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WO2003062832A1 - Procede et trousse pour examen systematique de la fertilite - Google Patents

Procede et trousse pour examen systematique de la fertilite Download PDF

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Publication number
WO2003062832A1
WO2003062832A1 PCT/GB2003/000229 GB0300229W WO03062832A1 WO 2003062832 A1 WO2003062832 A1 WO 2003062832A1 GB 0300229 W GB0300229 W GB 0300229W WO 03062832 A1 WO03062832 A1 WO 03062832A1
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Prior art keywords
egf
cells
antibody
female
sol
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Inventor
Helen Mardon
David Barlow
Ian Sargent
Kayta Chobotova
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Oxford University Innovation Ltd
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Oxford University Innovation Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1808—Epidermal growth factor [EGF] urogastrone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/18—Feminine contraceptives
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/485—Epidermal growth factor [EGF] (urogastrone)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • Heparin-binding epidermal growth factor is a member of the EGF growth factor family which has been implicated in a variety of biological processes such as wound healing, tumour growth, smooth muscle cell hyperplasia, angiogenesis and reproduction ([Higashiyama et al , Science, 251:936-9,1991]; reviewed in [Raab et al , Biochim Biophys Acta., 1333: F179-99, 1997] [Arkonac et al , Biol Chem., 10; 273(15) :9352, Apr 1998]) .
  • HB-EGF is expressed by many cell types [Abraham et al , Biochem Biophys Res Commun.
  • the extracellular domain can be released as a soluble form of HB-EGF (sol -HB-EGF) , probably as a result of the action of metalloproteinases [Higashiyama et al , Biol Chem., 267:6205-12, 1992] [Suzuki et al , Biol Chem., 272:31730-7, 1997] [Izumi et al , E bo J. , 17:7260-72, 1998] , although a substantial amount of transmembrane precursor remains uncleaved on the cell surface
  • Tm-HB-EGF is a juxtacrine growth factor with diverse functions in that it can either stimulate [Goishi et al, Mol Biol Cell., 6:967-80, 1995] [Higashiyama et al, J Cell Biol., 128:929-38, 1995] or suppress [Miyoshi et al, Biol Chem., 272:14349-55, 1997] [Iwamoto et al, Biol Chem., 274:25906-12, 1999] .the proliferation of the neighbouring cells, mediate cell attachment [Raab et al, Development., 112:637-45, 1996], serve as a survival [Takemura et al, Biol Chem., 272:31036-42, 1997] [Miyoshi et al , Biol Chem., 272:14349-55, 1997] or apoptosis
  • Sol-HB-EGF acts as a potent paracrine and autocrine growth factor and chemotactic agent for many cell types such as vascular smooth muscle cells, keratinocytes, fibroblasts and cardiomyocytes [Higashiyama et al, Science ' ., 251:936-9, 1991] [Wilson et al, Exp Eye Res., 59:665-78, 1994] [Kiso et al , Hepatology, 22:1584-90, 1995] [Hashimoto et al, Biol Chem., 269:20060-6, 1994] [Mishima et al , Acta Neuropathol Berl . , 96:322-8, 1998] [Elenius et al, Embo J., 16:1268-78, 1997].
  • HB-EGF may have an important function in reproduction in rodents.
  • the spatio-temporal expression of HB-EGF suggests a function in rodent uterine receptivity and implantation of the rodent blastocyst .
  • Expression of HB-EGF in the endometrium of rats is regulated by steroid hormones, progesterone and estradiol [Zhang et al, Endocrinology, 134:1089-94, 1994].
  • HB-EGF mRNA can be detected at the site of blastocyst apposition, prior to blastocyst implantation [Das et al , Development, 120:171-83, 1994] .
  • 32D cells engineered to express tm- HB-EGF adhere to mouse preimplantation blastocysts, but cells expressing sol-HB-EGF do not [Raab et al,
  • HB-EGF is expressed in the stroma of proliferating endometrium (day 5-14 of the cycle) and in the glandular and lumenal" epithelium where expression of HB-EGF increased during the period of "implantation window” [Yoo et al, Dev Genet., 21:102-8, 1997] [Leach et al , Clin Endocrinol Metab. , 84:3355-63, 1999].
  • Sol-HB-EGF also improves the development of mouse [Das et al, Development, 120:171-83, 1994] and in-vitro fertilised human [Martin et al, Hum Repod. , 13:1645-52, 1998] embryos to the blastocyst stage and subsequent hatching.
  • sol- and tm-HB- EGF are mediated by the receptors EGFR1 (HER1) and ErbB4 (HER4) .
  • Sol-HB-EGF binds and activates EGF-R1
  • HB-EGF binds to ErbB4 expressed on the trophectoderm of mouse blastocysts [Paria et al, Development, 126:1997-2005, 1999] .
  • sol-HB-EGF has been reported _to act as chemotactic factor, but not as a mitogen for cells expressing ErbB4
  • sol-HB-EGF is both chemotactic and mitogenic in cells expressing EGF-R1 [Elenius et al Embo J. 16:1268-78, 1997] .
  • the inventors have now elucidated models for human embryo implantation and endometrium proliferation that allow the dissection of specific molecular events that occur during the implantation/ proliferation process in the human.
  • the inventors have identified a function for both soluble and tm-HB-EGF in the proliferative activity of stromal cells of the human endometrium. Also, they have determined for the first time a function for the transmembrane form of HB-EGF in implantation of the human blastocyst .
  • the inventors have identified the specific HB-EGF receptors involved in blastocyst attachment and endometrium proliferation.
  • they have demonstrated that the function of HB-EGF in human embryo implantation is mediated via the receptor ErbB4 and that the proliferation of the human endometrium is mediated by EGF-R1.
  • the inventors have developed assays for screening compounds capable of modulating the activity of HB-EGF, both sol-HB-EGF and tm-HB-EGF, its receptors and its interactions therewith.
  • the elucidation of the mechanisms involved in human endometrium proliferation and blastocyst attachment has enabled the inventors to identify potential contraceptive or fertility agents, exhibiting specific activity in respect of the above target mechanisms.
  • the screened compounds are potential fertility and/or contraceptive agents. Diagnostic assays are also provided for determining the fertility status of a human female.
  • the invention provides a method for identifying a compound which modulates the binding of HB-EGF to an HB-EGF receptor and thereby identifying potential fertility enhancing and/or contraceptive agents, which method comprises: contacting the compound under test with an HB-EGF receptor or a biologically active portion thereof, in the absence or presence of HB-EGF, determining the binding .of HB-EGF to the receptor in the presence and absence of the test compound, determining the effect of the test compound on the binding of HB-EGF to said receptor and, thereby identifying a compound which modulates the binding of HB-EGF to said receptor.
  • the HB-EGF receptor is EGF-R1 or Erb- B4.
  • the assay can be a cell-free or cell-based.
  • the assay is carried out on physiologically-relevant cells expressing the desired EGF-R1 or Erb-B4 receptor.
  • the cell for example, can be a yeast cell or a cell of mammalian origin, such as human embryonal kidney cell line, HEK 293T. More preferably, the cell is an isolated human female endometrial cell, such as a stromal cell.
  • the method of the invention can advantageously be based upon a phosphorylation assay or upon a proliferation assay, both well known in the art..
  • Protocols for performing phosphorylation or proliferation assays are well known in the art, such as described in Sambrook et al . (Molecular Cloning: a Laboratory Manual, 1989) ; representative examples are given in the experimental part included herein.
  • the binding of ligands to the EGF family receptors, including EGF-Rl and Erb B4 results in activation of tyrosine kinase and in receptor phosphorylation.
  • Compounds which modulate activation of EGF-Rl or Erb-B4 can be identified by detecting the effect on receptor phosphorylation.
  • the phosphorylation assay involves immunoprecipitation of cell lysates (previously exposed to HB-EGF) with an anti- EGF-Rl or ErbB4 antibody followed by Western blotting of the immuno precipitate with an anti-phosphotyrosine antibody.
  • tissue culture plates (6 well-plate) are coated with isolated cells expressing the desired receptor or with cells expressing said receptor.
  • a sample containing the test compound in the absence or presence of recombinant HB-EGF (or a fragment of the protein which is still capable of binding to EGF-Rl or Erb-B4) is then added to the wells and the plates are incubated to allow time for binding of the test compound to the receptor.
  • the wells are then washed; the cells are lysed, antibodies specific for the receptors are added and complexes are collected.
  • Receptors which ' have become phosphorylated can be detected via the antibodies specific for the phosphorylated complex and the type of receptor can be identified via an antibody specific for said receptor; this can easily be done by immuno blotting or Western- blotting.
  • Western blotting is a well known technique for the analysis and identification or proteins.
  • the complexes are separated by polyacrylamide gel electrophoresis and then transferred to a cellulose membrane or chemically treated paper to which the proteins bind; preferably, the complexes are transferred to a PVDF (Polyvinylidene Difluoride) membrane.
  • PVDF Polyvinylidene Difluoride
  • Phosphotyrosines, EGF-Rl and/or Erb-B4 bound to the membrane are detected by overlaying the appropriate antibody, and bound specific antibodies detected by overlaying with a labelled secondary antibody followed by use of standard detection procedures for that label.
  • the secondary antibody may be an HPR-conjugated secondary antibody detected by standard chemi-immunescent detection procedures.
  • Antagonists will result in decreasing phosphorylation; agonists will result in increasing phosphorylation, as assessed by the intensity of the bands .
  • the method of the invention can also be based upon a proliferation assay.
  • the effects of agonists/antagonists of HB-EGF receptors can be identified by measuring the effects on cellular growth or increase, after treatment with the compound.
  • Cellular proliferation assays either directly measure effects on cell growth by counting the cells under microscope or using an electronic particle counter or indirectly, by measuring incorporation of radio-active cDNA precursors, by using chromogenenic dyes to quantitate total protein or by measuring the metabolic •activity of cellular enzymes.
  • the uptake of 3 [H] thymidine is a common method to indirectly determine cell number after treatment .
  • This method requires a pulse of H 3 thymidine followed by washing and counting in a scintillation counter.
  • the wells of microtiter plates are coated with cells expressing the desired receptor.
  • a sample containing the test compound in the absence or presence of recombinant HB-EGF (or fragment of the protein which is still capable of binding to EGF-Rl or Erb-B4) is then added to the wells and the plates are incubated to allow time for binding.
  • 3 [H] thymidine is added to each well, cells are washed and harvested.
  • the amount of incorporation of 3 [H] thymidine is determined by scintillation.
  • Antagonists will result in decreasing thymidine uptake; agonists will result in increasing thymidine uptake.
  • the above-described method can be used to screen for agonists or antagonists of EGF Rl and/or Erb B4 receptors, and hence for compounds potentially capable of enhancing or inhibiting fertility.
  • the screened compounds can respectively be useful as fertility enhancing agents or as contraceptive agents.
  • the invention further provides a method for identifying potential fertility enhancing and/or contraceptive agents, based on their capacity to bind HB-EGF receptors, which method comprises: expressing HB-EGF receptor proteins as dimers, contacting said dimers with the compound under test in the presence of a labelled tracer, said labelled tracer being subsequently detected by a conjugated counteragent , determining the binding of HB-EGF to said dimers in the presence and absence of the test compound, - . determining the effect of the test compound oh the binding of HB-EGF to said receptors and, thereby identifying a compound which modulates the receptor- ligand binding and therefore has potential as a fertility enhancing or contraceptive agent.
  • HB-EGF receptor is EGF-Rl and/or Erb B4.
  • the labelled tracer is biotinylated or iodinated HB-EGF; a preferred conjugated counteragent is streptavidin-HRP.
  • the invention further provides a method for identifying potential fertility enhancing and/or contraceptive agents, which method comprises: contacting an HB-EGF producing cell with a compound under test, determining the amount of tm-HB-EGF expressed in the presence and absence of the test compound, determining the effect of the test compound on the amount of tm-HB-EGF expressed and thereby identifying a compound which modulates the production of tm-HB-EGF.
  • the invention provides a method for identifying potential fertility enhancing and/or contraceptive agents which method comprises contacting an HB-EGF producing cell with a compound under test, determining the amount of soluble HB-EGF secreted in the presence and absence of the test compound, determining the effect of the test compound on the amount of soluble HB-EGF secreted and thereby identifying a compound which modulates the production of soluble HB-EGF.
  • soluble HB-EGF refers to -HB-EGF which has been released from any HB-EGF producing cell by the cell's natural mechanism and does not include HB-EGF which has been generated .from tm-HB-EGF by technical intervention of man.
  • Tm-HB-EGF as used herein means an HB-EGF which includes an amino acid sequence encoding the transmembrane domain or an HB-EGF from which all or substantially all the transmembrane domain has been cleaved by a technical intervention of man.
  • the HB-EGF producing cell is an isolated cell of mammalian origin, more preferably, - li ⁇
  • the cell is an isolated human female endometrial stromal cell.
  • the aforesaid compound screening assay will preferably be carried out in a multiwell plate as is well-known to a person skilled in the art.
  • the cells may be cultured for a suitable period, for example, from 24 to 27 hours in any tissue culture medium suitable for the purpose.
  • tissue culture medium suitable for the purpose.
  • DMEM medium is found to be useful, although other suitable media would be well-known to those skilled in the art.
  • the medium may be serum-free for part or all of the incubation period. In test wells the medium will include the compound under test while control wells will not. An inhibitor of protein translocation may be added to the wells.
  • the culture media may be harvested and the resultant potential HB-EGF containing solution tested.
  • Immunoassays are particularly advantageous for the purpose, making use of an antibody which recognises one or more epitopes of soluble HB-EGF, which antibody is conjugated to a molecule which facilitates identification by being a revealing label or being " suitable for attachment in a revealing label.
  • label may be radioactive, luminescent or flourescent or may be an enzyme .
  • HB-EGF may advantageously be based upon an enzyme-linked immunosorbent assay (ELISA) , well known in the art; protocols can be found for instance in Sambrook at al . (Molecular Cloning: a Laboratory Manual, 1989) ; representative examples are given in the experimental part included herein.
  • ELISAs provide sensitive and quantitative detection of specific antigens or antibodies. A variety of ELISA formats can be employed. Commercially available ELISAs are often based on the antibody-sandwich format or double-layer variation.
  • the sandwich ELISA generally requires two antibodies that are directed against a particular antigen. One antibody is passively adsorbed (coated) onto the surface of the wells of an ELISA plate.
  • the wells are then "blocked" with a nonspecific protein solution to keep background levels low.
  • the samples containing the antigen in solution are then added to the wells and incubated for a sufficient amount of time for the antigen to bind to the antibody immobilized on the plate.
  • the second antibody is added to the well. This second antibody binds to the immobilized antigen completing the sandwich.
  • the second antibody is detected with an enzyme conjugate specific for the second antibody. Alternatively, the second antibody itself can be labelled for subsequent detection.
  • the conjugated enzyme When the enzyme substrate is added to the wells in the final step, the conjugated enzyme, and therefore the antigen, is detected by observing the colorimetric, flourescent or chemiluminescent reaction products -in an appropriate ELISA plate reader.
  • antigen is bound to the plastic surface (test tubes, wells or beads) followed by the test sample containing antibody, then the enzyme conjugate. Incubation complexes with a suitable substrate results in a coloured product which may be measured spectrophotometrically.
  • the cells may be cultured in suitable media with or without test compounds as described above .
  • the media can include an inhibitor of protein translocation, for example monensin to prevent release of HB-EGF from the cell .
  • a suitable antibody specific for one or more epitopes of HB-EGF is added to the culture wells and the wells are washed whereby all but antibody bound to tm-HB-EGF is removed.
  • a secondary antibody is then added, the qualitative signal from which will correlate with the amount of tm-HB-EGF expressed.
  • wells are coated with an HB-EGF (or a biologically active portion thereof) , producing cell.
  • a sample containing the compound under test together with monensin is then added to the wells and the plates are incubated to allow time for specific modulation of the HB-EGF production.
  • the wells are blocked, for instance with a solution of BSA in PBS.
  • a tag of primary antibody is then added; a suitable labelled antibody for example is biotinylated anti HB- EGF. Many other tag molecules which are equally suitable for this purpose are know in the art and commercially available.
  • the wells are then washed and a secondary antibody with the appropriate specificity is added to the wells.
  • the enzyme substrate is then added and bound proteins are detected, leading to the determination of the amount of tm-HB-EGF production.
  • the cells are cultured in the presence or absence of test compound as described above.
  • EGF is cleaved from the molecule using a suitable proteolyte enzyme. This may be achieved by adding the enzyme to the wells following culture of the cells or by adding an inducer of the cell's naturally occurring proteases.
  • Suitable proteolytic enzymes which may be added include ADAM12 (Asahusa et al , 2002, Mature Med. 8., pp35-40) , MDC9/meltrin gamma/71DAM9 (Izumi et al, , 1998, EMBO J. 17, pp7260) or MMP3 (Suzuki et al , 1997, J. Biol. Chem. 272, pp31730-31737) .
  • Preferred enzymes are metalloproteases .
  • Suitable enzyme inducers are selected from phorbol ester PMA (Phorbol 12-myristate 13 acetate) , a metallo proteinase inducer, ionomycin and thapsigargin (Dehlefsen et al, (1998) , J. Cell Biochem, 69., ppl43-153) .
  • enzymic cleavage effectively converts tm-HB-EGF to a soluble form which then may be measured as described above for soluble HB-EGF.
  • test compounds may include, for example, compounds having a known pharmacological or biochemical activity, compounds having no such identified activity and completely new molecules or libraries of molecules such as might be generated by combinatorial chemistry.
  • Compounds which are nucleic acids, including naturally occurring nucleic acids and synthetic analogues, polypeptides or proteins are not excluded.
  • compound screening assays involve - .
  • Compounds which are identifiable as having potential pharmacological activity using the methods of the invention may be used as lead compounds in the further development of drugs with pharmaceutical potential or may themselves be formulated into pharmaceutical compositions.
  • the compounds screened in accordance with the present method can either increase or decrease HB-EGF production. They can respectively be useful as fertility agents or contraceptive agents.
  • the invention refers to the compounds identifiable by the above described methods .
  • the invention provides a method of producing a composition suitable for treating infertility in a female or suitable for use as a contraceptive agent in a female which comprises: a) carrying out any one of the compound screening methods described above; and b) formulating any compound identified as capable of modulation of ligand binding to an HB-EGF receptor or modulation of sol- and/or tm-HB-EGF production, into a pharmaceutical composition with a pharmaceutically acceptable carrier or diluent .
  • the invention relates to a method of inducing contraception in a- mammalian (preferably human) female which comprises administering to said female an inhibitor of sol- and/or tm-HB-EGF expression or an antagonist of an HB- EGF receptor.
  • Contraceptive compositions are also provided comprising said inhibitor or antagonist together with a pharmaceutically acceptable carrier of diluent .
  • Preferred receptors are HER1 and HER4.
  • the invention relates to a method of enhancing fertility in a mammalian (preferably human) female which comprises administering to said female an enhancer of sol- and/or tm-HB-EGF expression or an agonist of an HB-EGF receptor.
  • Pharmaceutical compositions are also provided comprising said enhancer or agonist together with a pharmaceutically acceptable carrier or diluent.
  • the use of said enhancer or agonist in the manufacture of a medicament for treatment of human female infertility is also provided.
  • the enhancer of sol- and/or tm-HB-EGF may be TNF ⁇ or TGF ⁇ shown in Example 8 herein to stimulate sol- and tm-HB-EGF expression in endometrial stromal cells.
  • contraceptive and pharmaceutical compositions of the present inventions are advantageously formulated for local application to the endometrium and suitable excipients for this purpose will be well- known to those skilled in the art.
  • a gel or viscous tablet is may be used for delivery to the endometrium or tot he vagina or cervix.
  • the female reproductive tract is more accessible, for specific drug delivery than many other organ systems .
  • the advantages of local application of therapeutic agents to the endometrium are that unwanted effects on other systems that might be induced by systemic drug delivery can be avoided, and the dose can be lower than with systemic administration of drugs.
  • the need is to deliver drugs directly into the uterus or into the vagina from which natural diffusion may occur.
  • Direct delivery to the uterine cavity is most readily achieved either by canulation to incorporate a molecule in a liquid medium as already occurs at embryo transfer in IVF with the use of transfer medium, or by instillation through the cervical canal by syringe placed at the cervix without canulation of the uterine cavity.
  • drug delivery to the upper vagina and cervix in the form of a viscous gel or tablet is a currently recognised route of drug delivery to this area.
  • the invention also provides a diagnostic test to assess fertility status in a female patient.
  • Said test comprises: determining the level of tm-HB-EGF in a isolated endometrial sample within implantation window time for the patient, correlating the appearance of tm-HB-EGF with fertility status.
  • the above test is aimed at assessing levels of tm-HB-EGF during the implantation window in the endometrium of women.
  • the term "implantation window” encompasses the window of time during which the uterine endometrium is receptive to the conceptus; in the human, this occurs in the secretory stage of the menstrual cycle. Implantation is defined as days 6-8 post the day of the luteinising hormone (LH) surge.
  • the implantation window can be estimated on the basis of a regular menstrual pattern as approximately seven days before the expected first day of the menstrual period; alternatively, it may be determined by using the mid-cycle LH urine test (Clear-Plan Styx) to identify LH surge, starting at day 12 until the surge is detected. Any woman with infertility should have had ovulation confirmed.
  • the level of tm-HB-EGF expression may be determined in the endometrial sample by direct staining of the tissue sample, for example with a suitably labelled antibody specific for one or more eptiopes of tm-HB-EGF.
  • the sample is obtained by pipette biopsy. A preferred method is described in Example 6 herein.
  • the level of tm-HB-EGF expression my be determined by any of the methods already described herein and in particular by using the method involving the release of the extracellular domain of tm-HB-EGF by proteolytic cleavage followed by measurement of the amount of the cleaved product.
  • the HB-EGF levels may be correlated with endometrial receptivity and likelihood of conception.
  • kits for assessing the fertility status of a human female which comprises: antibody specific for one or more epitopes of tm-HB-EGF conjugated to a molecule or element facilitating its detection and a proteolytic enzyme and/or a compound for inducing proteolytic enzyme expression in a cell.
  • the proteolytic enzyme or enzyme inducing agent may be any one of the suitable enzymes and inducing
  • the antibody is conjugated to a revealing label or to a molecule such as biotin facilitating attachment of a revealing label.
  • the revealing label is also an enzyme in which case the kit preferably also includes the substrate for said enzyme.
  • Figure 1 shows the expression of HB-EGF protein throughout the cycle. Tissues derived from proliferative (A, C) and secretory (B) endometrium were sectioned and stained with anti tm-HB-EGF antibodies. Control staining was performed with control peptide (D) . Lumenal surface is marked by arrow. Scale bars in A B and C are 125mm each, bar in C is 50mm; Figure 2 shows the expression of ErbB4 in human endometrium throughout the cycle. Tissues derived from proliferative (A and C) and secretory (B) endometrium were sectioned and stained with anti ErbB4 antibodies. Control staining was performed with blocking peptide (D) . Scale bars in A, B and D are 100mm each, bar in C is 50mm;
  • Figure 3 shows the expression of tm-HB-EGF and HB-EGF receptors, EGF-Rl and ErbB4 in cultures of stromal cells. Stromal cells were grown on cover slips, fixed and stained as described in Materials and
  • Proliferation was assessed by incorporation of 3 [H] -thymidine during the last four hours of incubation. Proliferation of stromal cells without CHO cells was taken as 100%. Displayed graph is a representation of a typical experiment which has been repeated at least three times. Each value shows mean of four replicas ⁇ SEM. Dark bars represent stromal cells from secretory endometrium, light bars, stromal cells from proliferative endometrium;
  • Figure 6 shows that HB-EGF induces phosphorylation of EGF-Rl.
  • Serum starved stromal cells were stimulated with either sol-HB-EGF (A) or tm-HB-EGF (B) .
  • Control cells were treated with medium alone. Lanes 1 and 3, control cells. Lanes 2 and 4, HB-EGF treated cells.
  • Whole cell lysates were prepared and immunoprecipitated with either anti-EGF-Rl or with anti-ErbB4.
  • Samples were separated by SDS-PAGE (7.5%) and subjected to Western blotting with either anti- phosphotyrosine antibodies (A and B) , or with anti- EGF-R1 antibodies (panel C, lanes 1 and 2) and anti- ErbB4 antibodies (panel C, lanes 3 and 4) ;
  • FIG. 7 shows that HB-EGF-induced proliferation of stromal cells is supressed by the EGF Rl tyrosine kinase inhibitor PD153035.
  • Stromal cells derived from secretory stage endometrium were incubated in serum- free medium in the presence of HB-EGF (lOng/ml) , PD153035 (2mM) or combination of both.
  • HB-EGF laspasmodifier
  • PD153035 2mM
  • Each bar represents mean + SEM of three independent experiments ;
  • Figure 8 shows that tm-HB-EGF is expressed on the apical surface of the luminal epithelium in the secretory stage of the cycle. Sections of human endometrium at different stages of the menstrual cycle stained for tm-HB-EGF (brown staining) .
  • B Mid-secretory (day 22) ;
  • C High magnification of mid-secretory endometrium showing apical staining for tm-HB-EGF on the luminal epithelium;
  • Figure 9 shows expression and purification of HB- EGF-Fc.
  • a SDS-PAGE of purified HB-EGF-Fc showing multiple bands between ⁇ 38-42 kDa.
  • Figure 10 shows that human blastocysts attach to HB-EGF-Fc in a solid phase assay. Blastocysts cultured on a coverslip pre-coated with HB-EGF-Fc. A) low and B) high magnification of an expanded blastocyst. Blastocyst cultured on HB-EGF-Fc in the presence of sol-HB-EGF did not attach but remained expanded;
  • Figure 11 shows that human blastocysts attach to CHO/HB-EGF cells expressing tm-HB-EGF.
  • A) Embryo cultured on CHO/HB-EGF attached and remained expanded at low and high magnification.
  • Figure 12 shows that CHO/HB-EGF express HB-EGF on the cell membrane.
  • FIG. 13 shows that human peri-implantation blastocysts express ErbB4 abundantly and EGF-Rl to a lesser extent on the surface of the trophectoderm adjacent to the inner cell mass.
  • EGF-Rl is localised in the inner cell mass, with some faint, punctate staining on the trophectoderm;
  • Figure 14 shows relative increase in levels of intracellular HB-EGF synthesised by endometrial stromal cells in response to 1 ⁇ g and 10 ⁇ g bFGF and TGF ⁇ ;
  • Figure 15 shows a standard curve for soluble HB- EGF when measured using a sandwich ELIZA in which the detection antibody is biotin-conjugated and steptavidin-HRP has been added together with TNB substrate;
  • Figure 16 shows the total HB-EGF expression levels (sol + tm) of endometrial cells when stimulated with TMF ⁇ , TGF ⁇ , EGF or bFGF;
  • Figure 17 shows the soluble HB-EGF expression level for endometrial cells when stimulated with TNF ⁇ or TGF ⁇ respectively.
  • Figure 18 shows the tm-HB-EGF expression levels when endometrial cells are stimulated with TNF ⁇ or TGF ⁇ respectively.
  • Example 1 Proliferation is mediated via EGF-Rl
  • Tissue samples were collected in accordance with the requirements of the Central Oxford Research Ethics Committee. Endometrial samples were obtained from patients aged 20-46 years undergoing hysterectomy for benign indications, who had a regular 26-33 day menstrual cycle, and who had received no hormonal medication in the preceding three months. The cycle stage of the endometrium was assessed according to Noye's criteria [Noyes et al , Fertility Sterility, 1:3-25, 1950] .
  • Endometrial tissues were obtained from eight women: four specimens were from proliferative and four secretory endometrium.
  • the endometrial stromal cells were isolated as described previously [Fernandez-Shaw et al, Hum. Repord. , 7:156-161, 1992]. Briefly, tissues were washed, minced into small pieces, digested with Type I collagenase (Worthington Biochemical Corporation, US) and filtered through two sieves (Lockertex, UK) . The filtrate containing stromal cells was centrifuged through a percoll gradient, the pellet washed with PBS and resuspended in culture medium.
  • Cell viability was estimated by trypan blue exclusion in a counting chamber (Improved Neubauer ARH) .
  • Cells were cultured in DMEM (Life Technologies Ltd., UK) supplemented with 10% foetal calf serum, 50IU/ml penicillin, and 50ng/ml streptomycin and passaged according to standard procedures. Cells between passages 2-6 were used in these experiments.
  • Stromal cells were plated (10 4 cells/well) in 96 well plate in DMEM supplemented with 10% FCS, lOOIU/ml penicillin and lOOng/ml of streptomycin. After 24 hours the medium was changed to serum-free and incubated for 18 hours. Cells were stimulated with either recombinant HB-EGF (R&D systems Ltd. , UK) or PD153035 (Calbiochem) for 24 hours. l ⁇ Ci H 3 -thymidine (Amersham Pharmacia Biotech, UK) was added to each well for the last 4 hours of incubation. Cells were washed 3 times in PBS, harvested, and the amount of incorporated H 3 -thymidine was determined using a b- ⁇ plate counter (Wallac Ltd., Finland) .
  • the nucleotide sequence of the construct was confirmed by automated DNA sequencing.
  • Stable transfectants CHO/TM HB-EGF cells were generated as described before [Bebbington et al , A Companion to Methods in Enzymology. , 2:136-145, 1991] .
  • a control cell line was generated using a similar protocol by which cells were transfected with the pEE14 vector alone (CHO/vector) .
  • Selection for- • amplification was performed in one step with 25 ⁇ M of MSX (Sigma Ltd., UK).
  • the fixed cells were washed twice in DMEM containing 10% FCS, scraped from the plate and resuspended in serum-free DMEM.
  • Fixed cells (10 5 ) were added to each monolayer of stromal cells in 96-well plates (plated at 10 4 cells/well) which had been previously serum-starved for 18 hours.
  • the co-cultures were incubated in serum-free medium for 24 hours and the thymidine incorporation assays were performed as described above.
  • Confluent stromal cells were starved in serum free medium for 18 hours, and then stimulated with lOOng/ml of HB-EGF or overlayed with fixed CHO/HB-EGF in serum-free DMEM for 3 min at 37°C Cells were placed on ice and washed two times with ice-cold PBS supplemented with 2mM orthovanadate. Cells were lysed for 10 min in 2mM orthovanadate, 2mM sodium fluoride, 2mM sodium pyrophosphate, 2mM PMSF, lOmg/ml leupeptin, lmg/ml pepstatin (RIPA buffer) .
  • the cell lysates were clarified by microcentrifugation at 14,000rpm at 4°C . Equal volumes of supernatants were mixed with 5mg of either anti-EGF-Rl mouse monoclonal (PharMingen, US) or anti-ErbB4 rabbit polyclonal (Santa Cruz
  • EGF-Rl and ErbB4 were detected in parallel blots hybridised with 2 ⁇ g/ml of rabbit anti- EGF-Rl (Santa Cruz Biotechnology Inc., US) or rabbit anti-ErbB4 (Santa Cruz Biotechnology Inc., US) antibodies correspondingly.
  • Tissue sections derived from proliferative and secretory stages of the cycle were stained with antibodies recognising tm-HB-EGF (Fig. 1) .
  • Staining for tm-HB-EGF in early- mid-secretory stage tissues was intense throughout the stroma and epithelium of both the basalis and functionalis layers of the endometrium (Fig. 1, B) .
  • High magnification secretory stage tissues revealed intense, membrane-associated staining for tm-HB-EGF (Fig IC) .
  • Fig IC membrane-associated staining for tm-HB-EGF
  • Staining with anti- HB-EGF pre-incubated with control peptide showed no cross-reactivity with tm-HB-EGF in any of the tissue samples (Fig. 1, D) .
  • HB-EGF can act as such a mediator and contribute to endometrial maturation by promoting the proliferation of endometrial stromal cells.
  • Cells derived from proliferative and secretory endometrium responded differently to sol-HB-EGF.
  • Present data demonstrate that cells from the proliferative endometrium do not proliferate in serum- free medium supplemented with HB-EGF.
  • addition of 5% serum elicits a three-fold higher HB- EGF-dependent growth response in these cell lines. This suggests that stromal cells from the proliferative endometrium require additional factors to elicit a HB-EGF-dependent response.
  • tm-HB-EGF endometrial stromal cells
  • Juxtacrine activity of tm-HB-EGF was determined in H 3 - thymidine incorporation assays in which cultures of endometrial stromal cells were overlaid with fixed CHO/HB-EGF, constitutively expressing full length HB_- EGF, and CHO/vector, a control cell line.
  • serum- free medium the CHO/HB-EGF preparation induced increase in H 3 -thymidine incorporation compared to CHO/vector in both proliferative and secretory stromal cells (Fig. 5) .
  • results demonstrate the proliferative potential of tm-HB-EGF.
  • tm-HB-EGF CHO cells can promote proliferation of cells derived from both, proliferative and secretory endometrium.
  • the relative activity of cells overexpressing tm-HB- EGF was less than two fold higher than that of the control cell line and therefore less than the activity of sol-HB-EGF in serum-free conditions.
  • tm-HB-EGF forms a complex with DRAP27, a homologue of human CD9 antigen which upregulates functional receptors and diphtheria toxin sensitivity in mouse cell lines [Iwamoto et al, Embo
  • HB-EGF expressed on the membrane of CHO cells is associated with other membrane molecules that could modulate the proliferative function of HB-EGF.
  • HB-EGF induces phosphorylation of EGF-Rl but not ErbB4 in endometrial stromal cells
  • HB-EGF is known to bind two receptors, EGF-Rl and ErbB4 [Higashiyama et al , Science., 251:936-9, 1991] [Elenius et al, Embo J. , 16:1268-78, 1997].
  • the binding of ligands to the EGF family receptors results in activation of tyrosine kinase and receptor phosphorylation [Alroy et al, FEBS Letters, 410:83- 86, 1997] .
  • HB-EGF has been shown previously to induce phosphorylation of EGF-Rl [Higashiyama et al , J Biol Chem., 267:6205-12, 1992] and ErbB4 [Elenius et al, Embo J. , 16:1268-78, 1997] in cells overexpressing these receptors.
  • EGF- Rl or ErbB4 mediates the activity of HB-EGF in the endometrial stromal cells.
  • Our data demonstrated that EGF-Rl but not ErbB4 was phosphorylated in response to soluble HB-EGF (Fig. 6, A) . Phosphorylation of ErbB4 was not manifested when cells were induced with soluble HB-EGF in media containing 10% of serum (results not shown) .
  • sol- and tm-HB-EGF increase the proliferative capacity of endometrial stromal cells, and that the proliferative effect of both sol- and tm-HB-EGF in human endometrial • stromal cells is mediated via EGFR1 and not ErbB 4.
  • Example 2 Implantation is mediated via Herb B4
  • Transfected CHO cells were generated as described above. Briefly, DNA encoding full length HB-EGF was cloned into the pEE14 vector vector (CellTech Ltd, UK) . The resulting construct (pEE14/tm-HB-EGF) or the original plasmid (pEE14) were transfected into CH0-K1. Clones overexpressing tm-HB-EGF (CHO/tm-HB-EGF) and control (CHO/vector) were selected in ' 25 ⁇ M of methioninesulfoximine (MSX, Sigma Ltd, UK) . Immunohistochemical detection of tm-HB-EGF in CHO cells was performed according to the protocol described previously.
  • CHO cells were plated on glass coverslips (Chance Propper Ltd, UK) , 2xl0 4 cells per coverslip and grown overnight. Cells were washed 3 times in PBS, fixed in 3% paraformaldehyde in PBS for five minutes at room temperature and permeabilised with permeabilisation buffer (lOmM HEPES, pH 7.4, 200mM sucrose, 3mM MgCl 2 , 50mM NaCl,
  • Triton-XlOO, 0.2% NaN 3 0.5% Triton-XlOO, 0.2% NaN 3 ) for five minutes at room temperature. Non-specific binding sites were blocked with 3% BSA in PBS for thirty minutes at room temperature. The cells were incubated for 1 hour at room temperature with goat anti- tm-HB-EGF antibodies at 1 ⁇ g/ml (Santa Cruz Inc., US). After PBS wash, rabbit anti-goat FITC-conjugated antibodies (Sigma Ltd, UK) were added at 1/75 dilution and incubation was carried on for one hour at room temperature . Coverslips were washed in PBS and mounted on Vectashield medium with DAPI (Vector Laboratories Inc., US) . The staining was observed under a LEITZ DMRBE fluorescent microscope (Leica Wetzlar, Germany) , images were received with the help of Openlab software (Improvision Inc, US) .
  • CHO/tm-HB-EGF and CHO/vector cells were plated on glass coverslips as described above. The following day, cells were washed with PBS and fixed with 3% paraformaldehyde in PBS for 5 min at room temperature . Sequentially cells were washed with DMEM containing 10%FCS and PBS.
  • the extracellular region of HB-EGF coding for amino acids 1-149 was amplified with the following primers 5' TAGAAGCTTCCACCAATGAAGCTGCTGCCGTCGGTGGT, 3' ACGGATCCACTTACCTGTTGGGAGGCTCAGCCCATGACA which introduced HindiII and optimised Kozak ' sequence [Kozak et al, Cell., 44:283-292, 1986] to the 5' end and BamHI sites and a splice donor sequence to the 3' end.
  • the amplified fragment was cloned into pIGl vector [Simmons, D. A. Hartley ed. , Oxford, IRL Press].
  • the nucleotide sequence of the construct was verified by automated DNA sequencing.
  • the pIGl/HB-EGF vector was transfected into the human kidney epithelial cell line 293T [Dubrige, Mol. Cell. Biol. 7:379-89, 1987] and soluble HB-EGF-Fc fusion protein was expressed and purified as described before [Hudson, J. Biol. Chem. 271:971-8, 1996] . Purified protein was analysed by 7.5% PAGE and by western blotting using standard protocols. Detection of HB-EGF on western blots was achieved by the use of goat anti-human HB-EGF antibodies (R&D Systems Ltd., UK) at 4 ⁇ M and rabbit anti-goat HRP-conjugated antibodies (Sigma Ltd. , UK) at 1/8000 dilution.
  • Embryos were then transferred into Nunc cryovials (Life Technologies, UK) in regular buffer (1.5M PrOH; 0.1M sucrose; 20%serum in PBS) (Medicult, UK) .
  • Embryos were frozen in a Planner Cryo 10 Series III freezer (Planner Products Ltd, UK) by cooling at 2°C per minute to-7°C and held for 10 minutes. They were then cooled to -30°C at 0.3°C per minute and held for 5 minutes before finally being cooled to -150°C at 50°C per minute and plunged into liquid nitrogen for storage .
  • Embryos were thawed at room temperature for
  • Embryos were observed with the use of a Leica MZ8 dissecting microscope.
  • The-- -embryos were cultured individually (I) or in groups (G) of 3 in 15ml of the medium until they reached the expanding blastocyst stage (day 6 or day 7) .
  • Embryo attachment assays Human blastocysts were transferred on tm-HB- EGF/CHO or HB-EGF-Fc coated coverslips and cultured in 500 ⁇ l of CSFM3 supplemented with 2.5% HAS.
  • the negative control for CHO-attachment experiments was coverslips coated with CHO/vector cell line.
  • the negative control for HB-EGF-Fc-attachment experiments was the addition of 2 ⁇ g/ml of sol-HB-EGF (R&D Systems Ltd., UK) .
  • the blastocysts were cultured for 24-48 hours and assessed for signs of attachment every 4-8 hours by gently shaking the culture dish three times for three seconds each. Observation was performed under the microscope at xlOO magnification.
  • Embryos were fixed, permeabilised and stained for ErbB4 and EGF-Rl using an anti-ErbB4 monoclonal antibody (clone HFR1 [Srinivasan et al, Clin. Cancer Res., 5:2877-
  • Blastocyst conditioned medium and controls (CSFM3 + 2.5%HSA) were assayed for hCG using a solid-phase, two site fluoro-immunometric assay and kit (Delfia hCG; Perkin Elmer Ltd., UK) .
  • the sensitivity of the assay is typically better than 0.5U/L, defined as "the ⁇ value of 2 standard deviations above the mean of the zero standard measurement values .
  • Each sample provided an estimate of the amount of hCG secreted by a single human blastocyst into 500 ⁇ l CSFM3 over a 24 to 48 hour incubation period. The hCG secretion by single embryos in 500 ⁇ l per 24hrs (mU/24hrs) was derived from this value .
  • Tm-HB-EGF is expressed on the apical surface of the luminal epithelium in the human endometrium during the window of implantation.
  • tm-HB-EGF The function of tm-HB-EGF on human embryo attachment was investigated by the use of tm-HB-EGF presented in two different forms: either as HB-EGF-Fc fusion protein, that was engineered to allow the presentation of the HB-EGF portion of the fusion protein in the same direction by binding to protein A- coated coverslip (2b) ; or expressed as trans-membrane surface protein in CHO cells (2c) .
  • tm-HB-EGF is expressed on the apical surface of the lumenal epithelium at the time of implantation
  • the function of tm-HB-EGF was investigated in functional assays for embryo attachment using human blastocysts (Figs. 9 and 10; Table 1) .
  • Recombinant HB-EGF-Fc fusion protein was expressed and purified as described above (Materials and Methods) . Analysis of the recombinant protein by
  • tm-HB-EGF The capacity of tm-HB-EGF expressed on the cell membrane to mediate the attachment of human blastocysts was determined (Fig. 11; Table 1).
  • blastocysts did not attach to either HB-EGF-Fc in the presence of sol-HB-EGF, or CHO cells transfected with vector alone, suggesting that the activity of HB-EGF in mediating blastocyst attachment is specific.
  • tm-HB-EGF in both modes of presentation, was found to significantly promote attachment of human blastocysts compared to the control coverslips.
  • the presented data demonstrate a function for HB- EGF in human blastocyst attachment.
  • None of the attached blastocysts exhibited trophoblast outgrowth or, in the CHO/tm-HB-EGF experiments, invasion of the cell layer.
  • One explanation for these results is that HB-EGF does mediate trophoblast spreading or invasion in vivo, but our model systems do not support these secondary implantation events.
  • fixation of the CHO cells prior to the transfer of blastocysts may have inhibited further dynamic responses to attachment, such as formation of complexes between HB-EGF and additional molecules.
  • a second explanation, as supported by the solid phase assays for tm-HB-EGF-fc function is that the function of tm-HB-EGF in implantation of the human blastocyst is to mediate the primary event of embryo attachment and that additional molecules are required for downstream processes to occur.
  • hCG The production of hCG is a useful indicator of the well-being of the embryo.
  • Levels of hCG secreted by the embryos used in the HB-EGF-Fc and CHO ' attachment assays above were measured to determine if sol- or tm-HB-EGF induced hCG production.
  • hCG production by human blastocysts cultured in vitro has been reported to reach peak concentrations around day ten [Dokras, Hum. Reprod 6:1143-51, 1991] [Woodward, Hum. Reprod. 8:1463-68, 1993] .
  • HB-EGF is not a primary inducer of hCG expression in these early stage embryos and that additional factors, such as those expressed by endometrial cells, may induce hCG by invading trophoblast in vivo.
  • ErbB4 and ErbBl are expressed on the surface of the trophectoderm of human peri-implantation blastocysts
  • the present results demonstrate unequivocally that the HB-EGF receptor, ErbB4 is abundantly expressed, and EGFR-1 to a much lesser extent, on the polar trophectoderm of hatched, peri-implantation human blastocysts. Thus it is shown that both the receptor and the ligand are appropriately expressed, on the endometrium and the blastocyst, respectively. Although both receptors for HB-EGF are expressed on the trophectoderm, ErbB4 is diffuse on the cell membrane and absent in the inner cell mass whereas EGF-Rl localisation is punctate and sparse on the trophectoderm, with higher levels in a diffuse pattern in the inner cell mass. These differential patterns of expression of ErbB4 and EGF-Rl suggest that the receptors may have different functions in the implanting embryo.
  • Phosphotyrosines are detected with antibody clone 4G10 (Upstate Biotechnology, US) .
  • EGF-Rl and ErbB4 were detected in parallel blots hybridised with 2 ⁇ g/ml of rabbit anti-EGF-Rl (Santa Cruz Biotechnology Inc., US) or rabbit anti-ErbB4
  • EGF-Rl EGF-Rl
  • HER2 Erb B2
  • HER4 Erb B4
  • cDNAs of extracellular domains of EGF-Rl, HER2 and HER4 are generated using RT-PCR on mRNA prepared from endometrial stromal cells or placenta.
  • Transfection is performed both with individual plasmid (EGF-Rl/pIGl, HER2/pIGl and HER4/pIGl and with a combination of two (EGF-Rl/pIGl and HER2/pIGl; EGF-Rl/pIGl and HER4/pIGl; HER4/pIGl and HER2/pIGl) .
  • This will allow expression of both homo- and hetero-dimeric receptor subunits.
  • Transfected cells are incubated in serum-free media for 3-4 days to allow accumulation of expressed soluble proteins. 4.
  • Fc-fusion proteins are purified by protein-A affinity column followed in the case of double plasmid transfection by EGF-Rl affinity column followed either by HER2 affinity column (to purify EGF- R1/HER2 hetero-dimers) or by HER4 affinity column (to purify EGF-R1/HER4 hetero-dimers) .
  • HER2/HER4 heterodimer is purified in similar manner using consecutively HER2 and HER4 affinity columns. Proteins are eluted from affinity columns with salt solution and dialysed.
  • binding assays include using immobilised homo- and ' hetero-dimers of EGF-Rl, HER2 and HER4 and ELISA and gel shift assays as follows:
  • Chosen receptor homo- or hetero-dimer is immobilised on protein-A coated onto the wells of a 96-well plate.
  • Receptor-binding of the tested ligand is assessed by comparison with the receptor binding of the known ligand (EGF or HB-EGF) in competition with the tracer: biotinylated EGF OR HB-EGF.
  • Biotinylated tracer bound to the receptor is detected using streptavidin-conjugated with HRP using standard procedure.
  • Receptors purified as described above can also be used to screen HB-EGF mutants or known or novel compounds for proliferation agonist/antagonist activity in bioassays as above.
  • Example 5
  • HB-EGF ELISA for measuring levels of HB-EGF synthesised by human endometrial stromal cells.
  • test compounds (10 ⁇ g/ml) and monensin (2.4 or 14.4 mM) and incubate for 24 hr at 37°C in DMEM F12 (minus phenol red), p/s, 2%FCS.
  • Peroxidase treatment treat cells for 30 min. at RT with 0.6% H 2 0 2 in 40% MeOH/PBS.
  • Primary antibody add biotinylated anti-HB-EGF at 100 ng/ml (1:2500 dilution). Leave for 1 hr at RT.
  • Secondary antibody add streptavidin-HRP at 1 ⁇ g/ml (1:1000 dilution) . Leave for 1 hr at RT.
  • Relative values are determined by dividing the amount of HB-EGF (OD readings at 490 nm) by th_e_ total number of cells (OD readings at 600 nm) .
  • Statistically significant bars in Figure 15 are asterisked on the graphs as determined using a 2- tailed, paired Student's T-test in Excel. '
  • bFGF Basic fibroblast growth factor
  • TGF- ⁇ Transforming growth factor alpha
  • the following diagnostic test is aimed to confirm whether HB-EGF is deficient during the implantation window in the endometrium of women with infertility or sub-fertility.
  • the implantation is defined as days 6- 8 post the day of the LH surge.
  • an endometrial sample suitable for carrying out the present test fulfills the following requirements: Patient should preferably have ovulation confirmed; the sample should preferably correspond to the "implantation window" time; generally the "implantation window” time can be based on a regular menstrual pattern as approximately seven days before the expected first day of the menstrual period; or, alternatively, it can be based on using the mid-cycle leutenising hormone (LH) urine test (Clear-Plan Styx) to identify LH surge, starting at day 12 (until the surge is identified) .
  • LH leutenising hormone
  • the sample can be taken by pipelle biopsy.
  • the isolated suitable endometrial sample is fixed in formaldehyde and processed for paraffin wax embedding .
  • Sections are cut and HB-EGF detected with the use of primary antibodies (goat anti-tm-HB-EGF (Santa Cruz) optimally diluted in PBS and applied to the sections for one hour at room temperature. Control staining is performed with the same antibodies pre-incubated with the corresponding control peptides.
  • primary antibodies goat anti-tm-HB-EGF (Santa Cruz) optimally diluted in PBS and applied to the sections for one hour at room temperature.
  • Control staining is performed with the same antibodies pre-incubated with the corresponding control peptides.
  • Sections are washed in three changes of PBS and incubated in peroxidase conjugated anti-goat or anti-mouse IgGs for one hour at room temperature.
  • ELISA 96-well plates are coated for 18-24 hours with 4mg/ml of goat anti human HB-EGF polyclonal abs (R&D, catalogue number AF-259-NA) . Plates are blocked with 1%BSA/PBS for 1 hour at room temperature. Cell conditioned media and rh-HB-EGF standards (500-Opg/ml) are incubated for 24-48 hours at 4°C . Following PBS/0.05% Tween-20 wash (3 times) captured HB-EGF is detected with goat anti human HB-EGF polyclonal abs, biotin conjugated (R&D, catalogue number BAF 259) at 125 ng/ml for 2 hours at room temperature.
  • R&D catalogue number AF-259-NA
  • HB-EGF cells remaining in 24 well plates were treated for 40 min with 1 ⁇ M of -phorbol ester PMA (phorbol 12-myristate 13 acetate) that activated metalloproteinases (1-6) and cleavage of pro-HB-EGF and generation of soluble HB-EGF (7) .
  • PMA phorbol 12-myristate 13 acetate
  • cleavage of pro-HB-EGF and generation of soluble HB-EGF (7) cleavage of pro-HB-EGF and generation of soluble HB-EGF (7) .
  • PMA phorbol 12-myristate 13 acetate
  • media was transferred to HB-EGF ELISA plate and soluble form of HB-EGF was detected as described above.
  • Example 8 Regulation of soluble and transmembane HB-EGF expression. Endometrial cells were plated in 24 -well plates and cultured until confluent in DMEM/10% FCS. Before stimulation cells were serum-starved overnight. Cells were stimulated by 10 ng/ml of TNF ⁇ and lng/ml of TGF ⁇ for 48 hours in serum-free DMEM (200 ⁇ l/well) in quadruplicates. Detection of soluble HB-EGF was performed as described above by transferring cell- conditioned media to ELISA plates (200 ⁇ l of supernatant from each of the 24 wells was distributed into 2 wells of the 96 well plate at 100 ⁇ l/well) .
  • HB-EGF expression cells were stimulated as above with 10 ng/ml of TNF ⁇ and lng/ml TGF ⁇ 10 ng/ml EGF and 10 ng/ml bFGF. After 48 hours of incubation PMA was added to the final concentration 1 ⁇ M into the conditioned medium and incubation was continued for 40 min. Conditioned medium was analysed by HB-EGF ELISA as described above .

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Abstract

L'invention concerne un procédé permettant d'évaluer l'état de fertilité d'une femelle mammifère. Ledit procédé consiste à déterminer le niveau de tm-HB-EGF dans un échantillon de tissu endométrial pendant une fenêtre d'implantation pour ladite femelle, et à mettre en corrélation l'apparence de tm-Hb-EGF avec l'état de fertilité. L'invention a également trait à des trousses de diagnostic permettant de mettre en oeuvre le procédé, ainsi qu'à des compositions contraceptives et pharmaceutiques contenant des modulateurs de la production de HB-EGF.
PCT/GB2003/000229 2002-01-21 2003-01-21 Procede et trousse pour examen systematique de la fertilite Ceased WO2003062832A1 (fr)

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EP2348318A1 (fr) 2010-01-21 2011-07-27 Equipo Ivi Investigación, S.L. Procédé de diagnostic pour la réceptivité endométriale
RU2504551C2 (ru) * 2007-09-26 2014-01-20 УЗ ФАРМА ГмбХ Белки, связывающие антиген фактор роста, подобный гепаринсвязывающему эпидермальному фактору роста
US10081840B2 (en) 2008-07-22 2018-09-25 Igenomix S.L. Gene expression profile as an endometrial receptivity marker
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US10081840B2 (en) 2008-07-22 2018-09-25 Igenomix S.L. Gene expression profile as an endometrial receptivity marker
WO2011061376A2 (fr) 2009-11-19 2011-05-26 Fundación Centro Nacional De Investigaciones Cardiovasculares Carlos Iii Utilisation de cd98 en tant que marqueur de réceptivité endométriale
EP2348318A1 (fr) 2010-01-21 2011-07-27 Equipo Ivi Investigación, S.L. Procédé de diagnostic pour la réceptivité endométriale
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US11129851B2 (en) 2014-06-17 2021-09-28 Igenomix S.L. Stem cell therapy in endometrial pathologies

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