WO2008047086A2

WO2008047086A2 - Biomarker

Info

Publication number: WO2008047086A2
Application number: PCT/GB2007/003913
Authority: WO
Inventors: William Atiomo; Robert Layfield; Catherine Hughes
Original assignee: The University Of Nottingham
Priority date: 2006-10-16
Filing date: 2007-10-15
Publication date: 2008-04-24
Also published as: WO2008047086A3; JP2010507093A; EP2076771A2

Abstract

A method of determining the Polycystic Ovarian Syndrome (PCOS) status of a subject, comprising the steps of (i) providing a sample of material obtained from a subject; (ii) determining the level of one or more of the following proteins: the complement C4 precursor protein; a cleavage fragment of the complement C4 precursor protein; and a haptoglobin; in the sample and (iii) comparing the levels determined with one or more reference values.

Description

BIOMARKER

The present invention relates to novel biomarkers for Polycystic Ovarian Syndrome (PCOS) , and to methods of determining the PCOS status in a subject using these novel biomarkers.

Women with Polycystic Ovary Syndrome (PCOS) often present to the clinician with a wide range of symptoms, the most common being sub/infertility with menstrual irregularities and/or problems associated with hyper androgenemia including acne, hirsutism and androgenic alopecia. Many of these women also suffer from obesity and may be at increased risk of diabetes, heart disease and endometrial cancer in later life. PCOS is thought to affect 5-10% of women of reproductive age in the U.K. Stein and Leventhal initially characterised the phenotype of PCOS in 1935 and significant advances have since occurred in the understanding of the pathophysiology, diagnosis, clinical presentation and treatment. Despite these advances, there is still no consistent diagnostic marker available and the development of effective treatments for this multifaceted syndrome remains a challenge.

There are currently three commonly advanced hypotheses relating to what causes PCOS, these involve (i) the hypothalamo-pituitary-ovarian axis (ii) insulin resistance and (iii) androgen levels. For example, it is thought that the endocrinological basis of PCOS stems from the hypothalamus where increased discharge of gonadotrophin-releasing hormone increases the pulse-frequency of luteinising hormone (LH) being released from the pituitary gland. The raised LH levels are thought to increase the production of androgens by ovarian thecal cells, thus increasing the levels of circulating free testosterone in affected women. However, LH hypersecretion is not universal in all subjects diagnosed with PCOS. In another example, insulin resistance is understood to increase androgen production by inhibiting sex hormone-binding globulin (SHBG) synthesis and acting synergistically with LH to directly stimulate theca cell androgen production. However, the lack of a precise aetiology for insulin resistance in PCOS and the presence of selective ovarian sensitivity in the presence of peripheral insulin resistance further reflects the challenges of PCOS. Hyperandrogenism appears to underlie all other manifestations of PCOS, again, the initial cause and molecular mechanisms of hyper androgenaemia are by no means clear.

The current diagnosis of PCOS is one of exclusion of other androgenic- based diseases following clinical identification of at least two of the following three criteria: (i) chronic oligo-/anovulation; (ii) clinical and/or biochemical signs of hyperandrogenism; and (iii) ultrasound evidence of polycystic ovaries. However, relative weightings of each criterion tend to vary depending on the clinician's medical background, again highlighting the clinical challenges of PCOS. Treatment strategies also vary depending on the patient's presenting features and desired outcome, tending towards symptom-based strategies for gynaecological or cosmetic improvements or simple weight management and life-style changes.

Thus there remains a need for a simple, rapid, reliable, reproducible method for diagnosing PCOS, and for suitable new therapeutic targets for the treatment/alleviation of the disease.

The present invention provides novel biomarkers useful in the diagnosis of PCOS, as well as methods and kits for using the biomarkers to diagnose PCOS and determine the PCOS status of a subject. According to a first aspect, the invention provides a method of determining the Polycystic Ovarian Syndrome (PCOS) status of a subject, comprising the steps of:

(a) providing a sample of material obtained from a subject; (b) determining the level of one or more of the following proteins: the complement C4 precursor protein; a cleavage fragment of the complement C4 precursor protein; and a haptoglobin; in the sample and

(c) comparing the levels' determined with one or more reference values.

Preferably the method allows the diagnosis of PCOS in a subject from the analysis of the proteins in a sample provided by the subject.

The complement C4 precursor protein, cleavage fragments of the complement C4 precursor protein and haptoglobins have been identified as biomarkers for PCOS, and are referred to herein as the biomarkers or the biomarker proteins.

These biomarkers may be used to determine the PCOS status of a subject, for example, to diagnose PCOS. The phrase "PCOS status" includes any distinguishable manifestation of the PCOS disease, including diseased and non-diseased. For example, PCOS status includes, without limitation, the presence or absence of disease, the risk of developing the disease, the stage of the disease, the progression of the disease (e.g. , progress of disease or remission of disease over time) and the effectiveness or response of a subject to treatment of the disease

Typically the method of the invention will be used in conjunction with the assessment of clinical symptoms. The C4 precursor protein is one component of the complement pathway, and is an important contributor to the immune response system. C4 plays a central role in the classical pathway of the complement system. The DNA sequence of C4 can be found as two isoforms in the GenBank database, these are accessible via accession numbers NM007293 for isoform A (http : //www . ncbi . nlm . nih . gov/entrez/viewer . f cgi?db = nucleotid e&val = 67190747) and NM001002029 for isoform B (http : //www . ncbi . nlm . nih . gov/entrez/viewer . f cgi?db = nucleotide&val = 50 345295) . The corresponding proteins have the accession numbers POCOL4 and POCOL5 (the protein sequence for the C4 precursor can be found at http : //www . ncbi . nlm . nih . gov/entrez/ viewer .f cgi?db = nucleotide&val = 67 190747 and the corresponding DNA sequence can be found at http : //www . ebi . ac . uk/cgi-bin/expasyf etch?K02403) . The protein sequence of each isoform is given in Figures 8 and 9.

The terms C4 protein and C4 precursor protein refer to the same protein, and are used interchangeably herein.

The one or more cleavage fragments of the complement C4 precursor protein may be selected from any of the group comprising the polypeptides: a) C4α; b) C4β; c) C4γ; d) C4a anaphylatoxin; e) C4b; f) C4α₃c fragment; g) C4αd; h) C4α₄c fragment; and i) a polypeptide with at least 75% amino acid sequence identity, more preferably at least 80%, 85%, 90% or 95% or more sequence homology, to the amino acid sequence of any of a) to h) .

Preferably at least one or the fragments a) to i) is detected in the method of the invention, more preferably at least 2, 3, 4, 5, 6, 7, 8, or 9 of the fragments a) to i) are detected.

The amino acid sequence of the cleavage fragments a) to h) can be found with reference to Figures 8 and 9.

The one or more haptoglobin proteins may be selected from the group comprising haptoglobin α and haptoglobin β. The amino acid sequence of both haptoglobins is given in Figure 10.

Proteins frequently exist in the body, and in samples derived there from, in a plurality of different forms. These forms can result from either or both of pre- and post-translational modification. When detecting or determining the level of a protein in a sample, the ability to differentiate between different forms of a protein depends upon the nature of the difference and the method used to detect or measure the protein level. For example, an immunoassay using a monoclonal antibody will detect all forms of a protein containing the epitope and will not distinguish between them. However, a sandwich immunoassay that uses two antibodies directed against different epitopes on a protein will detect all forms of the protein that contain both epitopes and will not detect those forms that contain only one of the epitopes.

In methods of the invention the assay method used to determine the level of one or more of the biomarker proteins preferably detects all forms of the specific biomarker protein. Preferably at least all biologically active forms of the specific biomarker protein are detected. Preferably, all forms of any of the biomarker proteins with at least 75% or more, preferably at least 80%, 85%, 90% or 95% or more, identity with the amino acid sequence of the biomarkers given in Figures 8, 9 and 10, will be detected in the method of the invention.

Methods of measuring polypeptide/protein identity are well known in the art. For example the UWGCG Package provides the BESTFIT program which can be used to calculate identity (e.g. used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395).

The PILEUP and BLAST algorithms can also be used to calculate homology or line up sequences (typically on their default settings), for example as described in Altschul S.F. (1993) J MoI Evol 36:290-300; Altschul, S, F et al (1990) J MoI Biol 215:403-10.

Software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).

The method of the invention preferably will allow the level of biomarker proteins with polymorphisms common in the general population to be detected. Polymorphisms can occur that do not affect the function of a protein, it is intended that the method of the invention will allow these to be detected.

The sample material obtained from the subject may comprise whole blood, blood serum, blood plasma, urine, fat tissue (adipose) , endometrial tissue, ovary tissue or any other bodily fluid or tissue. The level of the one or more of the complement C4 precursor protein, a cleavage fragment of the complement C4 precursor protein, and a haptoglobin, may be determined by any suitable assay which may comprise the use of any of the group comprising immunoassays, spectrometry, mass spectrometry, Matrix Assisted Laser Desorption/Ionization Time-of- Flight (MALDI-TOF) Mass Spectrometry, microscopy, northern blot, western blot, southern blot, isoelectric focussing, SDS-PAGE, PCR₁ RT-PCR, gel electrophoresis, protein microarray, DNA microarray, and antibody microarray, or combinations thereof. Preferably the level of the one or more biomarkers is determined using an immunoassay. An immunoassay uses an antibody or antibodies to a specific antigen to determine the levels of the antigen. In this case, an antibody or antibodies specific to one or more of the C4 precursor protein, or a cleavage fragment of the C4 precursor protein or a haptoglobin may be used. The immunoassay may be an enzyme linked immunoassay (ELISA) , a sandwich assay, a radioimmunoassay, a Western Blot, an immunoassay using a biosensor, an immunoprecipitation assay, an agglutination assay, a turbidity assay or a nephelometric assay.

The one or more antibodies may be synthetic, monoclonal, polyclonal, bispecific, chimeric or humanised. A chimeric antibody includes portions derived from different animals. Humanised antibodies are antibodies from non-human species having one or more complementarity determining regions from the non-human species and a framework region from a human immunoglobulin molecule. Chimeric and humanised antibodies can be produced by recombinant techniques well known in the art.

The one or more antibodies may comprise a tag or a label selected from the group comprising a radioactive, a fluorescent, a chemiluminescent, a dye, an enzyme, or a histidine tag or label, or any other suitable label or tag known in the art. The presence, and possibly even the level, of particular proteins in a sample may also be determined by using mass spectrometry techniques. Mass spectrometry techniques may be used to detect gas phase ions which correlate to specific proteins or parts of proteins, such as, trypsin peptides. Examples of mass spectrometers include time of flight, magnetic sector, quadruple filter, ion trap, ion cyclotron resonance, electrostatic sector analyser and hybrids of these.

The mass spectrometer may use laser desorption/ionisation.

MALDI (Matrix-assisted laser desorption/ionization) peptide mass fingerprinting may be used to identify the presence of particular biomarker proteins from 2D gel analysis and RPE/SDS PAGE analysis.

MALDI-MS and ANN analysis may be used to profile identified proteins and tryptic biomarker signatures for PCOS.

Preferably the reference values to which the determined levels of one or more of complement C4 precursor protein, a cleavage fragment of the complement C4 precursor protein, and a haptoglobin, are compared, are the levels observed in subjects who do not have any clinical symptoms of PCOS, so called "normal values" .

Alternatively, the reference values may be the previous values obtained for a specific subject. This kind of reference value may be used if the method is to monitor progression of a disease or to monitor the response of a subject to a particular treatment.

When the determined levels of one or more of complement C4 precursor protein, a cleavage fragment of the complement C4 precursor protein, and a haptoglobin, are compared with reference values, an increase or a decrease in the level of one or more of the biomarker proteins may be indicative of PCOS status of the subject.

More specifically an increase in the level of C4α₃c and/or the level of C4α₄c and/or the level of haptoglobin α and/or haptoglobin β may be indicative, or diagnostic, of PCOS.

A decrease in the level of the C4 precursor protein and/or C4α and/or C4β and/or C4γ and/or C4a anaphylatoxin may be indicative, or diagnostic, of PCOS.

The method of the invention may also be used to monitor disease progression and/or to monitor the efficacy of treatments administered to a subject. This may be achieved by analysing samples taken from a subject at various time points following initial diagnosis and monitoring the changes in the levels of biomarkers and comparing these levels to reference values.

In this case reference levels may include the initial levels of the biomarkers in the subject, or the levels of the biomarkers in the subject when they were last tested, or both.

Preferably the method of the invention is carried out in vitro .

The subject may be mammal, and is preferably a human, but may alternatively be a monkey, ape, cat, dog, cow, horse, rabbit or rodent.

According to another aspect, the invention provides one or more of the complement C4 precursor protein, a cleavage fragment of the complement

C4 precursor protein and a haptoglobin for use as a biomarker, or for use as part of a panel of biomarkers, to determine the PCOS status of a subject. Preferably a panel of biomarkers comprises at least 2, 3, 4, 5 or more of the biomarkers selected from the list comprising C4 precursor protein, C4α, C4β, C4γ, C4a anaphylatoxin, C4b, C4α₃c fragment, C4αd, C4cc₄c fragment, haptoglobin α and haptoglobin β.

According to another aspect of the invention there is provided a method of determining the PCOS status in a subject, comprising the steps of; (a) providing a sample of material obtained from a subject; (b) analysing proteins or tryptic peptides obtained from the sample using MALDI-MS;

(c) concluding that the presence of the six ions m/z 8674,

8668, 1351, 8727, 8673 and 6871 in a protein sample and/or the presence of the three ions m/z 2924, 3025 and 1977 in a tryptic peptide sample, is diagnostic of PCOS in the subject.

The six ions m/z 8674, 8668, 1351 , 8727, 8673 and 6871 and the three ions m/z 2924, 3025 and 1977 are biomarkers for PCOS.

According to another embodiment of the invention there is provided a kit for use in determining the PCOS status of a subject comprising instructions to analyse a sample for the presence of the six ions m/z 8674, 8668, 1351, 8727, 8673 and 6871 in a protein sample and/or the presence of the three ions m/z 2924, 3025 and 1977 in a tryptic peptide sample.

According to another embodiment of the invention there is provided a kit for use in determining the PCOS status of a subject comprising at least one agent for determining the level of one or more of the complement C4 precursor protein, a cleavage fragment of the complement C4 precursor protein and a haptoglobin. The agent may be an enzyme, an antibody, a nucleic acid, a protein probe or other suitable composition.

The agent for determining the level of the one or more PCOS biomarkers is preferably labelled. The kit may also comprise means for detecting the label.

The kit may comprise one or more capture agents for capturing one or more biomarker proteins the level of which is to be determined. The capture agent may be one or more antibodies.

The capture agent or the agent for determining the level of the one or more PCOS biomarkers may be attached to a solid support. The solid support may be a chip, a micro titre plate, a bead or a resin.

The kit may also comprise a wash solution or instructions for making a wash solution. The wash solution may, alone or in combination with the capture agent, allow efficient capture of a biomarker or biomarkers on a solid support for subsequent detection by, for example, mass spectrometry or immunoassay methods.

The kit may comprise instructions for suitable operational parameters in the form of a label or separate insert. The instructions may inform a consumer about how to collect the sample, and/or how to wash a capture agent, and/or details of the particular biomarkers to be detected.

The kit may comprise one or more biomarker samples, to be used as standard(s) for calibration and comparison. According to a yet further aspect, the invention provides the use of the determination of the levels of the C4 precursor protein and/or cleavage fragments of the C4 precursor protein and/or haptoglobin chains, with or without the detection of other PCOS biomarkers, as a means of assessing the PCOS status in an individual.

According to a further aspect, the invention provides a probe set capable of detecting one or more of the aforementioned PCOS biomarkers. The man skilled in the art will appreciate how to design suitable probes, which may, for example, be a synthetic molecule, an antibody, a nucleic acid or proteinacious in nature. The probe may also carry one or more labels to facilitate its detection; the label may, for example, be a radioactive, a chemiluminescent or a florescent label, but is not limited to these examples. Preferably the probe set contains at least two probes directed to different biomarker proteins.

According to another aspect, the invention provides a method of treating PCOS in a subject comprising administering to the subject an agent capable of modulating the level of one or more PCOS biomarkers in a cell, wherein said one or more biomarkers are selected from the group comprising the complement C4 precursor protein, a cleavage fragment of the complement C4 precursor protein and a haptoglobin. The agent may be an antisense or an interfering RNA molecule designed to inhibit or reduce expression of a biomarker protein in the cell.

Alternatively the agent may be an inhibitor of C4 precursor protein processing, or a composition which reduces complement activity.

According to a further aspect, the invention provides a method of determining the course of PCOS in a subject, or determining the efficacy of treatment administered to a subject with PCOS, comprising: (a) determining the level of one or more of the following proteins: the complement C4 precursor protein; a cleavage fragment of the complement C4 precursor protein; and a haptoglobin; in a sample obtained from a subject at a first time point;

(b) determining the level of one or more of the following proteins: the complement C4 precursor protein; a cleavage fragment of the complement C4 precursor protein; and a haptoglobin; in a sample taken from a subject at a second later time point; and

(c) comparing the first measurement and the second measurement, wherein the comparative measurements indicate the course of the PCOS.

This method may be used to compare sample taken at two or more time points.

According to a further aspect the invention provides a method of identifying compounds for treating PCOS comprising screening for one or more compounds that modulate the level of one or more of the complement C4 precursor protein, a cleavage fragment of the complement C4 precursor protein, and a haptoglobin, in vitro or in vivo.

Compounds suitable for therapeutic testing may be screened initially by identifying compounds which interact with one or more of the PCOS biomarkers listed herein. By way of example, screening might include recombinantly expressing a biomarker, purifying the biomarker, and affixing the biomarker to a substrate. Test compounds would then be contacted with the substrate, typically in aqueous conditions, and interactions between the test compound and the biomarker may be measured, for example, by measuring elution rates as a function of salt concentration. Certain proteins may recognize and cleave one or more of the biomarkers, in which case the proteins may be detected by monitoring the digestion of one or more biomarkers in a standard assay, e.g. , by gel electrophoresis of the proteins .

In a related embodiment, the ability of a test compound to inhibit the activity of one or more of the biomarkers may be measured. One of ordinary skill in the art will recognize that the techniques used to measure the activity of a particular biomarker will vary depending on the function and properties of the biomarker. For example, enzymatic activity of a biomarker may be assayed provided that an appropriate substrate is available and provided that the concentration of the substrate or the appearance of the reaction product is readily measurable. The ability of potentially therapeutic test compounds to inhibit or enhance the activity of a given biomarker may be determined by measuring the rates of catalysis in the presence or absence of the test compounds.

Test compounds capable of modulating the activity of any of the PCOS biomarkers may be administered to patients who are suffering from or are at risk of developing PCOS. For example, the administration of a test compound which increases the activity of a particular biomarker may decrease the risk of PCOS in a patient if the activity of the particular biomarker in vivo prevents the accumulation of proteins which cause PCOS. Conversely, the administration of a test compound which decreases the activity of a particular biomarker may decrease the risk of PCOS in a patient if the increased activity of the biomarker is responsible, at least in part, for the onset of PCOS.

A yet further aspect provides the use of the C4 precursor protein, C4α, C4β, C4γ, C4a anaphylatoxin, C4b, C4α₃c fragment, C4αd, C4α₄c fragment, haptoglobin α and haptoglobin β and/or other PCOS biomarkers as therapeutic targets for the treatment / alleviation of PCOS.

The skilled man will appreciate that preferred features of any one embodiment and/or aspect of the invention may be applied to all other embodiments and/or aspects of the invention.

The present invention will be further described in more detail, by way of example only, with reference to the following figures in which:

Figure 1 - shows the results of 2-dimensional gel electrophoresis illustrating the protein level changes between PCOS subjects and matched control subjects. The upper 10 images are silver-stained 2D gels in which the protein spots of interest are circled, and the difference in the levels of protein between PCOS subjects and control age/BMI-matched subjects can be seen. The lower 5 images are genereated using Delta 2D Image Analysis Software and are merged images of the corresponding gel pairs showing the same protein spot in a colorimetric display. Whilst the colours are not visible, the results show that the observed spot appears in four of the five gel pair merged images coloured orange indicating that it occurs only in the PCOS subjects. The spot was subsequently identified as C4α₃c;

Figure 2 - shows a quantitative analysis of the intensity of the

C4α₃c spot between age- weight matched PCOS subject-control subject gel pairs. The data was calculated using DELTA 2D image analysis software.

Figure 3 - shows a western blot analysis of 10 depleted serum samples using an antibody raised against an unknown C4α₄c peptide sequence. Figure 3 A shows western blot analysis of whole serum from the same sample set used in earlier 2DE studies. The blot was probed with a polyclonal anti-C4α antibody raised against epitopes in the C4α chain. The ~ 24 kDa band shown represents the C4α₄c fragment. Figure 3B shows quantitative analysis of the band intensity in Figure 3 A, determined using SynGene Gene Tools densitometry software. * P= 0.03;

Figure 4 - shows a schematic representation of the primary structure of the complement component C4 protein. The C4 precursor and a number of its cleavage fragments are shown. The C4 protein is shown to consist of three chains, which can all be cleaved from the single C4 precursor and folded to form the protein's functional structure. The α chain is the active element, when cleaved the C4a fragment is released to act as the anaphylatoxin. The C4b fragment forms a complex with C2 to continue the enzymatic cascade of the classical complement pathway and can also act as an opsonin. C4b is later cleaved into C4α3c, C4αd and C4α4c fragments, this inactivates the complement cascade (based on Nagasawa et al. , (1980) J Immunol 125(2): 578-82 and Hessing, (1991) Biochem J

277:581-92) . Note: t C4α₃c fragment identified in 2DE analysis as being up regulated in PCOS samples. Φ C4α₄c fragment shown by Western blot to also be up regulated in PCOS. * C4γ chain identified in the SDS PAGE analysis of the RP-SPE fractions as being down regulated in PCOS;

Figure 5 - shows RP-SPE analysis showing differences between

PCOS and matched controls. Figure 5 A shows a ~ 33kDa band present in 5/6 control samples and only very low level in 3/6 PCOS samples for fraction 12 only. There is potentially a similar sized band in fractions 4 and 5 but this is taken to be a different protein species. Figure 5B shows a ~ 40kDa band present in all PCOS samples at an increased level than in the control samples. This difference was most pronounced in fractions 8-12. Figure 5C shows a ~ 17kDa band present in 5/6 PCOS samples and only 2/6 control samples;

Figure 6 - shows a quantitative RP-SPE analysis showing differences between PCOS and matched controls. Figure 6 A shows the results of densitometric quantification of the ~ 40Kda band seen on RP-SPE (Figure 5B) . P = 0.001. Figure 6B shows the results of densitometric quantification of the ~ 17Kda band seen on RP-SPE (Figure 5C) ;

Figure 7 - illustrates representative MALDI-TOF mass spectra of:

Figure 7A serum peptides after tryptic digestion and from Figure 7B proteins after Ci₈ ZipTip clean-up from PCOS patients and control sera. This spectrum illustrates the observed differences between PCOS sera and control sera. Figure 7C shows the predictive capability of ANNs to recognise tryptic peptide profiles based on a 3 ion model and Figure 7D shows protein profiles based on a six ion ANNs model. The grey bars indicate control samples and the black bars indicate PCOS samples. A predicted value below 1.5 indicates a control sample, whilst a prediction greater than 1.5 indicates a PCOS sample;

Figure 8 (Sequence ID No: 1) - is the protein sequence of the C4 precursor protein of isoform A. More specifically, amino acids 20 to 675 are the C4β cleavage fragment, amino acids 680 to 1446 are the C4α cleavage fragment, amino acids 680 to 756 are the C4a anaphylatoxin cleavage fragment, amino acids 757 to 1446 are the C4b cleavage fragment, amino acids 757 to 956 are the C4α₃c cleavage fragment, amino acids 957 to 1333 are the C4αd cleavage fragment, amino acids 1334 to 1446 are the C4α₄c cleavage fragment, and amino acids 1454 to 1744 are the C4γ cleavage fragment;

Figure 9 (Sequence ID No: 2) - is the protein sequence of the C4 precursor protein of isoform B. The amino acids different to isoform A are underlined; and

Figure 10 (Sequence ID No: 3) - is the protein sequence of haptoglobin. Amino acids 19 to 161 are haptoglobin α and amino acids 162 to 406 are haptoglobin β .

MATERIALS AND METHODS

Subjects

The study comprised eleven women (aged 18-40 years) diagnosed with PCOS, and ten age-BMI matched controls. _N Women with PCOS were initially identified at the infertility, gynaecology or PCOS clinics run at the Queen's Medical Centre, Nottingham and were then approached verbally to participate in the study. Control women without PCOS were recruited by poster advertisements, from the same gynaecology clinics that women with PCOS were recruited from and also from female members of staff. Ethical approval for the study was granted by the Nottingham Local Research Ethics Committee and informed consent was obtained for each subject.

Clinical Assessment

Demographic details along with menstrual, gynaecological, obstetric and other medical histories were collected for each subject during a clinical interview. A regular menstrual cycle was defined as being between 21 and 35 days. The presence of acne and hirsutism was assessed and hirsutism defined as a Ferriman Gallwey score of > 7 (Ferriman and Gallwey, (1961) J Clin Endocrinol Metab 21 : 1440-7) . Subjects were excluded from the study if there was a history of thyroid disease, hyperprolactinaemia, recent delivery, miscarriage or surgery, a history of myocardial infarction, thrombosis or haematological disease or current use of sex steroid therapy. Various anthropometric measurements were also taken at this interview. Height and weight measurements were used to calculate Body Mass Index (BMI) , waist and hip circumferences were measured in centimetres and blood pressure was taken using an appropriately sized cuff with the patient in a sitting position. Following a clinical interview fasted blood samples were collected for each woman. For all control women and, where possible, in women with PCOS, samples were taken during the follicular phase of their menstrual cycle. A transvaginal pelvic ultrasound was also performed. The sonographer performing the ultrasound scans and the clinical chemistry department were blind to the clinical diagnosis of each subject. Blood samples were collected in BD vacutainer SST tubes for biochemical testing and BD vacutainer Fluoride tubes for glucose determination (both from BD Biosciences, Oxford, U.K.) . The samples were assayed for endocrines to measure blood glucose, insulin, lipid profile (triglycerides, total cholesterol and HDL cholesterol) and serum hormone profile (testosterone, SHBG, luteinising hormone (LH) , follicle stimulating hormone (FSH) , 17-hydroxyprogesterone and prolactin) .

Subjects were diagnosed with PCOS, using the Rotterdam criteria (Rotterdam ESHRE/ASRM Sponsored PCOS Consensus Workshop (2004) Fertil Steil 81 (1) : 19-25) if they presented with 2 or more of oligo- /anovulation, polycystic ovaries and clinical and/or biochemical signs of hyper androgenism. Hyper androgenism was defined clinically by hirsutism (Ferriman Gallwey score > 7) and biochemically by raised testosterone ( > 2nmol/L) and/or a raised ( > 10) free androgen index (testosterone/SHBG x 100) . Control subjects had regular 21-35 day menstrual cycles, no ultrasound evidence of polycystic ovaries or evidence of hyper androgenism.

Sample Preparation

Serum samples were collected concurrently over a 12 month period from 21 subjects. The samples were separated within an hour of collection from coagulated whole blood in SST tubes (obtained in parallel with samples for biochemical testing) by centrifugation at 4,000 rpm for 10 min at 4⁰C and stored at -8O⁰C until required. Samples were arbitrarily labelled to maintain anonymity as P- for PCOS samples and C- for controls with consecutive numbering in each group. PCOS and control women with highly similar age and BMI were matched in sample pairs.

Serum Depletion and Purification of Samples

For the initial study serum samples (15 μl) from five age-BMI matched PCOS-control pairs were simultaneously depleted of albumin and immunoglobulin G (IgG) using antibody-based columns according to the manufacturer's protocol (Albumin & IgG Removal Kit; Amersham Biosciences, Little Chalfont, Bucks, U.K.) . The resultant samples ( ~ 500 μl) were subjected to protein precipitation according to the manufacturer's protocol (2D clean-up kit; Amersham Biosciences) . The protein pellet was then resuspended at room temperature in solubilisation buffer (7M urea (Sigma, Poole, U.K.), 2M thiourea (Sigma) , 4% w/v 3-(cyclohexylamino)-l-proρanesulfonic acid (CHAPS; Amersham Biosciences) , 1OmM dithiothreitol (DTT; Fisher Scientific, Loughborough, Leics, U.K.) , 1% v/v BioLyte carrier ampholytes 3-10 (BioRad, Hercules, CA, U.S.A.)) , to a final concentration of 5 μg/μl, as determined by modified Bradford assay (Quick Start Protein Assay; BioRad) .

Isoelectric Focussing (IEF)

IEF was performed using a PROTEAN IEF Cell (BioRad) . ReadyStrip™ 11 cm immobilised pH gradient (IPG) strips, pH 3-10NL (BioRad) were rehydrated for 16 hours at 20°C with 188 μl IEF buffer (7M urea, 2M thiourea, 4% w/v CHAPS, 10OmM bis(2-hydroxyethyl) disulfide (HED; DeStreak reagent, Amersham Biosciences) , 0.5% v/v BioLyte carrier ampholytes 3-10) . Following strip rehydration 12 μl of sample (60 μg total protein) was loaded at the cathode end of each strip. An optimised IEF programme was then run at 2O⁰C applying 250 V for 15 min, linear ramping to 8,000 V over 2.5 h and focussing at 8,000 V for 50 kVh. All 10 serum depleted and purified samples were focussed simultaneously and the strips stored individually at -20°C until required.

Second Dimension SDS-Polyacrylamide Gel Electrophoresis (SDS- PAGE)

Each age and BMI matched PCOS-control pair of sample strips produced by IEF was thawed and equilibrated at room temperature, first for 15 min in equilibration buffer (0.37M Tris.HCl (pH 8.8; Invitrogen, Paisley, U.K.) , 6M urea, 2% w/v sodium dodecyl sulphate (SDS; Fisher Scientific) , 30% v/v glycerol (Sigma)) with 2% w/v DTT and subsequently for 15 min in equilibration buffer with 2.5% w/v iodoacetamide (Sigma) . SDS-PAGE was performed on uniform 13% acrylamide or 8-20% acrylamide gradient gels with the equilibrated strips sealed onto the top of the gels using 1% w/v low-melt agarose (BioRad) made in Ix gel running buffer (0.25M Tris, 1.86M glycine (Fisher Scientific) , 0.1% w/v SDS) . Electrophoretic separation of the two strips were run in parallel, and was performed at 22 rtiA per gel for 3.5 h at room temperature. Proteins in the gels were stained using a modified silver staining protocol (Plus One Silver Stain Kit; Amersham Biosciences) compatible with mass spectrometry (Y an et al. , 2000) with the development step performed at 4°C to improve spot resolution. Imaging of the stained gels was performed using Agfa Fotolook v3.0 and a Duoscan T1200 flatbed scanner followed by qualitative and quantitative analysis using DELTA 2D image analysis software (v3.3; Decodon, Greifswald, Germany) . Stained gels were stored in dH₂O at room temperature.

Western Blotting

Equal amounts (50 μg) of diluted (1 :10 in dH₂O) serum samples from the initial 10 samples were diluted 1:1 in gel loading buffer and denatured by heating at 95°C for 7 min. Samples were loaded on 5-20% acrylamide SDS-PAGE gels and separated by electrophoresis at 35 mA per gel for 2.5 h. Protein in the gels was then transferred to nitrocellulose membrane by blotting overnight at 40 mA. The membrane was blocked using 5 % w/v skimmed milk (Marvel, Premier International Foods (UK) Ltd, Lines, U.K.) in TBS-T (1OmM Tris.HCl (pH 7.5) , 15OmM sodium chloride (Fisher Scientific) , 0.05 % (v/v) Tween-20 (Sigma)) and probed with a polyclonal primary antibody raised against the complement component C4α chain (Santa Cruz, Heidelberg, Germany) followed by a horseradish peroxidase-conjugated secondary antibody (Dako, Ely, Cambs, U.K.) . Antibody binding was detected by chemiluminescence using an ECL detection kit (Amersham Biosciences) .

RP-SPE and SDS PAGE Serum Fractionation Serum fractionation of 6 PCOS and 6 control samples was performed using solid phase extraction (SPE) employing large pore (1000 A) polystyrenedivinylbenzene (PDVB) 25 mg resin (International Sorbent Technologies, mid-Glamorgan, U.K.) in reversed phase (RP) mode. Mass spectrometry grade mobile phase (Riedel de Haen, Sigma) was delivered, using an applied vacuum, at flow rates similar to lmL.min-1 and with the composition of ImL deliveries being adjusted volumetrically. Resin was solvated using 70% v/v acetonitrile (MeCN, Sigma) 0.1% v/v trifluoroacetic acid (TFA, Sigma) and equilibrated with aqueous 0.1% TFA. Serum samples (100 μl equating to ~ 7mg total protein) from the age-BMI matched PCOS-control pairs were applied to the solid phase columns. Binding of sample proteins was dependent upon molecular hydrophobicity (Badock et al. , 2001) . Columns were then washed with 0.1% v/v TFA and retained components were eluted in fractions using 5 to 100% v/v step-wise incrementing MeCN composition. Eluates were directly compatible with analysis by electrospray or MALDI-TOF mass spectrometry but in this instance were subjected to electrophoretic analysis subsequent to drying by centrifugal vacuum evaporation (BETA- RVC, Christ Gefriertrocknungsanlagen Gmbh, Osterode am Harz, Germany) and resuspension in 100 μl gel loading buffer (0.15M Tris.HCl (pH 6.8), 8M urea, 2.5% w/v SDS, 20% v/v glycerol, 10% v/v 2- mercaptoethanol (Sigma) , 3% w/v DTT, 0.1% w/v bromophenol blue (Sigma)) at room temperature for 3 hours. Samples were stored in the gel loading buffer at -20 °C.

SDS-PAGE Equal amounts (5 μl) of each serum sample fraction in gel loading buffer were denatured by heating at 95° C for 7 min. Samples were loaded on pre-cast 4-12% acrylamide bis-tris SDS-PAGE gels (Invitrogen) and separated by electrophoresis in 3-morpholinopropanesulfonic acid (MOPS; Invitrogen) at 200 mA per gel for 2.5 h. Protein in the gels was stained using filtered Coomassie blue (50% v/v methanol (Fisher Scientific) , 20% v/v acetic acid (Fisher Scientific) 0.12% w/v Brilliant blue-R250 (Sigma)) for 2.5 hr and then destained in 10% v/v acetic acid/10% v/v methanol solution to the desired intensity. Imaging of the stained gels was performed using Agfa Fotolook v3.0 and a Duoscan T1200 flatbed scanner followed by calibrated densitometric quantification (Gene Tools Quantification, SynGene) . Stained gels were stored in dH₂O at room temperature.

Peptide Mass Fingerprinting and Database Searching Individual spots with significantly different expression profiles in PCOS samples when compared to controls were excised from stained gels and destained. Proteins were trypsin digested in-gel and subjected to matrix- assisted laser desorption/ionisation time- of -flight mass spectrometry (MALDI-TOF MS) to obtain peptide mass fingerprints. The monoisotopic mlz values of the tryptic peptide ions were submitted to the Aldente software (http://www.expasy.org/tools/aldente/) and searched against the Human Swiss-Prot and TrEMBL databases to afford protein identification.

Statistical Analysis Differences between control and PCOS groups were assessed using the paired sample student's t-test. A two-tailed p value of <0.05 was considered to be statistically significant.

MALDI Mass Spectrometry Sample Preparation

Protein and peptide analysis

Sample preparation randomization was carried out prior to sample handling and analysis of the full set of 11 PCOS and 10 control serum samples. The same aliquot of serum diluted 1 in 10 with Q.I % TFA was used for protein and tryptic peptide analysis. Diluted serum 25μl was Ci₈ ZipTip fractionated according to manufacturer's instructions using the Xcise robotic system (Proteome systems, Shimadzu, UK) and the eluted proteins/peptides spotted together with SA, 10 mg/ml onto the MALDI- TOF MS target plate by the robotic system. The remaining elutate was carried forward for tryptic digestion. Briefly, the fractionated serum sample was combined with ammonium bicarbonate (16.6μL of 10OmM) , water (7.6μL) , and trypsin (1.3μL of 0.5μg/μL) and incubated at 37°C overnight. The reaction was quenched and the sample cleaned using C₁₈ ZipTip according to the manufacturer's instructions and spotted onto the MALDI target using the dried droplet method with CHCA (10mg/ml solution in 50% ACN + 0.1% TFA, LaserBio Labs, Cedex, France) . The target plate was analysed using the AXIMA-CFR + MALDI-TOF MS (Shimadzu, Manchester, UK) in linear mode using the raster option for proteins and reflectron and autoquality modes for tryptic peptides. A bovine serum albumin (BSA) control was used to ensure the efficiency of the digestion procedure and 0.1% TFA blank to ensure there was no contamination from the reagents or plate. Close external calibration was performed using protein calibration Proteomix3 and Proteomix2 for peptides (Laser Biolabs, Cedex, France) .

The resultant mass spectra were all examined visually and rejected outliers (having poor signal to noise) were removed from the data set before being processed for bioinformatic analysis.

ANN (Artificial Neural Network) Analysis

Data processing: The raw mass spectral data (m/z, intensities) obtained from MALDI-MS were exported as ASCII files and smoothed to yield rounded masses and intensities for the mass range of interest (m/z 1000- 25000) . These intensity values were subsequently used as inputs to the ANN models, developed using Statistica 7.0 (StatSoft Inc. Tulsa, USA) . The models developed were used to predict membership of each sample to one of two output classes: control (1) or PCOS (2) .

Model Architecture: Model architecture and network parameters are described in Lancashire et al (2005) Bioinformatics 21(10) :2191-9. Prior to training, samples were randomly divided into three subsets: training

(60%, n= 14), test (20%, n = 5) , and validation (20%, n = 5) . Training was conducted using 50 randomly extracted data splits, with the validation subset used to independently validate the model on blind data each time to ensure overfitting did not occur. Sensitivity analysis was conducted to identify those inputs having the greatest influence upon the model system with respect to classification performance. These inputs were then subjected to further training. This involved using a stepwise approach which enabled the identification of the optimal subset of biomarkers for class differentiation. The process was stopped when there was no further significant (p = < 0.001) improvement for three steps (ion additions to the optimal subset) on the independent validation set of data.

RESULTS The 21 women analysed in this study were all recruited from the same hospital clinics and were defined as PCOS or control using the Rotterdam criteria. Both the control and PCOS groups represented diverse but equally matched age and BMI ranges. The biometric, clinical and biochemical profiles for the women with PCOS were consistent with current consensus for PCOS patients in the literature. Table 1 shows raised mean hirsutism scores, testosterone levels, free androgen index (FAI) and LH/FSH ratios with lower mean levels of SHBG in a sample set of 5 women with PCOS compared to 5 control subjects. Also consistent with the consensus, PCOS women in this study, on average, had higher numbers of follicles and increased ovarian volumes compared to control women. However, there was much variability between individuals in each group, which reflects phenotypic population variability in both normal, and PCOS women. The only significant differences (P < 0.05) were seen in the SHBG levels, which were significantly lower in the PCOS women than the controls, and FAI, which was increased in the PCOS women. However, significant differences were also seen for increased testosterone levels, LH/FSH ratios, hirsutism score, number of follicles and ovarian volumes when a larger patient cohort (11 PCOS and 10 controls) from the same clinics was investigated (data not shown) .

Table 1

Comparison of demographic, anthropometric, biochemical and reproductive parameters in the two study groups (mean + SD) used in the RP --SPE study

PCOS Control P value

(n=5) (n=5) between groups

Age (y) 25.2 + 6.3=0 26.4 + 5.9 0.75

BMI (kg/m²) 31.6 + 7.34 26.4 + 5.5 0.24

WHR 0.83 + 0.07 0.89 + 0.24 0.61

Oligo/anovulation (%) 100 0 -

Hirsutism (F-G score) 9.8 + 7 6.2 + 5.2 0.39

Total T (nmol/L) 2.38 + 0.63 1.76 + 0.39 0.1

SHBG (nmol/L) 21.2 + 9.28 66.2 + 28.4 0.009*

FAI 13.1 + 6 3.4 + 2.28 0.009*

LH/FSH ratio 1.77 + 0.66 1.28+ 0.50 0.23

Follicles right ovary 22.75 + 10.2 11.4 + 6.14 0.07

Right ovarian volume 9.4 + 4.0 9.2 + 2.8 0.95

Follicles left ovary 18.75 + 4.3 9.8 + 3.96 0.01* left ovarian volume 7.8 + 1.6 3.62 + 1.14 0.003*

Note: PCOS = polycystic ovary syndrome; BMI = body mass index; WHR = waist(cm)/hip(cm) ratio; F-G = Ferriman & Gallwey; T = testosterone; SHBG = sex hormone-binding globulin; FAI = free androgen index; LH = luteinising hormone (IU/L); FSH = follicle stimulating hormone (IU/L).

•= statistically significant difference between PCOS and control groups

•One control woman had a Ferriman Galwey score of 14 but she had regular menstrual cycles and ultrasound features consistent with a control. A different control had isolated increased folicularity on ovarian ultrasound but normal androgens and regular menstrual cycles.

During the studies it was noted that one of the control patients (C4) had features which were borderline PCOS using some of the Rotterdam (2004) criteria. The C4 control and corresponding PCOS pair was thus removed from most of the studies. For the reversed phase-solid phase extraction study (RP-SPE) which was carried out subsequent to the 2DE study, the excluded pair was replaced by a new pair, consisting of one PCOS subject and one definite control.

EXAMPLE 1 - C4α3C as a Biomarker for PCOS

2D SDS PAGE separations were performed on an initial cohort of 5 PCOS and 5 control serum depleted samples, the results are depicted in Figure 1. When stained gels were compared a single protein spot that was visually noted to be present in all the PCOS sample gels but only the Cl control sample gel (Figure 1) .

The relevance of this protein was confirmed using the DELTA 2D image analysis software which generated equalised merged images of matched gel pairs, pseudo-colouring the control gel spots as blue and PCOS gel spots as orange so that equally expressed spots in the merged image appear black. In the majority of gel areas analysed the spot patterns were identical and black. However, the spot circled in the Figure 5 (lower five images) was found only in the PCOS samples and in one control sample (Cl) .

Image analysis software was then used to quantify spot volume as a percentage of total spot volume for the analysed gel area, thus allowing all gels to be directly compared. This confirmed the visual assessment. Figure 2 shows the quantification in graphical form. The greatest differential expression was seen in the C2-P3 sample pair, with spot intensity in the P3 gel being increased by a factor of ~ 10 compared to C2. The mean quantification values highlight the overall up-regulation of the spot intensity in PCOS samples over control samples by a factor of 4. Protein identification

The upregulated protein spot (Figure 1) was excised from the Pl, P3, P4 and P5 sample gels, pooled, trypsinised and the extracted peptides were analysed by MALDI-TOF MS.

The resultant 13 peptide ions, when searched against the Human Swiss- Prot and TrEMBL databases, were consistent matches of the complement component-4, (Swiss-Prot accession number P01028) , α chain, c fragment (C4α₃c) with 5 matched peptide ions, covering 15.2% of the C4α₃c peptide sequence. The predicted MW and pi of the C4α₃c sequence, as calculated using the ExPASy primary structure analysis software, were 32.9 kDa and 5.1 respectively. This corresponds reasonably with the spot position in the gel ( ~ 35 kDa, pi ~ 7.0) .

EXAMPLE 2 - C4α₄C as a Biomarker for PCOS

Western blot analysis of samples from PCOS and control subjects showed differential expression of C4 derived proteins. Whole serum from PCOS and control samples were probed with a polyclonal antibody raised against the αd and α₄c fragments of complement C4 precursor protein (Figure 3) . The resultant band at ~ 24 kDa indicative of the α₄c fragment, another of the C4b degradation products, was present in all samples (Figure 3A) but densitometric analysis of the band intensities (Figure 3B) revealed a significantly higher intensity (P = 0.03) in the PCOS samples compared to controls. In the age-BMI matched pairs the intensity of the band was higher in four of the five pairs. The pair in which the control sample band was more intense than the PCOS sample band (C4-P1) is believed to relate to a control sample who was borderline PCOS thereby explaining this anomaly. It was not possible to source an antibody specific to the C4α₃c fragment but the increase in C4α₄c is indicative of the proteolytic degradation of the C4b polypeptide which produces C4α₃c and C4α₄c (Figure 4) .

Example 3 - C4γ and Haptoglobin α and β Chains as Biomarkers for PCOS

Samples from 6 PCOS and 6 control subjects were analysed be RP- SPE/SDS-PAGE. The samples were from the 5 previous BMI and age matched PCOS and control subject pair and a further PCOS and control pair. Aliquots of serum samples from the subjects were prefractionated using reversed-phase solid phase columns and sequential elution with a stepped gradient of incrementing MeCN concentration. The resulting 12 samples for each elution fraction were then run side-by-side on SDS PAGE gels, one gel representing all 12 serum samples at the specified acetonitrile concentration, and stained with Coomassie blue. This enabled the protein composition of each fraction to be compared across the full set of age/BMI matched serum samples. For reasons outlined above, the data from a PCOS control pair (Pl and C4) was not taken into account in the quantitative data analysis.

Visual analysis of the stained gels noted three obvious differences in the banding pattern between PCOS and control samples (Figure 5A, 5B and 5C) . The first difference detected in the 70% MeCN fraction (Figure 5A) is for components at ~ 33kDa which are present in all the control samples, but observed at lower abundance, in only one of the five PCOS samples. Densitometric quantitation of these components was not possible owing to the low signal intensities.

The second difference between the two sample groups was one of abundance change in components present in all samples at ~ 4OkDa in the

42-70% MeCN fractions (Figure 5B), which was increased for all PCOS samples compared to the control samples in the six consecutive elution fractions. An additional instance of potential protein up regulation in PCOS serum was found across the gel analysis, set although most prominent in the 10-20% MeCN fractions. In this case a component at ~ 17kDa was present in four PCOS samples and only two control samples (Figure 5C) . Densitometric quantification for these two changes in Figures 5B and 5C is shown in graphical display in Figures 6A and 6B with the differential expression at ~ 40kDa (Figure 6A) having a significance value of 0.001.

Identification of differentially expressed proteins

The differential bands identified in the RP-SPE/SDS PAGE analysis were excised, pooled, trypsinised and analysed using MALDI-TOF MS. Interrogation of the Swiss-Prot and TrEMBL databases using measured peptide ions and derived sequences identified potential matches for all three observed differences. The ~ 33 kDa band (Figure 5B) extracted from the 70% MeCN fraction gel generated six peptide ions characteristic of the complement component C4γ chain, tandem MS/MS product ion analysis confirmed this match. The predicted MW of the C4γ chain, as calculated using the ExPASy primary structure analysis software, is ~ 33.5 kDa, which corresponds well with the observed band position following SDS PAGE.

The - 40 kDa band (Figure 5B and 6A) extracted from the 42% MeCN fraction gel yielded three peptide ions characteristic of the haptoglobin β chain, of which two were subsequently analysed by tandem LC-MS/MS product ion analysis to confirm this match. A further three peptide ions matched the α region of the haptoglobin precursor protein. The ~ 17 kDa band (Figure 5C and 6B) extracted from the 20 % MeCN fraction gel yielded six peptide ions characteristic of the haptoglobin α chain. Theoretical sequence derived molecular masses for the haptoglobin α chain sequence is 15.9 kDa, which corresponds well with the observed molecular weight following SDS PAGE, and for the β chain sequence is 27.3 kDa, with the precursor having a molecular weight of 43.6 kDa, which is a better match for the observed ~ 40 kDa band in the RP-SPE analysis. The presence of these bands in multiple fractions from the RP- SPE columns suggests that the protein can be differentially modified (e.g. by glycosylation) and so exhibit different affinities for the column matrix.

Example 4 - MALDI-MS Serum Protein/Peptide Biomarker Signatures for PCOS

This analysis was carried out on 21 serum samples, 11 from PCOS subject and 10 controls.

The most prominent spectral discriminatory pattern in PCOS compared with unaffected individuals are exemplified in Figures 7A and 7B respectively. However, to investigate the full dataset for both the protein and tryptic peptides a comprehensive bioinformatic ANN analysis was undertaken.

ANN analysis identified an optimal subset of three and six biomarker ions for protein and peptide datasets respectively. The six ions {m/z 8674, 8668, 1351, 8727, 8673, and 6871) correctly classified 90.4 % of the independent validation set of samples based on whether they were control or PCOS with a threshold of 0.5 (Figure 7C) . The three biomarker ions (2924, 3025 and 1977) identified from the digested peptide data successfully classified the independent validation subset of samples to an accuracy of 100 % (Figure 7D) .

Claims

1. A method of determining the Polycystic Ovarian Syndrome (PCOS) status of a subject, comprising the steps of: (a) providing a sample of material obtained from a subject;

(b) determining the level of one or more of the following proteins: the complement C4 precursor protein; a cleavage fragment of the complement C4 precursor protein; and a haptoglobin; in the sample and (c) comparing the levels determined with one or more reference values.

2. The method of claim 1 wherein the cleavage fragment of the complement C4 precursor protein is selected from the group comprising a) C4α; b) C4β ; c) C4γ; d) C4a anaphylatoxin; e) C4b; f) C4α₃c fragment; g) C4αd; j) h) C4α₄c fragment; and i) a polypeptide with at least 75% amino acid sequence identity to the amino acid sequence of any of a) to h) .

3. The method of claim 1 or 2 wherein the one or more haptoglobin proteins is selected from the group comprising haptoglobin α and haptoglobin β .

4. The method of any of claims 1 to 3 wherein the sample material is whole blood, blood serum, blood plasma, urine, fat tissue (adipose) , endometrial tissue, ovary tissue or any other bodily fluid or tissue.

5. The method of any preceding claims wherein the level of one or more of the complement C4 precursor protein, a cleavage fragment of the complement C4 precursor protein, and a haptoglobin, is determined using an assay selected from the group comprising immunoassay, spectrometry, mass spectrometry, Matrix Assisted Laser Desorption/Ionization Time-of- Flight (MALDI-TOF) Mass Spectrometry, microscopy, northern blot, western blot, southern blot, isoelectric focussing, SDS-PAGE, PCR, RT- PCR, gel electrophoresis, protein microarray, DNA microarray, antibody microarray, and combinations thereof.

6. The method of any preceding claim wherein the one or more reference values are the levels of one or more of the complement C4 precursor protein, a cleavage fragment of the complement C4 precursor protein, and a haptoglobin, observed in one or more subjects who does not have any clinical symptoms of PCOS.

7. The method of any of claims 1 to 5 wherein the one or more reference values are an earlier level of one or more of the complement C4 precursor protein, a cleavage fragment of the complement C4 precursor protein, and a haptoglobin, determined in the same subject who has provided the sample.

8. The method of any preceding claim wherein an increase or a decrease in the level of one or more of one or more of the complement C4 precursor protein, a cleavage fragment of the complement C4 precursor protein, and a haptoglobin is indicative of the PCOS status of the subject.

9. The method of claim 8 wherein an increase in the level of C4α₃c and/or the level of C4α₄c and/or the level of haptoglobin α and/or haptoglobin β in a sample compared to the reference value is indicative, or diagnostic, of PCOS.

10. The method of claim 8 wherein a decrease in the level of the C4 precursor protein and/or C4α and/or C4β and/or C4γ and/or C4a anaphylatoxin is indicative, or diagnostic, of PCOS.

11. The method of any preceding claims wherein the method is carried out in vitro .

12. The method of any preceding claims wherein the subject is a mammal.

13. The method of claim 12 wherein the mammal is a human.

14. A method of determining the PCOS status in a subject, comprising the steps of; (a) providing a sample of material obtained from a subject;

(b) analysing proteins or tryptic peptides obtained from the sample using MALDI-MS;

(c) concluding that the presence of the six ions m/z 8674, 8668, 1351, 8727, 8673 and 6871 in a protein sample and/or the presence of the three ions m/z 2924, 3025 and

1977 in a tryptic peptide sample, is diagnostic of PCOS in the subject.

15. A kit for use in determining the PCOS status of a subject comprising at least one agent for determining the level of one or more of the complement C4 precursor protein, a cleavage fragment of the complement C4 precursor protein and a haptoglobin.

16. The kit of claim 15 wherein the cleavage fragment of the complement C4 precursor protein is selected from the group comprising a) C4α; b) C4β; c) C4γ; d) C4a anaphylatoxin; e) C4b; f) C4α.,c fragment; g) C4αd; k) h) C4α₄c fragment; and i) a polypeptide with at least 75% amino acid sequence identity to the amino acid sequence of any of a) to h) .

17. The kit of claim 15 or 16 wherein the one or more haptoglobin proteins is selected from the group comprising haptoglobin α and haptoglobin β.

18. The kit of claim 15, 16 or 17 wherein the agent is selected from the group comprising an enzyme, an antibody, a nucleic acid, a protein probe and any other suitable composition.

19. The kit of any of claims 15 to 18 wherein the agent is labelled.

20. The kit of claim 19 further comprising means for detecting the label.

21. The kit of any of claims 15 to 20 further comprising instructions for suitable operational parameters in the form of a label or separate insert.

22. The kit of any of claims 15 to 21 further comprising a sample of one or more of the complement C4 precursor protein, a cleavage fragment of the complement C4 precursor protein and/or a haptoglobin to be used as standard(s) for calibration and comparison.

23. A method of treating PCOS in a subject comprising administering to the subject an agent capable of modulating the level of one or more of the complement C4 precursor protein, a cleavage fragment of the complement C4 precursor protein and a haptoglobin.

24. The method of claim 23 wherein the agent is an antisense or an interfering RNA molecule designed to inhibit or reduce expression of the complement C4 precursor protein, a cleavage fragment of the complement C4 precursor protein and a haptoglobin.

25. The method of claim 23 wherein the agent is an inhibitor of C4 precursor protein processing.

26. The method of claim 23 wherein the agent is a composition which reduces complement activity.

27. A method of determining the course of PCOS in a subject, or determining the efficacy of treatment administered to a subject with PCOS, comprising:

(a) determining the level of one or more of the following proteins: the complement C4 precursor protein; a cleavage fragment of the complement C4 precursor protein; and a haptoglobin; in a sample obtained from a subject at a first time point;

(c) comparing the first measurement and the second measurement, wherein the comparative measurements determine the course of the PCOS.

28. A method of identifying compounds for treating PCOS comprising screening for one or more compounds that modulate the level of one or more of the complement C4 precursor protein, a cleavage fragment of the complement C4 precursor protein, and a haptoglobin, in vitro or in vivo.

29. Use of the C4 precursor protein, C4α, C4β , C4γ, C4a anaphylatoxin, C4b, C4α₃c fragment, C4αd, C4α₄c fragment, haptoglobin α and/or haptoglobin β as therapeutic targets for the treatment/alleviation of PCOS.

30. A probe set comprising one or more probes capable of detecting one or more the complement C4 precursor protein, a cleavage fragment of the complement C4 precursor protein and a haptoglobin.

31. A probe set according to claim 30 wherein the probes are selected from the group comprising an antibody, nucleic acid or peptide.

32. The probe set of claim 30 or 31 wherein the probe is labelled.

33. The probe set of claim 30, 31 or 32 comprising two probes directed to two different proteins selected from the group comprising the complement C4 precursor protein, a cleavage fragment of the complement C4 precursor protein and a haptoglobin.

34. A kit for use in determining the PCOS status of a subject comprising instructions to analyse a sample for the presence of the six ions m/z 8674, 8668, 1351, 8727, 8673 and 6871 in a protein sample and/or the presence of the three ions m/z 2924, 3025 and 1977 in a tryptic peptide sample.