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  Methods of assessing the risk of reproductive failure by measuring telomere lengthUSPTO Application #: 20060141461Title: 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. (end of abstract) Agent: Edwards & Angell, LLP - Boston, MA, US Inventor: David L. Keefe USPTO Applicaton #: 20060141461 - Class: 435006000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic Acid The Patent Description & Claims data below is from USPTO Patent Application 20060141461. Brief Patent Description - Full Patent Description - Patent Application Claims
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; Continue reading... Full patent description for Methods of assessing the risk of reproductive failure by measuring telomere length Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Methods of assessing the risk of reproductive failure by measuring telomere length patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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