A PROGNOSTIC ASSAY

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

The present invention relates generally to a prognostic assay to determine a stage of labor or likelihood of initiation of labor in a pregnant, female mammal and in particular a human. The present invention further provides medical and veterinary protocols to assist a pregnant, female mammal, and in particular a human, given birth following identification of the stage of labor or likelihood of initiation of labor. A kit useful in a prognostic assay of labor is also contemplated by the present invention.

DESCRIPTION OF THE PRIOR ART

Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

Preterm birth is defined as birth occurring before completion of the 37th week of gestation (259 days). Worldwide, it remains the most significant problem facing contemporary obstetrics. In industrialised countries, preterm birth occurs in approximately 5-12% of all deliveries. In developing countries limited birth data exist. The estimated rate of preterm birth is thought to be as high as 25%, with infectious diseases such as malaria and HIV contributing to the high rates in these countries (Ayisi et al, AIDS 17:585-594, 2003). With the exception of France and Finland, no developed country has reported a decrease in the incidence of preterm birth (Robinson Seminars in Perinatology 25 (4): 204-214, 2001). Australian figures are comparable over the period 1997-2001, with the proportion of preterm deliveries ranging from 7.3% to 7.9% (Al-Yaman et al, Australian Institute of Health and Welfare: 1 -349, 2002).

The significance of preterm birth cannot be underestimated. It is the major cause of perinatal morbidity and mortality accounting for 85% of all early infant deaths (Norwitz Robinson Seminars in Perinatology 25(4):223-235, 2001). Advances in neonatology over the past several decades have resulted in increased survival rates, particularly for the extremely premature baby born between 23 and 28 weeks' gestation. Effective interventions for these newborns include access to neonatal intensive care facilities (Doyle, et al, Australian and New Zealand Journal of Obstetrics and Gynaecology 37 (3) /253-7, 1997), antenatal administration of corticosteroids (Foix-L 'helias et al, Archives of Disease in Childhood Fetal and Neonatal Edition 90(l):F46-4S, 2005) and use of exogenous surfactant therapy (Rushing and Ment Seminars in Perinatology 28(6):444-450, 2004). However, for these surviving extremely premature babies, there remains a significant rate of neonatal morbidity. Between 20 and 25% of survivors will have at least one major disability including long-term neurocognitive deficits, pulmonary dysfunction and ophthalmological disorders. The most prevalent disability affecting approximately 20% of survivors is impaired mental development (Lorenz, J. Seminars in Perinatology 25 (5 ):34S- 359, 2001). Data suggest that low birth weight (for which prematurity is the commonest cause) may also be linked to cardiovascular disease in adult life (Wilson, J Australian and New Zealand Journal of Obstetrics and Gynaecology 39:1-7, 1999; Leeson et al, Circulation 103 (9): 1264-1268, 2001).

Compared to a neonate delivered at term (defined as 37 completed to 42 weeks of gestation) there is a raised cost of health care for the preterm infant during the first 10 years of life (Petrou, S "The economic consequences of preterm birth during the first 10 years of life" 112(Suppl l):\Q-\5, 2005). For the severely disabled survivors of preterm birth, there are substantial societal costs beyond childhood. There are considerable emotional costs sustained by the infants, their families and carers. As such there remains a desperate need for an effective prevention of preterm birth.

The current approach to the management of preterm birth is twofold. First, interventions aimed at delaying preterm birth may be employed when a woman presents with symptoms of threatened preterm labor. Tocolytic drugs are utilised in this context to suppress uterine

activity, providing there is no evidence of infection or bleeding. With timely use, these drugs may delay delivery long enough for administration of maternal corticosteroids and permit transfer of the laboring woman to a tertiary perinatal facility. There remains controversy as to the effectiveness of tocolytic drugs in the prevention of preterm birth (Berkman et al, American Journal of Obstetrics and Gynecology 188: 1648-1659, 2003).

Secondly, screening tests may be employed to identify women at high-risk of preterm birth and permit an appropriate intervention aimed at reducing the incidence of preterm birth. A reduction in preterm birth has been reported with the use of antibiotic treatment in women with abnormal lower genital tract colonization (Ugwumada et al, Lancet 361(9362):9S3- 988, 2003) and prophylactic use of progesterone used in women at high risk of preterm birth (Sanchez-Ramos et al, Obstetrics and Gynecology 105 (2) /273-279, 2005). However, the screening tests in current use when applied to the general obstetric population are neither sensitive nor specific and the positive predictive values are relatively poor. As a consequence, this may lead to costly and sometimes unnecessary interventions without an impact upon the overall rate of preterm birth.

Early identification of women at risk of preterm birth provides the best chance of optimizing the current management of this disorder. The relative failure of current screening tests used to identify women at risk of preterm birth may reflect the relatively limited understanding of the complex, heterogeneous pathophysiology of preterm labor. Present knowledge of the exact pathways of both term and preterm labor remains incomplete. A common final pathway of cervical maturation, membrane rupture and myometrial activation occurs. Current research efforts are being focused upon finding predictive markers associated with this common final pathway of preterm labor. Whilst several well-recognized markers of labor have been studied in detail in the serum, amniotic fluid and cervico-vaginal fluid (CVF), there may be other novel markers in this final pathway that may improve the predictive ability of a screening test for preterm labor.

Proteomic applications in obstetrics have largely focused upon diagnosis of intra-uterine infection/inflammation and preterm prelabor rupture of the fetal membranes (PROM).

Insulin-like growth factor-binding protein- 1 (IGFBP-I) and calgranulin B identified by proteomic analysis of both amniotic fluid and serum, have been proposed as biomarkers of intra-amniotic infection (Gravett et al, Journal of the American Medical Association 292(4) :462-469, 2004). Neutrophil defensins-1 and -2 and calgranulins A and C have been proposed as biomarkers of intrauterine inflammation leading to pre term delivery (Buhimschi et al, British Journal of Obstetrics and Gynaecology 112(2): 173-181, 2005). Proteomic analysis of amniotic fluid and serum has identified agrin and perlecan as potential biomarkers of preterm PROM (Vuadens et al, Proteomics 3. 1521-1525, 2003). These heparin sulphate proteoglycans are unique to amniotic fluid and are present from the 17th to the 40th week of pregnancy.

Until the advent of the present invention, there have been no proteomic or metabolomic patterns identified in CVF particularly with respect to approaching labor, either term or preterm. There is a need, therefore, to identify markers present in the CVF that would be useful in the diagnosis of a stage of labor and in particular preterm labor that display a better positive predictive value than the current generation of biochemical markers used in clinical or veterinary practice.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

One aspect of the present invention contemplates a method for determining whether a pregnant, female mammal is likely to go into labor or to determine at which stage the labor has progressed said method comprising screening for levels of one or more biomarkers in cervico-vaginal fluid (CVF) isolated from said pregnant female mammal and comparing said levels to a control wherein changes in levels of one or more of said biomarkers is indicative of approaching, existing or stage of labor.

Another aspect of the present invention provides a method for determining whether a pregnant female mammal is likely to go into labor or to determine at which stage the labor has progressed, said method comprising measuring one or more proteomic and/or metabolomic markers in the CVF of said pregnant female mammal and comparing the levels of said markers with a control profile of markers wherein changes in the levels of said markers is indicative of approaching, existing or stage of labor.

Still another aspect of the present invention is directed to a method for detecting or predicting preterm labor in a pregnant female mammal said method comprising determining the levels of one or more proteomic and/or metabolomic markers in the CVF of said pregnant female mammal and comparing the levels to a control wherein changes in levels of the biomarkers is indicative of approaching or existing preterm labor.

Yet another aspect of the present invention is directed to determining whether a pregnant female human,is likely to go into labor or to determining at which stage the labor has progressed, said method comprising screening for levels of one or more biomarkers in the

CVF of said pregnant human female and comparing said levels to a control wherein

changes in levels of one or more of said biomarkers is indicative of approaching, existing or stage of labor.

Another aspect of the present invention provides a method for determining a stage of labor in a pregnant mammal, said method comprising screening for the presence or level of one or more biomarkers selected from a blood transport protein, a structural protein, a protein involved in fatty acid metabolism, a calcium-binding protein, an anti-inflammatory cytokine, a proteinase inhibitor or an enzyme involved in oxidative stress defence.

Still yet another aspect of the present invention is directed to a prognostic assay to detect labor or to determine the stage of labor in a pregnant mammal said assay comprising screening for the presence or level of one or more biomarkers selected from the list consisting of a blood transport protein, a structural protein, a protein involved in fatty acid metabolism, a calcium-binding protein, an anti-inflammatory cytokine, a proteinase inhibitor or an enzyme involved in oxidative stress defence.

Yet another aspect of the present invention provides a prognostic assay to detect or predict labor or a stage of labor in a pregnant female mammal including a pregnant human, said assay comprising screening for the levels of two or more biomarkers selected from blood transport proteins (e.g. albumin and transthyretin), a structural protein (e.g. β-actin), proteins involved in fatty acid metabolism (fatty acid binding protein or acetyl CoA- binding protein), a calcium-binding protein (e.g. annexin III), an anti-inflammatory cytokine (e.g. Interleukin-1 receptor antagonist), a proteinase inhibitor (e.g. α-1- antitrypsin, monocyte/neutrophil elastase inhibitor, squamous cell carcinoma antigen- 1 or cystatin A) and/or an enzyme involved in oxidative stress defence (e.g. thioredoxin, peroxiredoxin 2, glutathione-S-transferase P or copper-zinc superoxide dismutase) and comparing said profile to a control to thereby assess the presence of labor, a risk of development of labor or the stage of labor.

The present invention further provides a birthing protocol for a pregnant mammal said protocol comprising screening for labor or approaching labor by the method as described above and intervening in the birthing process to assist in delivery,

In a particular embodiment, the pregnant female mammal is a human. However, the present invention extends to veterinary and animal husbandry applications.

Whilst the present invention is particularly directed to proteomic and metabolomic markers in the CVF, one skilled in the art will readily appreciate that the subject invention could be practised using white blood cells, blood plasma, and/or serum.

The present invention further contemplates a kit for conducting the prognostic assay.

Abbreviations and terms used in this specification are defined in Table 1.

TABLE 1

Abbreviations/Terms

Abbreviaiioii/T|erm Definition ' .

2DE Two-dimensional electrophoresis

CVF Cervico-vaginal fluid

FABP Fatty acid binding protein

GST Glutathione-S-transferase

IGFBP-I Insulin growth factor-binding protein- 1

IL Interleukin

Metabolomic The complete metabolite complement of an organism, tissue, cell or body fluid

PROM Prelabor rupture of fetal membranes

Proteomic The complete protein complement of an organism, tissue, cell or body fluid

Prx2 Peroxiredoxin II

PTL Preterm labor

SCCA-I squamous cell carcinoma antigen 1

M/NEI monocyte/ neutrophil elastase inhibitor

GSTPl-I glutathione S-transferase P

CuZn-SOD CuZn-superoxide dismutase

ACBP acyl-CoA-binding protein

BRIEF DESCRIPTION OF THE FIGURES

Figure Ia is a photographic representation of a 2-dimensional electrophoresis (2DE) display of cervico-vaginal fluid (CVF) of a pregnant woman at 37 weeks gestation. Figure Ib shows the names of the identified proteins.

Figure 2 is a photographic representation of a 2DE display of CVF at 26-30 days prelabor versus term labor.

Figure 3 is a photographic representation of a 2DE display of CVF at 26-30 days prelabor (term) versus spontaneous PTL (1-4 days prior to preterm labor).

Figure 4 is a graphical representation showing Interleukin-1 receptor antagonist levels in cervico-vaginal fluids obtained from pregnant women stratified from day of spontaneous term labor. ^A demotes outliers; * group is significantly different from 0 days (p<0.05).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a prognostic assay to determine risk factors or diagnostic indicators of preterm, term or post-term labor in a pregnant mammalian female animal and in particular a pregnant human female. The prognostic assay is based on the identification of a set of biomarkers in biological fluid and in particular, cervico-vaginal fluid (CVF). The biomarkers undergo changes in levels (including presence or absence) relative to a control. Identification of the changes in levels provide a pattern or profile associated with a stage of labor being term, preterm or post-term. The control includes monitoring a subject's levels over time during the pregnancy or comparing the levels to normalized values. Appropriate therapeutic or medical protocols can be implemented once a subject is identified as being in an early or late stage of labor development. Examples of medical intervention depend upon gestation and include medicinal administration of tocolytic therapy or inducement of labor.

The biomarkers of the present invention include proteinaceous molecules from the proteome such as cytokines, growth factors or enzymes or metabolites from biochemical pathways of the metabolome which form a profile of markers stratifying a test subject on the stage of approaching or development of labor.

Useful biomarkers include blood transport proteins (e.g. albumin and transthyretin), a structural protein (e.g. β-actin), proteins involved in fatty acid metabolism (fatty acid binding protein or acetyl CoA-binding protein), a calcium-binding protein (e.g. annexin III), an anti-inflammatory cytokine (e.g. Interleukin-1 receptor antagonist), proteinase inhibitors (e.g. α-1 -antitrypsin, monocyte/neutrophil elastase inhibitor, squamous cell carcinoma antigen- 1 or cystatin A) and/or enzymes involved in oxidative stress defence (e.g. thioredoxin, peroxiredoxin 2, glutathione-S -transferase P or copper-zinc superoxide dismutase).

Particularly useful biomarkers include one or more of Interleukin-1 (IL-I) receptor antagonist, copper-zinc superoxide dismutase, glutathione-S-transferase P (GSTP), thioredoxin, peroxiredoxin II (Prx2), and cy statin A,.

Hence, the present invention provides a proteomic and metabolomic approach to diagnosis of labor in pregnant, female mammals, and in particular human females.

Accordingly, one aspect of the present invention contemplates a method for determining whether a pregnant, female mammal is likely to go into labor or to determine at which stage the labor has progressed said method comprising screening for levels of one or more biomarkers in cervico-vaginal fluid (CVF) isolated in said pregnant female mammal and comparing said levels to a control wherein changes in levels of one or more of said biomarkers is indicative of approaching labor or stage of labor.

In one particular embodiment, the present invention provides a method for determining whether a pregnant female mammal is likely to go into labor or to determine at which stage the labor has progressed, said method comprising measuring one or more proteomic and/or metabolomic markers in the CVF of said pregnant female mammal and comparing the levels of said markers with a control profile of markers wherein changes in the levels of said markers is indicative of approaching labor or stage of labor.

Still another embodiment of the present invention is directed to a method for detecting or predicting preterm labor in a pregnant female mammal said method comprising determining the levels of one or more proteomic and/or metabolomic markers in the CVF of said pregnant female mammal and comparing the levels to a control wherein changes in levels of the biomarkers is indicative of approaching preterm labor.

Yet another aspect of the present invention provides a method for determining a stage of labor in a pregnant mammal, said method comprising screening for the presence or level of one or more biomarkers selected from a blood transport protein, a structural protein, a protein involved in fatty acid metabolism, a calcium-binding protein, an anti-inflammatory

cytokine, a proteinase inhibitor or an enzyme involved in oxidative stress defence in a sample from said mammal and comparing presence or levels with a control wherein changes in the presence or absence or levels of the one or more biomarkers is indicative of approaching labor or a stage of labor.

Preferred biomarkers include but are not limited to blood transport proteins (e.g. albumin and transthyretin), a structural protein (e.g. β-actin), proteins involved in fatty acid metabolism (fatty acid binding protein or acetyl CoA-binding protein), a calcium-binding protein (e.g. annexin III), an anti-inflammatory cytokine (e.g. Interleukin-1 receptor antagonist), a proteinase inhibitor (e.g. α-1 -antitrypsin, monocyte/neutrophil elastase inhibitor, squamous cell carcinoma antigen- 1 or cystatin A) and/or an enzyme involved in oxidative stress defence (e.g.- thioredoxin, peroxiredoxin 2, glutathione-S-transferase P or copper-zinc superoxide dismutase).

Before describing the present invention in more detail, the following definitions and clarifications are provided.

Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

All scientific citations, patents, patent applications and manufacturer's technical specifications referred to hereinafter are incorporated herein by reference in their entirety.

The singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a biomarker" includes a single

biomarker, as well as two or more biomarkers; reference to "an assay" includes reference to a single assay or two or more assays; and so on.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

Reference to an "assay" includes performing one or more steps to determine the presence or level of a biomarker. The assay may not necessarily require all the steps needed to derive a particular data point on the presence or level of the biomarker but may provide data or samples for input into another assay. The term "prognostic" assay is used for the purposes of defining an assay which is predictive of an outcome, i.e. labor. The assay may also take the form of a microarray. In one embodiment, antibodies or other ligands to a target biomarker are immobilized to a solid support. CVF is then contacted with the microarray. Binding patterns of biomarkers to the solid support then provide a profile of presence or absence which is useful in assessing changes in the profile. There are many other forms of assay, including preparing samples for two-dimensional gel electrophoresis

(2DE) or an immunoassay.

Detection of levels of biomarkers is by any of a number of procedures including immunoassays, gel electrophoresis, spectrometric (including mass spectrometry) assays and matrix-affinity-based fractionations. The term "level" includes concentration.

The term "labor" is used in its broadest sense and covers inter alia childbirth, partus, partuition, accounchement, vaginal birth, childbearing, delivery and travail. A female mammal is considered to be in labor when the mammal experiences regular, strong uterine contractions accompanied by changes in the cervix, manifestating as effacement and dilatation.

The term "labor" is used to cover any stage of labor including a first stage which comprises onset (the beginning of contractions), latent onset (a gradual onset), active (when contractions become regular), rupture of amniotic sac phase, and transition phase (after rupture of the amniotic sac) and a second stage comprising postnate delivery.

Reference to "labor" includes labor that occurs at a preterm, term or post-term gestation.

The present invention permits an identification of any stage of approaching labor, including sub-stages of labor.

In a particular embodiment, the labor identified is preterm labor. Reference to "preterm labor" includes labor which initiates prior to the average gestation period for a particular mammal. Hence, for human females, preterm labor occurs prior to at least the 37 ⁽ week of pregnancy. Preterm labor occurs, therefore at about 7 weeks to about at 30 weeks including 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or at 30 weeks. "Post-term" labor would occur after at least the 42 ^nd week of pregnancy such as for 42 to 50 weeks including 42, 43, 44, 45, 46, 47, 48, 49 or at 50 weeks.

The present invention provides, therefore, a proteomic and metabolomic approach to diagnosis.

Unlike genetic studies that focus upon differential gene expression, proteomic analysis provides information on the gene products in cells, tissues and body fluids. Proteomics embodies one or more of a number of techniques that involves characterization of the protein complement of a tissue, cell or body fluid. It is a direct measure of proteins in terms of their presence and relative abundance. The commonest method employed to display the proteome is 2DE. Hundreds of proteins may be analyzed simultaneously by use of this technology. 2DE, therefore, permits direct comparisons to be made between different states of a cell or tissue (e.g. disease and non-disease) leading to identification of both qualitative and quantitative protein changes. The signature proteomic pattern of each condition can be compared and. differential protein expression or novel proteins may be

identified. Protein identification is achieved by excision of the protein spot of interest on the 2DE gel and subsequent characterisation by mass spectrometric analysis. Metabolomic analysis investigates metabolites generated in metabolic pathways. For example, an end product or an intermediate in the conversion of one compound to another via one or more enzymatic reactions. The presence and level of metabolites is an indication of the level and/or presence of particular enzymes or protein substrates. The present invention extends, however, to a range of other detection methods including immunoassays, gel- electrophoresis, spectrometric assay and matrix-affinity-based fractionations.

The present invention is particularly applicable to pregnant human females.

Hence, another aspect of the present invention includes determining whether a pregnant female human is likely to go into labor or determining to which stage the labor has progressed, said method comprising screening for levels of one or more biomarkers in the CVF of said pregnant human female and comparing said levels to a control wherein changes in levels of one or more of said biomarkers is indicative of approaching labor, or stage of labor.

Insofar as the invention provides a means for detecting or predicting preterm labor, this aspect is directed to a method for detecting or predicting preterm labor in a pregnant female human said method comprising determining the levels of one or more proteomic and/or metabolomic markers in the CVF of said pregnant female mammal and comparing the levels to a control wherein changes in levels of the biomarkers is indicative of approaching preterm labor.

However, the present invention is also applicable to non-human medicine. Hence, a "mammal" includes a human or other non-human primate, livestock animal, laboratory test animal, companion animal or captured wild animal.

A "non-human primate" includes a gorilla, marmoset, chimpanzee or monkey; a livestock animal includes a cow, sheep, goat, pig or horse; a laboratory test animal includes a mouse,

rat, guinea pig or hamster; a companion animal includes a dog or cat. The mammal may be referred to as the "subject", "individual", "patient" or "recipient".

Determining "levels" of a biomarker includes determining presence or absence and also includes determining an amount in terms of inter alia a mass per volume of fluid, mass per mass of another molecule or activity. As indicated above, determining levels includes determining concentrations.

The levels may be determined in CVF in situ or in. CVF isolated from the subject. Isolation of the CVF may be by any convenient means including syringe, pipette, sampling straw, syphon tube, needle or the like. An "applicator" approach is also contemplated. In this approach a solid support coated with ligands to selected biomarkers is brought into contact with the CVF of a subject. After appropriate visualization techniques (e.g. dyes, labeled antibodies, etc.), a determination can be made of the presence or absence or amount of the biomarker. In addition, the present invention extends to the detection of protomic and metabolmic markers in white blood cells, blood plasma and serum.

The "biomarker" represents a proteomic or metabolomic agent such as in a preferred embodiment, but not limited to, IL-I receptor antagonist, copper-zinc superoxide dismutase, glutathione- S -transferase (GST), thioredoxin, peroxiredoxin II (Prx2), and cystatin A. The levels (i.e. presence, absence or amount) of one or more biomarkers constitutes a profile of markers. In accordance with the present invention, the profile is determined in a subject's CVF and compared to a control, the control may be a comparison over time of the profile of biomarkers in the CVF of the same patient or the comparison may be with respect to normalized levels. Hence, the instant assay can stratify a subject based on changing levels of biomarkers or based on changed levels relative to normalized controls.

Hence, another aspect of the present invention provides a prognostic assay to detect or predict labor or a stage of labor in a pregnant female mammal including a pregnant human, said assay comprising screening for the levels of two or more biomarkers selected from

Interleukin — 1 (IL-I) receptor antagonist, copper-zinc superoxide dismutase, glutathione- S- transferase (GST), thioredoxin, peroxiredoxin II (Prx2), and cystatin A in isolated or in situ CVF to generate a profile of levels of said biomarkers and comparing said profile to a control to thereby assess the risk of development of labor or the stage of labor.

Reference to "approaching labor" means that labor is expected to begin (i.e. at least stage 1 of labor) within 10 days such as from within 30 minutes to 10 days. Examples of approaching labor initiation include labor initiating within 30 minutes, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days of the assay.

The results of the assay enable medical intervention or initiation of medical protocols, depending on gestation. At a preterm gestation medical intervention encompasses (but is not limited to) administration of maternal corticosteroid therapy, uterine tocolytic therapy and, transfer of the pregnant human female to a tertiary medical facility that provides neonatal intensive care in the event of preterm delivery. At a post-term gestation, knowledge of approaching labour permits unnecessary interventions including tests of fetal well-being such as ultrasound, labor induction and Caesarean section.

Hence, the present invention further provides the use of a profile of biomarkers or change in profile of biomarkers of the proteome or metaboloniic in CVF of a pregnant, female mammal in the prediction of initiation of labor.

Still another aspect of the present invention contemplates a method for the management of childbirth in a pregnant, female human prior to 37 weeks gestation, said method comprising screening for levels of one or more biomarkers in the CVF of said pregnant human female and comparing said levels to a control wherein changes in levels of one or more of said biomarkers is indicative of approaching labor, or stage of labor in determining whether a pregnant female human is likely to go into labor or to determining at which stage the labor has progressed, said method comprising screening for levels of one or more biomarkers in the CVF of said pregnant human female and comparing said

levels to a control wherein changes in levels of one or more of said biomarkers is indicative of approaching labor or stage of labor.

The present invention also extends to a kit. The kit may take many forms. In one form, the kit comprises compartments adapted to contain or receive one or more of a CVF sample, a reagent and/or a portal for measuring the level of the biomarker.

The present invention is further described by the following non-limiting Examples.

EXAMPLE l

Development of 2DE gel assay

This example involved the proteomic analysis and identification of proteins present in CVF at term. Initial computerized analysis of 2DE gels performed revealed some degree of variation between different women despite comparing identical gestations. As CVF is non- sterile this was to be expected. In order to overcome the issue of inherent variation, only those proteins consistently matched in a group of pregnant women at term were subjected to formal characterization (protein identification).

The process of 2DE includes obtaining and solubilizing and denaturing a sample from the CVF, undertaking a first-dimension separation based on protein charge and then a second- dimension separation based on molecular size. In the first-dimension separation the charged polypeptide subunits migrate under current until the overall charge is neutral (isoelectric point). The second dimensional separation is perpendicular to the first- dimension and provides a sieving action of proteins by polyacrylamide gel matrix under electric current.

Protein spots are visualized with Sypro Ruby (trade mark) fluorescent stain and gel images can be converted to a digital format using PDQuest (trade mark) Software.

Two-dimensional polyacrylamide gel electrophoresis allows complex mixtures of proteins to be reproducibly separated based on their charge (1st dimension) and molecular weight (2nd dimension). Proteins resolved by 2-DE can be stained with total protein stains (e.g. Coomassie, silver or SYPRO Ruby), or subsequently transferred to PVDF membranes and probed with monoclonal antibodies directed against any antigen or combination of antigens (Western blotting). The topography (or gel coordinates) of proteins resolved using this technique can be reproducibly compared between different gels by computer image analysis. This software allows differences between the proteomic map of different samples to be identified. Differential 'protein spots' can then be excised from the gel and characterised further using mass spectrometry and/or amino- or carboxy-terminal

microsequencing. For 1 st dimension protein separation, both IEF cells (Bio-Rad) and the IPGphor (Amersham-Pharmacia) are available. Gel imaging is performed using a FX-Pro Plus multi-imager (Bio-Rad) which allows fluorescent, isotopic and densitometric imaging. Gel analysis and differential spot determination is performed using the PDQuest 2-D Analysis Software (Bio-Rad).

CVF samples were separated in the first dimension by isoelectric focusing using pre-cast immobilised pH4-7 gradient (IPG) strips. IPG strips were loaded with 15μg total protein in rehydration buffer consisting of 7M Urea, 2M Thiourea, 10OmM DTT, 4% v/v CHAPS, 4OmM Tris, and 0.5% v/v ampholytes. Strips were actively rehydrated overnight and then focused for 35,000Vhr. IPG strips were then equilibrated for slab gel electrophoresis in buffer containing 5.5M Urea, 0.035M SDS, 0.3% glycerol, 0.1% v/v Tris/HCI, and 5mM TBP. Second dimension slab gel electrophoresis was performed using 8-16% gradient gels. Analytical gels were stained with SYPRO Ruby, washed and imaged using an FX imager. Composite 2-D gels were analysed using PD Quest software. Only those proteins that display a significant difference in abundance between study groups will be subjected to sequence analysis.

Differentially expressed proteins were characterized using peptide-mass fingerprinting mass spectrometry. Protein spots from 2-D gels were excised using an automated spot cutter in batches of 96 and washed in 60:40 5OmM NH4HC03:acetonitrile and then dried in a SpeedVac (Savant) for 25 minutes. Dried gel pieces were re-swollen in 15μl of

10mg/ml trypsin in 5OmM NH ₄ HCO ₃ at 4°C for 45 minutes and then incubated overnight in 15μl 5OmM NH ₄ HCO ₃ at 37 ⁰ C. Tryptic digest peptides are analyzed in a MALDI-ToF mass spectrometer (Bruker Daltonics). For proteins that cannot be identified in automated mode, peptide mixtures were concentrated and de-salted using a C 18 Zip-Tip (Millipore) and screened again via MALDI-ToF mass spectrometry. Proteins that cannot be identified at this stage were re-screened using a de novo sequencing approach utilising a LC Ion Trap for MS/MS amino acid sequence for up to 5 separate peptides.

Subject recruitment, subsequent gel analysis and spot excision were undertaken. In order to obtain optimal gels for software analysis, a standard quantity of total protein was loaded on to each gel. This resulted in technical difficulties in protein characterization given the relatively small amounts of protein present in each protein spot. When the 2DE gels were deliberately loaded with a higher total protein load, the resolution of the resulting gels was poor and several of the proteins of interest were not visualized. This highlighted a technical limitation of 2DE. With complex fluids containing many different proteins (such as CVF), as the protein load is increased, high-abundance proteins obscured the low-abundance proteins. Three different methods of mass spectrometric analysis were investigated in order to achieve accurate protein characterization. Fifteen proteins have been successfully identified and are displayed on a representative image of a 2DE gel (Figure 1 (b)).

EXAMPLE 2 Protein Characterization

Proteins identified in Example 1 were excised from the gel and analysed by a range of techniques including matrix-assisted laser desorption ionisation/time-of-flight mass spectrometry or capillary-liquid chromatography using reversed-phase HPLC coupled to electrospray tandem mass spectrometry using an ion-trap mass spectrometer. Figure l(a) shows the results of 2DE of CVF of a pregnant woman at 37 weeks gestation. Figure 1 (b) shows the proteins in the CVF at 37 weeks gestation.

The proteins identified were IL-I receptor antagonist, copper-zinc superoxide dismutase, Glutathione S-transferase P, Thioredoxin, Peroxiredoxin 2, Cystatin A, Fatty acid binding protein, beta-actin, squamous cell carcinoma antigen 1, monocyte/neutrophil elastase inhibitor, annexin III, acyl-CoA-binding protein, transthyretin precursor, albumin precursor, and alpha- 1 -antitrypsin. Results are shown in Table 2.

TABLE 2

Significant Spots In Labor

EXAMPLE 3 Analysis of CVF

The example investigated proteomic analysis of CVF to determine changes in protein expression in late pregnancy as labor approaches. Ten women were recruited at 36 weeks' gestation. These ten women provided between 2 and 5 serial CVF samples for analysis.

Serial CVF samples obtained from the same woman were utilized to overcome inherent sample variation. Extensive software analysis was used to determine the changes in protein expression between different time points leading up to and including labor. Utilizing paired t-tests between CVF samples obtained 30 days from labor and in labor, a number of proteins demonstrated significant differences in expression. Some of these changes are apparent 1 -2 days prior to spontaneous labor onset. These proteins have been selected for mass spectrometric characterization. The results are shown in Figure 2 and in

Table 2.

EXAMPLE 4 Analysis of CVF in preterm subjects

It is proposed that the proteins identified in Example 1 serve as markers of the final common pathway of labor. It is proposed that these proteins provide a guide for identifying preterm labor. The methodology is as in Example 2. Women were screened who labored spontaneously at a preterm gestation. CVF samples were obtained from 9 women at 26-30 days prior to spontaneous term labor and compared with CVF from 4 women obtained 1 -4 days prior to spontaneous preterm (21-36 weeks) labor. Four spots of significance were identified which were all down-regulated. The results are shown in Figure 3.

EXAMPLE 5 Analysis of IL-I receptor antagonist

Cervico-vaginal samples of pregnant women at term were screened for Interleukin-1 (IL-I) receptor antagonist using an ELISA kit (BioSource International, Carmarillo, California, USA) [Figure 4].

IL-I receptor antagonist demonstrates a significant decreasing trend towards labor (one way ANOVA, p=0.025). Using LSD post-hoc test, there is a tenfold significant decrease (p=0.033) in IL-I receptor antagonist level between 14+2 days and 0 days (in-labor). There is also a significant elevenfold difference between 7+2 and 0 days (p=0.004). IL-I receptor antagonist levels were decreased by sixfold between 2+1 days and 0 days (p=0.154).

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

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