WO2009075862A1

WO2009075862A1 - Method of assessing viability of embryos in vitro

Info

Publication number: WO2009075862A1
Application number: PCT/US2008/013602
Authority: WO
Inventors: Paul H. Pevsner; Frederick Naftolin; Jaime Grifo; Fred Licciardi
Original assignee: New York University School Of Medicine
Priority date: 2007-12-11
Filing date: 2008-12-11
Publication date: 2009-06-18

Abstract

The present invention provides methods for predicting the viability of an embryo or for predicting the likelihood of a negative outcome during pregnancy by identifying the presence or absence of or determining the amount of one or more pregnancy associated markers such as a human serum albumin, a serum albumin precursor, a gamma-aminobutyric acid receptor subunit or a tetratricopeptide repeat protein 9 in a sample. In many instances, the invention is applicable to embryos generated by in vitro fertilization techniques, for instance, to embryos developing in a growth media. The present invention further provides methods for determining the amount of a pregnancy associated markers such as a human serum albumin, a serum albumin precursor, a gamma-aminobutyric acid receptor subunit or a tetratricopeptide repeat protein 9 in a sample. The present invention also provides a diagnostic kit for predicting the viability of an embryo or for predicting the likelihood of a negative outcome during pregnancy by identifying the presence of or determining the amount of one or more pregnancy associated markers such as a human serum albumin, a serum albumin precursor, a gamma-aminobutyric acid receptor subunit or a tetratricopeptide repeat protein 9 in a sample.

Description

METHOD OF ASSESSING VIABILITY OF EMBRYOS IN VITRO

FIELD OF THE INVENTION

[0002] This invention relates to methods of determining whether a fetus is likely to become viable and whether a pregnancy is likely to have a positive or negative outcome.

BACKGROUND OF THE INVENTION

[0003] The first evidence of in vitro fertilization (IVF) dates back to the 1890's when embryos were transferred between rabbits in England. In the late 1950s rabbit oocytes were fertilized in vitro and subsequently transferred back to female rabbits. Based on these animal techniques, the procedures were applied to humans, and eventually the first "test-tube baby" was born in England on July 25, 1978 (Steptoe et al, Lancet 1978;2: 366). Subsequently, physicians around the world began performing IVF. The first successful IVF procedure in the United States was performed in 1981. Since then, IVF procedures have developed and success rates have increased dramatically. It is estimated that in the US, 1% of births are conceived after IVF.

[0004] According to statistics from a 1995 Center for Disease Control (CDC) survey, 6.1 million women aged 15-44 years in the US have an impaired ability to conceive and during that year, 9.3 million women sought fertility services. The Society of Assisted Reproductive Technology (SART) 2005 annual report indicates that 123,200 cycles of IVF were performed in women under the age of 43 in the US. This number includes the majority of fertility clinics in the country; however, a minority do not report to SART and therefore this number underestimates the total IVF cycles in the US for 2005.

[0005] In an IVF procedure, patients are started on ovarian stimulation protocols per their individual physician based on infertility etiology, physical exam, hormonal assays, and prior history. Specifically, patients are placed on down-regulated GnRH agonist (leuprolide) protocols, micro-dose leuprolide protocols, or GnRH-antagonist protocols with gonadotropins starting on day 2 or 3 of the menstrual cycle. The goal of ovarian hyperstimulation is to suppress a woman's endogenous ovulation and hyperstimulate the ovaries to make multiple oocytes without placing the woman at risk. Patients are monitored frequently with serum estradiol levels and transvaginal ultrasonography of the ovaries to assess follicular development. Gonadotropin doses are adjusted during the cycle to achieve adequate numbers of mature oocytes at retrieval while minimizing the risk of ovarian hyperstimulation syndrome. When lead follicles reach a mean diameter of 17-18 mm, human Chorionic Gonadotropin (hcG) 10,000 Iu is given and approximately 36 hours later, oocytes are collected by ultrasound guided, transvaginal aspiration.

[0006] Oocytes are immediately placed in Human Tubal Fluid media (HTF, Irvine Scientific, Irvine CA) supplemented with 6% Plasmanate (Plasma Protein Fraction (Human) 5%, USP, Bayer Co., Elkhart, IN) overlaid with Sage mineral oil (Cooper Surgical, Trumbull, CT). The partner's sperm is collected and washed on the day of retrieval. Oocytes are fertilized by standard insemination or intracytoplasmic sperm injection (ICSI) when indicated 4-6 hours post retrieval. Oocytes inseminated by ICSI are quickly placed in Quinn's Advantage^R Cleavage Media (Ql) (Cooper Surgical, Trumbull,CT). After fertilization, oocytes are incubated overnight. On the day following insemination, day 1 post retrieval, oocytes are assessed for fertilization. When 2 pronuclei (2pn) are observed, fertilization is confirmed. Embryos that are created after standard insemination are transferred to Ql media. Embryos are incubated and not reassessed until day 3 post retrieval. At this time, if only several viable embryos are available, embryo transfer is scheduled on this day. However, when rapidly developing good quality embryos exist in excess to the number that would be safely transferred (generally > 4 embryos), embryos are transferred to Quinn's Advantage¹¹ Blastocyst (Q2) media and cultured to day 5. On day 5 post retrieval, embryos are assessed and the most morphologically advanced embryos are selected for transfer to the uterus. Excess embryos are transferred into a fresh dish of Q2 and incubated overnight. On day 6, embryos that meet strict morphologic criteria are selected for cryopreservation .

[0007] Patients undergo embryo transfer using a soft tipped catheter. In most cases ultrasound guidance is used to deposit the embryos approximately lcm from the uterine fundus. The decision on the number of embryos to transfer is based on embryo morphology and patient characteristics (age, history). The final decision on the number to transfer is made after a discussion between physician and patient. Patients are instructed to take intramuscular progesterone injections until day 28 (day 14 established as day of oocyte retrieval) when the initial pregnancy test is obtained. In general, progesterone is continued until a fetal heartbeat is observed on ultrasound. Patients with a positive pregnancy test are followed for the development of a viable intrauterine pregnancy and for ectopic pregnancies. [0008] Human preimplantation embryos cultured in vitro are characterized by variable morphology and developmental potential. Following in vitro fertilization (IVF), an average of 25% of transferred embryos implant and, an average of 20% of women become pregnant. The established criteria for human embryo viability are histological patterns of 3 and 5 day embryos identified microscopically by experienced reproductive physicians and embryologists. Implant decisions made with these criteria can achieve successful implantation in half of the implants and a top rate of 50% live births. In vitro studies show that during the first six days of preimplantation development approximately 50% of human embryos arrest. Varying degrees of cytoplasmic fragmentation occurs in approximately 75% of human embryos. This is associated with reduced blastocyst formation and implantation. The causes, mechanism of fragmentation and precise reasons for early embryonic loss remain unknown. Cell death has been observed during preimplantation embryogenesis both in vivo and in vitro in a range of mammalian species. Cell death is prevalent in human blastocysts with approximately 75% of embryos having one or more dead cells on day 6. Cells and nuclei with the morphological features of apoptosis which include intracellular fragmentation, chromatin condensation, DNA fragmentation and phagocytosis have been identified in human preimplantation embryos. Apoptosis may be involved in early embryonic arrest, and cytoplasmic fragments are equivalent to apoptotic bodies, i.e., the end product of apoptosis. Suboptimal culture conditions may be implicated in preimplantation arrest and cell death. In particular, there is increasing evidence that growth factors play an important role in preimplantation development. For example, the preimplantation development of mouse embryos cultured in vitro is retarded when compared to that in vivo. Moreover, reduced incubation volume or cultures of embryos in groups improve preimplantation development. Furthermore, a range of polypeptide growth factor ligands and their receptors have been found to be expressed and produced in the reproductive tract or preimplantation embryo, including epidermal growth factor(EGF), transforming growth factor alpha (TGF-a), insulin receptor insulin-like growth factor I (IGF-I) and its receptor (IGF-IR). Generally, growth factors have been shown to promote blastocyst formation and development and increase cell number. Taken together, these findings suggest the presence of regulatory autocrine and paracrine pathways acting in vivo that may be 'diluted' or not present in the in vitro environment. (Hardy,K. et al, PNAS 2001, 98(4): 1655-1660; Spanos et al, Biology of Reproduction 2000, 63: 1413-1420).

[0009] The scope of IVF and other forms of assisted reproduction technologies (ART) in the United States is over 100 000 IVF cycles, and ART babies now account for approximately 0.6% of all births in this country. A small but important percentage of these children suffer from a variety of significant morbid congenital problems. Children conceived by ART have twice the rate of major birth defects as compared with babies conceived naturally. This risk persists even after adjusting for increased maternal age and increased incidence of multiple gestations with ART. A wide range of birth defects has been noted, including chromosomal abnormalities, musculoskeletal and cardiovascular defects, and low birthweight. (Li,Tao et al, Molecular Human Reproduction, 2005, 1 1(9):631-640).

[0010] The ability to identify the most viable chromosomal normal embryos for transfer is of fundamental importance to assisted reproduction techniques (ARTs). An early predictive assessment made in a non-invasive manner would allow ART clinics to identify the best embryo that would result in a live birth.

[0011] In the most recent Society of Assisted Reproductive Technology 2005 review, 37%, 29%, 20%, and 11% of IVF cycles in women >35, 35-37, 38-40, and 41-42 years, respectively, result in a live birth. At the NYU Fertility Center, 48%, 38%, 28%, and 12% of cycles in women <35, 35-37, 38-40, and 41-42 years, respectively, result in a live birth. Furthermore, 40% of these births in women <35 years at the NYU Fertility Center are twin deliveries. One of the greatest criticisms of assisted reproductive technologies (ART) is the high incidence of multiple and high order multiple gestations. Since 1980, there has been an over 80% increase in twins and 500% increase in triplet and higher order births. It is well established that fetuses and mothers of multiple and high-order multiple pregnancies are faced with increased morbidity and mortality. Grifo et al. recently described their clinic's progression to blastocyst transfer as a means to reduce the high-order multiple rate. (Munne et al, Fertil Steril. 2007;88:781-784) The ART community has addressed the need for more single embryo transfers (SET) but also recognizes the lowered pregnancy rates that may ensue. The ability to identify additional markers associated with embryo viability and competence has been the greatest challenge towards promoting SET. In a recent study of 3 and 5 day growth media, we have identified gi|223976 haptoglobin Hp2, mass 41717. (Keegan et al. Journal of the Society for Gynecologic Investigation. 2006/2; 13 : A61 -A 171 ) Two more proteins were identified in a recent report, gi|90108928 1 Chain H, Orally Available Factor7a Inhibitor, mass 28582, and gi| l 19573737 hCG 1793647 [Homo sapiens]Mass: 61 12. (Pevsner et al., British Mass Spectrometry Society; 2007). SUMMARY OF THE INVENTION

[0012] The present invention provides methods of predicting whether a fetus is likely to become viable by determining whether one or more pregnancy associated marker is present in a sample. The pregnancy associated marker may be a human serum albumin, a serum albumin precursor, a gamma-aminobutyric acid receptor subunit or a tetratricopeptide repeat protein 9. One, two, three or more of the pregnancy associated markers such as a human serum albumin, a serum albumin precursor, a gamma-aminobutyric acid receptor subunit or a tetratricopeptide repeat protein 9 may be present in the sample and may be identified. In some embodiments it is the absence or relative absence of one or more pregnancy associated markers that is predictive of the likelihood of a viable fetus.

[0013] The sample used for determining the presence of one or more pregnancy associated marker may be an in vitro growth medium, amniotic fluid, plasma, serum, urine or blood. The one or more pregnancy associated marker such as a human serum albumin, a serum albumin precursor, a gamma-aminobutyric acid receptor subunit or a tetratricopeptide repeat protein 9 may be identified by many methods well known to those of skill in the art including matrix assisted laser desorption ionization (MALDI) mass spectrometry. The pregnancy associated markers such as a human serum albumin, a serum albumin precursor, a gamma-aminobutyric acid receptor subunit or a tetratricopeptide repeat protein 9 may also be identified by contacting the sample with an antibody which specifically binds to the human serum albumin, a serum albumin precursor, a gamma-aminobutyric acid receptor subunit or a tetratricopeptide repeat protein 9 under conditions permitting formation of a complex between the antibody and the pregnancy associated marker, and optionally measuring the amount of complexes formed, thereby determining the amount of the pregnancy associated marker.

[0014] The methods may optionally include quantifying one or more of the pregnancy associated markers such as a human serum albumin, a serum albumin precursor, a gamma-aminobutyric acid receptor subunit or a tetratricopeptide repeat protein 9. The methods may further include comparing the amount of one or more pregnancy associated markers in the sample determined to be present in the sample with either (i) the amount determined for temporally matched, normal samples or (ii) the amount determined for one or more samples obtained from one or more nonpregnant subjects. The relative absence of one or more of the pregnancy associated markers such as a human serum albumin, a serum albumin precursor, a gamma-aminobutyric acid receptor subunit or a tetratricopeptide repeat protein 9 in the sample indicates that the embryo is relatively non viable and the relative abundance of one or more of the pregnancy associated markers such as a human serum albumin, a serum albumin precursor, a gamma-aminobutyric acid receptor subunit or a tetratricopeptide repeat protein 9 in the sample indicates that the embryo is relatively viable in some embodiments.

[0015] The methods of the present invention are applicable to determining viability of an embryo at many stages in its development, whether in vitro or in vivo. In most instances, the embryo is a mammal, especially a human, and in most instances the embryo is in a relatively early developmental stage within the first trimester of pregnancy. The embryo may be, for instance, three months, two months, one month, two weeks, one week, five days, three days or one day post fertilization. In some embodiments, the embryo is a product of in vitro fertilization. In some of these embodiments, the embryo has yet to be implanted and is growing in an in vitro growth media.

[0016] The present invention further provides a method of predicting pregnancy outcome in a subject. In some embodiments, it is a method of predicting the likelihood of a positive pregnancy outcome in a female subject comprising determining the presence of one or more pregnancy associated markers such as a human serum albumin, a serum albumin precursor, a gamma- aminobutyric acid receptor subunit or a tetratricopeptide repeat protein 9 in a sample. In other embodiments, it is a method of predicting the likelihood of a negative pregnancy outcome in a female subject comprising determining the absence or the relative absence as compared to normal samples or normal subjects of one or more pregnancy associated markers such as a human serum albumin, a serum albumin precursor, a gamma-aminobutyric acid receptor subunit or a tetratricopeptide repeat protein 9 in a sample. One, two, three, or more of the pregnancy associated markers such as human serum albumin, a serum albumin precursor, a gamma- aminobutyric acid receptor subunit or a tetratricopeptide repeat protein 9 may be present in the sample and may be identified. One, two, three, or more of the pregnancy associated markers such as a human serum albumin, a serum albumin precursor, a gamma-aminobutyric acid receptor subunit or a tetratricopeptide repeat protein 9 may be present in the sample and may be elevated in comparison to samples obtained from normal pregnant subjects.

[0017] The sample used for determining the presence of one or more pregnancy associated markers may be an in vitro growth media, amniotic fluid, plasma, serum, urine or blood. The one or more pregnancy associated markers such as a human serum albumin, a serum albumin precursor, a gamma-aminobutyric acid receptor subunit or a tetratricopeptide repeat protein 9 may be identified by many methods well known to those of skill in the art including Matrix assisted laser desorption ionization (MALDI) mass spectrometry. The pregnancy associated markers such as a human serum albumin, a serum albumin precursor, a gamma-aminobutyric acid receptor subunit or a tetratricopeptide repeat protein 9 may also be identified by contacting the sample with an antibody which specifically binds to the pregnancy associated marker under conditions permitting formation of a complex between the antibody and the pregnancy associated marker, and optionally measuring the amount of complexes formed, thereby determining the amount of the pregnancy associated marker in the sample.

[0018] The methods may optionally include quantifying one or more of the pregnancy associated markers such as a human serum albumin, a serum albumin precursor, a gamma-aminobutyric acid receptor subunit or a tetratricopeptide repeat protein 9. The methods may further include comparing the amount of pregnancy associated markers such as a human serum albumin, a serum albumin precursor, a gamma-aminobutyric acid receptor subunit or a tetratricopeptide repeat protein 9 in the sample determined to be present in the sample with either (i) the amount determined for temporally matched, normal samples or (ii) the amount determined for samples obtained from non-pregnant subject(s) when the sample is any other than an in vitro growth media. The relative absence of one or more of the pregnancy associated markers in the sample indicates that the likelihood of a negative outcome is greater than the normal, and the relative abundance of one or more of the pregnancy associated markers in the sample indicates that the likelihood of a positive outcome is greater than the normal.

[0019] The invention provides methods for determining whether a fetus is likely to be viable and methods for determining the relative likelihood of a positive or negative outcome for a pregnant woman comprising the steps of: (a) obtaining a biological sample from a pregnant woman or from a growth media in instances where the fetus is not in vivo; (b) measuring an amount of one or more pregnancy associated marker present in the biological sample; and (c) comparing the amount of one or more pregnancy associated markers with a predetermined value, whereby the amount of pregnancy associated marker relative to the predetermined value indicates whether a fetus is likely to be viable or the relative likelihood of a positive or negative outcome for a pregnant woman.

[0020] In one embodiment, the method further comprises measuring an amount of one or more pregnancy associated markers such as a human serum albumin, a serum albumin precursor, a gamma-aminobutyric acid receptor subunit or a tetratricopeptide repeat protein 9 in the biological test sample; and calculating the ratio of the amount of the one or more pregnancy associated markers such as a human serum albumin, a serum albumin precursor, a gamma-aminobutyric acid receptor subunit or a tetratricopeptide repeat protein 9 to the amount of other forms of pregnancy associated markers such as a human serum albumin, a serum albumin precursor, a gamma-aminobutyric acid receptor subunit or a tetratricopeptide repeat protein 9, whereby the ratio indicates whether a fetus is likely to be viable or the relative likelihood of a positive or negative outcome for a pregnant woman.

[0021] Further, the present invention provides a diagnostic kit for assessing viability of an embryo by determining the presence or absence or by quantifying the amount of one or more pregnancy associated markers such as a human serum albumin, a serum albumin precursor, a gamma-aminobutyric acid receptor subunit or a tetratricopeptide repeat protein 9 in a biological sample.

BRIEF DESCRIPTION OF THE FIGURES

[0022] Figure 1 is a MSMS (MALDI TOF TOF) of the biomarkers of competent embryos, a human serum albumin, a serum albumin precursor, a gamma-aminobutyric acid receptor subunit and a tetratricopeptide repeat protein 9.

DETAILED DESCRIPTION OF THE INVENTION

[0023] As used herein, the following terms mean as follows:

[0024] As used herein, "pregnancy associated marker" means any molecule, such as a protein, peptide or fragment thereof whose presence, absence or amount in absolute quantity or in quantity relative to other molecules may be used as evidence of whether an embryo is likely to be viable or whether a pregnancy is likely to have a positive or a negative outcome. Stated differently, a pregnancy associated marker is any molecule that may be used as a statistically significant predictor of viability or pregnancy outcome.

[0025] As used herein, a "predetermined value" is a standardized value based on a control. For example, a predetermined value can be based on an amount of pregnancy associated marker such as a human serum albumin, a serum albumin precursor, a gamma-aminobutyric acid receptor subunit or a tetratricopeptide repeat protein 9 that are present in a biological sample obtained from a pregnant woman who carries a normal fetus. In this embodiment of the invention, a fetus may be determined likely to be non-viable or a pregnancy deemed likely to have a negative outcome if the amount of one or more pregnancy associated markers is lower than the predetermined value. Similarly, a fetus may be determined likely to be viable or a pregnancy deemed likely to have a positive outcome if the amount of one or more pregnancy associated markers is higher than the predetermined value.

[0026] The term "amount" is used within the context of the analytical method used to measure the different pregnancy associated markers such as a human serum albumin, a serum albumin precursor, a gamma-aminobutyric acid receptor subunit or a tetratricopeptide repeat protein 9 and may reflect a number, a concentration, etc., depending upon the analytical method chosen to measure the pregnancy associated markers such as a human serum albumin, a serum albumin precursor, a gamma-aminobutyric acid receptor subunit or a tetratricopeptide repeat protein 9.

[0027] The term "relative abundance" means that a marker such as a peptide, polypeptide or protein is present in a biological sample in a higher concentration or in a greater amount than in a control or normal tissue biological sample. The term "relative absence" means that a marker such as a peptide, polypeptide or protein is present in a biological sample in a lesser concentration or in a lesser amount than in a control or normal tissue biological sample.

[0028] The term "biological sample," as used herein, generally refers to urine, saliva, serum, plasma, tears, or amniotic fluid as well as in vitro growth media.

[0029] The term "detecting" as used herein refers to identifying the presence of, identifying the presence of in relative amounts relative to another molecule or pregnancy associated markers such as a human serum albumin, a serum albumin precursor, a gamma-aminobutyric acid receptor subunit or a tetratricopeptide repeat protein 9 relative to a predetermined value, or quantifying in absolute amounts.

[0030] The term "positive outcome" in relation to pregnancy means delivery of a baby substantially free of genetic abnormalities or diseases. [0031] The term "negative outcome" in relation to pregnancy means failure to deliver a baby at all, abortion or delivery of a baby substantially impacted or afflicted by genetic abnormality or disease.

[0032] The established criteria for determining human embryo viability are histological patterns identified microscopically by experienced reproductive physicians and embryologists. Implant decisions made with these criteria can achieve successful implantation in half of the implants.

[0033] In some embodiments the methods provide for comparing the amount of pregnancy associated marker in a sample with either (i) the amount determined for temporally matched, normal pregnant subject(s) or (ii) the amount determined for non-pregnant subject(s), wherein amounts of the pregnancy associated marker in the sample similar to amounts of pregnancy associated marker in temporally matched pregnant samples indicates a positive outcome, amounts of early pregnancy associated marker in the sample similar to amounts of pregnancy associated marker in the non-pregnant samples indicates a negative outcome of pregnancy for the subject.

[0034] According to an embodiment of this invention, the sample may be a urinary sample, a sample of amniotic fluid or a blood sample. In one embodiment of this invention, the sample is an aggregate sample taken from at least two consecutive days. In an embodiment of this invention, the sample is a spot urine sample, a first morning void urine sample, or an aggregate sample of the first morning void urine samples for at least two consecutive days. In one embodiment of this invention, the antibody is labeled with a detectable marker. In an embodiment of this invention, the detectable marker is a radioactive isotope, enzyme, dye, magnetic bead, or biotin.

[0035] In addition, the present invention provides a method for determining the amount of pregnancy associated marker of in a sample comprising: (a) contacting the sample with an antibody which specifically binds to a pregnancy associated marker under conditions permitting formation of a complex between the antibody and the pregnancy associated marker; and (b) determining the amount of complexes formed thereby determining the amount of pregnancy associated marker in the sample.

[0036] Pregnancy associated markers can be directly measured, for example, using anti- marker antibodies in an immunoassay, such as a Western blot or ELISA. Pregnancy associated markers can be indirectly measured, for example, using a capture antibody that binds the pregnancy associated marker.

[0037] The methods of the invention can be used alone or in combination with any known test for assessing viability of a fetus or for determining prognosis for a pregnancy, including, but not limited to, a triple screen test (combination of maternal age with serum measurements of hCG, α- fetoprotein (AFP), and unconjugated estriol, unconjugated and/or conjugated estriol measurements, hCG assays, β-core fragment analyses, free β -subunit or free α -subunit analyses, PAPP-A or CA 125 analyses, α -fetoprotein analyses, inhibin assays, observations of fetal cells in serum, and ultrasound. The methods of the invention can be used to screen a biological sample collected during the first, second, or third trimester of pregnancy or before in vivo implant. In addition, the methods of the invention can be combined with any known diagnostic test during the first, second, or third trimester of pregnancy.

[0038] The amount of pregnancy associated markers in a biological sample can be determined using any method known in the art, including, but not limited to, immunoassays using antibodies specific for the pregnancy associated marker. Any assay that functions to qualitatively or quantitatively determine variations in sample concentrations of pregnancy associated markers from normal levels can be employed in the practice of the invention.

[0039] For example, a monoclonal anti-pregnancy associated marker antibody can be generated by immunizing a mouse with the pregnancy associated marker. Once an immune response is detected, e.g., antibodies specific for the pregnancy associated marker are detected in the mouse serum, the mouse spleen is harvested and splenocytes are isolated. The splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example, cells from cell line SP20 available from the American Type Culture Collection (ATCC). Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding the pregnancy associated marker. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.

[0040] Any type of fusion phage, monoclonal, or polyclonal antibodies can be used in immunoassays of the invention, so long as the antibodies can be used in a reproducible fashion as markers for various pregnancy associated markers or as measures of the different levels of pregnancy associated markers observed in normal and variant populations. [0041] In one embodiment, an amount of pregnancy associated marker can be measured using a capture antibody followed by a labeled secondary antibody using a strategy as described, for example, in U.S. Pat. No. 6,429,018, hereby incorporated by reference. The label on the secondary antibody can comprise any chemical, radioactive, lanthanide, colored dye, or genetic tag used in enzyme-linked immunosorbent assays (ELISAs), Western blots, and other sensitive and specific immunoassays and immunoradiometric assays using known methodology. These include conjugating the antibody with horseradish peroxidase or alkaline phosphatase that are easily measurable, typically using colorimetric, fluorometric or luminescent substrates. Genetic labels include firefly luciferase, employed because luciferase produces a bioluminescent molecule when incubated with its substrate, luciferin.

[0042] Matrix-assisted laser desorption/ionization (MALDI).

Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique used in mass spectrometry, allowing the analysis of biomolecules (biopolymers such as proteins, peptides and sugars) and large organic molecules (such as polymers, dendrimers and other macromolecules), which tend to be fragile and fragment when ionized by more conventional ionization methods. It is most similar in character to electrospray ionization both in relative softness and the ions produced (although it causes many fewer multiply charged ions). The ionization is triggered by a laser beam (normally a nitrogen laser). A matrix is used to protect the biomolecule from being destroyed by direct laser beam and to facilitate vaporization and ionization.

[0043] The matrix consists of crystallized molecules, of which the three most commonly used are 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), α-cyano-4-hydroxycinnamic acid (alpha-cyano or alpha-matrix) and 2,5-dihydroxybenzoic acid (DHB). A solution of one of these molecules is made, often in a mixture of highly purified water and an organic solvent (normally acetonitrile (ACN) or ethanol). Trifluoroacetic acid (TFA) may also be added. A good example of a matrix-solution would be 20 mg/mL sinapinic acid in ACN:water:TFA (50:50:0. l).The identity of suitable matrix compounds is determined to some extent by trial and error, but they are based on some specific molecular design considerations: they have a low molecular weight; they are acidic and act as a proton source to encourage ionization of the analyte; they have a strong optical absorption in the UV and efficiently absorb the laser irradiation; and they are functionalized with polar groups allowing use in aqueous solutions. [0044] The matrix solution is mixed with the analyte (e.g. protein sample). The organic solvent allows hydrophobic molecules to dissolve into the solution, while the water allows for water- soluble (hydrophilic) molecules to do the same. This solution is spotted onto a MALDI plate (usually a metal plate designed for this purpose). The solvents vaporize, leaving only the recrystallized matrix, but now with analyte molecules spread throughout the crystals. The matrix and the analyte are said to be co-crystallized in a MALDI spot.

[0045] The laser is fired at the crystals in the MALDI spot. The matrix absorbs the laser energy, and the matrix is ionized by this event. The matrix transfers part of its charge to the analyte molecules (e.g. protein) thereby ionizing them while still protecting them from the disruptive energy of the laser. Ions observed after this process consist of a neutral molecule [M] and an added or removed ion. Together, they form a quasimolecular ion, for example [M+H]⁺ in the case of an added proton, [M+Na]⁺ in the case of an added sodium ion, or [M-H]^" in the case of a removed proton. MALDI is capable of creating singly-charged ions, but multiply charged ions ([M+nH]ⁿ⁺) can also be created with electrospray. Note that these are all even-electron species. Ion signals of radical cations can be observed e.g. in case of matrix molecules and other stable molecules.

[0046] Atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) is an ionization technique (ion source) that in contrast to vacuum MALDI operates at normal atmospheric environment. In vacuum MALDI, ions are typically produced at 10 mTorr or less while in AP-MALDI ions are formed in atmospheric pressure.

[0047] AP-MALDI is used in mass spectrometry (MS) in a variety of applications including proteomics and drug discovery fields. AP-MALDI mass spectrometry is ofen used in proteomics, DNA/RNA/PNA, lipids, oligosaccharides, phosphopeptides, bacteria, small molecules and synthetic polymers, similar applications as available also for vacuum MALDI instruments.

[0048] The AP-MALDI ion source is easily coupled to an ion trap mass spectrometer or any other MS system equipped with ESI (electrospray ionization) or nanoESI source.

[0049] The type of a mass spectrometer most widely used with MALDI is the TOF (time-of- flight mass spectrometer) because of its large mass range. The TOF measurement procedure is suited to the MALDI ionization process since the pulsed laser takes individual 'shots' rather than working in continuous operation. MALDI-TOF instruments are typically equipped with an "ion mirror," deflecting ions with an electric field thereby doubling the ion flight path and increasing the resolution. Today, commercial reflectron TOF instruments reach a resolving power m/Δm of well above 20'0OO FWHM (full-width half-maximum, Δm defined as the peak width at 50% of peak height).

[0050] In proteomics, MALDI is used for identifying proteins isolated through gel electrophoresis: SDS-PAGE, size exclusion chromatography, and two-dimensional gel electrophoresis. One method used is peptide mass fingerprinting by MALDI-MS, or with post ionization decay or collision-induced dissociation.

[0051] IMS.

The history of IMS (Imaging Maldi Spectroscopy) began with single cell studies of Aplysia californica neurons by matrix-assisted laser desorption/ionization time-of flight mass spectrometry (MALDI/TOF-MS). This was probably the first direct tissue MALDI identification of peptides and tissue profiling based on ion density. (Garden et al, J Mass Spectrom 1996; 31 : 1 126-30) A refinement of this technique, imaging MALDI was described in human buccal mucosa, and rat pituitary and pancreas glands using two different approaches: direct targeting of the tissue itself and by analysis of blotted targets previously exposed to the tissue. (Caprioli et al, Anal Chem 1997; 69:4751-60) An "MS Image Tool," using the peptide neurotensin (peak at m/z 1674) significantly improved the data acquisition, speed of IMS, and utilization of the technique. (Stoeckli et al., JAm Soc Mass Spectrom 1999; 10:67-71) In a laser, capture microdissection (LCM) study of both tumor and normal human breast tissue fixed in ethyl alcohol, and stained with hematoxylin and eosin, normal tissue, carcinoma in situ, invasive carcinoma, and metastatic carcinoma could be distinguished by their different MALDI spectra. The introduction of "BioMap" software, by applying a baseline correction to the spectra and integrating over the peak of interest, demonstrated mouse brain images of amyloid β, and Aβ peptides. That report was proof of the principle that MALDI images of tissue could be obtained based upon the mass spectrometry mass/charge, m/z peak of interest. (Stoeckli et al, Nat Med 2001; 7:493-6) Subsequent profiling and IMS of normal mouse epididymis identified different protein activity (ion densities) throughout the sections. (Chaurand et al, Electrophoresis 2002; 23:3125-35)

[0052] The negative effects on IMS resolution from destructive tissue freezing artifacts, excessive dehydration due to ethanol fixation, paraformaldeyde cationization, embedding artifacts from OCT polymer and agar, and coarse matrix crystal size were first described in a report on spatial profiling of invertebrate ganglia. (Kruse et al, J Am Soc Mass Spectrom 2003; 14:752-9) In a follow-up report, they suggested a solution to the problem of freezing artifacts using glycerol and the related compounds ethane- 1, 2-diol and propane- 1, 2-diol to stabilize cellular membranes. (Rubakhin et al, Anal Chem 2003; 75:5374-80) A subsequent report suggested direct liquid nitrogen immersion of tissue in aluminum wrapping as a means of rapid fixation, but ignored the known consequences of freezer artifact. (Schwartz et al, J Mass Spectrom 2003; 38:699-708) Adjunctive histologic staining with methylene blue stained tissues on standard metal plates or indium-tin coated glass slides were shown to be compatible with IMS, but cresyl violet stain decreased IMS signal intensity. (Chaurand et al., Anal Chem 2004; 76:1145-55)

[0053] Tissue blotting with trypsin digestion for MSMS (mass spectrometry mass spectrometry using two mass spectrometers in tandem) data base analysis was shown to be useful in analyte localization, but was destructive to tissue morphology. (Bunch et al, Rapid Communications in Mass Spectrometry 2004; 18:3051-60) Others described a less destructive trypsin digest step to their prior tissue blotting technique for IMS. (Rohner et al, Mech Ageing Dev 2005; 126: 177-85) Another method, matrix-enhanced secondary ion mass spectrometry (ME) SIMS was described and used for direct molecular imaging of the ganglia of the freshwater snail, Lymnaea stagnalis. (Altelaar et al, Anal Chem 2005; 77:735-41) However, this technique presented significant limitations and was proved unsuitable for direct tissue imaging. A refinement of IMS, oversampling with complete sample ablation at each sample position on the target plate, provided significant resolution enhancement with a translation stage raster step size of 25 μM. A 40 μM object could now be resolved with a 100 μM laser. (Jurchen et al, Journal of the American Society for Mass Spectrometry 2005; 16: 1654-9)

[0054] Tissue treatments with organic solvents such as chloroform, acetone, hexane, toluene, or xylene were shown to be an effective and rapid method for signal enhancement in MALDI direct tissue profiling. (Lemaire et al, Analytical Chemistry 2006; 78:7145-53) These studies demonstrated that solvent treatments partially removed lipids from the tissue surface. Compared to previous studies with ethanol, chloroform/ xylene solvent, rinsing is more specific for lipid removal and does not generate derealization or extraction of most soluble peptides/proteins as tested by immuno-histochemistry experiments. Among all the tested solvents, chloroform and xylene produced the greatest increase in MALDI signal intensity and number of detected peptides/ proteins. However, this treatment does not reduce salt adducts as does alcohol treatment. The results suggest that it is possible to detect, after organic rinsing treatments, compounds, such as peptides/proteins present in the cytoplasm, that were masked by lipids in the tissue. [0055] Clench et al reported the development and application of a method using 9-aminoacridine as a matrix for negatively charged ions in MALDI imaging. (Burrell et al, J Exp Bot 2007; 58:757-63) Crossman demonstrated the need for thin sections to avoid differential extraction efficiency of matrix solvent in different tissues. (Crossman et al, Rapid Communications in Mass Spectrometry 2006; 20:284-90) Pevsner demonstrated direct cellular MALDI identification of proteins in fixed cells and tissues without freezer artifact, tissue corrosion by matrix solvents or the use of tissue blotting. This was confirmed in a later report. (Pevsner et al, Direct identification of proteins from cells and tissues using MALDI TOF. Anal Chem. ; Pevsner et al, J Soc Gynecol Investig 2006;13:Al-B10; Groseclose et al, J Mass Spectrom 2007;42:254-62)

[0056] Metal-assisted (MetA) secondary ion mass spectrometry (SIMS), a variation on SIMS as well as matrix-assisted laser desorption/ionization (MALDI) IMS can provide images from tissue, but the duration of these protocols were highly dependent on sample size and technique parameters. The duration of these studies averaged approximately 5 h. (Altelaar et al, Nat Protoc 2001; 2: 1 185-96)

[0057] Agar et al. studied multiple solvent/matrix combinations. However, the tissue sections at the electron microscopic level demonstrated both freezing artifact and structural distortion, indicating that their method disrupts normal subcellular structures such as mitochondria. (Agar et al, Matrix Solution Fixation: Histology-Compatible Tissue Preparation for MALDI Mass Spectrometry Imaging. Anal Chem, Published on World Wide Web September 7, 2007) Baluya et al reported a variation on inkjet-printed matrix application to tissue specimens previously described by Sloane et al (Baluya et al, Anal Chem 2007; 79:6862-7; Sloane et al, MoI Cell Proteomics 2002; 1 :490-9) The matrix application was of better quality and more reproducible than from specimens prepared by the electrospray and airbrush methods, but still was not completely uniform. A uniform method of tissue matrix application is sublimation. Sublimation is solvent free, rapid, and was successfully used to identify lipids in brain tissue, and more recently described for proteins or peptides. The challenge of IMS in formalin fixed paraffin embedded tissue was first addressed by and then by Pevsner and later by Stauber. (Hankin et al, J Am Soc Mass Spectrom 2007; 18: 1646-52; Pevsner et al "Microtubule Associated Proteins (MAP) and Motor Molecules: Direct Tissue MALDI Identification and Imaging," 2007; Pevsner et al, 2007, British Mass Spectrometry Society, Edinburgh, Scotland "Colon Cancer: Protein Biomarkers in Tissue and Body"; Pevsner et al, "Colorectal Carcinoma - Field Defects in SatelliteTissue." 2007, British Mass Spectrometry Society, Edinburgh, Scotland, Robinson College, Cambridge, UK; Puolitaival et al, JAm Soc Mass Spectrom 2008; 19:882-6; Lemaire et al, JProteome Res 2007; 6: 1295-305; 48. Stauber et al, J Proteome Res 2008; 7:969-78)

[0058] This invention is illustrated in the examples that follow. These examples are set forth to aid in understanding the invention but are not intended to and do not limit the invention as claimed.

Example 1

[0059] Two unique biomolecule analyte biomarkers of competent or high potential human embryos were obtained from five day embryo growth media, Quinn's II. These analytes were obtained from the growth media by extraction in an organic solvent with a cycling high pressure (using ProteoSolve and the Barocycler respectively (Pressure BioSciences, West Bridgewater, MA)), 35,000 psi, followed by centrifugation and lypholization of the supernatant to dryness, and rehydration with 100 μL of lOOmMolar ammonium bicarbonate. Trypsin lμg/ μL, 50: 1 sample to trypsin v/v was added, and the sample was digested for 12 hours at 37° C. The digested sample was passed through a Cl 8 micropipette tip filter, and loaded onto a LCMS for peptide separation and both MS and MSMS mass spectrometry. The peptide mass list data is entered into a protein database via MASCOT^® software, and the proteins identified. These proteins were not present in non-competent or low potential human embryo growth media.

[0060] The combination of high pressure protein extraction with an organic solvent and LCMS significantly increased the yield of peptides obtained from the growth media digest and identification of heretofore unidentified proteins in competent embryos, namely human serum albumin, a serum albumin precursor, a gamma-aminobutyric acid receptor subunit or a tetratricopeptide repeat protein 9. The combination of high pressure protein extraction with an organic solvent and LCMS increased the yield of peptides, and improved subsequent NCBInr data base identification of proteins. These proteins represent biomarkers of competent embryos.

Claims

We claim:

1. A method of predicting viability of an embryo comprising determining the presence of one or more pregnancy associated markers.

2. A method according to claim 1 wherein the pregnancy associated marker is selected from the group consisting of a human serum albumin, a serum albumin precursor, a gamma- aminobutyric acid receptor subunit and a tetratricopeptide repeat protein 9.

3. A method according to claim 1 wherein the sample is selected from the group consisting of an in vitro growth media, amniotic fluid, plasma, serum, urine and blood.

4. A method according to claim 1 wherein determining the presence of one or more pregnancy associated markers in a sample is performed by Matrix assisted laser desorption ionization (MALDI) mass spectrometry.

5. A method according to claim 1 wherein determining the presence of one or more pregnancy associated markers in a sample is performed by (a) contacting the sample with an antibody which specifically binds to a pregnancy associated marker permitting formation of a complex between the antibody and the pregnancy associated marker; and (b) measuring the amount of complexes formed, thereby determining the amount of the pregnancy associated marker in the sample.

6. A method according to claim 4 or 5 further comprising comparing the amount of pregnancy associated marker in the sample with either (i) the amount determined for temporally matched, normal samples or (ii) the amount determined for samples obtained from nonpregnant subject(s), wherein the relative abundance of the pregnancy associated marker in the sample indicates that the embryo is relatively viable.

7. A method according to claim 1 wherein the embryo is between about 3 and about 5 days old.

8. A method of predicting the likelihood of a positive pregnancy outcome in a female subject comprising determining the presence of one or more pregnancy associated markers in a sample.

9. A method according to claim 8 wherein the pregnancy associated marker is selected from the group consisting of a human serum albumin, a serum albumin precursor, a gamma- aminobutyric acid receptor subunit and a tetratricopeptide repeat protein 9.

10. A method according to claim 8 wherein the sample is selected from the group consisting of an in vitro growth media, amniotic fluid, plasma, serum, urine and blood.

1 1. A method according to claim 8 wherein determining the presence of one or more pregnancy associated markers in a sample is performed by Matrix assisted laser desorption ionization (MALDI) mass spectrometry.

12. A method according to claim 8 wherein determining the presence of one or more pregnancy associated markers in a sample is performed by (a) contacting the sample with an antibody which specifically binds to a pregnancy associated marker under conditions permitting formation of a complex between the antibody and the molecular pregnancy associated marker; and (b) measuring the amount of complexes formed, thereby determining the amount of the pregnancy associated marker in the sample.

13. A method according to claim 11 or 12 further comprising comparing the amount of pregnancy associated marker in the sample with either (i) the amount determined for temporally matched, normal pregnant samples or (ii) the amount determined for samples obtained from non-pregnant subject(s), wherein the relative abundance of the pregnancy associated marker in the sample indicates a greater than normal likelihood of a positive pregnancy.

14. A diagnostic kit for predicting viability of an embryo or for determining the likelihood of a positive outcome for a pregnancy comprising a means for detecting the presence or the quantity of a pregnancy associated marker and instructions correlating the presence or the quantity of the pregnancy associated marker with the likelihood that an embryo will become viable or that a pregnancy will result in a positive outcome.