U.S. patent application number 10/531964 was filed with the patent office on 2006-06-29 for methods of assessing the risk of reproductive failure by measuring telomere length.
This patent application is currently assigned to Women & Infants Hospital of Rhode Island. Invention is credited to David L. Keefe.
Application Number | 20060141461 10/531964 |
Document ID | / |
Family ID | 32110212 |
Filed Date | 2006-06-29 |
United States Patent
Application |
20060141461 |
Kind Code |
A1 |
Keefe; David L. |
June 29, 2006 |
Methods of assessing the risk of reproductive failure by measuring
telomere length
Abstract
The invention features a method of identifying oocytes with a
risk of reproductive failure and/or aneuploidy based on a telomere
length assay.
Inventors: |
Keefe; David L.; (Tampa,
FL) |
Correspondence
Address: |
EDWARDS & ANGELL, LLP
P.O. BOX 55874
BOSTON
MA
02205
US
|
Assignee: |
Women & Infants Hospital of
Rhode Island
Providence
RI
|
Family ID: |
32110212 |
Appl. No.: |
10/531964 |
Filed: |
December 7, 2005 |
PCT NO: |
PCT/US03/32672 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60419071 |
Oct 16, 2002 |
|
|
|
60452741 |
Mar 7, 2003 |
|
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Current U.S.
Class: |
435/6.12 |
Current CPC
Class: |
C12Q 1/6827 20130101;
A61B 17/435 20130101; C12Q 2600/156 20130101; C12Q 1/6827 20130101;
C12Q 1/6841 20130101; C12Q 1/6883 20130101; C12Q 1/68 20130101;
C12Q 1/6841 20130101; C12Q 1/6888 20130101; C12Q 2525/151 20130101;
C12Q 2525/151 20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method for determining the risk of reproductive failure in a
cell comprising: obtaining at least one chromosome from the cell;
measuring telomere length of the chromosome; and comparing the
measured length of the telomere to the standardized average length
of a control telomere; to thereby determine the risk of
reproductive failure in the cell.
2. The method of claim 1, wherein the cell is an oocyte, an oocyte
representative of a population of oocytes, a polar body from a
fertilized oocyte, or a polar body from an unfertilized oocyte.
3. The method of claim 2, wherein the cell is an oocyte.
4. The method of claim 1, wherein a labeled telomere-specific probe
is hybridized to the chromosome prior to measuring telomere length
of the chromosome.
5. The method of claim 4, wherein the probe is hybridized to
telomere repeats.
6. The method of claim 4, wherein the probe is peptide nucleic acid
(PNA)-labeled.
7. The method of claim 1, wherein the telomere is measured using
quantitative fluorescent in situ hybridization (Q-FISH)
analysis.
8. The method of claim 1 for use in in vitro fertilization
(IVF).
9-12. (canceled)
13. A method for determining the predisposition of an oocyte to
reproductive failure comprising: obtaining at least one chromosome
from the oocyte: measuring telomere length of the chromosome; and
comparing the measured length of the telomere to the standardized
average length of a control telomere; to thereby determine the
predisposition of the oocyte to reproductive failure.
14. The method of claim 13, wherein a labeled telomere-specific
probe is hybridized to the chromosome prior to measuring telomere
length of the chromosome.
15. The method of claim 14, wherein the probe is hybridized to
telomere repeats.
16. The method of claim 14, wherein the probe is peptide nucleic
acid (PNA)-labeled.
17. The method of claim 14, wherein the telomere is measured using
quantitative fluorescent in situ hybridization (Q-FISH)
analysis.
18. The method of claim 13, wherein the oocyte is representative of
a population of oocytes.
19. A method for selecting a fertilized oocyte with a low risk of
reproductive failure for in vitro fertilization, comprising:
obtaining at least one chromosome from the polar body of the
fertilized oocyte; measuring telomere length of the chromosome; and
comparing the measured length of the telomere to the standardized
average length of a control telomere; to thereby select a
fertilized oocyte with a low risk of reproductive failure for in
vitro fertilization.
20. A method of in vitro fertilization comprising: selecting a
fertilized oocyte according to the method of claim 19; and
implanting the selected fertilized oocyte in the subject.
21. The method of claim 20, wherein the subject is a human.
22. A method for optimizing the viability of an embryo comprising:
selecting a fertilized oocyte according to the method of claim 19;
and implanting the selected fertilized oocyte in a subject.
23. The method of claim 22, wherein the subject is a human.
24. A method for determining the risk of aneuploidy in a cell
comprising: obtaining at least one chromosome from the cell;
measuring telomere length of the chromosome; and comparing the
measured length of the telomere to the standardized average length
of a control telomere; to thereby determine the risk of aneuploidy
in the cell.
25. The method of claim 24, wherein the cell is selected from the
group consisting of an oocyte, an oocyte representative of a
population of oocytes, a polar body from a fertilized oocyte, and a
polar body from an unfertilized oocyte.
26. The method of claim 24, wherein a labeled telomere-specific
probe is hybridized to the chromosome prior to measuring telomere
length of the chromosome.
27. The method of claim 26, wherein the probe is hybridized to
telomere repeats.
28. The method of claim 26, wherein the probe is peptide nucleic
acid (PNA)-labeled.
29. The method of claim 26, wherein the telomere is measured using
quantitative fluorescent In situ hybridization (Q-FISH)
analysis.
30. The method of claim 26 for use in vitro fertilization
(IVF).
31. The method of claim 24 wherein said cell is in a population of
cells representative of said cell.
32. The method of claim 31 wherein the cell is an.
33. The method of claim 33 further comprising: hybridizing
telomere-specific probes to said chromosome; and performing
quantitative fluorescent in situ hybridization (Q-FISH)
analysis.
34. A method for selecting a fertilized oocyte with a low risk of
aneuploidy for in vitro fertilization, comprising: obtaining at
least one chromosome from the polar body of the fertilized oocyte;
hybridizing telomere-specific probes to said chromosome; performing
quantitative fluorescent in situ hybridization (Q-FISH) analysis;
measuring telomere length of the chromosome; and comparing the
measured length of the telomere to the standardized average length
of a control telomere; to thereby select a cell with a low risk of
aneuploidy.
35. A method for determining the predisposition of an oocyte to
anouploidy comprising: obtaining at least one chromosome from the
oocyte; measuring telomere length of the chromosome; and comparing
the measured length of the telomere to the standardized average
length of a control telomere; to thereby optimize the viability of
the embryo.
36. The method of claim 35, wherein a labeled telomere-specific
probe is hybridized to the chromosome prior to measuring telomere
length of the chromosome.
37. The method of claim 36, wherein the probe is hybridized to
telomere repeats.
38. The method of claim 36, wherein the probe is peptide nucleic
acid (PNA)-labeled.
39. The method of claim 35, wherein the telomere is measured using
quantitative fluorescent in situ hybridization (Q-FISH)
analysis.
41. The method of claim 35, for use in vitro fertilization.
42. The method of claim 35, wherein the oocyte is representative of
a population of oocytes.
43. A method of pre-implantation genetic testing to identify an
oocyte with a predisposition to aneuploidy comprising: obtaining at
least one chromosome from the oocyte; measuring telomere length of
the chromosome; and comparing the measured length of the telomere
to the standardized average length of a control telomere.
44. The method of claim 43, wherein a labeled telomere-specific
probe is hybridized to the chromosome prior to measuring telomere
length of the chromosome.
45. The method of claim 43, wherein the telomere is measured using
quantitative fluorescent in situ hybridization (Q-FISH)
analysis.
46. The method of claim 43 for use in vitro fertilization
(IVF).
47. The method according to claim 1, further comprising obtaining a
probe for hybridizing to the chromosome.
48. The method according of claim 47, wherein said probe is a
labeled telomere-specific probe.
49. The method according to claim 48, wherein the telomere specific
probe comprises a nucleic acid sequence identified by any one of
SEQ ID NOS: 1 through 10.
50. The method according to claim 48, wherein the telomere specific
probe comprises a nucleic acid sequence having at least about 80
percent sequence identity to any one of SEQ ID. NOS. 1 through
10.
51. The method according to claim 48, wherein the telomere specific
probe comprises a nucleic acid sequence having at least about 90
percent sequence identity to any one of SEQ ID. NOS. 1 through
10.
52. A kit for determining the risk of reproductive failure and/or
aneuploidy in a cell comprising reagents for preparing a
chromosomal spread from the cell or at least one cell in a
population of cells representative of said cell; labeled
telomere-specific repeat probes; reagents for performing
quantitative fluorescent in situ hybridization (Q-FISH) analysis on
the chromosomal spread; and instructions for measuring the length
of a telomere obtained from the chromosomal spread, or obtained
from a chromosome of said cell, and comparing the measured length
of the telomere to the standardized average length of a
control.
53. The kit of claim 52, wherein the chromosome is obtained from a
cell selected from the group consisting of an oocyte, an oocyte
representative of a population of oocytes, or the polar body from a
fertilized or unfertilized oocyte.
54. The kit of claim 52, wherein the probes are peptide nucleic
acid (PNA)-labeled.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 60/419,071, filed Oct. 16, 2002 and U.S.
Provisional Patent Application Ser. No. 60/452,741, filed Mar. 7,
2003. The entire contents of each of these patent applications are
hereby incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Telomeres are repeated sequences of DNA that cap the ends of
chromosomes and prevent the formation of end-to-end fusions. During
normal DNA replication, the ends of chromosomes, or telomeres, are
left unreplicated. This results in the loss of a small amount of
DNA from each chromosome with every cell cycle. This loss is
subsequently corrected by an enzyme known as telomerase. Telomerase
is a cellular enzyme, which is directed to the nucleotide
polymerization or maintenance of telomeres, and contains a complex
of protein components and an integral RNA component.
[0003] Vertebrate telomeres consist of tandem repeats of the
sequence TTAGGG and associated proteins, which cap the ends of
chromosomes and protect them from degradation and fusion (Blackburn
E. H., Cell 106: 661-673, Nature 408(6808): 53-6. 2001). Extensive
evidence has shown that telomere shortening and dysfunction in
cultured somatic cells leads to so-called replicative senescence
(Blackburn E. H., Nature 408(6808): 53-6, 2000). In turn, reversal
of telomere shortening by forced expression of telomerase rescues
cells from senescence and extends cell life span indefinitely
(Bodnar, A. G., M. Ouellette, et al., Science 279(5349): 349-52,
1998). (Vaziri and Benchimol et al., Curr Biol 8(5): 279-82.
1998).
[0004] Telomeres cap and protect the ends of chromosomes, establish
homologue pairing and enhance chiasmata formation during early
meiosis, and shorten with cell division and exposure to reactive
oxygen (ROS) to mediate cellular aging in somatic cells. Germ
cells, like stem cells and cancer cells, contain telomerase, a
reverse transcriptase which maintains telomeres by adding telomere
repeats to the 3 prime end of DNA, thus allowing germ cells to
largely bypass the senescence response exhibited by cells with
critically short telomeres. However, telomere elongation is
variable and stochastic in most cell types, and in females,
telomerase activity decreases during late meiosis, so telomere
length, determined during early development, provides a
developmental bottleneck. Germ cells with adequate telomere length
pass across generations, but the fate of eggs "stuck in the
bottleneck" by short telomeres is poorly understood.
[0005] Active telomerase, composed of a small RNA molecule, known
as the telomerase RNA (TR) and of a catalytic subunit, the
telomerase reverse transcriptase (TERT), is the primary enzyme for
maintaining the length of telomere repeats. Telomerase activity is
present during early oogenesis, but is almost absent during late
oogenesis and early preimplantation embryo development until the
morula stage of development. Thus telomere length in oocytes and
early embryos is established during early development. When
telomeres reach a critically short length, the cell undergoes cell
cycle arrest and apoptosis, so late oogenesis and early
preimplantation embryo development may represent a kind of
bottleneck for telomere length during development.
[0006] The RNA component of the human enzyme contains a short
region complementary to the human telomeric repeat sequence (Feng
et al. (1995) Science, 269:1236). Somatic cells lack telomerase
activity, and their telomeres have been found to shorten with cell
division both in vivo and in culture. In germ cells and embryos,
telomerase actively restores telomeres, so that the chromosomes do
not shorten progressively across generations. The short and
numerous cell cycles of replicating primordial germ cells and
oogonia during oogenesis, however, challenge telomerase to keep up
with the progressive telomere shortening. In mature oocytes and
early stage pre-implantation embryos, telomerase is down regulated
until the blastocyst stage of development.
[0007] The length of telomeres within the chromosomes of an oocyte
at fertilization can determine the resultant telomere length of the
embryo. Oocytes exhibit significant variability in telomere length.
Oogonia exit mitosis after variable numbers of cell cycles, making
the process of telomere shortening stochastic. This inevitable
variability in telomere length of chromosomes from oocytes and
embryos provides a cytogenetic mechanism to explain the most widely
accepted theory of chromosomal aberration or aneuploidy in
mammals--the production line hypothesis.
[0008] The production line hypothesis states that oogonia exiting
from mitosis late during oogenesis have traversed more cell cycles
than oogonia exiting from mitosis early during oogenesis. The late
exiting oogonia, therefore, have sustained more telomere shortening
than their earlier counterparts. The telomeres of oocytes from
late-exiting oogonia (late-ovulating oocytes) would be expected to
be shorter than those of oocytes from early-exiting oogonia
(early-ovulating oocytes).
[0009] Female germ cells enter meiosis and arrest at the diplotene
stage of prophase I during fetal development and remain arrested at
the germinal vesicle (GV) stage for weeks in mice and years in
humans until puberty, when the meiotic arrest is lifted in part by
gonadotropin stimulation. Pairing and genetic recombination of
homologous chromosomes, unique to meiosis, occurs at
leptotene/zygotene stages early during the first meiotic prophase,
during prenatal life. In fission yeast, plants and mammals,
chromosome pairing during leptotene/zygotense is promoted by a
process of telomere clustering at the nuclear envelope, called
bouquet formation, as chromosomes tethered at their telomeric ends
find their homologous partner based on their similar sizes. Bouquet
formation is thought to be a prerequisite for pairing and
recombination (and therefore chiasmata formation) of homologous
chromosomes before meiotic arrest (Bass, Riera-Lizarazu et al., J
Cell Sci, 113(Pt 6): 1033-42, (2000); de Lange T., Nature
392(6678): 753-4 (1998); Scherthan, Jerratsch et al, Mol Biol Cell
11(12): 4189-203 (2000); Scherthan, Weich et al., J Cell Biol.,
134(5): 1109-25 (1996); Tease C. and Fisher G., Chromosome Res.
6(4): 269-76 (1998)). With increasing maternal age in women,
meiotic chromosomes increasingly missegregate in human females,
leading to aneuploidy, failed implantation, miscarriage, and
increased rates of aneuploid offspring (Hassold, T., M. Abruzzo, et
al,. Environ. Mol. Mutagen. 28(3): 167-75 (1996)). Experiments in
mice indicate that checkpoints for meiotic chromosome behavior at
metaphase-to anaphase transition are less efficient in females
compared to males (Hunt, P., R. LeMaire, et al., Hum Mol Genet
4(11): 2007-12 (1995); LeMaire-Adkins, R., K. Radke, et al., J.
Cell. Biol. 139(7): 1611-9 (1997)).
[0010] TR.sup.-/- mice, which are deficient for the telomerase RNA
and lack telomerase activity, show progressive telomere shortening
with increasing mouse generations, eventually resulting in
telomere-exhausted chromosomes and chromosomal end-to-end fusions
(Blasco et al. (1997) Cell, 91:25-34; Gonzalez-Suarez et al (2000)
Nat. Genetic. 26:114-117; Herrera et al. (1999a) EMBO 18:1172-1181;
Herrera et al. (1999b) EMBO, 18:2950-2960; Lee et al. (1998)
Nature, 392:569-574; Rudolph et al. (1999) Cell, 96:701-712).
Telomerase deficiency in TR.sup.-/- mice leads to the disruption of
functional meiotic spindles and the misalignment of chromosomes
during meiotic division of oocytes in late generation mice. In
early generations, however, oocytes from TR.sup.-/- mice show no
appreciable telomere dysfunction, and exhibit normal chromosome
alignment during metaphase (Liu et al. (2002) Biol. Reprod.
64:204-210). Telomere dysfunction in late generation TR.sup.-/-
mice leads to various pathologies including defects in development,
growth, and immune function, as well as influences tumorigenesis.
Female fertility also decreases with increasing TR.sup.-/- mouse
generations, as evidenced by reduction in litter size, and
compromised embryo development, eventually resulting in sterility
(Herrera et al., 1999b; Lee et al., 1998).
[0011] Oocyte dysfunction is a major source of infertility and
failed treatment in infertile women, even when the egg and embryo
morphology appear normal. Chromosomal abnormalities are the leading
cause of oocyte dysfunction in aging women. Aneuploidy (trisomy and
monosomy), or the aberrant segregation of chromosomes during
meiosis, is the most commonly identified chromosomal abnormality in
humans, observed in at least 35% of first trimester miscarriages,
4% of stillbirths and 0.3% of live-borns (Hassold, et al. (2001)
Nat Rev Genet. 2(4):280-91). Recent studies in pre-implantation
embryos employing more sensitive technology suggest the presence of
even higher rates (up to 80%) of aneuploidies in human eggs (Munne
et al. (1999) Hum. Reprod. 14(9):2191-9; Volarcik et al. (1998) Hum
Reprod. 13(1): 154-160). Even some young women with multiple failed
attempts at in vitro fertilization (IVF) exhibit high rates of
aneuploidy in their oocytes and embryos. The clinical consequences
of aneuploidy can be catastrophic to both mother and fetus and any
attempt to prevent such an occurrence would have profound clinical
impact.
[0012] Chromosomal aneuploidy is associated with a large number of
genetic disorders that could be prevented or prepared for by
appropriate diagnosis, e.g., Verp et al. (1990) Chap. 7, in Filkins
and Russo, Eds., Human Prenatal Diagnosis. Such disorders include
Down's syndrome associated with chromosome 21 trisomy, Edward's
syndrome associated with chromosome 18 trisomy, Plateau's syndrome
associated with chromosome 13 trisomy, Tumer's syndrome associated
with an absence of an X chromosome (XO), Kleinfelter's syndrome
associated with an extra X chromosome (XXY), XYY syndrome, triple X
syndrome, and the like.
[0013] Some in vitro fertilization (IVF) centers have begun to
apply multi probe fluorescent in situ hybridization (FISH) to
screen oocytes and embryos for aneuploidy. The application of multi
probe FISH analysis for use in human IVF is limited, however,
because of the small number of chromosomes which can be studied in
a single cell at one time, the diversity of chromosomes susceptible
to aneuploidy, and the high rate of mosaics in human
pre-implantation embryos.
[0014] There is a need for a reliable assay to examine an oocyte's
(and the resulting embryo's) predisposition to reproductive failure
and/or aneuploidy. Clinical assays that can predict oocyte and
embryo developmental potential are needed to help women decide
whether to continue infertility treatments which depend on their
own eggs or desist and pursue alternatives, such as egg donation or
adoption. Such assays would also stave the rising epidemic of
multiple gestations associated with assisted reproductive
technologies by allowing the transfer of only one or two of the
most developmentally competent embryos after IVF. Finally, reliable
assays that could predict oocyte and embryo developmental potential
would help prevent the creation of babies with debilitating
aneuploidies, such as Down's Syndrome.
SUMMARY OF THE INVENTION
[0015] The invention is based in part on the application of the
quantitative fluorescent in situ hybridization (Q-FISH) assay to
measure telomere length in oocytes and polar bodies from women as a
means to predict risk of reproductive failure and aneuploidy in
embryos and/or offspring, as well as to predict meitotic spindle
morphology in oocytes and polar bodies. Meitotic spindle imaging in
oocytes is described in published PCT international application WO
02/00013. It has been found that telomere shortening in animals
induces not only cytogenic abnormalities but also cell cycle arrest
and apoptosis. Aneuploidy can result in cell death and
developmental arrest in pre-implantation embryos, as well as a
consequent risk of miscarriage and risk of fertility and aneuploidy
in any resulting offspring. Accordingly, telomere length can be
easily used as an indicator of reproductive failure and/or
aneuploidy.
[0016] Thus, in one aspect the invention provides a method for
determining the risk of reproductive failure in a cell comprising:
[0017] obtaining at least one chromosome from the cell; [0018]
measuring telomere length of the chromosome; and [0019] comparing
the measured length of the telomere to the standardized average
length of a control telomere; to thereby determine the risk of
reproductive failure in the cell.
[0020] In a related aspect, the invention provides a method for
determining the risk of reproductive failure in an oocyte
comprising: [0021] obtaining at least one chromosome from at least
one oocyte in a population of oocytes representative of said
oocyte; [0022] measuring telomere length of the chromosome; and
[0023] comparing the measured length of the telomere to the
standardized average length of a control telomere; to thereby
determine the risk of reproductive failure in the oocyte.
[0024] In another related aspect, the invention provides a method
for determining the risk of reproductive failure in a subject
comprising: [0025] obtaining from said subject at least one
chromosome from at least one oocyte in a population of oocytes
representative of said oocyte; [0026] measuring telomere length of
the chromosome; and [0027] comparing the measured length of the
telomere to the standardized average length of a control telomere;
to thereby determine the subject's risk of reproductive
failure.
[0028] In yet another related aspect, the invention provides a
method for determining the risk of reproductive failure in an
oocyte comprising:
[0029] obtaining at least one chromosome from at least one oocyte
in a population of oocytes representative of said oocyte; [0030]
hybridizing telomere-specific probes to said chromosome; [0031]
performing quantitative fluorescent in situ hybridization (Q-FISH)
analysis; [0032] measuring telomere length of the chromosome; and
[0033] comparing the measured length of the telomere to the
standardized average length of a control telomere; to thereby
determine the risk of reproductive failure in the oocyte.
[0034] The invention also provides a method for determining the
predisposition of an oocyte to reproductive failure comprising:
[0035] obtaining at least one chromosome from the oocyte; [0036]
measuring telomere length of the chromosome; and [0037] comparing
the measured length of the telomere to the standardized average
length of a control telomere;
[0038] to thereby determine the predisposition of the oocyte to
reproductive failure.
[0039] In another aspect, the invention features a method for
selecting a fertilized oocyte with a low risk of reproductive
failure for in vitro fertilization, comprising: [0040] obtaining at
least one chromosome from the polar body of the fertilized oocyte;
[0041] measuring telomere length of the chromosome; and [0042]
comparing the measured length of the telomere to the standardized
average length of a control telomere; to thereby select a
fertilized oocyte with a low risk of reproductive failure for in
vitro fertilization.
[0043] In a related aspect, the invention provides a method of in
vitro fertilization comprising: [0044] selecting a fertilized
oocyte by obtaining at least one chromosome from the polar body of
the fertilized oocyte; [0045] measuring telomere length of the
chromosome; [0046] comparing the measured length of the telomere to
the standardized average length of a control telomere; and [0047]
implanting the selected fertilized oocyte in the subject.
[0048] In yet another related aspect, the invention provides a
method for optimizing the viability of an embryo comprising: [0049]
selecting a fertilized oocyte by obtaining at least one chromosome
from the polar body of the fertilized oocyte; [0050] measuring
telomere length of the chromosome; [0051] comparing the measured
length of the telomere to the standardized average length of a
control telomere; and [0052] implanting the selected fertilized
oocyte in the subject.
[0053] The invention also features a method for determining the
risk of aneuploidy in a cell comprising: [0054] obtaining at least
one chromosome from the cell; [0055] measuring telomere length of
the chromosome; and [0056] comparing the measured length of the
telomere to the standardized average length of a control telomere;
to thereby determine the risk of aneuploidy in the cell.
[0057] In one aspect, a method for determining the risk of
aneuploidy in a cell is provided, comprising: [0058] obtaining at
least one chromosome from at least one cell in a population of
cells representative of said cell; [0059] measuring telomere length
of the chromosome; and [0060] comparing the measured length of the
telomere to the standardized average length of a control telomere;
to thereby determine the risk of aneuploidy in the cell.
[0061] In a related aspect, the invention provides a method for
determining the risk of aneuploidy in an oocyte comprising: [0062]
obtaining at least one chromosome from at least one oocyte in a
population of oocytes representative of said oocyte; [0063]
measuring telomere length of the chromosome; and [0064] comparing
the measured length of the telomere to the standardized average
length of a control telomere; to thereby determine the risk of
aneuploidy in the cell.
[0065] In yet another related aspect, the invention provides a
method for determining the risk of aneuploidy in an oocyte
comprising: [0066] obtaining at least one chromosome from at least
one oocyte in a population of oocytes representative of said
oocyte; [0067] hybridizing telomere-specific probes to said
chromosome; [0068] performing quantitative fluorescent in situ
hybridization (Q-FISH) analysis; [0069] measuring telomere length
of the chromosome; and [0070] comparing the measured length of the
telomere to the standardized average length of a control telomere;
to thereby determine the risk of aneuploidy in the cell.
[0071] The invention also features a method for selecting a
fertilized oocyte with a low risk of aneuploidy for in vitro
fertilization, comprising: [0072] obtaining at least one chromosome
from the polar body of the fertilized oocyte; [0073] hybridizing
telomere-specific probes to said chromosome; [0074] performing
quantitative fluorescent in situ hybridization (Q-FISH) analysis;
[0075] measuring telomere length of the chromosome; and [0076]
comparing the measured length of the telomere to the standardized
average length of a control telomere; to thereby select a cell with
a low risk of aneuploidy.
[0077] A related aspect of the invention is a method for
determining the predisposition of an oocyte to aneuploidy
comprising: [0078] obtaining at least one chromosome from the
oocyte; [0079] measuring telomere length of the chromosome; and
[0080] comparing the measured length of the telomere to the
standardized average length of a control telomere; to thereby
optimize the viability of the embryo.
[0081] The invention also features a method of pre-implantation
genetic testing to identify an oocyte with a predisposition to
aneuploidy comprising: [0082] obtaining at least one chromosome
from the oocyte; [0083] measuring telomere length of the
chromosome; and [0084] comparing the measured length of the
telomere to the standardized average length of a control
telomere.
[0085] In yet another aspect, the invention provides a kit for
determining the risk of reproductive failure and/or aneuploidy in a
cell comprising reagents for preparing a chromosomal spread from
the cell or at least one cell in a population of cells
representative of said cell; labeled telomere-specific repeat
probes; reagents for performing quantitative fluorescent in situ
hybridization (Q-FISH) analysis on the chromosomal spread; and
instructions for measuring the length of a telomere obtained from
the chromosomal spread, or obtained from a chromosome of said cell,
and comparing the measured length of the telomere to the
standardized average length of a control.
BRIEF DESCRIPTION OF THE DRAWINGS
[0086] FIG. 1 is a graph showing telomeric lengths from subjects at
risk for reproductive failure. Eggs from unsuccessful cycles have
shorter telomeres
[0087] FIG. 2 is a plot showing telomere lengths were highly
correlated between eggs and polar bodies.
DETAILED-DESCRIPTION OF THE INVENTION
Definitions
[0088] Before further description of the invention, certain terms
employed in the specification, examples and appended claims are,
for convenience, collected here.
[0089] The term "aneuploidy" is intended to mean a condition
wherein a cell contains an abnormal number of chromosomes. The term
also encompasses the condition wherein individual genes are present
in abnormal quantity, or wherein fragments of individual genes are
present in abnormal quantity. An abnormal number of chromosomes is
a number greater than or less than the normal diploid number. In
humans, aneuploidy is defined as any deviation from the normal
human diploid number of 46 chromosomes.
[0090] The term "cell" is intended to include any eukaryotic cell,
such as a mammalian somatic or germ line cell. The cell of the
invention contains DNA, which includes telomeres. In one
embodiment, the cell is obtained from a human. In another
embodiment, the cell is an oocyte. In yet another embodiment of the
invention, a cell is from a population of cells and is
representative of the cells in that population. In still another
embodiment, the cell of the invention is a somatic cell.
[0091] The term "chromosome" is intended to encompass a long
structure composed of DNA and associated proteins. Chromosomes
consist of nucleic acids. Each animal species has a defined number
of chromosomes found in individual cells, referred to as the
diploid number. The diploid number refers to the two copies of each
homologous chromosome. Egg and sperm cells contain haploid numbers
of chromosomes, or one copy of each homologous chromosome. The
diploid number is restored at fertilization with the union of the
sperm and egg. The term "chromosomal material" is intended to
include any number of chromosomes, or portion thereof, taken from
an organism. In one embodiment of the invention, chromosomes are
from an oocyte. In another embodiment, chromosomes of the invention
are from the polar body of a fertilized or unfertilized egg.
[0092] The term "embryo" includes any animal in the early stages of
growth and development following fertilization up to and including
the blastocyst stage. An embryo is characterized as having
totipotent cells that are nondifferentiated. In contrast, somatic
cells of an individual are cells of the body that are
differentiated and not totipotent. A "pre-implantation embryo"
refers to a fertilized oocyte with two pronuclei (up to and
including a blastocyst), which is not yet implanted in the lining
of the female reproductive tract. In general, the pre-implantation
embryo contains between about 2 and about 8 cells, although these
ranges may vary among species. Typically, the quality assessment
for a human or a mouse embryo is performed on an embryo comprising
between 2 and 8 cells (i.e., the embryo is assessed between about
18 and about 24 hours post-fertilization).
[0093] A "nucleic acid molecule" refers to the phosphate ester
polymeric form of ribonucleosides (adenosine, guanosine, uridine or
cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine,
deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"),
or any phosphoester analogs thereof, such as for example,
phosphorothioates and thioesters, in either single stranded form,
or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and
RNA-RNA helices are possible. The term nucleic acid molecule, and
in particular DNA or RNA molecule, refers only to the primary and
secondary structure of the molecule, and does not limit it to any
particular tertiary forms. Thus, this term includes double-stranded
DNA found, inter alia, in linear or circular DNA molecules (e.g.,
restriction fragments), plasmids, and chromosomes. In discussing
the structure of particular double-stranded DNA molecules,
sequences may be described herein according to the normal
convention of giving only the sequence in the 5' to 3' direction
along the nontranscribed strand of DNA (i.e., the strand having a
sequence homologous to the mRNA). A "recombinant DNA molecule" is a
DNA molecule that has undergone a molecular biological
manipulation.
[0094] Homologous nucleic acid sequences, when compared, exhibit
significant sequence identity or similarity. The standards for
homology in nucleic acids are either measures for homology
generally used in the art by sequence comparison or based upon
hybridization conditions. The hybridization conditions are
described in greater detail below.
[0095] As used herein, "substantial homology" in the nucleic acid
sequence comparison context means either that the segments, or
their complementary strands, when compared, are identical when
optimally aligned, with appropriate nucleotide insertions or
deletions, in at least about 50% of the nucleotides, generally at
least 56%, more generally at least 59%, ordinarily at least 62%,
more ordinarily at least 65%, often at least 68%, more often at
least 71%, typically at least 74%, more typically at least 77%,
usually at least 80%, more usually at least about 85%, preferably
at least about 90%, more preferably at least about 95 to 98% or
more, and in particular embodiments, as high at about 99% or more
of the nucleotides. Alternatively, substantial homology exists when
the segments will hybridize under selective hybridization
conditions, to a strand, or its complement, typically using a
sequence or fragment derived from any one of SEQ ID NOS: 1 through
10. Typically, selective hybridization will occur when there is at
least about 55% homology over a stretch of at least about 14
nucleotides, preferably at least about 65%, more preferably at
least about 75%, and most preferably at least about 90%. See
Kanehisa (1984) Nuc. Acids Res. 12:203-213.
[0096] Percent identity and similarity between two sequences
(nucleic acid or polypeptide) can be determined using a
mathematical algorithm (see, e.g., Computational Molecular Biology,
Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., eds., M Stockton Press, New York, 1991).
[0097] The term "fluorescent in situ hybridization" or "FISH," as
used herein interchangeably, is intended to mean a nucleic acid
hybridization technique which employs a fluorophor-labeled probe to
specifically hybridize to, and thereby facilitate visualization of,
a target nucleic acid. In general, in situ hybridization, including
FISH, is useful for determining the distribution of a nucleic acid
in a nucleic acid-containing sample such as is contained in, for
example, tissues at the single cell level. Such techniques have
been used for karyotyping applications, as well as for detecting
the presence, absence and/or arrangement of specific genes
contained in a cell. FISH involves the use of nucleic acid probes
to determine if a particular nucleotide sequence is present in the
chromosomal DNA of particular cells. The terms "staining" or
"painting" are used interchangeably, and include hybridizing a
probe to a chromosome or segment thereof, such that the probe
reliably binds to the targeted chromosomal material therein and the
bound probe is capable of being visualized. Such methods are well
known to those of ordinary skill in the art and are disclosed, for
example, in U.S. Pat. No. 5,225,326; U.S. patent application Ser.
No. 07/668,751; and PCT WO 94/02646, the entire contents of which
are incorporated herein by reference.
[0098] The oligonucleotide probe in FISH is labeled with a
fluorophor (fluorescent "tag" or "label") according to standard
practice. The fluorophor can be directly attached to the probe
(i.e., a covalent bond) or indirectly attached thereto (e.g.,
biotin can be attached to the probe and the fluorophor can be
covalently attached to avidin; the biotin-labeled probe and the
fluorophor-labeled avidin can form a complex which can function as
the fluorophor-labeled probe in the method of the invention).
Fluorophors that can be used in accordance with the method of the
invention are well known to those of ordinary skill in the art.
These include 4,6-diamidino-2-phenylindole (DAPI), fluorescein
isothiocyanate (FITC) and rhodamine (see, for example, U.S. Pat.
No. 4,373,932, for a list of exemplary fluorophors that can be used
in accordance with the methods of the invention). The existence of
fluorophors having different excitation and emission spectrums from
one another permits the simultaneous visualization of more than one
target nucleic acid in a single fixed sample.
[0099] The term "hybridization" includes the process by which two
complementary nucleic acid molecules anneal to one another.
Hybridization is a general technique in which the complementary
strands of deoxyribonucleic acid (hereinafter "DNA") molecules,
ribonucleic acid (hereinafter "RNA") molecules, and combinations of
DNA and RNA are separated into single strands and then allowed to
renature or reanneal and reform base-paired double helices. In a
preferred embodiment of the invention, in situ hybridization is
used which makes possible the detection and localization of
specific nucleic acid sequences directly within an intact cell or
tissue without any extraction of nucleic acids whatsoever.
[0100] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences that are significantly
identical or homologous to each other remain hybridized to each
other. Preferably, the conditions are such that sequences at least
about 70%, more preferably at least about 80%, even more preferably
at least about 85% or 90% identical to each other remain hybridized
to each other. Such stringent conditions are known to those skilled
in the art and can be found in Current Protocols in Molecular
Biology, Ausubel et al., eds., John Wiley & Sons, Inc. (1995),
sections 2, 4 and 6. Additional stringent conditions can be found
in Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold
Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), chapters 7, 9
and 11.
[0101] The term "in situ hybridization" is intended to mean a
nucleic acid hybridization technique which employs a labeled probe
to specifically hybridize to and thereby, facilitate visualization
of, a target nucleic acid. In situ hybridization is performed by
denaturing the target nucleic acid so that it is capable of
hybridizing to a complementary probe contained in a hybridization
solution. The fixed sample may be concurrently or sequentially
contacted with the denaturant and the hybridization solution. Thus,
in one embodiment, the fixed sample is contacted with a
hybridization solution which contains the denaturant and at least
one oligonucleotide probe. The probe has a nucleotide sequence at
least substantially complementary to the nucleotide sequence of the
target nucleic acid. Optimization of the hybridization conditions
for achieving hybridization of a particular probe to a particular
target nucleic acid is well within the level of the person of
ordinary skill in the art.
[0102] Briefly, fluorescence in situ hybridization (FISH) first
involves fixing the sample to a solid support and preserving the
structural integrity of the components contained therein by
contacting the sample with a medium containing at least a
precipitating agent and/or a cross-linking agent. Exemplary agents
useful for "fixing" the sample are paraformaldehyde,
methanol:acetic acid, and Bouin's solution. Alternative fixatives
are well known to those of ordinary skill in the art. According to
standard practice for performing FISH, the hybridization solution
optionally contains one or more of a hybrid stabilizing agent, a
buffering agent and a selective membrane pore-forming agent.
Following hybridization and subsequent washing, the signal from the
fluorescently labeled probe is visualized using imaging techniques
well-known to one of ordinary skill in the art.
[0103] As used herein, the term "in vitro fertilization" or "IVF"
is intended to mean fertilization of an egg with sperm outside of a
subject. This procedure is often used as a treatment for
infertility. Briefly, eggs are retrieved from a subject and mixed
with sperm in a culture dish to allow for fertilization. After a
period of incubation, usually two or three days, the newly formed
embryos are transferred back to the subject. Examples of conditions
for which this technique is used include damaged or absent
Fallopian tubes, endometriosis, male factor infertility and
unexplained infertility.
[0104] The term "labeled" is used herein to indicate that there is
some method to visualize the bound probe, whether or not the probe
directly carries some modified constituent. "Label" means a
chemical used to facilitate identification and/or quantitation of a
target substance. Illustrative labels include fluorescent (e.g.,
FITC or rhodamine), phosphorescent, chemiluminescent, enzymatic,
and radioactive labels, as well as chromophores. The term label can
also refer to a "tag" that can bind specifically to a labeled
molecule. For instance, one can use biotin as a tag and then use
avidinylated or streptavidinylated horseradish peroxidase (HRP) to
bind to the tag, and then use a chromogenic substrate (e.g.,
tetramethylbenzamine) to detect and visualize the presence of HRP.
In a similar fashion, the tag can be an epitope or antigen (e.g.,
digoxigenin), and an enzymatically, fluorescently, or radioactively
labeled antibody can be used to bind to the tag for visualization
purposes. In one embodiment of the invention, the probes are
peptide nucleic acid (PNA)-labeled probes, and are labeled
according to manufacturer's protocols (Applied Biosystems, Inc.,
Framingham, Mass.).
[0105] The term "oocyte" includes a female germ cell. Oocytes occur
in both immature and mature states. "Immature" refers to oocytes
that are viable but incapable of fertilization without additional
growth or maturation. "Mature" refers to oocytes that have been
ovulated from the ova of a female and are capable of being
fertilized. Mature also refers to immature oocytes that have been
exposed to appropriate hormones or agents to render them capable of
fertilization by sperm. Oocytes recovered from "unstimulated"
follicles or ovaries are natural oocytes obtained from follicles or
ovaries that were not treated with any gonadotropins or other
hormones or agents to stimulate maturation of the oocytes. Oocytes
recovered from "stimulated" ovaries may be either mature or
immature. Subjective criteria to estimate the viability and
maturity of the ovum can be done microscopically after removal of
the ovum from the follicle, and includes assessing the number and
density of surrounding granulosa cells, the presence or absence of
the germinal vesicle, and/or the presence or absence of the first
polar body. In one embodiment of the invention, the oocyte is
ovulated from a female subject and obtained for use in IVF. In
another embodiment of the invention, the oocyte is immature and is
matured in vitro for use in IVF treatment. "Spare" oocytes are
those obtained from a female subject for use in IVF procedures but
not used for fertilization. Spare oocytes can be obtained from
fertile females who are donating their eggs for IVF. In one
embodiment of the invention, representative oocytes are chosen from
a population of oocytes for the telomere length assay. These
representative oocytes may be, for example, spare oocytes.
[0106] The term "optimizing the viability of the embryo" is
intended to mean maximizing the chance of successful embryonic
development. Successful development of the embryo is characterized
by the embryo being free of congenital defects. In one embodiment
of the invention, the viability of the embryo is optimized by
selecting oocytes which are unlikely to exhibit aneuploidy.
[0107] As used herein, the term "peptide nucleic acid" or "PNA"
means any oligomer, linked polymer or chimeric oligomer, comprising
two or more PNA subunits (residues). The term "peptide nucleic
acid-labeled" or "PNA-labeled," used interchangeably herein, refers
to a probe labeled with a non-naturally occurring polyamide, which
can hybridize to a nucleic acid (DNA or RNA) with sequence
specificity (U.S. Pat. No. 5,539,082; Egholm et al. (1993) Nature
365:566-568). Examples of uses of PNA-labeled probes include the
detection of rRNA in ISH and FISH assays (WO95/32305; WO97/18325),
the analysis and detection of mRNA, the analysis and detection of
viral nucleic acids, and the analysis and detection of centromeric
sequences in human chromosomes and human telomeres (Lansdorp et al.
(1996) Human Mol. Genetics, 5: 685-691; WO 97/14026). A PNA-labeled
probe has also been used to detect human X chromosome specific
sequences (WO 97/18325).
[0108] The term "predisposition to aneuploidy" is intended to mean
that there is a chance that aneuploidy will occur in a cell because
conditions are favorable for chromosomal missegregation. In one
embodiment, embryos that have a predisposition to aneuploidy are
those embryos which develop from an oocyte with telomeres that are
abnormal in length. In another embodiment, embryos with a
predisposition to aneuploidy are those from oocytes that are
produced by females that are likely to produce oocytes with
abnormally short telomeres.
[0109] The terms "predisposition for reproductive failure" or "risk
of reproductive failure" are used interchangeably throughout the
specification. The term are intended to mean that the telomeres in
the oocytes of a female are shorter than the standard size,
resulting in a high probability that the fertilized oocytes will
undergo cell cycle arrest shortly after fertilization. The term
"reproductive failure" indicates a history of recurrent spontaneous
abortion, unexplained infertility or implantation failure following
in vitro fertilization and embryo transfer. The term also includes,
for example, chromosomal abnormalities, depletion of oocytes,
embryonic cell cycle arrest, failure of the embryo to implant, and
apoptosis Telomerase activity is present during early oogenesis,
but is almost absent during late oogenesis and early
preimplantation embryo development until the morula stage of
development. Thus telomere length in oocytes and early embryos is
established during early development. When telomeres reach a
critically short length, the cell undergoes cell cycle arrest and
apoptosis, so late oogenesis and early preimplantation embryo
development may represent a kind of bottleneck for telomere length
during development. In one embodiment, female patients that have a
predisposition for reproductive failure are those female patients
with oocytes containing chromosomes with telomeres that are
abnormal in length. In another embodiment, female patients that
have a predisposition for reproductive failure are identified as
having oocytes with abnormally short telomeres.
[0110] The term "pre-implantation genetic testing" is intended to
mean testing of a certain genetic locus or loci of a cell which is
administered prior to implantation of a fertilized oocyte or
oocytes by IVF procedures. In one embodiment of the invention,
pre-implantation genetic testing is performed on the polar body of
a fertilized or unfertilized oocyte. In another embodiment of the
invention, pre-implantation genetic testing is performed on an
oocyte or oocytes which are representative of a population of
oocytes.
[0111] The term "probe" includes an oligonucleotide designed to
hybridize specifically to a target nucleic acid, wherein the
hybridization of the probe to the target can be detected. The probe
is labeled as described above so that its binding to the target can
be visualized. The probe is produced from a source of the target
nucleic acid sequence, for example, a collection of clones or a
collection of polymerase chain reaction (PCR) products comprising
the target sequence, or portion thereof. Prior to hybridization,
the source nucleic acid may be processed in some way, for example,
by removal of repetitive sequences or blocking them with unlabeled
nucleic add with complementary sequence, so that hybridization with
the resulting probe produces staining of sufficient contrast on the
target. Telomere-specific staining of the current invention is
accomplished by using nucleic acid probes that hybridize to
sequences specific to a telomere target. An example of a telomeric
probe of the invention is a FITC-labeled peptide nucleic acid (PNA)
probe (Applied Biosystems, Framingham, Mass.) comprising the
sequence CCCTAACCCTAACCCTAA (SEQ ID NO: 1). Other examples of
probes that identify telomeres include any one of SEQ ID NOS: 2
through 10.
[0112] The term "quantitative fluorescent in situ hybridization" or
"Q-FISH" is intended to mean the process by which results from the
FISH assay are quantified. In one embodiment of the invention,
results from the FISH analysis are analyzed using digital
fluorescent microscopes to measure the fluorescent signal from
probes directed to telomeric DNA sequences. This analysis is then
used to quantify the relative length of a telomeres on a chromosome
from a cell. The fluorescent signal from the hybridized probe is
indicative of the telomere length of the chromosome. For example,
the absence of signal would indicate extremely short or absent
telomeres on the chromosomes. A low signal would indicate shortened
telomeres, while a medium range signal would indicate an average
length telomere. Finally, an intense, strong signal would indicate
extra long telomeres relative to the average length.
[0113] The term "standardized average length" is intended to mean
an average telomere length which is used to determine whether the
telomere length of a cell of interest is abnormal. The standardized
average length of a cell is determined by measuring the telomere
length of a control cell or taking the average telomere length of a
population of control cells. In one embodiment of the invention,
oocytes which are known to have fertility are used to determine the
standardized average length. For example, one or more oocytes
obtained from a woman who has demonstrated fertility and who is
known to have oocytes which are not predisposed to aneuploidy, can
be used to determine the standardized average length of oocytes. In
another example, spare eggs and polar bodies obtained from young
egg donors, whose eggs generate pregnancies, can be used to
determine the standardized average length.
[0114] The term "telomere" is intended to mean the modified end of
an eukaryotic chromosome which contains repeated sequences of DNA.
In humans, telomeres are composed of many kilobases of simple
tandem 5'-TTAGGG repeats (Moyzis et al. (1988) Proc. Natl. Acad.
Sci. U.S.A. 85:6622). During DNA synthesis, the termini of the
chromosomes are not fully replicated (Watson (1972) Nature New
Biology 239:197,) by the action of DNA polymerase. Incomplete
replication occurs at the 3' end of each of the two template
strands of the chromosome, because the RNA primer needed to
initiate synthesis in effect masks the 3' end of the template. The
RNA primer is degraded after strand synthesis, and, as there are no
additional sequences beyond the 3' end of the template to which
primers can anneal, the portion of the template to which the RNA
primer hybridized is not replicated. In the absence of other
enzymes, the chromosome is thus shortened with every cell
division.
[0115] The terms "telomeric DNA" or "telomeric region," used herein
interchangeably, are intended to mean chromosomal DNA located on
the ends of a chromosome. Telomeres consist of a tandem repeat
array of a short sequence, which may be desired for experimental
manipulation including, e.g., contacting with a probe or primer.
For convenience, human telomeric region and human telomeric repeat
sequences are typically referred to herein for illustrative
purposes. This illustrative use is not intended to limit the
invention, and those of skill in the art will recognize that the
present methods can be used to measure telomere length of telomeres
from any organism.
[0116] The term "telomere length" is intended to mean the
approximate physical measurement of the length of all telomeric
repeat sequences at the end of a chromosome relative to a
standardized control. The mean telomere length of a population of
cells can provide a standardized average length by which to
determine if a telomere is long or short. An abnormal telomere
length, as defined herein, is a telomere length which is greater
than or less than the standardized average length of a
telomere.
[0117] The term "telomere length assay" is intended to mean a
method by which the ends of chromosomes or telomeres of a cell are
measured. Measurement of telomere length in cells can predict risk
of aneuploidy. The method of the invention uses a telomere length
assay to predict the risk of aneuploidy in oocytes in order to
minimize the chance of fertilizing oocytes which may give rise to
embryos which will be predisposed to cell cycle arrest and embryo
death. For example, telomere length of chromosomes from polar
bodies (which contain mirror images of chromosomes still in the
oocyte) and/or from spare oocytes, provides an estimate of the risk
of aneuploidy in resulting embryos. Likewise, the method of the
invention uses a telomere length assay to select oocytes/fertilized
oocytes with a low risk of aneuploidy, thereby increasing the
likelihood of successful implantation and normal offspring.
[0118] In one embodiment of the invention, the telomere length
assay is performed using telomeres from chromosomal spreads from
unfertilized oocytes and/or polar bodies from fertilized or
unfertilized oocytes. In one embodiment of the invention, telomere
length is determined using Q-FISH analysis, wherein PNA-labeled
telomere repeat probes are hybridized to telomeric DNA in a cell
and analyzed by quantitative FISH using digital microscopy and
integrated optical density of the fluorescence signal. Digital
imaging technology is readily available in any molecular biology
laboratory and most IVF centers.
[0119] The term "telomere repeats" is intended to mean tandem
repeats of a specific nucleotide sequence found within the
telomeres at the end of chromosomes. In humans and other
vertebrates, the telomere repeats are commonly tandem repeats of
the sequence TTAGGG. The number of these tandem repeat sequences,
present at the ends of a chromosome, determine the telomere length
of a chromosome.
[0120] The invention provides methods for determining the risk of
reproductive failure and/or aneuploidy in a cell comprising
obtaining at least one chromosome from the cell and measuring the
telomere length of the chromosome to thereby determine the risk of
aneuploidy in the cell. In accordance with the invention, cells
include an oocyte, an oocyte representative of a population of
oocytes, a polar body form a fertilized oocyte or a polar body from
an unfertilized oocyte.
[0121] The telomere is preferably detected by a labeled
telomere-specific probe which is hybridized to the chromosome prior
to measuring telomere length of the chromosome. Preferably, the
probe is hybridized to telomere repeats. In one embodiment, the
probe is peptide nucleic acid (PNA)-labeled, preferably
FITC-labeled (CCCTAA).sub.3 (SEQ ID NO: 10).
[0122] In accordance with the invention, the telomere is
advantageously measured using quantitative fluorescent in situ
hybridization (Q-FISH) analysis, although any other measuring
methods know in the art may be used.
[0123] In one embodiment of the invention, the oocyte selected is
representative of a population of oocytes and a probe is obtained
for hybridizing to the chromosome of the oocyte. In one embodiment
of the invention, the probe is a labeled telomere-specific probe
comprising a nucleic acid sequence identified by any one of SEQ ID
NOS: 1-10. In other embodiments, the nucleic acid sequence
comprises a sequence having at least about 80 percent sequence
identity to any one of SEQ ID. NOS. 1 through 10; more preferably,
the nucleic acid sequence comprises a sequence having at least
about 90 percent sequence identity to any one of SEQ ID. NOS. 1
through 10; still more preferably the nucleic acid sequence
comprises a sequence having at least about 100 percent sequence
identity to any one of SEQ ID. NOS. 1 through 10.
[0124] In general, the invention provides assays to determine the
predisposition of cells, for example, an oocyte to reproductive
failure and/or aneuploidy in order to optimize the implantation and
viability of the embryo. In another embodiment, the invention
provides a method of pre-implantation genetic testing for screening
oocytes with a predisposition to aneuploidy. Oocytes which have a
predisposition to aneuploidy are more likely to give rise to
embryos with developmental defects. The oocytes are screened for
use in in vitro fertilization. To optimize the overall success of
in vitro fertilization and to maximize the chance of a normal
embryo, oocytes with abnormally long or short telomeres are not
fertilized and/or implanted in the patient undergoing IVF
treatment. In one embodiment, telomere lengths are determined by
Q-FISH analysis, using PNA-labeled probe, quantitative digital
microscopy, and integrated optical density of the fluorescence
signal.
[0125] In particular, the invention features a method for
distinguishing oocytes which have a predisposition to reproductive
failure and/or aneuploidy in order to optimize the viability of an
embryo for in vitro fertilization treatment. In order to assay
oocytes for their predisposition to aneuploidy, chromosomes are
obtained from the oocytes and/or polar bodies to analyze the length
of their telomeres. In one embodiment of the invention, an oocyte
is selected for the telomere length assay from a population of
oocytes. The telomere length of the selected oocyte is
representative of the population of oocytes. The representative
oocyte can be a spare oocyte which is not intended for use in IVF.
In another embodiment, a polar body from a fertilized or
unfertilized oocyte is obtained, and its chromosomes are used in
the telomere length assay of the invention. Polar bodies contain
mirror images of chromosomes from the oocyte from which they are
obtained. Polar bodies and oocytes are obtained through techniques
known to those of ordinary skill in the art of in vitro
fertilization procedures.
[0126] Based on the teachings of the invention, telomeric DNA from
oocytes and/or polar bodies can be used to predict the
developmental potential of an embryo based on the correlation
between telomere length and a predisposition of the oocyte or polar
body to aneuploidy. Standard molecular biology techniques known to
one of ordinary skill in the art can be used to determine the
length of a telomere, including, for example, quantitative
polymerase chain reaction (PCR) (Cawthon (2002) Nucleic Acids
Research, 30:1-6). In situ techniques can also be used to measure
telomere length, including, for example, primed in situ labeling
(PRINS) and fluorescent in situ hybridization (FISH) (Therkelsen,
et al. (1995) Cytogenet. Cell Genetic. 68:115-118; Pinkel et al.
(1986) PNAS USA, 83:2934-2938). To better quantify telomere length
using FISH, quantitative FISH (Q-FISH) can be performed according
to standard protocols, and those described herein. Q-FISH analysis
is enhanced through the use of PNA-labeled probes, which are
commercially available (see DAKO (Denmark), Applied Biosystems
(Framingham, Mass.)).
[0127] In another embodiment, a female's predisposition to
reproductive failure is determined by the length of the telomeric
ends of a chromosome. An oocyte is selected for the telomere length
assay from a population of oocytes. The telomere length of the
selected oocyte is representative of the population of oocytes. The
representative oocyte can be a spare oocyte which is not intended
for use in IVF. In another embodiment, a polar body from a
fertilized or unfertilized oocyte is obtained, and its chromosomes
are used in the telomere length assay of the invention.
[0128] In one embodiment, telomere length is measured using Q-FISH
analysis, which is described below. First, chromosomes are obtained
from a cell or cells of interest, and are morphologically
preserved. The chromosome sample, or chromosome spread, is prepared
according to standard techniques such that individual chromosomes
remain substantially intact and typically comprise metaphase
spreads or interphase nuclei. Cells described in the methods of the
invention include polar bodies and oocytes. Cells can be
morphologically preserved by fixation using conventional methods
(see for example, A. G. Everson Pearce (1980) Histochemistry
Theoretical Applied, 4th Ed., Churchill Livingstone, Edinburgh, for
details relating to the general techniques for preparing and fixing
tissue).
[0129] The cells of the invention can be fixed on a support, such
as slides or filters. For example, oocytes can be fixed by spinning
small volumes of cells onto slides. Chromosomal preparations or the
chromosome spread can also be pre-treated on the support.
Pretreatments include, but are not limited to, RNAse treatment to
remove endogenous RNA, protease treatment to increase the
accessibility by digesting protein surrounding the telomeres, and
detergent treatment when it is suspected that lipid membrane
components have not been extracted by other procedures.
[0130] The chromosome spread is then treated with a
telomere-specific probe. The probe is a nucleic acid, or analog
thereof, which is capable of hybridizing to telomeric DNA. Nucleic
acid analogs differ from natural DNA in that they do not have a
deoxyribose or ribose backbone. In one embodiment of the invention,
probes are constructed from the nucleic acid analog called peptide
nucleic acid (PNA) which contains a polyamide backbone (described
in WO 92/2070). PNA probes have been used in FISH to study
telomeres in hematopoietic cells (Lansdorp et al. (1996)), as well
as in multicolor FISH experiments detecting telomere sequences
(Taneja et al. (2001) Genes, Chrom., and Cancer 30:57-63). Other
examples of analogs which can be used to make probes include
analogs having cyclic backbone moieties comprising furan or
morpholine rings, or acyclic backbone moieties (WO 86/05518).
Determination of whether a probe hybridizes to a telomeric sequence
can be accomplished by hybridizing the probe to a nucleic acid
molecule comprising multiple copies of the telomeric repeat
sequences using the hybridization media and conditions described
herein.
[0131] In one embodiment of the invention, the probe for detecting
and quantitating the length of a telomere in a chromosome is a
PNA-labeled probe having the following sequence: CCCTAACCCTAACCCTAA
(SEQ ID NO: 1). In another embodiment, the probe for measuring the
length of a telomere contains the repeat sequence TTAGGG (SEQ ID
NO: 2), CCCTAA (SEQ ID NO: 3), CCCCAA (SEQ ID NO: 4), CCCCAAAA (SEQ
ID NO: 5), CCCACA (SEQ ID NO: 6), CCCTAAA (SEQ ID NO: 7), CCCCT
(SEQ ID NO: 8), CCCTAA (SEQ ID NO: 9), or FITC-labeled
(CCCTAA).sub.3 (SEQ ID NO: 10).
[0132] In another embodiment of the invention, the telomere
specific probe comprises a nucleic acid sequence having at least
about 80 percent sequence identity to any one of SEQ ID. NOS. 1
through 10; more preferably, at least about 90 percent sequence
identity to any one of SEQ ID. NOS. 1 through 10; still more
preferably, at least about 100 percent sequence identity to any one
of SEQ ID. NOS. 1 through 10.
[0133] To determine the percent identity of two nucleic acid
sequences, the sequences are aligned for optimal comparison
purposes (e.g., gaps can be introduced in one or both of a first
and a second amino acid or nucleic acid sequence for optimal
alignment and non-homologous sequences can be disregarded for
comparison purposes). In a preferred embodiment, the length of a
reference sequence aligned for comparison purposes is at least 30%,
preferably at least 40%, more preferably at least 50%, even more
preferably at least 60%, and even more preferably at least 70%,
80%, or 90% of the length of the reference sequence. The
nucleotides at corresponding nucleotide positions are then
compared. When a position in the first sequence is occupied by the
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position (as used herein
nucleic acid "identity" is equivalent to nucleic acid "homology").
The percent identity between the two sequences is a function of the
number of identical positions shared by the sequences, taking into
account the number of gaps, and the length of each gap, which need
to be introduced for optimal alignment of the two sequences.
[0134] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In a preferred embodiment, the percent
identity between two nucleotide sequences is determined using the
GAP program in the GCG software package (available at online
through the Genetics Computer Group), using a NWSgapdna.CMP matrix
and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1,
2, 3, 4, 5, or 6. A preferred, non-limiting example of parameters
to be used in conjunction with the GAP program include a Blosum 62
scoring matrix with a gap penalty of 12, a gap extend penalty of 4,
and a frameshift gap penalty of 5.
[0135] In another embodiment, the percent identity between two
amino acid or nucleotide sequences is determined using the
algorithm of Meyers and Miller (Comput. Appl. Biosci. 4:11-17
(1988)) which has been incorporated into the ALIGN program (version
2.0 or version 2.0U), using a PAM120 weight residue table, a gap
length penalty of 12 and a gap penalty of 4.
[0136] The probe used in the method of the invention is preferably
labeled with one or more detectable substances. In a preferred
embodiment of the invention, the probe of the invention is labeled
with a detectable substance so that formed hybrids can be
visualized microscopically after the in situ hybridization
procedure. Visualization can be achieved either directly or
indirectly depending on the nature of the label used for the probe.
Detectable substances which can be used in direct visualization
methods of the probe include, but are not limited to, fluorophores,
isotopes, and chemiluminescent compounds. Examples of isotopes
include iodine I.sup.125, I.sup.131 or tritium. Examples of
fluorophores which can be used to label the probe of the invention
include fluorescein-isothiocyanate (FITC), tetramethyl rhodamine
isothiocyanate (TRITC), amino-methyl coumarin acetic acid (AMCA)
(Molecular Probes, Eugene, Oreg.), Texas Red (Molecular Probes,
Eugene, Oreg.), and carboxymethylindocyano dyes such as Cy2, Cy3,
or Cy5 (Biological Detection Systems, Pittsburgh, Pa.). An example
of a chemiluminescent material is luminol. The probe of the
invention can be labeled with any of these detectable substances
using methods conventionally known in the art.
[0137] The probe can also be labeled with a detectable substance
which is detected in an indirect method. The probe can be labeled
with a detectable substance so that formed hybrids are visualized
after reacting with an element (e.g., a substrate, an antibody,
etc.) which results in a detectable signal. Detectable substances
which may be used in indirect methods include enzymes (e.g.,
horseradish peroxidase, alkaline phophatase, .beta.-galactosidase,
or acetylcholinesterase) and haptens (e.g., biotin, digoxigenin).
For example, if biotin is used as the detectable substance, in situ
hybridized sequences are detected using (strept)avidin or
anti-biotin antibodies. The probe may be labeled with enzymes and
haptens using conventional methods known in the art, for example,
in Pattersen et al. (1993) Science, 260:976-979.
[0138] The probe of the invention can also be labeled with more
than one detectable substance. For example, the probe may be
labeled with two or more fluorophores. The probe may also be
labeled with substances detectable for both direct and indirect
detection methods. The method of the invention can also be
practiced using differently labeled probes. For example, the method
may use three probes having the same nucleic acid sequence but each
labeled with a different detectable substance, such as three
different fluorophores.
[0139] In another embodiment, the method of the invention is
performed using a probe and a counterstain. A counterstain can be
used to visualize another feature of the cell (e.g., nucleic acids,
cell walls, nuclei, etc.), and includes, for example, DAPI and
Hoechst stains. In accordance with the invention, a counterstain
can be used to visualize the complete chromosome to compare with
the visual signal from the telomere-specific probe.
[0140] The detectable substance of the probe may provide a
colorimetric, photometric, radiometric, etc. signal which can be
detected by a wide variety of means. For example, the method of the
invention could employ a suitable detectable device capable of
detecting a change in absorption density, a change in fluorescence,
or radioactive emission, or a shift in the absorbance of a
characteristic .lamda..sub.max, for example, the .lamda..sub.max of
the detectable molecule, as detailed in the Examples which
follow.
[0141] In a one embodiment of the invention, the probe is labeled
with a fluorophore such as fluorescein-isothiocyanate (FITC),
tetramethyl rhodamine isothiocyanate (TRITC), amino-methyl coumarin
acetic acid (AMCA) (Molecular Probes, Eugene, Oreg.), Texas Red
(Molecular Probes, Eugene, Oreg.), or carboxylmethylindocyano dyes
such as Cy2, Cy3, or Cy5 (Biological Detection Systems, Pittsburgh,
Pa.). Images of fluorophore labeled probes hybridized to target
telomeric nucleic acid sequences can be created using conventional
techniques known in the art (see also Nederlof, et al (1992)
Cytometry 13:839-845). In a preferred embodiment, the images are
formed electronically, and most preferably are digital (discussed
further below).
[0142] The probe is hybridized to telomeric DNA by incorporating
the probe into an appropriate hybridization medium. Generally,
hybridization medium has a low ionic strength and typically
contains a buffer, denaturing agent and a blocking reagent.
Examples of buffers include TRIS and HEPES. Examples of suitable
denaturing agents include formamide and DMSO. A blocking reagent is
any reagent which substantially blocks non-specific binding of the
probe.
[0143] The hybridization medium comprising the telomeric-specific
probe is applied to the chromosomal DNA. The hybridization probe
and the chromosomal DNA are denatured simultaneously by heat or pH
treatment. The probe and telomeric DNA are hybridized under
stringent conditions.
[0144] Following the in situ hybridization process, the image of
the hybridized to a detectable probe is captured using imagery
equipment standard in a molecular biology or IVF laboratory. In
brief, instrumentation needed to create a digital image consists of
a microscope system, a detector for collection of images, and
computer hardware and software for image analysis.
[0145] In one embodiment of the invention, fluorescence microscopy
is used to quantitatively analyze the FISH results. For
fluorescence microscopy, objective lenses are generally used with
high magnification and high numerical aperture, since these
characteristics determine the spatial resolution and light
collecting properties. For example, a Leitz Dialux epifluorescence
microscope equipped with a 100 W mercury-arc lamp and a Neofluor
1,40 NA oil objective can be used to measure fluorescence, and the
filter used for selection of the fluorophore may be as follows:
PL450-SP490 (excitation filter), DM510 (dichroic mirror), and
LP515-SP560 (emission filters). A detector for imaging the FISH
results should be selected so that it has sufficient sensitivity to
detect the fluorescing signals obtained by FISH, for example,
detection of fluorescing signals that can be differentiated from
background fluorescence, as detailed in the Examples which follow.
Ideally the detector also provides a linear response to a wide
range of wavelengths, has a high signal to noise ratio, and has
wide dynamic range and little geometric aberrations.
[0146] For Q-FISH analysis, the intensity of a spot may be
calculated as the integrated intensity of all pixels within the
area of the spot corrected for the background. The length of the
telomere is quantitated from the specific fluorescent intensity of
all the pixels within the area of the spot (telomeric region)
corrected for the background. Methods for determining telomere
length quantitatively by analyzing FISH results are described in
Zijlmans et al. (1997) PNAS 94:7423-7428. Digital images are
recorded and a computer program is used for image analysis.
[0147] In general, computer image analysis is used to optimize
signal to noise ratio of telomere fluorescence from human oocytes
and polar bodies. Telomeres can be imaged using a number of
commercially available computer image analysis programs. An example
of a commercially available analysis program is the MetaMorph.RTM.
Imaging System program (Universal Imaging Corporation (Downingtown,
Pa.). Software, which can be programmed to integrate intensity and
area of telomere fluorescence, is also available in the public
domain; e.g., NIH provides image analysis software at no cost. It
will be appreciated that macro programs can also be written for
commercially available software, which will further optimize the
signal to noise ratio of telomere fluorescence from human oocytes
and polar bodies.
[0148] Calibration of the Q-FISH technique for telomere length is
performed using known telomere lengths, including, for example,
genetically engineered artificial telomeres. Genetically engineered
artificial telomeres can be generated based on the determined
standardized average length. In another example, chromosome spreads
from mouse oocytes from strains known to have long and/or short
telomeres can also be used to calibrate telomere length. Examples
of mouse strains known to have abnormal length telomeres include
the TR.sup.-/- strain (Blasco, et al. (1997) Cell 91:25-34) and the
ATM.sup.-/- strain (Hande et al. (2001) Hum. Mol. Genet.
10:519-528).
[0149] Telomeres are quantified as being average, longer, or
shorter than the determined standard telomere length of the cell
whose telomeres were analyzed. The standard telomere length of a
cell can be determined by the results obtained from Q-FISH analysis
of a control telomere. Measurement of telomere length is further
described in Martens et al. (1998) Nature Genetics 18:76-80. Cells
whose chromosomes are determined to have abnormal length telomeres
are likely to give rise to aneuploidy. In one embodiment of the
invention, an oocyte which is analyzed by Q-FISH and is determined
to have abnormal length telomeres, is not used for in vitro due to
a predisposition to aneuploidy. An oocyte with a predisposition to
aneuploidy is not likely to give rise to a viable embryo, and would
not be chosen by a clinician for in vitro fertilization purposes.
In another embodiment, a polar body is analyzed by Q-FISH analysis
for a predisposition to aneuploidy.
Exemplification
[0150] The invention is further illustrated by the following
examples which should not be construed as limiting.
EXAMPLES
Materials and Methods
[0151] Spare human eggs (N=43) matured from GV stage oocytes in
vitro, retrieved for attempted intracytoplasmic sperm injection,
were donated by consenting patients (N=24) undergoing ART for
infertility. FISH analysis of telomere length and extraction of
clinical characteristics and outcomes were performed by separate
investigators blinded to each others' findings.
[0152] Cumulus cells were removed by pipetting after brief
incubation in 0.03% hyaluronidase. GV oocytes were matured in vitro
for 24 to 48 hours until they reached MII stage of development.
Analysis of Telomeric Function Using Quantitative Fluorescence In
Situ Hybridization (Q-FISH) with Telomere Probe
[0153] Q-FISH has become the method of choice for examination of
both telomere length and loss in single cells (Zijlmans, Martens et
al. 1997, PNAS USA, 94: 7423-7428.). Chromosome spreads were
prepared by a hypotonic treatment of oocytes or spermatocytes with
1% sodium citrate for 20 min, followed by fixation in
methanol:acetic acid (3:1), and air dried. FISH with FITC-labeled
(CCCTAA).sub.3 (SEQ ID NO: 10) peptide nucleic acid (PNA) probe
(Applied Biosystems, Framingham, Mass.) was performed according to
the manufacturer's protocol. Chromosomes were counter-stained with
0.2 .mu.g/ml Hoechst 33342. Embryos were mounted onto a glass slide
in Vectashield mounting medium (Vector Laboratories, Burlingame,
Calif.). Telomeres were detected with a FITC filter using a Zeiss
fluorescence microscope (Axioplan 2 imaging) and images were
captured by an AxioCam using AxioVision 3.0 software.
[0154] Quantitative fluorescence in situ hybridization (Q-FISH):
Telomere FISH was performed on chromosome spreads from oocytes or
polar bodies, as previously described (Hande, Samper et al. 1999,
J. Cell Biol. 144(4): 589-601). Telomeres were denatured at
80.degree. C. for 3 min and hybridized with FITC-labeled
(CCCTAA).sub.3 (SEQ ID NO: 10) peptide nucleic acid (PNA) probe
(Applied Biosystems, Framingham, Mass.), washed and mounted in
Vectashield mounting medium added with 0.5 .mu.g/ml DAPI. For
quantitative measurement of telomere length, telomere fluorescence
intensity was integrated using the TFL-TELO program (Poon, Martens
et al. 1999, Cytometry, 36: 267-278), kindly provided by P.
Lansdorp, and calibrated with fluorescence beads. Telomere length
(kb) was estimated based on the relative fluorescence intensity,
using standard cell lines with known telomere length (McIlrath,
Bouffler et al. 2001, Cancer Res., 61(3): 912-5).
Example 1
Calculation of Control for Telomere Length Assay
[0155] To determine the standardized average length of a telomere
against which to compare telomere length in oocytes from women with
reproductive problems, telomere length is determined from spare
oocytes and polar bodies of control oocytes. Telomere lengths are
determined by the number of tandem repeats of a specific nucleotide
sequence found within the telomeres at the end of chromosomes. In
humans and other vertebrates, the telomere repeats are commonly
tandem repeats of the sequence TTAGGG.
[0156] Control oocytes and/or polar bodies are obtained from donors
undergoing IVF to donate their oocytes to women with egg
infertility. For example, oocytes can also be retrieved for control
studies from women undergoing intracytoplasmic sperm injection
(ICSI) for severe male factor infertility or egg donation. Other
factors considered for women donors include having a day three FSH
values less than 9 IU/ml, inhibin B levels greater than 45 and
estradiol levels less than 80 .mu.g/ml, at least 12 eggs retrieved
after COH with standard gonadotropin dosing, and no known diagnosis
of female infertility. Oocytes are retrieved by transvaginal needle
aspiration from the ovary, performed under gentle intravenous
sedation after controlled ovarian hyperstimulation (COH), per
standard protocols. Methods for obtaining eggs for egg donation are
described in Klein, et al. (2002) Best Pract. Res. Clin. Obstet.
Gynaecol. 16:277-291.
[0157] Human oocytes are cultured according to standard, published
clinical protocols. Protocols for culturing human oocytes can also
be found throughout the literature, including, for example, Quinn
et al. (1998) Fertil. Steril. 69:399-402.
[0158] Polar bodies from control oocytes are obtained and used as a
control according to whether the oocyte from which they arose went
on to form normal babies after the resulting embryos are
transferred into the uterus of the recipient women. Polar bodies
are biopsied according to standard protocols, as described, for
example, in Verlinsky et al. (1996) J. Assist. Reprod. Genet.
13(2): 157-162. Briefly, for biopsy of polar bodies, first the egg
or oocyte was placed on the holding pipette of a micromanipulator
under 40 X objective on an inverted microscope. The zona was
broached by laser, acid tyrodes, pronase or mechanical dissection.
The polar body was teased out with gentle suction into a biopsy
pipette affixed to the second micromanipulator. The polar body was
then released onto a slide where a chromosome spread was made. The
first polar body was obtained before fertilization, while the
second was obtained after.
Example II
Comparative Telomeric Lengths Between Dysfunctional Oocytes and
Normal Oocytes
[0159] For comparative purposes, oocytes are also obtained from
women who are known to have dysfunctional oocytes. Eggs are
retrieved by transvaginal aspiration under light intravenous
sedation after controlled ovarian hyperstimulation (COH).
Dysfunctional eggs are retrieved from women undergoing ICSI for
mild male factor and/or egg factor infertility, who have day three
FSH values greater than 14 IU/ml, inhibin B levels less than 16 and
estradiol levels greater than 80 .mu.g/ml, five or fewer eggs
retrieved after COH with high dose (at least FSH 450 IU/day)
gonadotropin dosing, and known diagnosis of female infertility.
[0160] Once control and dysfunctional oocytes and/or polar bodies
are obtained, the telomeres from the oocytes and polar bodies are
quantitatively analyzed to determine their average telomere length.
Telomere length of control oocytes and polar bodies is
quantitatively analyzed through Q-FISH analysis, as described in
the materials and methods section and example III.
Example III
Analysis of Telomere Length Using Quantitative Fluorescence In Situ
Hybridization (Q-FISH)
[0161] Q-FISH analysis is used to determine the telomere length of
chromosomes from oocytes. Oocytes are obtained from a patient who
is interested in determining the risk of aneuploidy in her oocytes.
From the batch of oocytes obtained from the ova, a spare oocyte or
oocytes are taken for Q-FISH analysis. Results obtained for spare
oocytes are representative of the population of oocytes in the
batch. Control oocytes are obtained from a woman with demonstrated
fertility and are prepared according to the methods described in
the materials and methods section and Example I. Control oocytes
are analyzed prior to Q-FISH analysis of the oocyte of interest or,
alternatively, are prepared for Q-FISH analysis alongside the
experimental spare oocyte(s). Average telomere length from control
oocytes is the standardized control to which the spare oocytes are
compared. Chromosomal spreads are prepared by hypotonic treatment
of oocytes with 1% sodium citrate for 20 min, followed by fixation
in methanol:acetic acid (3:1). Chromosomal spreads are then allowed
to air dry.
[0162] Fluorescence in situ hybridization (FISH) is performed
according to the manufacturer's protocol with FITC-labeled peptide
nucleic acid (PNA) probe (Applied Biosystems, Framingham, Mass.)
comprising the sequence CCCTAACCCTAACCCTAA (SEQ ID NO: 1). The
telomere specific probe is hybridized under stringent conditions to
the chromosomal spreads. Chromosomes are then counterstained with
Hoechst 33342 at a concentration of 0.2 .mu.g/ml. Chromosomes are
mounted onto a glass slide in Vectashield mounting medium (Vector
Laboratories, Burlingame, Calif.), and analyzed using fluorescence
microscopy.
[0163] Telomere length is determined by quantitative digital
microscopy and integrated optical density of the fluorescence
signal. The signal from the FITC-labeled probe hybridized to the
telomeres is detected using a Zeiss fluorescence microscope
(Axiophot) with a FITC filter. Images of fluorescing telomeres are
captured using an AxioCam digital microscope camera using
AxioVision 2.0 software. Commercially available software, including
MetaMorph (Universal Imaging Corporation.TM., Downingtown Pa.), is
used to integrate the fluorescent intensity and area. The digital
images are then used for quantitative analysis of telomere length
based on criteria described in Zijlmans et al. (1997). Background
is subtracted and integrated fluorescence intensity in individual
telomeres of chromosome spreads is measured to indicate the length
of telomeres (Zijlmans et al., 1997; Romanov, et al. Nature.
409:633-637 (2001)). The intensity of the fluorescence from each
telomere is expressed in "Telomere Fluorescence Units" or "TFUs"
and plotted on a graph for comparative purposes. Determined
telomere lengths of the spare oocytes are compared to the results
obtained for the control oocytes to establish whether the spare
oocyte telomere lengths are abnormal. If the determined length of
the oocyte's telomeres is found to be abnormal in comparison to the
control or standardized average length, the batch of oocytes is
considered at risk for aneuploidy and not fertilized for IVF
purposes. If, however, the telomere length of the spare oocytes is
found to be comparable to the control, the population of oocytes is
not considered at risk for aneuploidy and is used for IVF
procedures. Telomere lengths are determined by the number of tandem
repeats of a specific nucleotide sequence found within the
telomeres at the end of chromosomes. In humans and other
vertebrates, the telomere repeats are commonly tandem repeats of
the sequence TTAGGG.
[0164] One of ordinary skill in the art will recognize that the
above-mentioned procedure for determining telomere length by Q-FISH
analysis, can be repeated for a polar body or polar bodies which
are obtained from fertilized or unfertilized oocytes. Polar bodies
are obtained through conventional methods known to one of ordinary
skill in the art.
Example IV
Comparative Study of Age and Telomere Length in Unfertilized
Eggs
[0165] To show that telomere length is a predictor of the outcome
of IVF and embryo transfer procedures, telomere length was
determined in women of various ages who were undergoing IVF.
Unfertilized human eggs (n=43) were obtained from consenting donors
who had undergone WVF treatment. Clinical characteristics and
outcomes were also obtained from patients' charts. Telomere lengths
from the unfertilized eggs were measured by Q-FISH analysis.
Telomere length was then compared with pregnancy outcome and
analyzed by t test or logistic regression.
[0166] The results show that telomere maximum (19.3.+-.3.1 k.b. vs.
13.9.+-.3.28 k.b., p<0.01) and mean (7.5.+-.1.17 k.b. vs.
6.2.+-.1.69 k.b.) lengths were significantly longer and standard
deviation greater (4.4.+-.0.96 vs. 3.5.+-.1.12) in eggs from
patients who went on to become pregnant, compared to those who
failed attempts at pregnancy. Minimum telomere length and number of
missing telomere signals did not differ between groups. In
addition, clinical predictors of fertility, including patients'
age, baseline follicle stimulating hormone (FSH) level, egg number,
body mass index, ovarian stimulation protocol, numbers of previous
IVF cycles, diagnosis, or embryo morphology did not differ
significantly between groups with these sample sizes.
[0167] In sum, increased egg telomere length predicts favorable
reproductive outcome in infertile women undergoing IVF. Telomere
length provided a better predictor of pregnancy outcome following
IVF than patient age itself or other clinical parameters, including
when telomere length was measured only in spare eggs.
Example V
Analysis of Telomere Length in Oocyte for In Vitro
Fertilization
[0168] Upon obtaining a population of spare oocytes, Q-FISH
analysis is performed, as described above in the materials and
methods section and Example III, to determine the length of the
oocyte's telomeres. The length of telomeres within the population
is assumed to be similar, therefore allowing the ordinarily skilled
artisan to determine whether the population of oocytes might be at
risk for aneuploidy.
Example VI
Analysis of Telomere Length in Polar Body Prior to Implantation in
a Subject
[0169] Upon obtaining the polar body from an oocyte, Q-FISH
analysis is performed, as described above in the materials and
methods section and Example III, to determine telomere length of
the polar body. Determining the telomere length allows the
ordinarily skilled artisan to assess the risk of aneuploidy, and
subsequently whether the oocyte is likely to give rise to an
abnormal embryo.
Example VII
Detection of TTAGGG Repeats at Chromosome Telomeric Ends
[0170] To detect the presence of TTAGGG repeats at the chromosome
ends, the number of repeats comprising the telomeric ends determine
the length of the telomeres, telomeric FISH was performed on oocyte
metaphase spreads using a fluorescent FITC-labeled (CCCTAA).sub.3
(SEQ ID NO: 10) peptide nucleic acid (PNA) probe, which is able to
detect 200 bp of TTAGGG repeats at the telomeres. FISH was employed
to measure telomere length in chromosomes spread from spare eggs
matured from the GV stage after aspiration from consenting subjects
undergoing ART and ICSI (FIG. 1).
[0171] Forty-three eggs were donated from 21 women, with 23 eggs
coming from pregnant cycles and 20 from non-pregnant cycles. 10
(47.6%) of the women became pregnant and 11 (52.4%) did not. The
clinical characteristics of these patients did not differ
significantly between the pregnant and non-pregnant groups at the
sample size studied (Table 1). The embryo morphology score was
slightly worse in the non-pregnant group (p<0.04), although
after correction for multiple comparisons this difference was only
marginally significant. TABLE-US-00001 TABLE 1 Clinical
Characteristics and Pregnancy Outcomes: Pregnant Not Pregnant
Variable (mean .+-. SD) (n = 10 women) (n = 20 women) P-Value Age
34.2 .+-. 5.45 35.5 .+-. 3.24 0.52 No. Oocytes 15 .+-. 7.96 17.4
.+-. 12.96 0.61 No. Cleaved (d.2) 8.3 .+-. 5.2 4.5 .+-. 5.8 0.14
No. Frozen 3.9 .+-. 3.2 2.3 .+-. 4.7 0.37 Embryo Morphology 7.4
.+-. 1.22 6.2 .+-. 0.84 0.04 Pt. BMI 28.1 .+-. 4.17 28.4 .+-. 8.19
0.11 D. 3 FSH 6.5 .+-. 1.79 7.2 .+-. 3.25 0.56 Diagnosis 0.3 Cycle
No. 0.44
[0172] Telomere lengths were normally distributed. Although
clinical characteristics did not distinguish the pregnant vs. the
non-pregnant group, nearly all measures of telomere length did
differ significantly between the pregnant and non-pregnant groups
(t-test) (Table 2). Mean and maximum telomere lengths were greater
in oocytes from women who became pregnant compared to those who did
not become pregnant. Variation in telomere length, measured by
standard deviation, also was significantly greater in oocytes from
women who became pregnant compared to those who did not (F test).
No women became pregnant in this study who had a mean telomere
length in any spare oocytes less than 6.32 k.b. Minimum telomere
length did not differ between groups, presumably because Q-FISH is
not as accurate a measure of telomere length in the very short
range compared to measurement of longer telomeres. TABLE-US-00002
TABLE 2 Telomere Length Metrics and Pregnancy Outcomes: Pregnant
Not Pregnant Variable (mean .+-. SD) (n = 23) (n = 20) P-Value Mean
Length (k.b) 7.5 .+-. 1.17 6.2 .+-. 1.69 0.01 Max. Length (k.b)
19.3 .+-. 3.1 13.9 .+-. 3.28 0.01 Variation (S.D.) 4.4 .+-. 0.96
3.5 .+-. 1.12 0.01 Min. Length (k.b.) 0.87 .+-. .87 0.93 .+-. 0.74
0.89
[0173] Telomere lengths were highly correlated between polar bodies
and oocytes (n=8; R2=97.8%) (FIG. 2). Thus, telomere length
measured in the polar body should provide a highly accurate
estimate of telomere lengths in embryos later transferred to
patients.
Incorporation by Reference
[0174] The contents of all references, patents and published patent
applications cited throughout this application, as well as the
figures and the sequence listing, are incorporated herein by
reference.
Equivalents
[0175] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments and methods described
herein. Such equivalents are intended to be encompassed by the
scope of the following claims.
* * * * *