of between
10 and 800, more preferably of between 15 and 700, typically of
between 20 and 500. Primers typically are shorter single-stranded
nucleic acids, of between 10 to 25 nucleotides in length, designed
to perfectly or almost perfectly match a nucleic acid of interest,
to be amplified. The probes and primers are "specific" to the
nucleic acids 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. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).
[0056] The nucleic acid primers or probes used in the above
amplification and detection method may be assembled as a kit. Such
a kit includes consensus primers and molecular probes. A preferred
kit also includes the components necessary to determine if
amplification has occurred. The kit may also include, for example,
PCR buffers and enzymes; positive control sequences, reaction
control primers; and instructions for amplifying and detecting the
specific sequences.
[0057] In a particular embodiment, the methods of the invention
comprise the steps of providing total RNAs extracted from cumulus
cells and subjecting the RNAs to amplification and hybridization to
specific probes, more particularly by means of a quantitative or
semi-quantitative RT-PCR.
[0058] In another preferred embodiment, the expression level is
determined by DNA chip analysis. Such DNA chip or nucleic acid
microarray consists of different nucleic acid probes that are
chemically attached to a substrate, which can be a microchip, a
glass slide or a microsphere-sized bead. A microchip may be
constituted of polymers, plastics, resins, polysaccharides, silica
or silica-based materials, carbon, metals, inorganic glasses, or
nitrocellulose. Probes comprise nucleic acids such as cDNAs or
oligonucleotides that may be about 10 to about 60 base pairs. To
determine the expression level, a sample from a test subject,
optionally first subjected to a reverse transcription, is labelled
and contacted with the microarray in hybridization conditions,
leading to the formation of complexes between target nucleic acids
that are complementary to probe sequences attached to the
microanarray surface. The labelled hybridized complexes are then
detected and can be quantified or semi-quantified. Labelling may be
achieved by various methods, e.g. by using radioactive or
fluorescent labelling. Many variants of the microarray
hybridization technology are available to the man skilled in the
art (see e.g. the review by Hoheisel, Nature Reviews, Genetics,
2006, 7:200-210)
[0059] In this context, the invention further provides a DNA chip
comprising a solid support which carries nucleic acids that are
specific to the genes listed in table A or B.
[0060] Other methods for determining the expression level of said
genes include the determination of the quantity of proteins encoded
by said genes.
[0061] Such methods comprise contacting the sample with a binding
partner capable of selectively interacting with a marker protein
present in the sample. The binding partner is generally an antibody
that may be polyclonal or monoclonal, preferably monoclonal.
[0062] The presence of the protein can be detected using standard
electrophoretic and immunodiagnostic techniques, including
immunoassays such as competition, direct reaction, or sandwich type
assays. Such assays include, but are not limited to, Western blots;
agglutination tests; enzyme-labeled and mediated immunoassays, such
as ELISAs; biotin/avidin type assays; radioimmunoassays;
immunoelectrophoresis; immunoprecipitation, etc. The reactions
generally include revealing labels such as fluorescent,
chemiluminescent, radioactive, enzymatic labels or dye molecules,
or other methods for detecting the formation of a complex between
the antigen and the antibody or antibodies reacted therewith.
[0063] The aforementioned assays generally involve separation of
unbound protein in a liquid phase from a solid phase support to
which antigen-antibody complexes are bound. Solid supports which
can be used in the practice of the invention include substrates
such as nitrocellulose (e. g., in membrane or microtiter well
form); polyvinylchloride (e. g., sheets or microtiter wells);
polystyrene latex (e.g., beads or microtiter plates);
polyvinylidine fluoride; diazotized paper; nylon membranes;
activated beads, magnetically responsive beads, and the like.
[0064] More particularly, an ELISA method can be used, wherein the
wells of a microtiter plate are coated with an antibody against the
protein to be tested. A biological sample containing or suspected
of containing the marker protein is then added to the coated wells.
After a period of incubation sufficient to allow the formation of
antibody-antigen complexes, the plate(s) can be washed to remove
unbound moieties and a delectably labeled secondary binding
molecule added. The secondary binding molecule is allowed to react
with any captured sample marker protein, the plate washed and the
presence of the secondary binding molecule detected using methods
well known in the art.
[0065] Alternatively an immunohistochemistry (IHC) method may be
preferred. IHC specifically provides a method of detecting targets
in a sample or tissue specimen in situ. The overall cellular
integrity of the sample is maintained in IHC, thus allowing
detection of both the presence and location of the targets of
interest. Typically a sample is fixed with formalin, embedded in
paraffin and cut into sections for staining and subsequent
inspection by light microscopy. Current methods of IHC use either
direct labeling or secondary antibody-based or hapten-based
labeling. Examples of known IHC systems include, for example,
EnVision.TM. (DakoCytomation). Powervision(R) (Immunovision.
Springdale. Ariz.), the NBA.TM. kit (Zymed Laboratories Inc., South
San Francisco, Calif.). HistoFine(R) (Nichirei Corp, Tokyo,
Japan).
[0066] In particular embodiment, a tissue section (e.g. a sample
comprising cumulus cells) may be mounted on a slide or other
support after incubation with antibodies directed against the
proteins encoded by the genes of interest. Then, microscopic
inspections in the sample mounted on a suitable solid support may
be performed. For the production of photomicrographs, sections
comprising samples may be mounted on a glass slide or other planar
support, to highlight by selective staining the presence of the
proteins of interest.
[0067] Therefore IHC samples may include, for instance: (a)
preparations comprising cumulus cells (b) fixed and embedded said
cells and (c) detecting the proteins of interest in said cells
samples. In some embodiments, an IHC staining procedure may
comprise steps such as: cutting and trimming tissue, fixation,
dehydration, paraffin infiltration, cutting in thin sections,
mounting onto glass slides, baking, deparaffination, rehydration,
antigen retrieval, blocking steps, applying primary antibodies,
washing, applying secondary antibodies (optionally coupled to a
suitable detectable label), washing, counter staining, and
microscopic examination.
[0068] The invention also relates to a kit for performing the
methods as above described, wherein said kit comprises means for
measuring the expression level the levels of the genes of Tables A
or B that are indicative whether the oocyte or the embryo is
competent.
[0069] 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.
EXAMPLES
A NON-INVASIVE TEST FOR ASSESSING EMBRYO POTENTIAL BY GENE
EXPRESSION PROFILES OF HUMAN CUMULUS CELLS
Example 1
[0070] Material & Methods:
[0071] Patients and IVF treatment: In this retrospective study,
normo-responder patients (n=30) aged of 30.9 years .+-.2.5 and
referred to our centre for ICSI (Intra Cytoplasmic Sperm Injection)
for male infertility factor were studied. Patients were stimulated
with a combination of GnRH agonist or antagonist with recombinant
FSH (GonalF, Puregon; respectively of Merck-Serono and Organon) or
with hMG (Menopur, Ferring). Ovarian response was evaluated by
serum estradiol level and ultrasound examination to monitor
follicle development. Retrieval of oocytes was performed 36 hours
after hCG administration (5000 IU), under ultrasound guidance.
[0072] Assessment of embryo quality: On day 2 and 3
postmicroinjection, the quality parameters of individually cultured
embryo were evaluated using the number of blastomeres and the
degree of fragmentation as criteria (grade 1-2: equally sized
blastomeres and 0-20% fragmentation, grade 3-4: no equally sized
blastomeres and more than 20% fragmentation. A top-quality embryo
was defined on day 3 as 6-8 cells, equally sized blastomeres and no
fragmentation. One or two embryos were transferred on day 3 after
oocyte retrieval. Clinical pregnancy was evaluated two and six
weeks after embryo transfer based respectively on serum Beta-hCG
and ultrasound examination (presence of gestational sac with heart
beat).
[0073] Cumulus cells: All cumulus cells (CC) samples were frozen on
egg collection day. Then, one to 3 CC samples per patient were
randomly selected for microarray analysis. A total of 50 CC samples
were collected from 50 single oocytes and analyzed individually: 34
CC from grade 1-2 embryos (n=20 patients), 11 CC from grade 3-4
embryos (n=10 patients) and 5 CC from unfertilized oocytes (n=5
patients) (Table 1).
TABLE-US-00002 TABLE 1 The Characteristics of cumulus cells samples
in this study 30 patients 5 Patients 45 CC 5 CC G1/2 (34 CC) G3/4
(11 CC) cumulus cells from P+ P- NT unfertilized oocyte (5 CC)
chips nbr 18 16 11 5 patients nbr 11 9 10 5 CC nbr 18 16 11 5 CC:
cumulus cells, P+: cumulus cells from embryos with positive
pregnancy outcome, P-: cumulus cells from embryos without pregnancy
outcome, G1/2: cumulus cells from grade 1-2 embryos, G3/4: cumulus
cells from grade 3-4 embryos, NT: no transfer.
[0074] The data analysis was performed under double blind
conditions in which pregnancy outcome was disclosed only after
microarrays were hybridized. Regarding pregnancy outcome, the 45 CC
from fertilized oocytes included 16 CC from grade 1-2 embryos that
did not result into pregnancy (n=9 patients), 18 CC associated with
a positive pregnancy outcome (n=11 patients) and 11 CC from grade
3-4 embryos that were not transferred. Cumulus cells were stripped
immediately following oocyte recovery (<40 h post hCG
administration). Cumulus cells were mechanically removed and washed
in culture medium and immediately frozen at -80.degree. C. in RLT
RNA extraction buffer (RNeasy kit, Qiagen, Valencia, Calif., USA)
before RNA extraction.
[0075] Granulosa cells: An independent group of normo responder
patients (n=8) (age 34.8 years .+-.3.2) referred for ICSI program
for male infertility factor was selected for granulosa cells
collection (8 samples). Immediately after oocyte recovery,
follicular fluids from matures follicles (>17 mm) of the same
patient were pooled, after removal of the cumulus oocyte complex
and diluted in 1/3 volume of HBSS solution (BioWhittaker) in 50 ml
batches, representing one sample. Granulosa cells purification was
adapted from the protocol by (Kolena et al., 1983). Following a 20
min. centrifugation at 500 g in swinging buckets, granulosa cells
were collected on a Ficoll cushion (12 ml Lymphocyte separation
medium, BioWhittaker). They were successively washed in HBSS and
PBS, incubated 5 min. in blood lysis buffer (KHCO.sub.310 mM,
NH.sub.4Cl 150 mM, EDTA 0.1 mM) to remove red blood cells, counted
and pelleted in PBS before lysis in RLT buffer (Quiagen) and
storage at -80.degree. C. The number of follicular puncture and the
number of purified granulosa cells ranged from 6 to 12 and from 2
10.sup.6 to 9 10.sup.6 respectively.
[0076] Complementary RNA (cRNA) preparation and microarray
hybridization: CC and granulosa cells RNA was extracted using the
micro RNeasy Kit (Qiagen). The total RNA quantity was measured with
a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies Inc.,
Delaware, USA) and RNA integrity was assessed with an Agilent 2100
Bioanalyzer (Agilent, Palo Alto, Calif., USA). cRNA was prepared
with two rounds of amplification according to the manufacturer's
protocol "double amplification" (Two-Cycle cDNA Synthesis Kit,
Invitrogen) starting from total RNA (ranging from 70 ng to 100 ng).
cRNA obtained after the first amplification ranged from 0.1
.mu.g/.mu.l to 1.9 .mu.g/.mu.l and after the second amplification
ranged from 1.6 .mu.g/.mu.l to 4.5 .mu.g/.mu.l.
[0077] Labelled fragmented cRNA (12 .mu.g) was hybridized to
oligonucleotide probes on an Affymetrix HG-U133 Plus 2.0 array
containing 54 675 sets of oligonucleotide probes ("probeset") which
correspond to =30 000 unique human genes or predicted genes. Each
cumulus and granulosa sample was put individually on a microarray
chip.
[0078] Data processing: Scanned GeneChip images were processed
using Affymetrix GCOS 1.4 software to obtain an intensity value and
a detection call (present, marginal or absent) for each probeset,
using the default analysis settings and global scaling as first
normalization method, with a trimmed mean target intensity value
(TGT) of each array arbitrarily set to 100. Probe intensities were
derived using the MAS5.0 algorithm. This algorithm also determines
whether a gene is expressed with a defined confidence level or not
("detection call"). This "call" can either be "present" (when the
perfect match probes are significantly more hybridized than the
mismatch probes, p-value <0.04), "marginal" (for p-values
>0.04 and <0.06) or "absent" (p-value >0.06). The
microarray data were obtained in our laboratory in agreement with
the Minimal Information about a Microarray Experiment MIAME
recommendations (Brazma et al. 2001).
[0079] Data analysis and visualisation: Significant Analysis of
microarrays (SAM) (Tusher et al., 2001)
(http://www-stat.stanford.edu/-tibs/SAM/) was used to identify
genes whose expression varied significantly between sample groups.
SAM provides mean or median fold change values (FC) and a false
discovery rate (FDR) confidence percentage based on data
permutation (mean fold change >2 and FDR <5%). Array analysis
allowing the comparison of gene expression profile. between cumulus
cell samples and granulosa cell samples is first based on the
significant RNA detection (detection call "present" or "absent")
and then, submitted to a SAM (Significant Analysis of microarrays)
to identify genes whose expression varied significantly between
sample groups. To perform the comparison of gene expression profile
between cumulus cell samples according embryonic quality and/or
pregnancy outcome. a non-supervised selection of probesets using a
variation coefficient (CV .gtoreq.40%) and a Absent/Present
"detection call" filter was performed before the SAM. To compare
profile expression of cumulus cells from altered (grade 3-4) and
good (grade 1-2) embryonic development, or from embryos leading, or
not, to a pregnancy, we performed an unsupervised classification
with both principal component analysis (PCA) and hierarchical
clustering (de Hoon et al., 2004; Eisen et al., 1998). The PCA
involved original scripts based on the R statistics software
through the RAGE web interface (http://rage.montp.inserm.fr) (Reme
et al., 2008). Hierarchical clustering analysis based on the
expression levels of varying probes were performed with the CLUSTER
and TREEVIEW software packages. To uncover functional biological
networks and top canonical pathways, we imported gene expression
signatures into the Ingenuity Pathway's Analysis (IPA) Software
(Ingenuity Systems, Redwood City, Calif., USA).
[0080] Quantitative RT-PCR analyses: For qRT-PCR analysis, 10 CC
samples used in the microarray experiments were selected according
to their pregnancy outcome (5 CC samples associated to a negative
outcome and 5 to a positive outcome corresponding to 10 patients).
Labelled cRNA (1 .mu.g) from the patient was used to generate first
strand cDNA. These cDNAs (5 .mu.l of a 1/10 dilution) were used for
real-time quantitative PCR reactions according to the
manufacturer's recommendations (Applied Biosytems). The 20 .mu.l
reaction mixture consisted of cDNA (5 .mu.l), 1 .mu.M of primers
and 10 .mu.l of Taqman Universal PCR Master Mix (Applied
Biosystem). The amplification was measured during 40 cycles with an
annealing temperature at 60.degree. C. The amount of PCR product
produced in every cycle step of the PCR reaction is monitored by
TaqMan probe. A threshold is set in the exponential phase of the
amplification curve, from which the cycle number ("Ct" for "Cycle
Threshold") is read off. The Ct-value is used in the calculation of
relative mRNA transcript levels. Effectiveness (E) of the PCR was
measured. This effectiveness is obtained by a standard curve
corresponding to the primers used. Quantitative reverse
transcriptase polymerase chain reaction (QRT-PCR) was performed
using the ABI Prism 7000 sequence detection system (Applied
Biosystems) and normalized to PGKI for each sample using the
following formula: E.sub.tested
primer.sup..DELTA.Ct/E.sub.PGKI.sup..DELTA.Ct(E=10.sup.-1/slope),
.DELTA.Ct=Ct control-Ct unknown, control=one CC sample of the
non-pregnant group). Each sample was analysed in duplicate, and
multiple water blanks were included with the analysis.
[0081] Results
[0082] Gene expression profile of CC according to embryo outcome:
To identify a gene expression profile in CC that correlated with
embryo outcome, we established a gene expression signature for each
outcome category: CC of unfertilized oocytes, CC from oocytes that
resulted in embryo development but extensive fragmentation (grade
3-4), and CC from oocytes that resulted in embryo development with
no or limited fragmentation (grade 1-2). Granulosa cells samples
were taken as a reference tissue (control). Indeed, granulosa cells
are cells closely related to CC as opposed to other adult tissues.
The use of this reference tissue lowered the number of
differentially expressed genes related to crude lineage
differences, thus facilitating the identification of subtle
variation in the CC/oocyte interplay. A SAM analysis showed that
2605 genes were upregulated in the unfertilized group, 2739 in the
grade 3/4 group and 2482 in the grade 1-2 group with a FDR <5% .
Conversely, 4270, 4349 and 4483 genes, were downregulated,
respectively. These lists of genes were then intersected to
determine their overlap. While 449 up and 890 down expressed genes
were in common in all three groups, each category displayed a
specific gene expression profile. Interestingly, 860 up-regulated
genes, including for example Galanin and Gap Junction AS (GJA5).
and 1416 down-regulated genes, including HLA-G and EGRI were
specifically modulated in cumulus cells associated to a good
morphological embryonic quality. It must be noted that although the
grade 1-2 group displayed a strong gene expression profile, this
group was heterogeneous regarding to pregnancy outcome and included
18 CC samples associated with embryos that resulted in pregnancy
(including 4 twin pregnancies) but also 16 CC samples associated
with embryos that failed to give rise to pregnancy.
[0083] Gene expression profile of CC according to pregnancy
outcome: CC samples were therefore compared according to the
pregnancy outcome. A SAM analysis delineated a "pregnancy outcome"
list of 630 genes that varied significantly (FDR <5%) between
the two group of patients (pregnancy versus no pregnancy). PCA and
hierarchical clustering confirmed that this 630 gene list indeed
segregated a majority of CC samples associated with pregnancy from
from those associated with no pregnancy. Of note, genes from the
"pregnancy outcome" list were predominantly upregulated in samples
associated with a good outcome. The "pregnancy outcome" expression
signature was particularly marked in a sub-group of 10 CC samples
from embryos associated to the "pregnancy" group.
[0084] Functional annotation of the Pregnancy Outcome gene list: To
investigate biological processes correlated to embryo achieving
pregnancy. Ingenuity and Pubmed databases were used to annotate the
630 genes from the "pregnancy outcome" gene list. Among genes whose
overexpression is associated with pregnancy, the most significantly
overrepresented pathways were "oxidative stress", "TR/RXR
activation", "G2/M transition of the cell cycle", "xenobiotic
metabolism" and "NFKappaB" signalling. Among these pathways, the
most representative genes were interleukins, chemokines, adptator
proteins and kinases: IL1Beta (.times.4.5 in pregnancy samples
versus no pregnancy, P=0.001), IL16 (.times.4.8, P=0.001), IL8
(.times.2.6, P=0.007), ILIRN (.times.2.1, P=0.0051), IL17RC
(.times.3.6, P=0.001), TIRAP (.times.8.0, P=0.001), CXCL12
(.times.3.1, P=0.001), CCR5 (.times.2.6, P=0.0051), and PCK1
(.times.3.4, P=0.001). Strikingly, numerous genes involved in the
regulation of apoptosis were significantly modulated in CC samples
from oocytes resulting in a pregnancy. These genes were BCL2L11
(.times.6.9, P<0.001), CRADD (.times.2, P=0.0036), NEMO
(.times.4.6, P<0.001), BCL10 (.times.3.1, P=<0.001), SERPINB8
(.times.9.1, P<0.001). and TNFSF13 (.times.2.5, P=0.0038),
[0085] On the other hand, genes associated with no pregnancy were
correlated with the following pathways: G2/M DNA damage and
checkpoint regulation of the cell cycle, "Sonic hedgehog", "IGF-1",
"complement system" and "Wnt/Beta-catenin" Representative genes
correlated with no pregnancy included NFIB (.times.0.3,
P<0.001), MAD2L1 (.times.0.4, P<0.001) and IGFIR (.times.0.4,
P<0.001).
[0086] Candidate genes expressed in CC for embryo potential: The
SAM analysis of CC according to pregnancy outcome identified the 45
genes of Table A that are biomarkers for embryo potential that
would differentiate between oocytes that produced embryos resulting
in a pregnancy versus those that did not result in pregnancy based
on genes expression of CC analysis. QRT-PCR was used to confirm
independently the microarray data. We analyzed the differential
expression of 36 up-regulated genes and 9 down-regulated genes
between CC from grade 1-2 embryos did not achieve pregnancy and CC
from grade 1-2 embryos achieving pregnancy.
[0087] Conclusion:
[0088] In most mammalian species including human, the cumulus cells
which surrounds the oocyte are still present at the time of
fertilization in the oviduct and remain until embryonic
implantation. Extracellular matrix remodelling within and around
the cumulus probably plays a key role in both of these steps. In
this respect, we identified 45 genes expressed in cumulus cells
that are biomarkers for embryo potential and pregnancy outcome. In
our study, we demonstrated that genes expression profile of CC
which surrounds oocyte correlated to different outcomes, allowing
the identification of a specific expression signature of embryos
developing toward pregnancy. In conclusion, we found a differential
gene expression between human cumulus cells from oocytes resulting
in different pregnancy outcome from patients referred for ICSI or
IVF. Our results indicate that analysis of cumulus cells
surrounding the oocyte is a non-invasive approach for embryo
selection. Typically CC can be collected immediately after oocyte
pick-up, the CC can be analyzed with a genomic test (G-test) to
assess the potential of the embryo, and the embryo can be then be
selected for fresh replacement based on the G-test results.
Example 2
[0089] In order to test the reliability of the 45 gene list, we
conducted a prospective study including young (<36 years) normal
responder patients referred to our centre for ICSI for male
infertility. The embryo selection occurred either according to the
gene expression profile in CCs (group 1) or to morphological
aspects (group 2 used as control). For each group, two embryos were
replaced. For the first 60 patients (30 patients/group), on egg
collection day, in group I, each CC sample was collected
individually and processed for gene expression analysis. CC samples
(n=267) were analyzed. Quantitative RT-PCR analysis was performed
to measure the relative abundance of the transcripts of interest
genes in CCs, and expression data for all biomarkers were obtained
from all samples. All patients in both groups had a fresh embryo
transfer on day 3. The comparison between the 2 groups reveals
significant differences for implantation and ongoing pregnancy
rates/pick up (40.0% vs. 26.7% and 70.0% vs. 46.7; p<0.05,
respectively). We noted 5 twin pregnancies in group I versus 0 in
the group 2 used as control. In addition, we observed that there
was no relationship between morphological aspects and the CC gene
expression profile. On the basis of the analysis of 267 CC samples,
we noted 27% of CCs express genes which predict for embryos able to
achieve pregnancy, 42% of CCs did not, 31% of CCs showing gene
expression for early arrest of embryo development.
[0090] Selected candidate biomarkers are listed in the below Table
B. Table B presents the set of genes in cumulus cells were able to
predict different clinical conditions: (A) pregnancy, (B) absence
of pregnancy and (C) early embryo arrest.
TABLE-US-00003 TABLE B set of predictive genes Gene Gene Symbol
Gene name ID A: genes whose overexpressions are predictive of
pregnancy PCK1 phosphoenolpyruvate carboxykinase 1 (soluble) 5105
ADPRH ADP-ribosylarginine hydrolase 141 CABP4 calcium binding
protein 4 57010 SLAMF6 SLAM family member 6 114836 CAMTA1
calmodulin binding transcription activator 1 23261 CSPG2
Chondroitin sulfate proteoglycan 2 (versican) 1462 PRF1 perforin 1
(pore forming protein) 5551 B: genes whose overexpressions are
predictive of embryos unable to implant FOSB FBJ murine
osteosarcoma viral oncogene homolog B 2354 NLGN2 neuroligin 2 57555
PDE5A phosphodiesterase 5A, cGMP-specific 8654 PLA2G5 phospholipase
A2, group V 5322 GPC6 glypican 6 10082 EGR3 early growth response 3
1960 C: genes whose overexpressions are predictive of early embryo
arrest NFIB nuclear factor I/B 4781 NFIC nuclear factor I/C
(CCAAT-binding transcription factor) 4782 IGF1R insulin-like growth
factor 1 receptor 3480 G0S2 G0/G1 switch 2 50486 GIRK5 glutamate
receptor, ionotropie, kainate 5 2901 RBMS1 RNA binding motif,
single stranded interacting protein 1 5937
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References