FILING DATA

[0001] This application is associated with and claims priority from Australian Provisional Patent Application No. 2015902891, filed on 21 July 2015, entitled "Glycoforms Biomarkers", the entire contents of which, are incorporated herein by reference.

BACKGROUND FIELD

[0002] The present invention relates to an assay for determining mammalian endometrial or uterine receptivity for an embryo in order to facilitate successful implantation of the embryo by natural or artificial means. The assay further discriminates between fertile and infertile mammalian females at particular secretory phases.

DESCRIPTION OF THE PRIOR ART

[0003] Bibliographic details of the publication referred to by author in this specification are collected alphabetically at the end of the description.

[0004] 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.

[0005] Successful embryo implantation is important, not only due to the cost of the procedure, but more importantly to the psychological and emotional wellbeing of the potential mother. Whilst embryo health remains an important factor, equally or even more influential on a successful outcome is the endometrium being in a receptive state. This may be referred to as endometrial or uterine receptivity. However, developing a reliable test for such receptivity has remained elusive. [0006] There have been some proteomic studies in secreted fluid from the endometrium, referred to as the secretome (Casado-Vela et al (2009) J. Proteome Res 8: 4622-4632; Hannan et al (2009) J. Proteome Res 8: 1099-1103). Some of these investigations employ differential display studies of either proliferative and secretory phases (Chen et al (2009) J Proteome Res 8: 2032-2044; DeSouza et al (2005) Proteomics 5: 270-281; Dominguez et al (2009) Hum. Reprod 24: 2607-2617; Li et al (2011) Fertil Steril 95: 1161-1163; Rai et al (2010) Proteomics Clin Appl 4: 48-59) or direct comparison of fertile and infertile cohorts (Hannan et al (2010) J Proteome Res 9: 6256-6264). Whilst these studies have yielded interesting data, the main findings are multiple dysregulated forms of proteins as depicted on 2 dimensional gels as "spot trains" (for example see Packer et al (1998) Electrophoresis 19: 1872-1882; Rudd et al (1997) Crit Rev Biochem Mol Biol 32: 1-100).

[0007] Dysregulated protein forms can occur by any number of means including alterations in amino acid sequence or post translational changes. However, quantitation of a single dysregulated protein is problematic. One reason for this is that antibodies frequently are incapable of distinguishing between different post translationally modified forms of the same protein. The alterative is to use an array map or mass-spectrometric analysis which requires specialized equipment and skills, is costly and requires lengthy time frames.

[0008] There is clearly a need to develop a more efficacious assay for biomarker identification of uterine receptivity in natural and assisted reproduction.

SUMMARY

[0009] The present invention teaches the development of an assay to identify and validate markers of uterine receptivity for an embryo. Reference to "uterine receptivity" and "endometrial receptivity" are used interchangeably herein to determine the state of a uterus most receptive to successful implantation of a healthy embryo by either natural or by assisted reproduction means. The assay samples uterine lavage for particular glycoforms of one or more proteins indicative of endometrial (or uterine) receptivity.

[0010] A multiplex endometrial protein capture antibody-endometrial protein-lectin assay is taught herein to detect particular glycoforms of endometrial proteins in uterine lavage samples. Conventional sandwich ELISAs use a protein-specific primary capture antibody combined with a secondary antibody carrying a detectable label such as horseradish peroxidase or biotin. In accordance with the present invention, the secondary antibody is replaced by a lectin which provides a means to identify whether a specific glycoform is bound to the primary antibody. The lectin forms a glycoconjugate between the protein glycoform and the lectin. Hence, taught herein are particular endometrial protein-lectin binding pairs or glycoconjugates which are indicative of endometrial receptivity.

[0011] The present invention provides an assay to identify and validate glycoform biomarkers in endometrial lavage which are indicative of a receptive or non-receptive uterine environment for successful natural or assisted implantation of an embryo to or near to term. The glycoforms can also discriminate between fertile and infertile female subjects and particular secretory phase.

[0012] In an embodiment the protein-lectin pairs or glycoconjugates are identified which discriminate fertile from infertile women during mid-secretory phase of a natural cycle and those which discriminate pregnancy outcome among woman undergoing assisted reproduction.

[0013] Accordingly, one aspect of the present invention provides an assay to identify an endometrial protein glycoform biomarker in uterine lavage from a female mammal which is indicative of a level of receptivity of an endometrium for a likely successful or likely unsuccessful embryo implantation, the assay comprising selecting an endometrial protein having a glycoform which specifically binds to a lectin forming an endometrial protein- lectin binding pair, capturing the protein on a solid support using an immobilized primary antibody specific for the protein and then detecting the glycoform of the captured protein using the lectin specific for the glycoform wherein the lectin is labeled with a reporter molecule capable of being detected or binding a labeled antibody specific for the lectin which antibody is labeled with a reporter molecule capable of being detected. The implantation may be natural or assisted (e.g. by in vitro fertilization [IVF]).

[0014] Another aspect contemplates the use of a endometrial protein-lectin binding pair, wherein the lectin binds to a specific glycoform of the endometrial protein in the manufacture of an assay to detect uterine receptivity or otherwise for an embryo implant in a female mammal.

[0015] The present assay applies to human female subjects as well as non-human mammalian female animals. Hence, the assay has human clinical and veterinary applications.

[0016] Yet another aspect herein enables a method of assisted reproduction a method of assisted reproduction in a female mammal the method comprising selecting an endometrial protein having a glycoform which specifically binds to a lectin forming a protein-lectin pair, capturing the protein on a solid support using an immobilized primary antibody specific for the protein and then detecting the glycoform of the captured protein using the lectin specific for the glycoform wherein the lectin is labeled with a reporter molecule capable of being detected or binding a labeled antibody specific for the lectin which antibody is labeled with a reporter molecule capable of being detected wherein the presence or absence or level of the protein-lectin pair is indicative of whether an embryo implant should proceed.

[0017] Further enabled herein is a method of determining a female mammal that is likely to fall pregnant, the method comprising selecting an endometrial protein having a glycoform which specifically binds to a lectin forming a protein-lectin pair, capturing the protein on a solid support using an immobilized primary antibody specific for the protein and then detecting the glycoform of the captured protein using the lectin specific for the glycoform wherein the lectin is labeled with a reporter molecule capable of being detected or binding a labeled antibody specific for the lectin which antibody is labeled with a reporter molecule capable of being detected wherein the presence or absence or level of the protein-lectin pair is indicative of a receptive endometrial for pregnancy.

[0018] Endometrial proteins contemplated herein are defined in the subject specification and are referred to by the abbreviations ORMl, MUC5B, A2M-like (A2M-L) protein and TAFI (Table 1). The present invention extends to non-human equivalents of these proteins if they do not exist in such mammal. In addition, other potential proteins of interest are listed in Tables 19-23 of Example 9. Lectin abbreviations used herein include CONA, DBA, DSL, ECL, GSLI, GSLII, JAC, LEL, PSA, RCA, SBA, SJA, STL, ULEX, VVA and WGA (Table 2).

[0019] Particular protein-lectin biding pairs include ORMl -DBA, ORM1-CONA, A2M- L-WGA, glycodelin-RCAI, glycodelin-DBA TAFI-DBA and MUC5B-CONA.

Table 1

Endometrial protein abbreviations

Protein Abbreviation

Orosomucoid 1 ORM 1

Mucin 5B MUC5B

Glycodelin Glycodelin

Alpha-2-macroglobulin-like protein A2M-L-protein

Thrombin activatable fibrinolysis inhibitor TAFI Table 2

Lectin Abbreviation

Concavalin A CONA

Dolichos biflorus agglutinin DBA

Datura Stramonium lectin DSL

Erythrina cristagalli lectin ECL

Griffonia (Bandeiraea) simplicifolia lectin .1 GSL I

Griffonia (Bandeiraea) simplicifolia lectin II GSL II

Jacalin JAC

Lycopersicon esculentum lectin LEL

Pi sum sativum agglutinin PSA

Ricinus communis agglutinin RCA

Glycine max agglutinin. SBA

Sophora japonica agglutinin SJA

Solanum tuberosum lectin STL

Ulex europaeus agglutinin I ULEX

Vicia villosa agglutinin VVA

Triticum vulgaris agglutinin WGA

BRIEF DESCRIPTION OF THE FIGURES

[0020] Some Figures contain colored depictions. Copies of colored Figures can be obtained from the patentee.

[0021] Figure 1 is a graphical representation of a histochemical evaluation of endometrial tissue performed using DBA lectin. Tissue from fertile (blue) and infertile (pink) women were collected during proliferative (Pro), early stage (ES), mid-secretory (MS) and late secretory (LS) phases. Relative staining intensity was scored in arbitrary units and the mean (n=4) intensity for each tissue type calculate as shown.

[0022] Figure 2 is a graphical representation of pooled lavage samples from early secretory (A), infertile early secretory (B), fertile mid-secretory (C) and infertile mid- secretory (D) run on 12% w/v SDS-PAGE and stained with coomassie (Left). The samples were subject to western blot using DBA lectin (Right).

[0023] Figure 3 is a graphical representation of lavage samples collected during the mid- secretory phase of women with proven fertility (F-MS) and those with known infertility (IF-MS) and assayed for alpha-2-macroglobulin (Luminex assay). Results for each group are presented as mean +/- standard deviation. No significant difference was found.

[0024] Figures 4A to C are graphical representations of uterine lavage analyzed for the concentrations of three protein glycoforms. The women were classified according to cycle stage, early secretory (es) and mid-secretory (ms), and fertility status, fertile (F) and primary infertile (pif). Results were analyzed by Kruskal-Wallis between groups of women.

[0025] Figures 5A to D are graphical representations showing concentrations of CONA reactive glycoforms of ORMl and MUC5B analyzed in uterine lavage samples collected from naturally cycling fertile and primary infertile women (left side) during early (ES) and mid-secretory (MS) phases, and from IVF treated infertile women at oocyte collection human chorionic gonadotropin + 2 days (hCG+2). IVF stimulated women were grouped according to outcome of pregnancy (anta P val) or No pregnancy (ana NP val) following fresh embryo transfer in the same cycle. Results are expressed as Flurescence Intensity (FI). Comparison was made between groups using Mann -Whitney analysis.

[0026] Figure 6 is a photographic representation of a protein gel image from SDS TGX stainfree gel flushing pools from proliferative fertile, proliferative infertile, secretory fertile and secretory infertile (lanes 1-4, respectively) and equal volume loading (lanes 5-8, respectively).

DETAILED DESCRIPTION

[0027] 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 method step or groups of elements or integers or method steps but not the exclusion of any other element or integer or method step or group of elements or integers or method steps.

[0028] As used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus for example, reference to "a biomarker" or "a glycoconjugate" includes a single biomarker or glycoconjugate as well as two or more biomarkers or glycoconjugates; reference to "an embodiment" includes a single embodiment, as well as two or more embodiments; reference to "the disclosure" includes single and multiple aspects taught by the disclosure and so forth. Aspects taught and enabled herein are encompassed by the term "invention". All such aspects are enabled within the width of the present invention. Any variants and derivatives contemplated herein are encompassed by "forms" of the invention.

[0029] The present invention is predicated in part on the identification of endometrial proteins having a particular glycosylation pattern referred to herein as a "glycoform". The presence or absence or level or a particular endometrial protein or the ratio of levels of two or more endometrial proteins is indicative of the likelihood or otherwise of a successful embryo implantation whether by natural or assisted reproduction means. The glycoform is detected by a lectin which binds to a particular glycosylation pattern on the protein of interest to form a glycoconjugate. Hence, reference is made herein to an endometrial protein-lectin binding pair or glycoconjugate which is the lectin which binds to a particular glycoform of a selected protein. Shortened expressions such as "protein-lectin", "protein- lectin pair" and "glycoconjugate" have the same meaning as "endometrial protein-lectin binding pair".

[0030] Consequently, developed herein is an assay to identify endometrial protein-lectin binding pairs. Further developed is the validation of protein-lectin pairs using a modified ELISA which determines the presence, absence, level or ratio of levels of endometrial proteins in uterine lavage at early, mid or late secretory phase (or a sub-phase in between) which enables a determination of a favorable environment in which to implant by natural or artificial means an embryo. On the assumption the embryo is healthy, if the endometrium comprises a favorable protein environment based on the glycoform of one or more proteins, then a successful embryo implantation is the likely outcome.

[0031] Hence, enable herein is an assay to identify an endometrial glycoform biomarker in uterine lavage from a female mammal which is indicative of a level of receptivity of an endometrium for a likely successful or likely unsuccessful embryo implantation, the assay comprising selecting an endometrial protein having a glycoform which specifically binds to a lectin forming an endometrial protein-lectin binding pair, capturing the protein on a solid support using an immobilized primary antibody specific for the protein and then detecting the glycoform of the captured protein using the lectin specific for the glycoform wherein the lectin is labeled with a reporter molecule capable of being detected or binding a labeled antibody specific for the lectin which antibody is labeled with a reporter molecule capable of being detected.

[0032] Further enabled herein is a method of determining a female mammal that is likely to fall pregnant, the method comprising selecting an endometrial protein having a glycoform which specifically binds to a lectin forming a protein-lectin pair, capturing the protein on a solid support using an immobilized primary antibody specific for the protein and then detecting the glycoform of the captured protein using the lectin specific for the glycoform wherein the lectin is labeled with a reporter molecule capable of being detected or binding a labeled antibody specific for the lectin which antibody is labeled with a reporter molecule capable of being detected wherein the presence or absence or level of the protein-lectin pair is indicative of a receptive endometrial for pregnancy.

[0033] Whilst the subject assay is particularly exemplified with respect to human females, the concept of the present invention equally applies to assessing the receptivity of any non-human female mammal undergoing assisted reproduction or natural cycling. Such non-human mammals include horses, cattle, sheep, pigs, deer and zoo-housed mammals including those endangered such as gorillas, monkeys, orangutans and the Tasmanian devil. Hence, the present assay has both human clinical and veterinary applications. The assay is also useful in the monitoring of natural cycles in mammalian females such as human female subjects.

[0034] In an embodiment, however, the assay is used to assess the receptivity of a human female. Accordingly taught herein is an assay to identify an endometrial protein glycoform biomarker in uterine lavage from a human female which is indicative of a level of receptivity of an endometrium for a likely successful or likely unsuccessful embryo implantation, the assay comprising selecting an endometrial protein having a glycoform which specifically binds to a lectin forming an endometrial protein-lectin binding pair, capturing the protein on a solid support using an immobilized primary antibody specific for the protein and then detecting the glycoform of the captured protein using the lectin specific for the glycoform wherein the lectin is labeled with a reporter molecule capable of being detected or binding a labeled antibody specific for the lectin which antibody is labeled with a reporter molecule capable of being detected.

[0035] Further enabled herein is a method of determining a human female that is likely to fall pregnant, the method comprising selecting an endometrial protein having a glycoform which specifically binds to a lectin forming a protein-lectin pair, capturing the protein on a solid support using an immobilized primary antibody specific for the protein and then detecting the glycoform of the captured protein using the lectin specific for the glycoform wherein the lectin is labeled with a reporter molecule capable of being detected or binding a labeled antibody specific for the lectin which antibody is labeled with a reporter molecule capable of being detected wherein the presence or absence or level of the protein- lectin pair is indicative of a receptive endometrial for pregnancy.

[0036] Hence, the present invention provides:

(i) an endometrial protein present or absent or at a particular level in early, mid or late secretory phase uterine lavage;

(ii) the endometrial protein or ratios of levels of two or more endometrial proteins predicts a receptive or non-receptive uterine environment for successful embryo implantation by natural or artificial means;

(iii) the endometrial protein comprises a particular glycosylation pattern referred to herein as a glycoform; and

(iv) a lectin which specifically binds to that glycoform to form a glycoconjugate enabling detection and/or quantification of the presence, absence or level of one protein or ratio of two or more proteins enabling a predication to be made on the likelihood or otherwise of a successful embryo implantation.

[0037] As indicated above, the assay applies not only to assisted reproduction but also to the likelihood of otherwise of a natural pregnancy. Also as indicated above, a female mammal includes a human female and a non -human female mammal. A "human female" may also be referred to herein as a "woman". Generally, the female mammal is of baby bearing age but the assay may also be useful in assessing menopausal changes and hormonal changes, especially those changes leading to a predisposition to a uterine cancer.

[0038] The sample is referred to as a "uterine lavage" or "endometrial lavage" and both terms may be used interchangeably to assess uterine receptivity for embryo implantation. The term "lavage" is not to imply any limitation as to the means of obtaining a uterine sample which includes an endometrial sample. Conveniently, a uterine lavage is the least invasive. The term "biological sample" may also be applied to the uterine/endometrial sample. The assay includes micro-arrays, macro-arrays and nano-arrays on planar or spherical solid supports. A "sample" includes but is not limited to from 0.5 μΐ to 50 ml.

[0039] The term "successful" means an embryo which goes to term resulting in a live birth whether natural or by caesarean section as well as the embryo developing to an age where a baby can be delivered pre-term, generally by caesarean section.

[0040] The "likeliness" or "likelihood" of a successful implantation is relative but factors such as health of the embryo, physical condition of the female subject and other factors not necessarily related to the developing fetus do affect the outcome. In an embodiment, on the assumption the embryo is healthy, the implantation procedure does not adversely effect the embryo or endometrium and the female subject remains in a healthy state, the selected endometrial protein glycoform can successfully discriminate between a fertile female from a non-fertile female in early, mid or late secretory phase. Furthermore, pregnancy outcome can be predicted with an at least 75% success rate. By "at least 75%" means at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%). This applies to natural and assisted reproduction.

[0041] The endometrial protein can be referred to as glycoform, glycomarker, biomarker, glycoform biomarker, predictor, marker, indicator or any such term applying to a protein present in endometrial lavage. The term "glycolconjugate" applies to a protein-lectin bonded pair. The protein need not solely reside in the endometrial lavage but its presence, absence or level or its ratio of levels with at least one other protein needs to be correlated with uterine receptivity or non-receptivity for embryo implantation.

[0042] Examples of endometrial proteins, include but are not limited to orosmucoid 1 (ORM1), mucin 5B (MUC5B), glycodelin, alpha-2-macroglobulin (A2M)-like (A2M-L) protein and thrombin activatable fibrinolysis inhibitor (TAFI). Other Examples are provided in Tables 19 to 23 of Example 9. Examples of suitable lectins include concanavalin A (CONA), dolichos biflorus agglutinin (DBA), datura stramonium lectin (DSL), erythrina cristagalli lectin (ECL), griffonia (bandeiraea) simplicifolia lectin I (GSLI), griffonia (bandeiraea) simplicifolia lectin II (GSLII), jacalin (J AC), lycopersicon esculentum lectin (LEL), pisum sativum agglutinin (PSA), ricinus communis agglutinin (RCA), glucine max agglutinin (SB A), sophora japonica agglutinin (SJA), solanum tuberosum lectin (STL), ulex europaeus agglutinin I (ULEX), vicia villosa agglutinin (VVA) and triticum vulgaris agglutinin (WGA).

[0043] Examples of particular endometrial protein-lectin binding pairs include ORM1- DBA, ORMl-CONA, A2M-L-WGA, glycodelin-RCAl, glycodelin-DBA, TAFI-DBA and MUC5B-CONA.

[0044] In a particular embodiment, the endometrial protein-lectin binding pairs are A2M- L-WGA, ORM1-DBA and glycodelin-RCAl .

[0045] Accordingly, enabled herein is an assay to identify an endometrial protein glycoform biomarker in uterine lavage form a female mammal which is indicative of a level of receptivity of an endometrium for a likely successful or likely unsuccessful embryo implantation, the assay comprising selecting an endometrial protein from the group consisting of ORM1, MUC5B, glycodelin, A2M-like protein and TAFI from a human female or their equivalent in a non-human mammal having a glycoform which specifically binds to a lectin selected from the group consisting of CONA, DBA, DSL, ECL, GSLI, GSLII, JAC, LEL, PSA, RCA, SBA, SJA, STL, ULEX, VVA and WGA forming an endometrial protein-lectin binding pair, capturing the protein on a solid support using an immobilized primary antibody specific for the protein and then detecting the glycoform of the captured protein using the lectin specific for the glycoform wherein the lectin is labeled with a reporter molecule capable of being detected or binding a labeled antibody specific for the lectin which antibody is labeled with a reporter molecule capable of being detected.

[0046] In an embodiment the protein is selected from A2M-like protein, ORMl and glycodelin and the corresponding lectin specific for the particular glycoform is WGA, DBA and RCA1, respectively.

[0047] Monoclonal or polyclonal antibodies may be used as the capture antibody for the endometrial protein or if needed the "tertiary" antibody. A tertiary antibody is used to bind to a lectin portion of a glycoconjugate. The use of monoclonal antibodies in an immunoassay is particularly useful because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art.

[0048] Whilst a modified ELISA is useful, there is a wide range of other immunoassay techniques available as can be seen by reference to U.S. Patent Nos. 4,016,043, 4,424,279 and 4,018,653.

[0049] The following is a description of one type of assay. An unlabeled antibody is immobilized on a solid substrate and the sample to be tested for the endometrial protein brought into contact with the bound antibody. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-protein complex, a lectin specific to the glycoform of the protein, labeled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-protein-labeled letin. Any unreacted material is washed away, and the presence of the protein glycoform is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of protein. This generalized technique is well known to those skilled in the art as would be any of a number of variations. In addition, a "tertiary antibody" may also be used to detect the lectin if the lectin is not labelled.

[0050] In these assays, a primary antibody having specificity for the instant protein is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, spheres, discs of microplates, or any other surface suitable for conducting an immunoassay. Microspheres are particularly useful. The binding processes are well known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the solid phase-antibody complex is washed in preparation for the test sample. An aliquot of the lavage to be tested is then added to the solid phase antibody and incubated for a period of time sufficient (e.g. 2-120 minutes or where more convenient, overnight) and under suitable conditions (e.g. for about 20°C to about 40°C) to allow binding of any subunit present in the lavage. Following the incubation period, the reaction vessel is washed and dried and incubated with a lectin specific for the glycoform of the protein. The lectin is generally linked to a reporter molecule which is used to indicate the binding of the protein to the primary antibody.

[0051] There are many variations to this assay. One particularly useful variation is a simultaneous assay where all or many of the components are admixed substantially simultaneously. Furthermore, binding of an antibody to a lectin may be employed to detect the lectin. [0052] By "reporter molecule" as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules. Examples of suitable fluorophores well known. In the case of an enzyme-based reporter molecule, an enzyme is conjugated to the lectin, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta- galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labeled lectin is added to the immobilized antibody- protein complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-protein- lectin. The substrate will react with the enzyme linked to the lectin, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of protein which was present in the sample. Again, the present disclosure extends to a substantially simultaneous assay.

[0053] Alternately, fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to lectins without altering their binding capacity. When activated by illumination with light of a particular wavelength, the flurochrome-labeled lectin adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic color visually detectable with a light microscope. The fluorescent labeled lectin is allowed to bind to the first antibody-protein complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the protein of interest. Immunofluorescence and enzyme immunoassay techniques are both very well established in the art. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.

[0054] There are a range of other detection systems which may be employed including colloidal gold and all such detection systems are encompassed by the present disclosure.

[0055] The assay enabled herein may be automated or semi -automated for high throughput screening or for screening for a protein glycoforms from the one or multiple subjects. The automation is conveniently controlled by computer software.

[0056] The present disclosure further contemplates therefore web-based and non-web- based systems where data on the receptivity of a subject are provided by a client server or other architecture platform to a central processor which analyses and compares to a control and optionally considers other information such as patient age, weight and other medical conditions and then provides a report, such as, for example, a risk factor for embryo implantation failure. Hence, business method is provided whereby uterine lavage is collected in transportable tubes which is then analyzed for protein glycoforms at a defined location and the results then sent in the form of an electronic report via a client server or other architecture platform to a clinical care provider.

[0057] Hence, knowledge-based computer software and hardware also form part of the present disclosure. This facilitates clinical care to ascertain whether a subject has a degree of endometrial receptivity sufficient to expect a likely successful pregnancy.

[0058] In a further embodiment, the present disclosure enables kits for use with the methods described above. In one embodiment, an glycoconjugate kit is contemplated.

[0059] The immunodetection reagents of the kit may take any one of a variety of forms, including those detectable labels that are associated with or linked to the given protein glycoform, and detectable labels that are associated with or attached to lectin. Exemplary lectins are listed in Table 2.

[0060] The components of the kits may be packaged either in aqueous media or in lyophilized form. [0061] The container means of any of the kits generally includes at least one vial, test tube, flask, bottle, syringe or other container means, into which the testing lavage is placed, and generally, suitably aliquoted. Where a lectin is provided, the kit will also generally contain a second, third or other additional container into which this lectin may be placed. The kits taught by the present disclosure also typically include a means for immobilizes the protein of interest and any other reagents.

[0062] The present specification is also enabled for the use of an endometrial protein- lectin binding pair, wherein the lectin binds to a specific glycoform of the endometrial protein in the manufacture of an assay to detect uterine receptivity or otherwise for an embryo implant in a female mammal such as a human female.

[0063] The assay may be used in a method of assisted reproduction in a female mammal the method comprising selecting an endometrial protein having a glycoform which specifically binds to a lectin forming a protein-lectin pair, capturing the protein on a solid support using an immobilized primary antibody specific for the protein and then detecting the glycoform of the captured protein using the lectin specific for the glycoform wherein the lectin is labeled with a reporter molecule capable of being detected or binding a labeled antibody specific for said lectin which antibody is labeled with a reporter molecule capable of being detected wherein the presence or absence of the protein-lectin pair is indicative of whether an embryo implant should proceed.

[0064] In an embodiment, the female mammal is a human female. In another embodiment, the assay is used to predict receptivity for a natural pregnancy.

EX AMPLE S

[0065] Aspects taught and enabled herein are further described by the following non- limiting Examples.

EXAMPLE 1

Development of lectin-modified ELSA

[0066] A primary antibody to a particular protein identified in uterine lavage is immobilized to a solid support. The solid support may be any support including a microsphere, microparticle or the side of a reaction vessel. The primary antibody is immobilized via its constant domains in the Fc region. A lavage sample is brought into contact with the immobilized primary antibody for a time and under conditions sufficient for a protein to which the antibody is specific binds. Unbound material and superfluent lavage fluid is washed away. A selected lectin specific for a glycoform of the captured protein is then added. The lectin is labeled with a detectable marker or label such as streptavidin, horseradish peroxidase or detection is via a tertiary antibody specific for the captured lectin. By using various lavage samples from fertile and infertile women, a particular glycoform of the protein which either facilitates successful implantation or is associated with embryo failure is identified.

[0067] Where microspheres are employed, the microspheres are transferred to a microcentrifuge tube and pelleted at 8000 g. The supernatant is then removed and the beads suspended in 100 mm sodium phosphate, pH 6.2, by Vortex agitation and sonication. Conjugation reagents (e.g., sulfo- HS) and EDC are added to the microspheres and incubated with gentle mixing followed by centrifugation. Supernatant is then removed. The pelleted beads are resuspended in 50 mm MES, pH 5.0 and washed twice in the same buffer by repeated pelleting and re-suspension. The selected antibody is added to the microspheres and incubated with mixing. To optimize coupling, initially a titration of various antibody contractions is used.

[0068] The microspheres are pelleted and PBS/Tween 20 buffer added to block further reaction sites. Antibody binding is confirmed by incubating the microspheres with serial dilutions of a phycoerythrin anti-species IgG detection antibody in the wells of a pre- wetted filtration plate. After suctioning off the excess liquid and washing away inbound detection antibody, the microspheres are resuspended and screened for the IgG detection antibody.

[0069] Preliminary assessment of proteins of interest is made using lavage samples from individuals with proven fertility or infertility. In each group, approximately 5-6 samples collected at early/mid secretory phase are used. The samples are each mixed with the microsphere beads conjugated with antibody to the protein of interest in a prewetted 96- well filtration plate and incubated. Replicate wells of each sample/microsphere mixture are prepared to permit secondary detection with both a biotin conjugated antibody to the protein, and with the identifying lectin also biotinylated. Following incubation of microspheres and sample, the liquid is suctioned off, and the wells washed by three applications of wash buffer, with suctioning in between. The secondary agent (antibody- biotin or lectin-biotin) is then applied and incubated. After further washing, the detection reagent, streptavidin-phycoerthythrin is added to all wells. Following incubation and exhaustive washing, the microspheres are resuspended in Bioplex sheath fluid and analysed on a Bioplex-200 instrument. The detected signal quantified as fluorescence intensity will be determined for each well. A blank well for each microsphere/secondary reagent in which sample is substituted with buffer is used to assess any background binding occurring between the secondary reagent and the immobilized capture antibody.

[0070] A graphical and t-test of results for fertile and infertile individuals is made for the total (antibody detected) and individual glycoform (lectin detected) results. Further verification studies of the most promising biomarkers are performed using larger sample cohorts. EXAMPLE 2

Proteomic analysis of glycoconjugates

[0071] Pooled uterine lavage fluid collected from women, of known fertility or infertility, during their early-mid secretory phase is used. The two pools, fertile (F) and Infertile (IF) are utilized in parallel during all stages of analysis. Initial separation is performed by incubating each pool with a specific agarose bound lectin contained in microspin columns. The unbound protein fraction is removed following centrifugation, and used subsequently for repeated procedures on other agarose lectins. Following exhaustive washing of the spin columns with buffer, the bound glycoproteins (protein glycoforms) are eluted by incubation with a simple sugar complementary to the lectin, e.g. for DBA lectin n- acetylglucosamine is added to elute the captured glycoprotein. The isolated protein fraction is precipitated and resuspended in buffer ready for proteomic analysis.

[0072] The protein from F and IF pools captured using two different lectins is labeled using ICPL-4 reagent (Kellermann (2008) Methods mol boil 424: 113-123). Using ICPL-4 it is possible to label four proteomic states i.e. 4 samples for simultaneous quantitation (Lottspeich and Kellermann (2011) Methods Mol Biol 753: 55-64; Schmidt et al (2005) Proteomic S 5: 4-15). ICPL provides a top down approach labeling the protein prior to separation and enzymatic digestion. ICPL contains four isotopically labeled variants which respectively add a recognisable mass-shift relative to the native isotopic form. Thus, with each of four samples labeled with a unique variant, they may be combined for analysis.

[0073] Once each isolate is labeled it is separated using OFFGEL separation. Conventional 2D electrophoresis performs an initial isoelectric focussing (pi separation) with a secondary molecular weight separation of a complex protein mixture. OFFGEL has been demonstrated to provide improved proteomic coverage. Using OFFGEL the initial isoelectric separation is performed in the liquid phase. In brief, a rehydrated IPG strip is sealed within the well frame and the diluted solution (ICPL-4 labeled combination of four lectin isolates) added equally to all wells and sealed to prevent evaporation. High voltage is applied to the IPG strip and the proteins migrate until they reach a position where the pH matches their pi. After separation the proteins remain within their respective wells until transferred for further analysis.

[0074] The contents of each well are further separated by HPLC before the proteins are digested with Glu-C and analyzed using maldi-TOF spectrometry. At this stage the ICPLquant software (Brunner et al (2010) Proeomics 10: 315-326) is used to analyze the obtained spectra to identify the proteins present but also to make a quantitative comparison of each ICPL label for each identified protein, thus providing quantitative detail as to the abundance of a glycoform. Hence, at this stage it will identify glycoforms significantly dysregulated between the paired F and IF isolates for each lectin.

EXAMPLE 3

Lectin binding to endometrial epithelium changes with cycle phase

[0075] A preliminary histochemical screen with a number of lectins on endometrial tissue taken at different phases of the menstrual cycle confirms that some lectins demonstrate a phase dependent binding to endometrial tissue (Figure 1).

EXAMPLE 4

Endometrial lectin binding changes with fertility status

[0076] A study has also demonstrated that the intensity of binding for some lectins, and hence concentration of glycoconjugates within the endometrium differs in infertile women, notably during the receptive mid-secretory phase, and importantly within the secretory sub-structure of the endometrium i.e. the luminal epithelium and glands.

EXAMPLE 5

Cyclical and fertility-related lectin binding is also seen within the endometrial secretome

[0077] To determine if the cyclic lectin binding to endometrial tissue is reflected in binding to proteins of the endometrial secretome, Western blots are performed with individual lectins on uterine lavage pooled from fertile and infertile women during the early and mid-secretory phase of their cycle. The blots of fluid from infertile women clearly show greater amounts and differing band profiles of proteins binding DBA that have been secreted from the endometrium, indicative of an altered glycosylation profile approaching and during the critical receptive phase (Figure 2).

EXAMPLE 6

Conventional ELISA does not detect change in glycoforms

[0078] To illustrate the difficulty of utilizing identified dysregulated glycoforms as biomarkers in conventional ELISA, a comparative 2D-DIGE study of fertile and infertile women during the mid-secretory phase was further examined (Hannan et al (2010) Supra). The authors identified a number of protein spots as differentially expressed, including a number of highly abundant proteins (e.g. alpha-2-macroglobulin, antithrombin III and alphal -antitrypsin). They performed an immunohistochemical study for alpha-2- macroglobulin; however staining intensity between fertile and infertile tissue failed to confirm their initial proteomic data, though they were able to confirm an elevated expression in secretory as opposed to proliferative tissue of fertile women. To follow through on these data, a number of endometrial lavages were collected from fertile and infertile women during the mid-secretory phase and quantified the alpha-2-macroglobulin present using a commercial Luminex assay kit (Figure 3). No significant dysregulation was apparent, a finding attributable to the assay in effect quantifying all forms of the protein and not the one glycoform of interest.

EXAMPLE 7

[0079] A selection of antibody lectin pairs was tested to determine their ability to discriminate mid-secretory lavage samples from fertile and primary idiopathic infertile women. The antibody lectin pairs tested were listed in Table 3. A number of these displayed some significance in their discrimination of fertile and infertile endometrium (Table 3). Table 3

Data from protein Lectin pairs of interest

Protein Lectin J R alue Significant

Orosrrtuccid 1 DBA [ 0.030 Yes*

Orosmucoid 1 ConA 1 0,069 Yes

Mucin 5B ConA 0.0-93 Yes

G!ycode!in RCA1 J 0.0p4 Yes **

G!ycodeitn DBA [ 0.134 o

A2M-iike protein WGA1 I 0.004 Yes** 1

TAF! DBA !n progress TBD

[0080] In this Example, the aim was to further maximize the performance of these biomarkers, prior to screening of a larger cohort of lavages (n = 40) [EXAMPLE 8] comprising fertile secretory v infertile early and mid-secretory phase lavages. The data are analyzed by t-test to determine significance between groups for each individual marker, together with ROC curve analyses to provide diagnostic performance data (sensitivity and specificity). In addition combinations of the markers are assessed using multivariate logistic modelling, using a leave-one out cross-validation. The resulting ROC curve analysis determine the diagnostic efficiency of combined biomarkers. Use of a leave-one out cross-validation modelling negates the need for separate 'model' and 'test' sets (and thus increased sample numbers) while still providing a robust diagnostic signature.

[0081] The most successful markers are tested on lavage samples from fertile egg donors on LH+2 to determine the impact of their hormone treatments, and on samples taken on the same day from women at the time of embryo pickup. The biomarker test results for the IVF cohorts would then be input into the signature generated previously to predict whether the endometrium is classified as receptive. These predicted results correlate with the actual result (confirmed pregnancy or otherwise) to determine the reliability of the biomarkers for determining receptivity in IVF cycles. These results are reported as a diagnostic accuracy of the biomarker tests for prediction of endometrial suitability. Method and Rationale

Patient Cohorts

[0082] The patient cohorts were selected to provide a detailed assessment of the ability of protein glycoform assays to determine endometrial receptivity. A larger sample set of 40 subjects comprised:

10 fertile mid-secretory phase (i.e. receptive)

10 infertile mid-secretory phase (i.e. non-receptive)

10 fertile early-secretory phase (i.e. non-receptive)

10 infertile early-secretory phase (i.e. non-receptive)

[0083] The inclusion of the early secretory phase fertile women within this sample set provides an indication whether the protein glycoform expression is mid-secretory phase receptive specific, given that during the early secretory phase it is expected that the endometrium will be non-receptive. Further the inclusion of early secretory phase infertile women would indicate if there is a dys-synchrony in their endometrial receptivity.

[0084] Additional analysis is performed on lavage samples collected from infertile women undergoing IVF treatment. The lavage is collected at hCG+2 (oocyte collection day) with patients being segregated according to cycle outcome i.e. pregnancy or no pregnancy. All women in this cohort received GnRH antagonist regimen. This sample cohort can be considered early secretory phase and thus essentially non-receptive; however consideration has been given to the knowledge that in women undergoing IVF there may be an advancement of the receptive phase of the endometrium. Additionally, it is unknown as to the exact trigger for glycosylation changes in the endometrium: thus the IVF hormonal protocols may impact on the glycosylation profiles. The cohort comprised;

GnRH antagonist with outcome Pregnancy

GnRH antagonist with outcome No Pregnancy

Standardization

[0085] All assays are standardized using a pool of uterine flushing collected cross-cycle from secondary infertile and unknown fertility women. The standard is deemed as having a concentration of 1000 arbitrary units for each assay. Standard curves are fitted to a 5 parameter fit (or 4-parameter where insufficient points were available). Antibody and lectin concentrations are listed in Table 4 below.

Assay conditions

Table 4

Assay conditions for each antibody-lectin assay

Statistical Analysis

[0086] Data are analyzed by ANOVA with individual pairings further analyzed using Dunns comparison of means to indicate significance. Mann-Whitney analysis of significant group pairs is performed. Secondary ROC analysis is additionally performed.

[0087] Three assays (A2M-L-WGA, ORM1-DBA and Glycodelin-RCAl) had achieved significant results in initial testing with a small cohort of mid-secretory fertile and infertile women (Table 3). These three assays, A2M-L-WGA, ORM1-DBA and Glycodelin-RCAl, when tested with the extended cohort (n=40) again demonstrated significant difference in ANOVA analysis (p= 0.036*, 0.003** and 0.004**, respectively). Dunns comparison indicated significance between fertile and infertile women during the mid-secretory phase (Figure 4). Mann-Whitney analysis confirmed significance between mid-secretory fertile and infertile populations for A2M-L-WGA (p=0.0472*), ORM1/DBA (p=0.0054**) and Glycodelin-RCAl (p=0.0007***). For all three cases the infertile mid-secretory phase samples were raised. Further, the pregulation from early to mid-secretory phase among infertile women was itself significant for ORM1/DBA and A2M-L-WGA (p= 0.0024** and 0.0286* respectively). In all three assays no significant differences between the fertile and infertile populations during the early secretory phase was seen, and likewise there was no altered expression among the fertile population between early and mid-secretory phases. It was noted that in the ORMl-DBA assay, with the exception of the infertile mid- secretory group, a significant proportion of samples did not give measurable values, indicative that this particular glycosylation has a high degree of specificity.

[0088] ROC analysis of the three assays performance in discriminating fertile and infertile mid-secretory women was performed using DeLong method. Results for each assay showed significant discrimination by ROC analysis.

[0089] Spearman correlation of the three glycoform markers was performed on the mid- secretory samples, with fertile and infertile analyzed separately (Table 5). For the fertile samples a strong correlation (r=0.811, p=0.008) was seen between A2M-L-WGA and Glycodelin-RCAl . However in the infertile population this correlation was not so clearly evident (r=0.622, p=0.060) however A2M-L-WGA and ORMl-DBA did correlate (r=0.833, p=0.005).

Table 5

Spearman correlation analysis was made between assays for fertile and infertile cohorts collected during the mid-secretory phase

[0090] In these analyses, the uterine lavage was collected from both fertile and infertile women during natural menstrual cycles. These three assays were further applied to cohorts of infertile women undergoing IVF treatment. In these women, lavage was collected at the time of oocyte harvest. This is effectively the early secretory phase being just post- ovulation. The women sampled were undergoing GnRH antagonist treatment and were classified according to their cycle outcome, i.e. pregnancy, or no pregnancy following fresh embryo transfer in the same cycle. Statistical analysis found no significant difference between these groups classified by outcome. This is unsurprising given an earlier observation that these markers showed mid-secretory and not early secretory discrimination of fertility.

[0091] Multivariate modelling of the three assays was performed using WEKA software, with the mid-secretory data input. Results with the mid-secretory fertile and infertile data produced 100% correct classification, and an AUC of 1.000. However, no test data set is available; given it is the infertile mid-secretory women who differ in expression levels of the three assays, the use of the early secretory data cannot be used as a test set of non- receptivity. Cross validation was performed in two ways; 10-fold cross validation produced 70% correct classification with AUC=0.715, and leave-one out cross validation produced 65% correct classification with AUC = 0.700. Clearly this dataset is small and a larger dataset and independent test set would provide for a more robust signature.

[0092] Analysis of a further three protein glycoforms (ORM1-CONA), MUC5B-CONA and Glycodelin-DBA) on a small cohort of mid-secretory women had indicated that while there was some discrimination of fertile and infertile women this was not achieving a level of significance (Table 3). These protein glycoforms were further explored on the extended sample set of 40 samples, mid and early secretory, fertile and infertile women. For the two assays utilizing CONA, a significant background issue continues to make use of the assay difficult. The background arises from cross-reaction of the catcher antibodies own glycosylations with the CONA lectin. Analysis was thus performed on the fluorescence intensity as opposed to the calculated arbitrary units/mL concentration. This has the effect of reducing the level of difference as FI is not a direct linear relationship with concentration.

[0093] In the ORM1-CONA assay no significant difference was seen between fertile and infertile women during either the early or mid-secretory phase (Figure 5); however there was a significant change between cycle phases of both fertile (0.0003***) and infertile women (0.0115*). A similar pattern of discrimination was observed for the Muc5B-CONA assay with significance between cycle phases of fertile (0.0068**) and infertile (0.0089) women. Analysis of the antagonist cycle IVF patient cohort revealed a significant difference between outcomes of 'Pregnancy' and 'No pregnancy' for both the ORMl/ConA (0.0754) and Muc5B/ConA (0.0335) assays. This cycle phase data provide further evidence that glycosylation patterns do indeed alter during the receptive phase of the endometrium. Despite in the natural cycles no significant difference between fertile and infertile being evident in either early or mid-secretory phases, the data from IVF stimulation cycles does indicate a difference at hCG+2 (early secretory equivalent), adding to existing data that suggest a different response to the stimulation program by individual women.

[0094] Correlation analysis of ORM1-CONA and Muc5B-CONA showed no correlation among patient groups. Data are shown in Figure 5. EXAMPLE 8

Identification of lection panel

[0095] A selection of lectins with differing carbohydrate binding specificities were identified for testing and are listed in Table 6;

Table 6

Lectins anel

Immunohistochemical Evaluation

[0096] A comparison was made of lectin binding to proliferative (non-receptive) and secretory (receptive) endometrial tissue. Each lectin listed in Table 6 was optimized according to the protocol in EXAMPLE 2, to determine a suitable concentration of lectin for examination of tissues selected from panels of fertile and infertile women (Table 7 and Table 8). In addition for each tissue a negative control in which the lectin was inhibited by pre-incubation with an appropriate blocking sugar was included. Staining of glandular and luminal epithelial cells for each tissue was scored on a scale of 0-3 assessing intensity and distribution. Scoring is based within a single independent set of slides, i.e. a score of 2 in an individual lectin is not representative of a score of 2 in a second lectin. Tissue panels

Table 7

Tissue panels from fertile women for IHC studies of lectin binding

Table 8

Tissue panels from infertile women for IHC studies of lectin binding

WesternBlot Analysis

[0097] Westernblot analysis of fertile and infertile proliferative and early-mid secretory phase uterine flushings was conducted with the panel of lectins (Table 9). For this purpose four pool samples were prepared (fertile proliferative, infertile proliferative, fertile early- mid secretory and infertile early-mid secretory). These pools comprised the following individual samples pooled in equal volumes. Western blotting was performed as described in Example 1. Table 9

Individual uterine flushings pooled for western blot analysis

[0098] Each pooled was assayed for protein concentration using BCA protein assay. Each sample was assayed in duplicate at three dilution (neat, 1 :4, 1 :9), and the assay repeated by two operators. Mean protein concentration was determined for each pool, and the results are shown in Table 10.

Table 10

Protein determined using BCA assay for flushings pools are shown in ug/ml. Values shown are means from duplicate assays performed at three sample dilutions, and replicated by two operators.

Results

[0099] Table 11 summarizes the determined optimal concentrations of lectin and inhibitor sugar used in immunohistochemistry studies. The appropriate dilution of individual lectins for immunohistochemistry was determined in initial studies. For each lectin an appropriate neutralizing sugar and its' concentration required to achieve full inhibition of lectin binding to tissue for use as a negative control was also determined. Table 12 summarizes the optimal concentrations for western blotting with each lectin. The appropriate dilution of individual lectins for western blotting was determined in initial studies.

Table 11

Optimal concentration of lectin and inhibitor sugar

Table 12

Optimal concentration of lectin for Western blotting

[0100] Immunohistochemistry and western blotting results are presented for each individual lectin. Photographic images are produced representative of the tissue study results. Graphical representations of staining of Luminal and Glandular epithelia are also generated. Western blots show obvious differences between fertile and infertile, proliferative and secretory pools. For each lectin two development time images are produced, the second longer exposure providing comparison of some of the lower abundance proteins, while the shorter exposure picture allows visual comparison of the highly abundant proteins.

[0101] A representative protein gel image is shown in Figure 6. The gel is loaded in similar manner as was performed for each western blot, thus the four sample pools are shown at equal protein and equal volume loading. [0102] IHC studies show demonstrable CONA recognition throughout the cycle, however, it is seen to elevate in the mid to late secretory phase in fertile women. This elevation is less clear in the infertile cohort.

[0103] DBA staining within the luminal epithelia was sparse or zero in the proliferative phase tissues of both fertile and infertile individuals. The staining increased throughout the secretory phase and was apically positioned within the cells. The infertile women displayed greater expression, and this was indeed also true within the glandular epithelium where highly intense staining throughout the majority of glands was visible by the mid secretory phase, and visible secretions within the glands also noted. Western analysis though reactivity was weak, revealed two bands which appeared uniquely within the mid- secretory infertile pool.

[0104] Prolific staining of the apical surface of the luminal epithelia throughout the menstrual cycle. Staining also present in most glands throughout the cycle, and appearing as secretions within glands in the secretory phase tissues. No observable expression difference between fertile and infertile women. Western analysis identified a band of approximately 70kDa present in the fertile secretory pool.

[0105] Immunohistochemistry showed a consistent level of staining throughout the cycle and regardless of fertility status within the apical surface of luminal and glandular epithelium. Western analysis found only a weak signal making analysis difficult however there appeared to be no observable differences between pool samples.

[0106] Weak staining of tissue epithelium was seen, but there appeared no pattern to expression across the cycle and no evidence of altered expression between fertile and infertile cohorts. Western analysis showed very poor reactivity with GSLI.

[0107] GSLII appears to bind more strongly to secretory phase tissue from fertile women, compared with proliferative phase tissue. In addition there is an indication that infertile women showed less reactivity during the secretory phase. However, western analysis showed very poor reactivity with GSLII.

[0108] Immunohistochemistry indicates that in fertile women there is a rise in jacalin reactive sugars during the secretory phase compared to the proliferative. However, the infertile population appears to have a higher proliferative expression, thus obscuring any altered levels in the secretory phase. In Western analysis JAC showed good resolution . There is no clear specific staining below 50kDa. Of note is the 180kDa band which is visible only in the Fertile Proliferative pool. Additionally, a 150kDa band is visible in the Infertile secretory pool, however, it is not visible where less protein is loaded. A 50kDa band is visible in infertile pools. It is worth noting that overall staining is darker in the Fertile Proliferative pool even with equal protein loading suggestive of a greater expression of jacalin recognized sugars overall.

[0109] LEL binding reaches a maximum in the mid secretory phase of fertile women, evident in both the glandular and luminal epithelium. However there appeared no clear pattern of difference between fertile and infertile women. Western analysis identified two small molecular weight bands (21 and 22kDa) and a larger 70kDa band, prominent in all groups except the Infertile proliferative pool. Additionally, longer exposure identified a 45kDa band visible only in the Infertile proliferative pool.

[0110] Staining of glandular epithelium appears to increase in the mid secretory phase of the cycle, and there is some indication that this elevation is not so strong in the infertile cohort. There is, however, extensive stromal staining throughout the cycle. PSA Western blotting did not show differences between the pooled groups. The differences observed between groups, appear to be dependent on protein amount leaded at equal volume and are not seen with equal protein loading.

[0111] Immunohistochemical analysis found RCA-1 while strong in the proliferative phase luminal epithelium declined through to a lowest level in the mid-secretory phase of the fertile women's cycle before rising again in the late secretory. However, the infertile cohort appeared to reach its low point in the early secretory phase, and be recovering by the mid secretory. Glandular expression did not show such a clear cyclic pattern. Western blot analysis found RCA-I shows good resolution of several bands. There is no specific staining of proteins <50kDa. Some bands are apparently restricted to particular groups, however, most bands differ mainly in intensity between groups rather than being unique between samples. Most notably, three bands are prominent in all groups but appear very faint at 130 and 1 lOkDa or absent at 50kDa in the Fertile Secretory pool.

[0112] Tissue analysis showed a cyclic expression pattern of SB A binding sugars notably in the glandular epithelium, where in fertile women a trough occurs at the early secretory stage, followed by an increase in the mid secretory phase. These is a suggestion that the infertile cohort may show a somewhat reduced expression throughout the cycle. In western analysis of sample pools there was no clearly visible difference in binding of the SBA lectin.

[0113] Staining of tissue with SJA was restricted to some areas of luminal epithelium and glandular epithelium however the staining was very weak. This absence of SJA binding sites was likewise reflected in western analysis where there was little signal at all.

[0114] STL showed a very uniform level of staining of tissues regardless of stage or fertility status, though actual signal strength was quite strong. In western analysis however despite a lengthy imaging of 30 minutes very little signal could be observed, perhaps indicative that the STL recognized sugars are primarily on non-secreted proteins within the tissue.

[0115] ULEX showed a very disparate binding to tissues throughout the cycle with both high and low binding samples in all stages of the cycle. ULEX blotting did not show differences between the pooled groups. The differences observed between lanes, appear to be dependent on protein loading as they are not evident in lanes where equal protein is loaded.

[0116] A small amount of luminal epithelial staining in proliferative tissue was visible but did not show any discernable increase during progression of the secretory phase. The glandular epithelia did however show a clear elevation of binding in the mid and late secretory phases of the normal fertile women. This was replicated in the infertile cohort.

[0117] There was no clear pattern to WGA binding to tissue regardless of fertility status or stage of cycle. In the western blot analysis the area of most interest is in the region around 150kDa. Analysis revealed two distinct bands in the Fertile Proliferative pool, one large band in the Infertile Secretory pool, while the Fertile Secretory pool showed very little staining in this region. Conversely, a 75kDa band is relatively strong in the Fertile Secretory pool in comparison with the other groups.

EXAMPLE 9

Proteomic identification of protein glycoforms

[0118] This Example relates to the proteomic identification of protein glycoforms showing unique or dysregulated expression in uterine lavage samples between fertile and infertile women in the early-midsecretory phase.

[0119] Three lectins, CONA, RCA-1 and WGA, that capture glycoproteins were selected for further investigation based on the data the previous Examples. These were applied to pooled secretory phase lavage from fertile and infertile women. Subsequently CONA analyses were repeated using individual samples. This allowed more detailed analysis of early and mid-secretory phases individually.

[0120] The initial results using pooled lavage found for each lectin, a number of dysregulated glycoproteins showing either unique or greater than 2-fold change in detected protein between fertile and infertile groups. For WGA these were 46 of 113 proteins, for RCA, 43 of 103 proteins and for CONA, 50 of 118 proteins identified.

[0121] Subsequent analysis of individual samples using CONA lectin revealed 73 of 125 protein dysregulated, 68 of 116 proteins in early secretory samples, and 64 of 96 proteins in mid secretory samples. It should be noted that the mass-spectrometry used in this analysis identifies only proteins with an appropriate N-linked carbohydrate moiety, that used for the lavage pools identifies proteins with both O and N linkages.

Proteomics Method 1

[0122] Two pools of uterine flushings were prepared, one of fertile and the other of primary infertile women. Each pool comprised samples of early secretory (n=3) and mid secretory (n=3) phase lavage, based on pathology staging of endometrial biopsies taken at the time of lavage collection. The pooled samples were each loaded onto a fresh spin column of lectin beads, incubated and then extensively washed to remove unbound proteins. The lectin bound proteins were then competitively eluted using an appropriate sugar solution. A second elution with acid, and final elution with SDS were performed to ensure that all proteins bound by the lectin were eluted for analysis. SDS-PAGE and western blot using the lectin of interest were performed on non-binding and elution fractions from the isolation. The sugar eluate was trypsin digested for subsequent MS analysis.

[0123] Starting and elution fractions for each isolation were analysed by SDS-PAGE and western blot with the lectin, to determine efficiency of the capture/elution steps.

[0124] This process captures intact proteins from the uterine lavage samples, and by eluting with sugars, lectin binding of both N and O linked glycosylations is reversed. In the MS analysis post trypsin digestion, it is the non-glycosylated regions of the isolated proteins which are identified.

[0125] This process was applied to three lectins; WGA, RCA-1, and CONA. Proteomics Method 2

[0126] Individual samples of early and mid secretory staged uterine lavage samples from fertile and infertile women were trypsin digested, then mixed with the lectin of choice, and separated using a filter that isolated lectin bound glycosylated peptides from those not carrying an appropriate sugar moiety. The lectin captured peptides were released from the lectin enzymatically using PNGase cleavage and identified using MS.

[0127] This process isolates and identifies only the peptide region carrying the actual glycosylation of interest, allowing analysis of changes to specific glyosylation sites. In addition, this method can provide for future sequential lectin absorptions as only peptides, not all regions of the protein, are selectively bound at each stage.

[0128] This method was applied to CONA. Proteomics Data Analysis

[0129] For both proteomics methods, analysis used Scaffold software to identify proteins which were either absent from one cohort vs the other, or showed >2-fold change in expression (based on normalized spectra count), either up or down regulated. Fisher test for Exactness was also applied and the significance value (p) reported.

Results

Wheat Germ Agglutinin

[0130] From the 300μg protein in each of the pooled secretory flushings, 20^g (6.8%) was recovered by sugar elution from the fertile pool and 23^g (7.8%) from the infertile pool. Some protein (8^g) was measured in the acid elution from the fertile but not the infertile samples. Both samples were run on ID PAGE. A 16kDa band is visible in both stain-free gels, but is absent in the lectin blots indicating it is not WGA-specific.

[0131] The results are summarized in Tables 13 to 18.

Table 13

WGA Proteomics Method 1

Ricinus Agglutinin

[0132] From the 290 ig of protein in the fertile secretory flushings, 25.2 g (8.7%) was recovered in the sugar elution. From the 553 μg of protein in the infertile secretory flushings, 42^g (7.7%) was recovered in the sugar elution. A small amount of protein ^g) was measured in the acid elution of the fertile flushing isolation. Following analysis ID PAGE, this is visible as a 28kDa band in the stain-free gel, but is absent in the lectin blot indicating it is not RCA-specific.

Table 14

RCA1 Proteomics Method 1

Concavalin A

[0133] From the 29C^g of protein in the fertile secretory flushings, 19^g (6.8%) was recovered in the sugar elution. From the 553 μg of protein in the infertile secretory flushings, 46^g (8.5%) was recovered in the sugar elution. ID PAGE analyses were conducted. Unbound proteins are seen in the stain-free gel images in both groups. There is a small amount of CONA-specific 50kDa protein visible in the fertile pool lectin blot and in the infertile pool lectin blot. All isolated CONA-specific glycoproteins were eluted in the sugar solution in both the fertile and the infertile isolations.

[0134] 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 steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any to or more of said steps of features. Table 15

CON A Proteomics Method 1

Number of Proteins

Total Proteins Identified in Experiment 118

Unique 'Fertile' Proteins 24

Unique 'Infertile' Proteins 16

>2 up-regulated in infertile vs fertile groups 13

>2 fold down-regulated in infertile vs fertile groups 7

Table 16

CONA Proteomics Method 2 (individual samples analyzed)

Number of Proteins

Total Proteins Identified in Experiment 125

Unique 'Fertile' Proteins 14

Unique 'Infertile' Proteins 39

>2 up-regulated in infertile vs fertile groups 22

>2 fold down-regulated in infertile vs fertile groups 8

Secretory Fertile (n=6) v Secretory Infertile (n=6)

Table 17

Early Secretory Fertile (n=3) v Early Secretory Infertile(n=3)

Number of Proteins

Total Proteins Identified in Experiment 116

Unique 'Fertile' Proteins 12

Unique 'Infertile' Proteins 15

>2 up-regulated in infertile vs fertile groups 12

>2 fold down-regulated in infertile vs fertile groups 29 Table 18

Mid Secretory Fertile (n=3) v Mid Secretory Infertile(n=3)

Protein lists are shown in Tables 19 to 23. Identifications are color coded as shown:

Unique id ntifcation.: in Fertile aiVi l ?.

>2-f k1 dOs ^f .-!i-»eguL¾iecl in infertile

Unique id rtif dt urn inter tii*r ample

Table 19

WGA Protein Analysis Results (Proteomics method 1)

; « III I ls ill■III

r. 111 III m mmk

mm

-

- ill iii mm III mm 111 βΐ ... mm

iliiiiiiiiiiii !!!!!!ill ; > ' ί

RCA-1 Protein Analysis Results (Proteomics method 1)

-47 -

Table 20

CONA Protein Analysis Results (Proteomics method 1)

Illiji ss

111 mmm mmmmm m i

7

: ::

r 11 ...111

: ^"

- -. ^■■

111 m Si iss:::^::::

- im m ^ * s iiiii:

- mm

l :¾¾¾:*¾: mm : ... ~ ii: i l 1 iSi :: : :¾ u *:

fSs iP 1:β llit is

111 iiiiiii lllll

mm mm mm •N 1111 mm ·- ^■ ■∞**# m

mmm a. 111: IB mmm mmm i!ii!li 111:: iiSSii

mm m

*

flSS

111 "3 s¾g m v. mm

- mm z 11

~ mm m - m

Table 23

Concavalin A Proteomics Method 2 Mid Secretory Fertile v Mid Secretory Infertile Results

lis β m - ! mm mmm -.: mimsm BIBLIOGRAPHY:

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