WO2007030880A1 - Diagnostic de l’endométriose - Google Patents

Diagnostic de l’endométriose Download PDF

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Publication number
WO2007030880A1
WO2007030880A1 PCT/AU2006/001347 AU2006001347W WO2007030880A1 WO 2007030880 A1 WO2007030880 A1 WO 2007030880A1 AU 2006001347 W AU2006001347 W AU 2006001347W WO 2007030880 A1 WO2007030880 A1 WO 2007030880A1
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WIPO (PCT)
Prior art keywords
endometriosis
level
sample
nitric oxide
metabolite
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PCT/AU2006/001347
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English (en)
Inventor
Martin Graham Healey
Gregory Edward Rice
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The University Of Melbourne
The Royal Women's Hospital
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Priority claimed from AU2005905043A external-priority patent/AU2005905043A0/en
Application filed by The University Of Melbourne, The Royal Women's Hospital filed Critical The University Of Melbourne
Publication of WO2007030880A1 publication Critical patent/WO2007030880A1/fr

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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/364—Endometriosis, i.e. non-malignant disorder in which functioning endometrial tissue is present outside the uterine cavity

Definitions

  • the present invention relates to methods for the diagnosis or prognosis of endometriosis.
  • the invention relates especially to the use of blood based markers such as nitric oxide or metabolites thereof and/or cytokines for the diagnosis or prognosis of endometriosis.
  • the invention also relates to arrays and kits for use in these methods of diagnosis or prognosis of endometriosis.
  • Endometriosis is one of the most common gynecological disorders, affecting up to 15% of women within reproductive age (Eskenazi and Warner, 1997). It is closely associated with severe pelvic pain, dysmenorrhea, dyspareunia, infertility and several other symptoms such as intraperitoneal bleeding, back pain, constipation and/or diarrhea. It is a major threat to physical, psychological and social integrity of the patients.
  • endometrial cells results from the presence of endometrial cells outside of the uterine cavity.
  • endometriosis involves the establishment and subsequent sustained growth of endometrial cells at ectopic sites, most commonly the pelvic peritoneum and ovaries, following retrograde menstruation (Thomas and Prentice, 1992).
  • Implantation of autologous non-malignant ectopic tissue is a unique phenomenon suggesting that an abnormal host response may be present in women who develop this disease. This theory is supported by the fact that only a minority of women will develop the disease in spite of the common occurrence of retrograde menstruation as a source of endometrial tissue.
  • stage II stage II mild, stage III moderate, stage IV severe
  • stage IV severe stage IV severe
  • stage II stage II mild, stage III moderate, stage IV severe
  • stage IV severe stage IV severe
  • early or minimal endometriosis which can involve micro-lesions
  • microscopic endometriotic lesions were not detected laparoscopically.
  • diagnosis of endometriosis by surgical procedures is difficult, costly and invasive in some cases, several physicians and patients tend to avoid it or at least seriously delay it.
  • the length of time between the onset of symptoms and the diagnosis can be as long as 8 to 12 years.
  • the possibility to diagnose endometriosis at an early stage would certainly improve the efficacy of the treatments, and reduce dramatically the number of years during which patients endure acute or chronic pain.
  • Imaging methods such as transvaginal ultrasound and magnetic resonance imaging have been designed for the diagnosis of endometriosis.
  • these techniques can only be reliable for the detection of large (>1 cm diameter) endometrioma lesions detected among a very small proportion of patients with endometriosis.
  • the high cost of these techniques has limited their use for the diagnosis of endometriosis.
  • Serum proteins such as CA- 125 and placental protein- 14 have been proposed as diagnostic markers for endometriosis. Elevated levels of CA-125 have been observed in serum, menstrual effluent and peritoneal fluid of patients with endometriosis. However, these markers, when used alone, are of very limited value for a diagnosis test. Indeed, these markers are not suitable for screening or diagnostic purposes because they provide poor sensitivity. Furthermore, levels of CA-125 and placental protein- 14 vary according to several factors such as the assay, the stage of the disease and the menstrual cycle. Finally these markers are known to be modulated by conditions other than endometriosis.
  • markers present in blood that can be used to effectively discriminate between the presence and absence of endometriosis.
  • These markers can be used as the basis for a non-invasive diagnostic or prognostic test for endometriosis, preferably to reduce the exposure of women with pain but without endometriosis to the risk of diagnostic surgery. They can also be used to monitor the extent or progression of endometriosis in a given patient.
  • results of a diagnostic test of the present invention can be compared to previous results of the same test applied to a sample obtained from the patient prior to treatment or as part of a general health screening.
  • the blood based markers of the present invention are thus also well-suited to evaluate the efficacy of treatment decisions, such as drugs or surgery.
  • the present invention provides a method of diagnosing endometriosis or a predisposition to endometriosis in a patient, the method comprising analysing a blood sample obtained from the patient to determine: the level of nitric oxide or a metabolite of nitric oxide in the sample; and comparing said level in the sample to a control level of nitric oxide or a metabolite of nitric oxide, wherein a level in the sample that is different to the control level is indicative of endometriosis.
  • the blood sample may be whole blood, plasma or serum or any other suitable blood fraction.
  • the blood sample is serum.
  • the method comprises determining the level of a metabolite of nitric oxide.
  • the metabolite of nitric oxide may be any metabolite such as nitrite, nitrate or nitrotyrosine.
  • the metabolite is nitrotyrosine.
  • the method further comprises analysing the blood sample from the patient for the level of or biological activity of one or more cytokines in the sample selected from the group consisting of IL-4, IL-5, IL-7, IL-IO, IL-17, MCP, MIP- l ⁇ and G-CSF and comparing said level of or activity of the one or more cytokines in the sample to a control level, wherein a level or activity in the sample that is different to the control level is a further indication of endometriosis. This may be achieved, for example, by measuring the expression patterns of IL-4, IL-5, IL-7, IL-IO, IL-17, MCP, MIP- l ⁇ or G-CSF at the level of protein and/or mRNA.
  • the method may involve detecting a transcript associated with IL-4, IL-5, IL-7, IL-10, IL-17, MCP, MIP- l ⁇ or G-CSF in the sample, the method comprising contacting the biological sample with a polynucleotide that hybridizes to a sequence encoding IL-4, IL-5, IL-7, IL-10, IL-17, MCP, MIP-I ⁇ or G-CSF.
  • the polynucleotide hybridizes selectively to the sequence encoding IL-4, IL-5, IL-7, IL-10, IL-17, MCP, MIP- l ⁇ or G-CSF.
  • the polynucleotide is at least 80% identical to a sequence as shown in any one of SEQ ID NOs :1 to 8.
  • the percentage identity to a sequence disclosed in any one of SEQ ID NOs: 1 to 8 is at least about 85% or 90% or 95%, and still more preferably at least about 98% or 99%.
  • the polynucleotide is a nucleic acid probe or primer that hybridizes under high stringency to a sequence disclosed in any one of SEQ ID NOs: 1 to 8.
  • the method comprises: i) contacting a sample of nucleic acid from a blood sample of the patient with a nucleic acid probe that hybridizes to a sequence disclosed in any one of SEQ ID NOs: 1 to 8 under stringent conditions that allow the formation of a complex between the patient nucleic acid and the probe; ii) contacting a control sample with said probe under the same conditions used in step i); and iii) detecting the presence of complexes in said samples, wherein detection of levels of the complex in the patient sample that differ from levels of the complex in the control sample is indicative of endometriosis.
  • the method may involve contacting the patient and control samples with an array of at least two different nucleic acid molecules, preferably probes, wherein each nucleic acid molecule hybridizes to a sequence encoding a cytokine selected from the group consisting ofIL-4, IL-5, IL-7, IL-IO, IL- 17, MCP, MIP- l ⁇ and G-CSF, and detecting the presence of complexes on the array.
  • a cytokine selected from the group consisting ofIL-4, IL-5, IL-7, IL-IO, IL- 17, MCP, MIP- l ⁇ and G-CSF
  • the method comprises: i) contacting a sample of nucleic acid from a blood sample of a patient with one or more nucleic acid primers that hybridizes to a sequence disclosed in any one of SEQ ID NOs :1 to 8 under stringent conditions that allow the formation of a complex between the nucleic acid and the primers; ii) contacting a control sample with said primer under the same conditions used in step i); iii) amplifying the patient and control sample nucleic acid using the primers; and iv) detecting the level of the amplified nucleic acid in both patient and control samples, wherein detection of levels of amplified nucleic acid in the patient sample that differ significantly from levels of the amplified nucleic acid in a control sample is indicative of endometriosis.
  • the method comprises: i) contacting a sample from the patient with at least one binding agent that binds to a nitric oxide metabolite under conditions that allow for the formation of a complex comprising the binding agent and the nitric oxide metabolite; ii) contacting a control sample with said binding agent under substantially the same conditions used in step i); and iii) detecting the formation of complexes in the patient and control samples, wherein the detection of levels of complexes in the patient sample that differ from levels of complexes in the control sample is indicative of endometriosis.
  • the binding agent may be any polypeptide or compound that has binding affinity for a relevant nitric oxide metabolite or cytokine.
  • Particularly preferred binding agents are antibodies.
  • the antibody is an anti-nitrotyrosine antibody.
  • the binding agent is bound to a solid phase support.
  • the method comprises contacting the patient and control samples with an array of at least two different binding agents, wherein at least one binding agent binds to a metabolite of nitric oxide and at least one binding agent binds to a cytokine selected from the group consisting of IL-4, IL-5, IL-7, IL-IO, IL-17, MCP, MIP- l ⁇ and G-CSF 5 and detecting the formation of one or more complexes on the array.
  • a cytokine selected from the group consisting of IL-4, IL-5, IL-7, IL-IO, IL-17, MCP, MIP- l ⁇ and G-CSF 5
  • the method of the present invention further comprises analysing the blood sample obtained from the patient for the level or biological activity of one or more markers selected from IL-2, IL-6, IL-8, IL- 12, IL-13, TNF ⁇ , IFN ⁇ , GM- CSF 5 CA-125, CA19-9, soluble intercellular adhesions molecule-1 (sICAM-1), LH 5 FSH, Estradiol, a transferrin antibody and a 2-HS glycoprotein antibody.
  • markers selected from IL-2, IL-6, IL-8, IL- 12, IL-13, TNF ⁇ , IFN ⁇ , GM- CSF 5 CA-125, CA19-9, soluble intercellular adhesions molecule-1 (sICAM-1), LH 5 FSH, Estradiol, a transferrin antibody and a 2-HS glycoprotein antibody.
  • the method of the present invention comprises analysing the blood sample for the level or biological activity of the following: a metabolite of nitric oxide, preferably nitrotyrosine, IL-4, IL-6, IL-8, IL-IO, IL-5, IL-7, IL-12 and IL-13.
  • a metabolite of nitric oxide preferably nitrotyrosine, IL-4, IL-8, IL-IO, IL-7, IL-12 and IL-13.
  • the method of the present invention comprises analysing the blood sample for the level or biological activity of the following: a metabolite of nitric oxide, preferably nitrotyrosine, IL-4, IL-8, TNF ⁇ , IFN ⁇ and MCP-I.
  • a metabolite of nitric oxide preferably nitrotyrosine, IL-4, IL-8, TNF ⁇ , IFN ⁇ and MCP-I.
  • the method of the present invention comprises analysing the blood sample for the level or biological activity of the following: a metabolite of nitric oxide, preferably nitrotyrosine, IL-2, IL-10, IL-7, IL- 17, MCP-I and GM-CSF.
  • a metabolite of nitric oxide preferably nitrotyrosine, IL-2, IL-10, IL-7, IL- 17, MCP-I and GM-CSF.
  • the method of the present invention comprises analysing the blood sample for the level or biological activity of the following: a metabolite of nitric oxide, preferably nitrotyrosine, IL-2, IL-10 and GM-CSF.
  • a metabolite of nitric oxide preferably nitrotyrosine, IL-2, IL-10 and GM-CSF.
  • the method of the present invention comprises analysing the blood sample for the level or biological activity of the following: a metabolite of nitric oxide, preferably nitrotyrosine, IL-6, GM-CSF, IFN ⁇ , IL-5, IL-7, IL-12, and MIP- l ⁇ .
  • a metabolite of nitric oxide preferably nitrotyrosine, IL-6, GM-CSF, IFN ⁇ , IL-5, IL-7, IL-12, and MIP- l ⁇ .
  • the present invention further provides a method of diagnosing endometriosis or a predisposition to endometriosis in a patient, the method comprising analysing a blood sample obtained from the patient to determine: i) the level of nitric oxide or a metabolite of nitric oxide in the sample; and/or ii) the level or biological activity of one or more cytokines in the sample selected from the group consisting of IL-4, IL-5, IL-7, IL-10, IL- 17, MCP, MIP- l ⁇ and G-CSF; and comparing said level and/or biological activity in the sample to a control level and/or biological activity, wherein a level and/or biological activity in the sample that is different to an equivalent level and/or biological activity in the control is indicative of endometriosis.
  • the method comprises analysing the sample for the level or biological activity of IL- 17.
  • the diagnostic/prognostic methods described above further comprise analysing at least one clinical risk factor of the patient.
  • suitable clinical risk factors to be analysed include current age, age of menarche (when menses started), total number of pregnancies, number of miscarriages, number of terminations of pregnancy, number of children delivered, age when first became pregnant, length of time since last pregnancy, length of time using the combined oral contraceptive pill, previous or current use of an intra-uterine contraceptive device, length of time using an intra-uterine contraceptive device, current use of hormonal medication (such as contraceptive pill, Depo-Provera, minipill, zoladex, synarel, duphaston, danazol, oral provera), family history of endometriosis, height, weight, ethnic background, skin colour, hair colour, weekly intake of alcohol, cigarette usage, coffee consumption, tea consumption, amount of exercise, length of menstru
  • the present invention also provides a method of monitoring the therapeutic effect of treatment of endometriosis in a patient, the method comprising analysing blood samples obtained from the patient over time to determine: the level of nitric oxide or a metabolite of nitric oxide in the sample; wherein a change in a level in the samples over time is indicative of the therapeutic effect of the treatment.
  • the method further comprises analysing the blood samples obtained from the patient over time to determine: the level of or biological activity of one or more cytokines selected from the group consisting of IL-4, IL-5, IL-7, IL-IO, IL-17, MCP, MlP-l ⁇ and G-CSF, wherein a change in a level or biological activity in the samples over time is indicative of the therapeutic effect of the treatment.
  • cytokines selected from the group consisting of IL-4, IL-5, IL-7, IL-IO, IL-17, MCP, MlP-l ⁇ and G-CSF
  • the present invention also provides an array of at least two different nucleic acid molecules for the diagnosis or prognosis of endometriosis, wherein each nucleic acid molecule hybridizes to a sequence encoding a cytokine selected from the group consisting of IL-4, IL-5, IL-7, IL-IO, IL-17, MCP, MlP-l ⁇ and G-CSF.
  • a cytokine selected from the group consisting of IL-4, IL-5, IL-7, IL-IO, IL-17, MCP, MlP-l ⁇ and G-CSF.
  • the array comprises at least three, more preferably at least four, five, six seven, eight or nine different nucleic acid molecules, wherein each nucleic acid molecule hybridizes to a sequence encoding a cytokine selected from the group consisting of IL-4, IL-5, IL-7, IL-IO, IL-17, MCP, MlP-l ⁇ and G-CSF.
  • a cytokine selected from the group consisting of IL-4, IL-5, IL-7, IL-IO, IL-17, MCP, MlP-l ⁇ and G-CSF.
  • the array further comprises at least one additional nucleic acid molecule that hybridizes to a sequence encoding IL-2, IL-6, IL-8, IL- 12, IL- 13, TNF ⁇ , IFN ⁇ , GM-CSF, CA- 125, CAl 9-9, soluble intercellular adhesions molecule-1 (sICAM-1), LH, FSH, Estradiol, a transferrin antibody or a 2-HS glycoprotein antibody.
  • sICAM-1 soluble intercellular adhesions molecule-1
  • the present invention also provides an array of at least two different binding agents for the diagnosis or prognosis of endometriosis, wherein each binding agent binds to a metabolite of nitric oxide or to a cytokine selected from the group consisting of IL-4, IL-5, IL-7, IL-10, IL-17, MCP, MIP- l ⁇ and G-CSF.
  • the array comprises at least three, more preferably at least four, five, six seven, eight or nine different binding agents, wherein each binding agent binds to a metabolite of nitric oxide or a cytokine selected from the group consisting of IL-4, IL-5, IL-7, IL-IO, IL-17, MCP, MIP- l ⁇ and G-CSF.
  • each binding agent binds to a metabolite of nitric oxide or a cytokine selected from the group consisting of IL-4, IL-5, IL-7, IL-IO, IL-17, MCP, MIP- l ⁇ and G-CSF.
  • the array further comprises at least one additional binding agent that binds to a polypeptide selected from the group consisting of IL-2, IL-6, IL-8, IL-12, IL-13, TNF ⁇ , IFN ⁇ , GM-CSF, CA-125, CA19-9, soluble intercellular adhesions molecule-1 (sICAM-1), LH, FSH or Estradiol.
  • a polypeptide selected from the group consisting of IL-2, IL-6, IL-8, IL-12, IL-13, TNF ⁇ , IFN ⁇ , GM-CSF, CA-125, CA19-9, soluble intercellular adhesions molecule-1 (sICAM-1), LH, FSH or Estradiol.
  • the array further comprises at least one polypeptide that binds to a transferrin antibody or a 2-HS glycoprotein antibody.
  • the present invention also provides a kit for the diagnosis or prognosis of endometriosis comprising a first container containing at least one nucleic acid molecule that hybridises under stringent conditions with a nucleic acid encoding a polypeptide selected from IL-4, IL-5, IL-7, IL-IO, IL-17, MCP, MIP-I ⁇ and G-CSF.
  • the at least one nucleic acid molecule is a probe.
  • the at least one nucleic acid molecule is a primer useful for amplifying a nucleic acid encoding a polypeptide selected from IL-4, IL-5, IL-7, IL-IO, IL-17, MCP, MlP-l ⁇ and G-CSF.
  • the kit further comprises an enzyme useful for amplification.
  • the present invention also provides a kit comprising one or more binding agents that bind to a metabolite of nitric oxide and/or one or more polypeptides selected from IL-4, IL-5, IL-7, IL-IO, IL-17, MCP, MlP-l ⁇ and G-CSF.
  • the kit further comprises a reagent useful for the detection of a binding reaction between said binding agent and said metabolite or polypeptide.
  • Figure 1 The ratios of medians for cases and controls of substances measured in serum. This figure illustrates the ratios of medians between cases and controls for the 19 substances with inflammatory functions that were assayed in serum.
  • the Mann- Whitney U test was used to assess significant differences (case vs control) and these are annotated as # - significant ( ⁇ 0.05).
  • the scale of the Y axis is logarithmic.
  • the IL-12 ratio (2.9 vs 0.0) could not be displayed as it's value ( ⁇ ) is an infinite point on a logarithmic scale.
  • the present invention provides a method of diagnosing endometriosis or a predisposition to endometriosis in a patient, the method comprising analysing a blood sample obtained from the patient to determine: the level of nitric oxide or a metabolite of nitric oxide in the sample; and comparing said level in the sample to a control level of nitric oxide or a metabolite of nitric oxide, wherein a level in the sample that is different to the control level is indicative of endometriosis.
  • diagnosis and variants thereof such as, but not limited to, “diagnose”, “diagnosed” .or “diagnosing” shall not be limited to a primary diagnosis of a clinical state, but should be taken to include any primary diagnosis or prognosis of a clinical state or diagnosis of recurrent disease.
  • predisposition to endometriosis shall be taken to mean that a subject is susceptible to a form of endometriosis or is more likely to develop endometriosis than a normal subject or a normal population of subjects.
  • the present invention also provides a method of monitoring the therapeutic effect of treatment of endometriosis in a patient, the method comprising analysing blood samples obtained from the patient over time to determine: the level of nitric oxide or a metabolite of nitric oxide in the sample; wherein a change in a level in the samples over time is indicative of the therapeutic effect of the treatment.
  • the blood sample used in the methods of the present invention may be whole blood, plasma or serum or any other suitable blood fraction.
  • the blood sample is serum.
  • the blood sample is serum.
  • the blood sample comprises cells.
  • the blood sample is whole blood.
  • the diagnostic or prognostic methods of the present invention involve a degree of quantification to determine levels or biological activity of the various markers present in patient serum samples. Such quantification is readily provided by the inclusion of appropriate control samples.
  • internal controls are included in the methods of the present invention.
  • a preferred internal control is one or more serum samples taken from one or more healthy individuals.
  • the term "healthy individual” shall be taken to mean an individual who is known not to suffer from endometriosis, such knowledge being derived from clinical data on the individual, including, but not limited to, a different endometriosis diagnostic assay to that described herein.
  • the present invention is particularly useful for the early detection of endometriosis, it is preferred that the healthy individual is asymptomatic with respect to symptoms associated with endometriosis.
  • control when internal controls are not included in each assay conducted, the control may be derived from an established data set.
  • Data pertaining to the control subjects are preferably selected from the group consisting of:
  • a data set comprising measurements of the level or biological activity of the marker in question for a typical population of subjects known to have endometriosis
  • a data set comprising measurements of the level or biological activity of the marker in question for the subject being tested wherein said measurements have been made previously, such as, for example, when the subject was known to be healthy or, in the case of a subject having endometriosis, when the subject was diagnosed or at an earlier stage in disease progression;
  • a data set comprising measurements of the level or biological activity of the marker in question for a healthy individual or a population of healthy individuals; and 4. a data set comprising measurements of the level or biological activity of the marker in question for a normal individual or a population of normal individuals.
  • the term "typical population” with respect to subjects known to have endometriosis shall be taken to refer to a population or sample of subjects diagnosed with endometriosis that is representative of the spectrum of endometriosis patients. This is not to be taken as requiring a strict normal distribution of morphological or clinicopathopathological parameters in the population, since some variation in such a distribution is permissible.
  • a "typical population” will exhibit a spectrum of endometrial diseases at different stages of disease progression. It is particularly preferred that a "typical population” exhibits the expression characteristics of a cohort of subjects as described herein.
  • normal individual shall be taken to mean an individual displaying a normal level or biological activity of the marker in question.
  • data obtained from a sufficiently large sample of the population will normalize, allowing the generation of a data set for determining the average level or biological activity of a particular marker.
  • the method for diagnosis or prognosis of endometriosis comprises assessing the level of nitric oxide (NO) or a metabolite thereof in a blood sample, preferably serum, from a patient.
  • a blood sample preferably serum
  • an elevated level of NO or a metabolite thereof in a blood sample of a patient when compared to a control sample is indicative of endometriosis. Determination of the level of nitric oxide or a metabolite of nitric oxide
  • NO is synthesized from three kinds of isoforms of nitric oxide synthase (NOS). Endothelial NOS (eNOS) and neuronal NOS (nNOS) are known as constitutive NOS (cNOS) and are expressed all the time.
  • the third isoform is called inducible NOS (iNOS).
  • Inflammatory NO is thought to be synthesized by iNOS. Under normal conditions iNOS is not expressed, but it can be upregulated by a number of factors involved in inflammation, including interleukins, interferon-gamnia, TNF ⁇ and LPS resulting in the production of abundant amounts of NO.
  • NO can be measured either directly or indirectly using any of a number of techniques. Methods for the measurement of cellular NO secretion are reviewed in Stone et al. (2005). There are several methods that have been reported for the continuos detection of NO in biological samples. NO- sensitive microsensors, such as Clark-type and porphyrinic electrodes (Malinski and Taha, 1992; Vallance et ah, 1995), have been used to measure NO, and a porphyrinic electrode has been applied in vivo in humans (Vallance, et ah, 1995). More recently, in vivo measurements of NO have been undertaken in animals using a catheter-type NO sensor placed directly into the coronary sinus to measure plasma NO levels (Neishi et ah, 2005).
  • a further example includes a spectrophotometric assay wherein NO reacts with ferrous oxyhaemoglobin, and the resultant formation of methaemoglobin is measured by an increase in absorbance at 401 nm (Archer, 1993; Murphy and Noack, 1994).
  • Other sensitive chemiluminescent methods have also been developed for the detection of nitrate and nitrite (for example, see Yang et ah, 1997, Demoncheaux et ah, 2003).
  • metabolites refers to metabolites or end-products of NO.
  • examples of metabolites of NO include nitrite and nitrate. Quantitation of these stable anions can be used to indirectly determine the amount of NO originally present in a sample.
  • the Griess reaction is the most commonly used nitrite-based assay for the determination of NO levels. NO assays are performed using either a two-step assay or a three-step lactate dehydrogenase (LDH) assay. In both methods, the first step is the reduction of nitrate into nitrite by nitrate reductase. In the final step, Griess Reagent converts the nitrite into a purple-colored azo compound, which is quantitated by spectrophotometer at absorbance at 540 nm.
  • LDH lactate dehydrogenase
  • nitrotyrosine is the preferred marker.
  • N ⁇ 2 -modified amino acids such as nitrotyrosine, nitrophenylalanine and nitrotryptophan is described in WO 96/04311 and WO 98/29452.
  • These patent applications disclose the sequence independent detection of a nitrotyrosine or a nitrotryptophan residue in a protein or in its free form using an antibody, which specifically recognizes the nitro-group.
  • antibodies are preferably used to detect nitrotyrosine, nitrophenylalanine or nitrotryptophan.
  • NO levels can also be measured indirectly by determining the level of expression of NOS, and in particular iNOS.
  • iNOS reverse transcription-polymerase chain reaction
  • RT-PCR reverse transcription-polymerase chain reaction
  • antibodies targeted against iNOS can be used to detect the presence of iNOS using techniques such as Western blotting and ELISA
  • the method for diagnosis or prognosis of endometriosis comprises assessing the level of one of the following cytokines in a blood sample, preferably serum, from a patient.
  • Interleukin-4 (IL-4)
  • IL-4 is an anti-inflammatory cytokine produced by T helper type 2 cells (Th 2 cells) (as well as mast cells, basophils and B cells) and is crucial for naive T cell differentiation into Th 2 cells.
  • Th 2 cells T helper type 2 cells
  • IL-4 (along with IL-13) has a role in allergic inflammation, signalling the change by B cells from producing IgM to IgG 4 and then IgE (Vercelli, 2000). It also inhibits T helper type 1 cell (ThI cell) responses including reduction in IFN ⁇ production (Hamilton et al., 1999).
  • ThI cell T helper type 1 cell
  • IL-4 can both inhibit and stimulate NK cell activity, depending on the co-stimulus.
  • IL-4 and IL-13 activate macrophages.
  • IL-4 shifts leukocyte recruitment from granulocytes to T cells and eosinophils (Nelms et ah, 1999).
  • the sequence of human IL-4 is provided in SEQ ID No:l.
  • Interleukin-5 (IL-5)
  • IL-5 is a pro-inflammatory cytokine that is predominantly secreted by Th-2 cells, activated eosinophils and mast cells (Adachi and Alam, 1998; Lalani et ah, 1999). There are two cell lines that IL-5 acts upon, eosinophils and basophils. The effects of IL-5 include; inducing B cells to differentiate into eosinophils, priming eosinophils for degranulation, stimulating degranulation, chemotaxis and inhibiting apoptosis. It also induces increased cellular adherence (Adachi and Alam, 1998). There is a significant overlap in activities between the cytokines IL-5, IL-3 and GM-CSF.
  • IL-5 IL-3 and GM-CSF are themselves structurally similar (Lalani et al., 1999).
  • the production of IL-5 is unregulated by IL-2, histamine, and IgE, while IL-IO and TGF ⁇ reduce it (Lalani et al., 1999).
  • IL-5 Probably the most common disease states in which IL-5 plays a role are allergic processes including asthma, and some parasitic infestations (Lalani et al., 1999).
  • the sequence of human IL-5 is provided in SEQ ID No:2.
  • Interleukin-7 (IL-7)
  • IL-7 has a role in the development, differentiation, maturation and survival of both B and T lymphocytes (Al-Rawi et al., 2003). It is recognised to be a necessary cytokine for both T and B-cell development (Akashi et ah, 1998). In addition, IL-7 stimulates NK cells to lymphokine activated killer activity, allowing them to kill cells resistant to NK cytotoxicity (Naume and Espevik, 1994). IL-7 acts via the IL-7 receptor that has two chains, a unique ⁇ chain and a ⁇ c chain that it shares with IL-2, IL-4, IL-9 and IL- 15. There is increasing evidence that IL-7 inhibits both B cell and T cell apoptosis (Stoddart et al., 2000). The sequence of human IL-7 is provided in SEQ ID No:3. Interleukin-10 (IL-IO)
  • IL-IO is best recognised as an anti-inflammatory cytokine, limiting and terminating the inflammatory response. At least part of this action is via inhibition of NF- ⁇ B activity, resulting in down-regulation of pro-inflammatory cytokine production.
  • IL-IO has a regulatory effect on T cells, B cells, NK cells, mast cells and granulocytes. IL-IO is produced predominantly by macrophages but also by Th2 cells, monocytes, B cells, eosinophils and keratinocytes (Asadullah et ah, 2003). Stimuli for IL-IO release include TNF ⁇ , adrenaline, noradrenaline, bacterial wall components and parasites (Wilder and Elenkov, 1999).
  • IL-10 promotes B cell proliferation and differentiation, which is needed for mucosal defence, parasite defence and neutralising bacterial toxins (Asadullah et ah, 2003). Allergic eosinophilic inflammation involving IL-5 is suppressed by IL-10. There is some evidence emerging that IL-10 also has an initial pro-inflammatory role by enhancing the function of NK cells.
  • the sequence of human IL-10 is given in SEQ ID No:4.
  • Interleukin-17(A) is one of a family of six cytokines that have been recently discovered and named IL-17A-F (Moseley et ah, 2003; Kolls and Linden, 2004). There are currently five IL- 17 receptors described. IL- 17 is secreted mainly by activated T cells (CD4+) but also by neutrophils, T cells (CD8+) and astrocytes. IL- 17 has a proinflammatory effect, stimulating the increased production of COX-2, PGE 2 , IL-8, IL-6, GM-CSF, G-CSF, IL- l ⁇ , TNF ⁇ and inducible nitric oxide synthase.
  • IL- 17 The actions of IL- 17 include recruiting and activating neutrophils, stimulating fibrosis, angiogenesis and inducing abscess formation in intraperitoneal sepsis. Elevated levels of IL- 17 have been found in rheumatoid arthritis, psoriasis, multiple sclerosis, asthma and inflammatory bowel disease.
  • the sequence of human IL- 17 is provided in SEQ ID No:5.
  • MCP-I Monocyte chemotactic protein- 1
  • MCP-I is a chemokine of the C-C type, that attracts monocyte/macrophages, memory T cells, glial cells (Geppert, 2003), eosinophils (Wuyts et ah, 2003), and neutrophils (Wan et ah, 2003).
  • MCP-I production is induced by TNF ⁇ (Boekhoudt et ah, 2003), IL-I ⁇ , IL-12, bFGF, thrombin (Bachli et ah, 2003), activated protein C, hyperoxia (Reale et ah, 2003), and mechanical shearing stress on endothelial cells.
  • MCP-I is felt to have an important role in a number of chronic disease processes including atherosclerosus, restenosis post-angioplasty, cerebral ischaemia, allergic inflammation (Gonzalez-Espinosa et al., 2003) and rheumatoid arthritis (Dawson et al., 2003).
  • MCP-I has also been shown to be angiogenic (Salcedo et al., 2000) and arteriogenic - able to increase collateral blood flow (van Royan et al., 2003).
  • the sequence of human MCP- 1 is provided in SEQ ID No:6.
  • Macrophage inflammatory protein- I ⁇ (MlP-l ⁇ )
  • MIP- l ⁇ is a chemokine of the C-C type, with many similarities to MIP- l ⁇ and RANTES.
  • the major role for MIP- l ⁇ is as a chemoattractant for monocyte/macrophages, but it also can act as a chemokine for dendritic cells (produced from stimulating monocytes with IFN ⁇ ), antigen activated eosinophils, and T lymphocytes with a greater attraction for CD4+ than CD8+ cells (Maurer and von Stebut, 2004).
  • the only receptor that MlP-l ⁇ binds, CCR5, is shared with MIP-Ia, RANTES, and the HIV-I retrovirus. This means MIP-I ⁇ can act to inhibit HIV-I T cell infection.
  • MIP- l ⁇ include monocyte/macrophages, T lymphocytes and neutrophils. Factors reported to stimulate MIP- l ⁇ release include IL- l ⁇ , Substance P, LPS, IFN ⁇ and fibrinogen. In addition to its chemotactic role, MIP- l ⁇ is also a pyrogen. MIP- l ⁇ has been shown to be elevated in both acute inflammatory situations such as blunt traumas and chronic inflammatory conditions like rheumatoid arthritis and tuberculosis. Overall, it is considered to be a Th-I cell type cytokine (and shows reduced intracellular levels in bronchial lavages from women with asthma). The sequence of human MIP- l ⁇ is provided in SEQ ID No:7.
  • G-CSF Granulocyte colony stimulating factor
  • G-CSF induces the production and growth of neutrophils both in a basal state (maintaining normal numbers in the peripheral bloodstream) and in active states of infection and inflammation (Metcalf and Nicola, 1983).
  • Monocyte/macrophages, lymphocytes, fibroblasts and endothelial cells all produce G-CSF, under the stimulus of TNF, IL-I and IFN ⁇ (Sallerfors, 1994).
  • G-CSF acts via its own specific cell-surface receptor. In addition to stimulating neutrophil growth, G-CSF also helps to mobilise neutrophils, primes them for superoxide release, and acts as to inhibit their apoptosis (Basu et al, 2002).
  • the sequence of human G-CSF is provided in SEQ ID No:8.
  • the method for diagnosis or prognosis of endometriosis comprises assessing the level of expression of a gene or gene product, such as a cytokine gene or a cytokine, or its biological activity.
  • the method of the invention identifies any variation in the expression of particular genes; the variation may be the result of a mutation or polymorphism in the sequence of the gene whose expression is aberrant.
  • the presence of a mutation may result in either a decreased or an increased amount of gene transcription, for example, through altering the activity of a promoter or enhancer that governs regulation of the gene, by altering mRNA stability or by altering the efficiency of transcription termination.
  • Variation in gene expression may also result from aberrant regulation of the gene, for example, as a result of altered levels or activity of a regulatory protein.
  • typical mechanisms include altered promoter or enhancer activity, such as by affecting the binding of a regulatory protein to the promoter or enhancer site, and altered mRNA stability.
  • the aberrant expression of a gene is manifested physiologically through alterations in the biological activity of the protein encoded by the gene (the gene product).
  • biological activity is meant the activity of the wild type gene product and may comprise a binding activity, a structural activity or a stimulatory activity.
  • wild type is meant the phenotype that occurs naturally in the majority of the population of the species and which is manifested in healthy tissue.
  • the biological activity of the gene product may be either increased, decreased or regulated abnormally in the blood sample of a patient with endometriosis relative to its activity in healthy tissue.
  • the altered biological activity of gene products in blood samples from patients with endometriosis will typically be due to changes in the amounts of gene product expressed in the blood sample.
  • Diagnosis may also be made by monitoring gene expression itself, or by monitoring levels of gene product expressed from the gene whose aberrant expression is associated with endometriosis.
  • any suitable technique that allows for the quantitative assessment of the level of expression of a specific gene in a tissue may be used. Comparison may be made by reference to a standard control, or to a control level that is found in healthy tissue.
  • levels of a transcribed gene can be determined by Northern blotting, and/or RT-PCR. With the advent of quantitative (real-time) PCR, the number of gene copies present in any RNA population can accurately be determined by using appropriate primers for the gene of interest.
  • Levels of a plurality of transcribed genes can be now monitored by hybridisation on gene arrays that contain nucleic acid sequences from all the genes of interest, immobilised on a solid surface. The nucleic acid may be labelled and hybridised on a gene array, in which case the gene concentration will be directly proportional to the intensity of the radioactive or fluorescent signal generated in the array.
  • hybridization refers to the association of two nucleic acid molecules with one another by hydrogen bonding. Factors that affect this bonding include: the type and volume of solvent; reaction temperature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase molecule to the solid support (e.g., Denhardt's reagent or BLOTTO); the concentration of the molecules; use of compounds to increase the rate of association of molecules (e.g., dextran sulphate or polyethylene glycol); and the stringency of the washing conditions following hybridization (see Sambrook et a ⁇ . Molecular Cloning; A Laboratory Manual, Second Edition (1989)).
  • the inhibition of hybridization of a complementary molecule to a target molecule may be examined using a hybridization assay; a substantially homologous molecule possessing a greater degree of homology will then compete for and inhibit the binding of a completely homologous molecule to the target molecule under various conditions of stringency, as taught in Wahl and. Berger (1987) and Kimmel (1987).
  • Stringency refers to conditions in a hybridization reaction that favour the association of very similar molecules over association of molecules that differ.
  • High stringency hybridisation conditions are defined as, e.g., overnight incubation at 42 0 C in a solution comprising 50% formamide, 5 x SSC (150 mM NaCl 5 15 mM trisodium citrate, pH8.0), 50 mM sodium phosphate (pH7.6), 5 x Denhardts solution, 10% dextran sulphate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 x SSC at approximately 65 0 C.
  • Low stringency conditions involve the hybridisation reaction being carried out at 35 0 C.
  • the conditions used for hybridization in the methods of the present invention are those of high stringency.
  • selective hybridization or “selectively hybridize” or “specific hybridization” refers to an interaction of two nucleic acid molecules that occurs and is stable under moderately stringent or highly stringent conditions.
  • selective hybridization preferentially occurs, for example, between an oligonucleotide and a target nucleic acid molecule, and not substantially between the oligonucleotide and a nucleic acid molecule other than the target nucleic acid molecule, including not with nucleic acid molecules encoding related but different members of a gene family.
  • an oligonucleotide useful as a probe or primer that selectively hybridizes to a target nucleic acid molecule is at least about 12 to 15 nucleotides in length, generally at least about 18 to 20 nucleotides in length, usually at least about 21 to 25 nucleotides in length, and particularly about 26 to 35 nucleotides in length or more.
  • a nucleic acid, molecule hybridizes with a target nucleic acid molecule at least two times the background and more typically more than 10 to 100 times background.
  • RNA may be isolated from the blood sample to be analysed using conventional procedures, such as are supplied by QIAGEN technology. This RNA is then reverse-transcribed into DNA using reverse transcriptase and the DNA molecule of interest may then be amplified by PCR techniques using specific primers.
  • Hybridisation or amplification assays such as, for example, Southern or Northern blot analysis, immunohistochemistry, single-stranded conformational polymorphism analysis (SSCP) and PCR analyses are among techniques that are useful in this respect.
  • target or probe nucleic acid may be immobilised to a solid support such as a microtitre plate, membrane, polystyrene bead, glass slide or other solid phase.
  • endometriosis or susceptibility to endometriosis may be diagnosed by contacting nucleic acid isolated from patient blood samples with a nucleic acid probe under stringent conditions that allow the formation of a hybrid complex between the nucleic acid probe and the gene implicated in endometriosis and detecting the presence of a hybrid complex in the samples.
  • a nucleic acid probe For use as a diagnostic agent, it may be preferable to label the nucleic acid probe to aid its detection. This level of detection is compared to control levels, such as, for example, gene levels from a healthy specimen or a standard control; detection of altered levels of the hybrid complex from the patient tissue is indicative of endometriosis.
  • Techniques, such as antisense technology may also be used to assess gene expression levels in this manner; the degree of specific binding of oligonucleotide may be assessed as an indication of the level of gene expression.
  • a protein or an immunogenic fragment or epitope thereof is detected in a patient sample, wherein the level of the protein or immunogenic fragment or epitope in the sample is indicative of endometriosis.
  • the method comprises contacting a biological sample derived from the patient with a binding agent capable of binding to an endometriosis-associated protein described herein or an immunogenic fragment or epitope thereof, and detecting the formation of a complex between the binding agent and the endometriosis-associated protein.
  • the binding agent is an antibody.
  • the binding agent binds selectively to an endometriosis-associated protein.
  • binding agent that binds to an endometriosis-associated polypeptide and not generally to other polypeptides unintended for binding.
  • the binding agent is capable of binding an endometriosis- associated polypeptide in the presence of excess quantities of other polypeptides, and tightly enough (i.e. with high enough affinity) that it provides a useful tool for diagnosing endometriosis.
  • the parameters required to achieve such specificity can be determined routinely, using conventional methods in the art.
  • the binding agent binds to an endometriosis-associated polypeptide at least two times the background and more typically 10 to 100 times background.
  • an antibody against an endometriosis-associated protein or epitope thereof is detected in a patient sample, wherein the level of the antibody in the sample is indicative of endometriosis.
  • the method comprises contacting a biological sample derived from the subject with an endometriosis-associated protein or an antigenic fragment eg., a B cell epitope or other immunogenic fragment thereof, and detecting the formation of an antigen-antibody complex.
  • Preferred detection systems contemplated herein include any known assay for detecting proteins or antibodies in a biological sample isolated from a human subject, such as, for example, SDS/PAGE, isoelectric focussing, 2-dimensional gel electrophoresis comprising SDS/PAGE and isoelectric focussing, an immunoassay, a detection based system using an antibody or non-antibody ligand of the protein, such as, for example, a small molecule (e.g. a chemical compound, agonist, antagonist, allosteric modulator, competitive inhibitor, or non-competitive inhibitor, of the protein).
  • the antibody or small molecule may be used in any standard solid phase or solution phase assay format amenable to the detection of proteins.
  • Optical or fluorescent detection such as, for example, using mass spectrometry, MALDI-TOF, biosensor technology, evanescent fiber optics, or fluorescence resonance energy transfer, is clearly encompassed by the present invention.
  • Assay systems suitable for use in high throughput screening of mass samples, particularly a high throughput spectroscopy resonance method e.g. MALDI-TOF, electrospray MS or nano- electrospray MS, are particularly contemplated.
  • Immunoassay formats are particularly preferred, eg., selected from the group consisting of, an immunoblot, a Western blot, a dot blot, an enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay.
  • Modified immunoassays utilizing fluorescence resonance energy transfer (FRET) 5 isotope-coded affinity tags (ICAT), matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) 5 electrospray ionization (ESI), biosensor technology, evanescent fiber-optics technology or protein chip technology are also useful.
  • FRET fluorescence resonance energy transfer
  • ICAT isotope-coded affinity tags
  • MALDI-TOF matrix-assisted laser desorption/ionization time of flight
  • ESI electrospray ionization
  • the assay is a semi-quantitative assay or quantitative assay.
  • Standard solid phase ELISA formats are particularly useful in determining the concentration of a protein or antibody from a variety of patient samples.
  • such an assay involves immobilising a biological sample comprising antibodies against the endometriosis-associated protein or an immunogenic fragment thereof, onto a solid matrix, such as, for example a polystyrene or polycarbonate microwell or dipstick, a membrane, or a glass support (e.g. a glass slide).
  • a solid matrix such as, for example a polystyrene or polycarbonate microwell or dipstick, a membrane, or a glass support (e.g. a glass slide).
  • an antibody that specifically binds an endometriosis-associated protein is brought into direct contact with the immobilised biological sample, and forms a direct bond with any of its target protein present in said sample.
  • an immobilized endometriosis-associated protein or an immunogenic fragment or epitope thereof is contacted with the sample.
  • the added antibody or protein in solution is generally labelled with a detectable reporter molecule, such as for example, a fluorescent label (e.g. FITC or Texas Red) or an enzyme (e.g. horseradish peroxidase (HRP)), alkaline phosphatase (AP) or ⁇ - galactosidase.
  • a detectable reporter molecule such as for example, a fluorescent label (e.g. FITC or Texas Red) or an enzyme (e.g. horseradish peroxidase (HRP)), alkaline phosphatase (AP) or ⁇ - galactosidase.
  • a second labelled antibody can be used that binds to the first antibody or to the isolated/recombinant antigen.
  • the label is detected either directly, in the case of a fluorescent label, or through the addition of a substrate, such as for example hydrogen peroxide, TMB, or toluidine, or 5-bromo-4-chloro-3-indol-beta- D-galaotopyranoside (x-gal).
  • Such ELISA based systems are particularly suitable for quantification of the amount of a protein or antibody in a sample, such as, for example, by calibrating the detection system against known amounts of a standard.
  • an ELISA consists of immobilizing an antibody that specifically binds an endometriosis-associated protein on a solid matrix, such as, for example, a membrane, a polystyrene or polycarbonate microwell, a polystyrene or polycarbonate dipstick or a glass support.
  • a patient sample is then brought into physical relation with said antibody, and the antigen in the sample is bound or 'captured'.
  • the bound protein can then be detected using a labelled antibody. For example if the protein is captured from a human sample, an anti-human antibody is used to detect the captured protein. Alternatively, a third labelled antibody can be used that binds the second (detecting) antibody.
  • cytokines can be assayed in a multiplex format.
  • a suitable multiplex cytokine assay is the Bio-Plex Human cytokine 17-Plex panel (Bio- Rad, catalogue No: 171 -A 11171).
  • This cytokine assay is a capture sandwich immunoassay, wherein antibody specifically directed against a cytokine of interest is covalently coupled to colour-coded 5.6 ⁇ m polystyrene beads.
  • a biotinylated detection antibody specific for a different epitope on the cytokine is added to the beads.
  • the reaction mixture is detected by the addition of stre ⁇ tavidin-PE.
  • Each specific reaction is identified and quantified by bead colour and fluorescence and the magnitude of the reaction is measured using fluorescently labelled reporter molecules associated with each target protein.
  • the presence of antibodies against the endometriosis-associate protein or an immunogenic fragment thereof is detected using a radioimmunoassay (RIA).
  • RIA radioimmunoassay
  • the basic principle of the assay is the use of a radiolabeled antibody or antigen to detect antibody antigen interactions.
  • an antibody that specifically binds to an endometriosis-associated protein can be bound to a solid support and a biological sample brought into direct contact with said antibody.
  • an isolated and/or recombinant form of the antigen is radiolabeled and is brought into contact with the same antibody. Following washing the amount of bound radioactivity is detected.
  • any antigen in the biological sample inhibits binding of the radiolabeled antigen the amount of radioactivity detected is inversely proportional to the amount of antigen in the sample.
  • Such an assay may be quantitated by using a Standard curve using increasing known concentrations of the isolated antigen.
  • such an assay may be modified to use any reporter molecule, such as, for example, an enzyme or a fluorescent molecule, in place of a radioactive label.
  • any reporter molecule such as, for example, an enzyme or a fluorescent molecule, in place of a radioactive label.
  • Western blotting is also useful for detecting an endometriosis-associated protein or an immunogenic fragment thereof.
  • an assay protein from a biological sample is separated using sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) using techniques well known in the art and described in, for example, Scopes (1994).
  • SDS sodium dodecyl sulphate
  • SDS-PAGE polyacrylamide gel electrophoresis
  • Separated proteins are then transferred to a solid support, such as, for example, a membrane or more specifically PVDF membrane, using methods well known in the art, for example, electrotransfer.
  • This membrane may then be blocked and probed with a labelled antibody or ligand that specifically binds an endometriosis- associated protein.
  • a labelled secondary, or even tertiary, antibody or ligand can be used to detect the binding of a specific primary antibody.
  • High-throughput methods for detecting the presence or absence of antibodies, or alternatively endometriosis-associated protein or an immunogenic fragment thereof are particularly preferred.
  • MALDI-TOF is used for the rapid identification of a protein. Accordingly, there is no need to detect the proteins of interest using an antibody or ligand that specifically binds to the protein of interest. Rather, proteins from a biological sample are separated using gel electrophoresis using methods well known in the art and those proteins at approximately the correct molecular weight and/or isoelectric point are analysed using MALDI-TOF to determine the presence or absence of a protein of interest.
  • Biosensor devices generally employ an electrode surface in combination with current or impedance measuring elements to be integrated into a device in combination with the assay substrate (such as that described in U.S. Patent No. 5,567,301).
  • An antibody or ligand that specifically binds to a protein of interest is preferably incorporated onto the surface of a biosensor device and a biological sample isolated from a patient (for example serum) contacted to said device.
  • a change in the detected current or impedance by the biosensor device indicates protein binding to said antibody or ligand.
  • Some forms of biosensors known in the art also rely on surface plasmon resonance to detect protein interactions, whereby a change in the surface plasmon resonance surface of reflection is indicative of a protein binding to a ligand or antibody (U.S. Patent No. 5,485,277 and 5,492,840).
  • Biosensors are of particular use in high throughput analysis due to the ease of adapting such systems to micro- or nano-scales. Furthermore, such systems are conveniently adapted to incorporate several detection reagents, allowing for multiplexing of diagnostic reagents in a single biosensor unit. This permits the simultaneous detection of several epitopes in a small amount of body fluids.
  • Evanescent biosensors are also preferred as they do not require the pretreatment of a biological sample prior to detection of a protein of interest.
  • An evanescent biosensor generally relies upon light of a predetermined wavelength interacting with a fluorescent molecule, such as for example, a fluorescent antibody attached near the probe's surface, to emit fluorescence at a different wavelength upon binding of the diagnostic protein to the antibody or ligand.
  • the proteins, peptides, polypeptides, antibodies or ligands that are able to bind specific antibodies or proteins of interest are bound to a solid support such as for example glass, polycarbonate, polytetrafluoroethylene, polystyrene, silicon oxide, metal or silicon nitride.
  • a solid support such as for example glass, polycarbonate, polytetrafluoroethylene, polystyrene, silicon oxide, metal or silicon nitride.
  • This immobilization is either direct (e.g. by covalent linkage, such as, for example, Schiff s base formation, disulfide linkage, or amide or urea bond formation) or indirect.
  • Methods of generating a protein chip are known in the art and are described in for example U.S. Patent Application No. 20020136821, 20020192654, 20020102617 and U.S. Patent No. 6,391,625.
  • an antibody or ligand may be captured on a microfabricated polyacrylamide gel pad and accelerated into the gel using microelectrophoresis as described in Arenkov et al. (2000).
  • a protein chip is preferably generated such that several proteins, ligands or antibodies are arrayed on said chip. This format permits the simultaneous screening for the presence of several proteins in a sample.
  • a protein chip may comprise only one protein, ligand or antibody, and be used to screen one or more patient samples for the presence of one polypeptide of interest. Such a chip may also be used to simultaneously screen an array of patient samples for a polypeptide of interest.
  • a sample to be analysed using a protein chip is attached to a reporter molecule, such as, for example, a fluorescent molecule, a radioactive molecule, an enzyme, or an antibody that is detectable using methods well known in the art.
  • a reporter molecule such as, for example, a fluorescent molecule, a radioactive molecule, an enzyme, or an antibody that is detectable using methods well known in the art.
  • biomolecular interaction analysis-mass spectrometry is used to rapidly detect and characterise a protein present in complex biological samples at the low- to sub-fmole level (Nelson et al., 2000).
  • One technique useful in the analysis of a protein chip is surface enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS) technology to characterise a protein bound to the protein chip.
  • ESI electron spray ionisation
  • protein chips are particularly amenable to multiplexing of detection reagents. Accordingly, several antibodies or ligands each able to specifically bind a different peptide or protein may be bound to different regions of said protein chip. Analysis of a biological sample using said chip then permits the detecting of multiple proteins of interest.
  • the samples are analysed using ICAT, essentially as described in US Patent Application No. 20020076739.
  • This system relies upon the labelling of a protein sample from one source (i.e. a healthy individual) with a reagent and the labelling of a protein sample from another source (i.e. a tuberculosis patient) with a second reagent that is chemically identical to the first reagent, but differs in mass due to isotope composition. It is preferable that the first and second reagents also comprise a biotin molecule. Equal concentrations of the two samples are then mixed, and peptides recovered by avidin affinity chromatography. Samples are then analysed using mass spectrometry.
  • any difference in peak heights between the heavy and light peptide ions directly correlates with a difference in protein abundance in a biological sample.
  • the identity of such proteins may then be determined using a method well known in the art, such as, for example MALDI-TOF, or ESI.
  • a diagnostic or prognostic assay described herein may be a multiplexed assay.
  • the term “multiplex”, shall be understood not only to mean the detection of two or more diagnostic or prognostic markers in a single sample simultaneously, but also to encompass consecutive detection of two or more diagnostic or prognostic markers in a single sample, simultaneous detection of two or more diagnostic or prognostic markers in distinct but matched samples, and consecutive detection of two or more diagnostic or prognostic markers in distinct but matched samples.
  • matched samples shall be understood to mean two or more samples derived from the same initial biological sample, or two or more biological samples isolated at the same point in time.
  • a multiplexed assay may comprise an assay that detects several antibodies and/or epitopes in the same reaction and simultaneously, or alternatively, it may detect other one or more antigens/antibodies in addition to one or more antibodies and/or epitopes.
  • an assay is antibody or ligand based, both of these antibodies must function under the same conditions.
  • nucleic acid arrays are generating a large number of powerful tools for the study of DNA and RNA variation. These methods, based on techniques pioneered by Schena et al. (1995) and Fodor et al. (1991) facilitate the evaluation of variations in the nucleic acid sequence of DNA or RNA samples and so allow the identification and genotyping of mutations and polymorphisms in these sequences. Recent advances in this technology include those reported by Brown and Botstein (1999), Hacia et al. (1999) and Wang et al. (1998) and are reviewed generally in Nature Genetics 21, supplement 1 (January 1999). Many of these techniques are applicable to the present invention, including improvements in microarray technology that will undoubtedly be developed over the coming years. Gene arrays containing certain pathways (Clontech; Atlas Select Human Tumor Arrays) and gene families (Clontech, Atlas Select Human Tumor Arrays, R&D Systems' Cytokine Expression Array) are becoming commercially available even now.
  • RNA or DNA to DNA chips allows monitoring expression of mRNAs or the occurrence of polymorphisms in genomic DNA in a high-throughput manner.
  • Comparison of expression between two samples (healthy, diseased) on filter arrays may be performed by comparing healthy and diseased RNA samples to separate duplicate filters. Alternatively, a single filter may be used that must be stripped and hybridized sequentially.
  • Direct comparison of gene expression in two samples can be achieved on glass arrays by labelling the two samples with different fluorophores. This technique allows the evaluation of repression of gene expression as well as induction of expression.
  • the two fluorescently-labeled cDNAs are then mixed and hybridised on a single slide array. Glass arrays have the advantage of allowing the simultaneous analysis of two samples on the same array under the same hybridisation conditions.
  • Gene arrays containing sequences of genes implicated in endometriosis will allow high- throughput screening of individuals for diagnostic purposes or tailor-made treatments. Additionally, such arrays may allow evaluation of the success or failure of a drug treatment in the event that induced gene products are useful as surrogate markers.
  • a recent report describes the phenotypic diversity of breast tumours, captured using cDNA microarrays. These arrays provided a distinctive molecular portrait of each tumour and allowed the classification of the tumours into subtypes distinguished by their differences in their gene expression patterns (see Peron et al., 2000).
  • Arrays of polynucleotides whose sequences correspond to, or are complementary to the sequences of genes encoding endometriosis-associated proteins form a further aspect of the invention.
  • Such an array should include at least two nucleic acid molecules, wherein each of said nucleic acid molecules either corresponds to the sequence of, is complementary to the sequence of, or hybridises specifically to one of the genes herein implicated in endometriosis.
  • suitable genes include those encoding IL-4, IL-5, IL-7, IL-10, IL-17, MCP, MlP-l ⁇ and G-CSF
  • Nucleic acid molecules for the detection of multiple gene types may be included on arrays according to this aspect of the invention.
  • Such an array may contain nucleic acid molecules that either correspond to the sequence of, are complementary to the sequence of, or hybridise specifically under high stringency conditions to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 or more of the genes implicated in endometriosis by the method of the first aspect of the invention.
  • arrays according to the invention contain nucleic acid molecules which consist of between twelve and fifty nucleotides, more preferably, between fifteen and thirty-five nucleotides.
  • Protein arrays form a further aspect of the invention, that are useful for the diagnosis of endometriosis and also for the identification of additional molecules that are implicated in this disease.
  • Recent developments in the field of protein and antibody arrays allow the simultaneous detection of a large number of proteins.
  • Low-density protein arrays on filter membranes such as the universal protein array system (Ge, 2000) allow imaging of arrayed antigens using standard ELISA techniques and a scanning charge- coupled device (CCD) detector on an optically flat glass plate containing 96 wells.
  • Immuno-sensor arrays have also been developed that enable the simultaneous detection of clinical analytes.
  • One embodiment of this aspect of the invention therefore provides an array of antibodies, said array comprising at least two different antibody species, wherein each antibody species is immunospecif ⁇ c for a gene product of a gene implicated in endometriosis by the method of the present invention.
  • immunospecif ⁇ c means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.
  • antibody refers to both polyclonal antibodies and monoclonal antibodies, to intact molecules as well as fragments thereof, such as Fab, F(ab')2 and Fc, which are capable of binding to the antigenic determinant in question. Such antibodies thus bind to the polypeptides identified according to the first aspect of the invention.
  • a further embodiment of this aspect of the invention also provides an array of polypeptides, said array comprising at least two polypeptide species, wherein each polypeptide species comprises a gene product of a gene implicated in endometriosis, or is a functional equivalent or a fragment thereof.
  • Functionally-equivalent polypeptides may be polypeptides that are homologous to the polypeptides explicitly identified herein. Two polypeptides are said to be "homologous", as the term is used herein, if the sequence of one of the polypeptides has a high enough degree of identity or similarity to the sequence of the other polypeptide. "Identity" indicates that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences.
  • Similarity indicates that, at any particular position in the aligned sequences, the amino acid residue is of a similar hydrophobicity and charge type between the sequences. Degrees of identity and similarity can be readily calculated by those of skill in the art. Natural biological variants and mutants are therefore homologues as this term is used herein.
  • polypeptides of the first aspect of the invention have a degree of sequence identity with the polypeptide explicitly identified, or with active fragments thereof, of greater than 50%. More preferred polypeptides have degrees of identity of greater than 60%, 70%, 80%, 90%, 95%, 98% or 99%, respectively.
  • fragments refers to a polypeptide having an amino acid sequence that is the same as part, but not all, of the amino acid sequence of the polypeptides explicitly identified, or with one of its functional equivalents.
  • the fragments should comprise at least n consecutive amino acids from the sequence and, depending on the particular sequence, n preferably is 7 or more (for example, 8, 10, 12, 14, 16, 18, 20 or more). For instance, small fragments may form an antigenic determinant.
  • Such fragments may be "free-standing", i.e. not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region.
  • nucleic acids or proteins may be included on the array that have been identified as having a role in endometriosis. Alternatively, and particularly as the technology in this area develops, such that more nucleic acid molecules or polypeptides may be included on a single array, comprehensive arrays may be designed that include a large number of genes or protein types.
  • kits that are useful for diagnosing endometriosis.
  • kits may be suitable for detection of nucleic acid species, or alternatively may be for detection of a gene product, as discussed above.
  • kits may contain a first container such as a vial or plastic tube or a microtiter plate that contains an oligonucleotide probe.
  • the kits may optionally contain a second container that holds primers.
  • the probe may be hybridisable to DNA whose aberrant expression is associated with endometriosis and the primers are useful for amplifying this DNA.
  • Kits that contain an oligonucleotide probe immobilised on a solid support could also be developed, for example, using arrays (see supplement of issue 21(1) Nature Genetics, 1999, the references cited therein and the references cited above).
  • nucleic acid primers may be included in the kit that are complementary to at least a portion of a gene that encodes a protein associated with endometriosis.
  • the set of primers typically includes at least two oligonucleotides, preferably four oligonucleotides, that are capable of specific amplification of DNA.
  • Fluorescent-labelled oligonucleotides that will allow quantitative PCR determination may be included (e.g. TaqMan chemistry, Molecular Beacons). Suitable enzymes for amplification of the DNA, will also be included.
  • Control nucleic acid may be included for purposes of comparison or validation.
  • Such controls could either be RNA/DNA isolated from healthy tissue, or from healthy individuals, or housekeeping genes such as ⁇ -actin or GAPDH whose mRNA levels are not affected by endometriosis disease.
  • kits For detection of gene product, antibodies will most typically be used as components of kits. However, any agent capable of binding specifically to the gene product will be useful in this aspect of the invention. Other components of the kits will typically include labels, secondary antibodies, substrates (if the gene is an enzyme), inhibitors, co-factors and control gene product preparations to allow the user to quantitate expression levels and/or to assess whether the diagnosis experiment has worked correctly. Enzyme-linked immunosorbent assay-based (ELISA) tests and competitive ELISA tests are particularly suitable assays that can be carried out easily by the skilled person using kit components.
  • the kit comprises:
  • the kit comprises:
  • kits further comprises means for the detection of the binding of an antibody, fragment thereof or a ligand to an endometriosis-associated protein.
  • a reporter molecule such as, for example, an enzyme (such as horseradish peroxidase or alkaline phosphatase), a substrate, a cofactor, an inhibitor, a dye, a radionucleotide, a luminescent group, a fluorescent group, biotin or a colloidal particle, such as colloidal gold or selenium.
  • a reporter molecule is directly linked to the antibody or ligand.
  • a kit may additionally comprise a reference sample.
  • a reference sample comprises a peptide that is detected by an antibody or a ligand.
  • the peptide is of known concentration.
  • Such a peptide is of particular use as a standard. Accordingly various known concentrations of such a peptide may be detected using a prognostic or diagnostic assay described herein.
  • a kit comprises means for protein isolation (Scopes, 1994).
  • the clinical risk factors that could aid in diagnosing endometriosis include age, age of menarche, total number of pregnancies, number of miscarriages, number of terminations of pregnancy, number of children delivered, age at first pregnancy, length of time since last pregnancy, length of time using the combined oral contraceptive pill, previous or current use of an intra-uterine contraceptive device, length of time of use of an intra-uterine contraceptive device, current use of hormonal medication (contraceptive pill, Depo-Provera ® , minipill, Zoladex ® , synarel, duphaston, danazol, oral provera), family history of endometriosis, height, weight, ethnic background, skin colour, hair colour, weekly intake of alcohol, cigarette usage, coffee consumption, tea consumption, amount of exercise, length of menstrual cycle, length of menses, menstrual cycle regularity, previous diagnosis of endometriosis, previous diagnosis of polycystic ovarian syndrome (PCOS), and sexual intercourse during men
  • Alcohol intake in patients with endometriosis has been assessed in three studies. Signorello et al. (1997) in a case control study of women with infertility related endometriosis detected a trend towards increased risk of endometriosis with increasing alcohol consumption, but this did not achieve statistical significance as the sample size was small. Grodstein et al. (1994) also assessed women with infertility and endometriosis in a case control study. She found both endometriosis related infertility and ovulatory factor infertility significantly associated with alcohol consumption.
  • Cigarette smoking has been suggested to be protective against endometriosis. There is speculation that this may be due to lower levels of endogenous oestrogens seen in smoking women. Such an association has been demonstrated in a case control study of women with infertility (and women admitted for delivery as controls) (Cramer et al.,
  • Ovulation dysfunction has been hypothesised to result in altered local concentrations of hormones, producing a milieu that allows development of endometriosis, especially in the form of endometrial cysts.
  • Brosens et a (1978) and Donnez and Thomas (1982) used presence of an ovarian ostium in the luteal phase as a measure of ovulation in women with raised progesterone in luteal phase. Absence of ovarian ostium was diagnostic of a luteal unruptured follicle (LUF). An association was found between LUF and the presence of endometriosis.
  • Polycystic ovarian syndrome is another form of ovulation dysfunction, and was investigated by Bryer et al. (1994) in women having laparoscopy for infertility. There was found to be a positive association between polycystic ovaries and endometriosis.
  • Filer & Wu (1989) performed a case control study on women with infertility. They found a significant association between sex during the menses and increased risk of endometriosis. In addition, they noted a trend towards higher risk with higher frequency of menses coitus.
  • Pregnancy provides a woman with a nine-month period of amenorrhoea.
  • the assumption has been made that the fewer periods a woman has (in this case due to pregnancy), the less her risk of endometriosis. This would be consistent with both the retrograde menstruation theory of causation and the coelomic metaplasia theory.
  • the majority of studies assessing parity have shown a fall in risk with rising parity (Candiani et al., 1991; Sangi-Hagheykar and Poindexter, 1995; Parazzini and Ferraroni, 1993; Moen and Schei, 1997; Gruppo italiaino per Io studio dell'endometriosi, 1994).
  • the last two studies used multivariate analysis, reducing the likelihood of the presence of confounding variables such as infertility.
  • OCP oral contraceptive pill
  • IUCD intrauterine contraceptive device
  • Example 1 Biological Samples
  • the resulting supernatant (serum) was removed and aliquots of 0.5-1.0 ml were placed in Eppendorf tubes and then stored at -2O 0 C.
  • any peritoneal fluid present in the pelvis was aspirated and placed in a sterile container on ice and then transported to the laboratory.
  • the fluid was placed in Eppendorf tubes in aliquots of 0.5-1.0 ml and stored at -2O 0 C.
  • Prior to assay samples were thawed and centrifuged at 12,000 rpm for 2 minutes, and the supernatant was retained for assay.
  • An ELISA plate was coated with nitrated-BSA (bovine serum albumin). It was washed and blocked to prevent nonspecific binding of protein.
  • Equal volumes of (i) sample or standard (ii) rabbit anti- nitrotyrosine-BSA antibody were mixed in a 1 ml tube and incubated for 3 hours. This allowed the antibody to bind to nitrated proteins.
  • the plate was washed and TMB substrate added for colour development.
  • the reaction was stopped by adding H 2 SO 4 .
  • the colour response was assessed using a microplate reader at a wavelength of 450nm. A curve of best fit was calculated from the standard measures and unknown samples were read off this curve. AU samples and standards were tested in duplicate.
  • the 17 cytokines were assayed according to manufacturers instructions (17-plex, Bio- Rad Laboratories Inc., California, USA).
  • a 96-well filter microplate was pre-wet with 100 ⁇ l of assay buffer (supplied with kit) in each well. The fluid was then removed. 50 ⁇ l of bead stock was added to each well and the fluid removed. A wash was performed with 100 ⁇ l of wash buffer (supplied with the kit) in each well and the fluid then was removed. 50 ⁇ l of either standard or sample was added to each well and the microplate was sealed, covered in aluminium foil, then shaken on an orbital microplate shaker for 30 seconds at 1,100 rpm, then 30 minutes at 300 rpm. Following this the microplate was incubated overnight at 4°C.
  • the protein content of serum samples was determined.
  • a stock solution of BSA (2 mg/ml) was diluted with RPMI Medium 1640 (serum free) to produce a set of 6 serial standards (0.06 to 2 mg/ml).
  • RPMI Medium 1640 serum free
  • 5 ⁇ l of standard or sample was placed in each well.
  • Standards and samples were measured in duplicate.
  • a volume of 250 ⁇ l of Coomassie's Reagent was added to each well. Colour development was assessed using a microplate reader at a wavelength of 595 nm. Using the standard measures a curve of best fit was calculated and unknown samples were read off this curve.
  • the 17-plex cytokine assay was performed twice. For each cytokine the results from the two runs were compared and the sets of results were combined for further analysis. Comparison of cases and controls was performed with the Mann- Whitney U test because most of the substances assayed did not have normal distributions.
  • phase of menstrual cycle was modelled using linear regression.
  • the normalised results of an individual substance assayed and the phase of cycle were entered, then backward removal of variables performed to optimise the model.
  • the cytokine and nitrotyrosine data that were subject to multivariate analysis were combined with demographic and/or clinical factors, and the combined data were used to model tests for diagnosing endometriosis.
  • the variables included in the modelled tests for diagnosis of endometriosis are provided in Table 1, below.
  • the markers tested in each model are indicated by a "+".
  • Hormone treatment Y/N was used as a categorical variable (ie 0 or 1), whereas age at operation, age of menarche, weight and the biological markers were used as continuous variables in the modelling. Each of the continuous variables had a weighted influence on the final result when determining a diagnosis of endometriosis.
  • the statistical packages JMP v5.1 (SAS Institute Inc.) and SPSS vl l.5 were used in the analysis of each diagnostic model. As will be understood by those skilled in the art, the weighting of each variable and the threshold value for determining a diagnosis of endometriosis are dependant on the method of analysis chosen.
  • Results of cytokine and nitrotyrosine assays in serum have been summarised as ratios of medians (endometriosis subjects versus controls), and are shown in Figure 1.
  • Each substance assayed was tested for correlations with all other substances assayed within serum.
  • subjects with endometriosis showed more strong correlations between substances assayed in serum than controls.
  • Multivariate analysis of the data has also identified several markers that are suitable for combining in a diagnostic test for endometriosis. This includes substances that on univariate analysis are not obviously significantly increased or reduced in the presence of endometriosis, but are when allowance is made for the influence of other potentially confounding markers. Additionally, the inclusion of demographic and/or clinical data has also proven useful for the diagnosis of endometriosis.

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Abstract

La présente invention concerne des procédés de diagnostic ou de pronostic de l’endométriose. L’invention concerne en particulier l’utilisation de marqueurs sanguins tels que l’oxyde nitrique ou des métabolites de ce dernier et/ou des cytokines pour le diagnostic ou le pronostic de l’endométriose. L’invention concerne également des jeux d’échantillons et des kits destinés à être utilisés dans le cadre de ces procédés de diagnostic ou de pronostic de l’endométriose.
PCT/AU2006/001347 2005-09-13 2006-09-13 Diagnostic de l’endométriose WO2007030880A1 (fr)

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Cited By (1)

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CN114144674A (zh) * 2019-07-22 2022-03-04 豪夫迈·罗氏有限公司 P物质作为用于子宫内膜异位症的非侵入性诊断的血液生物标志物

Non-Patent Citations (5)

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Title
BEDAIWY M.A. AND FALCONE T.: "Laboratory testing for indometriosis", CLINICA CHIMICA ACTA, vol. 340, 2003, pages 41 - 56, XP003009905 *
BEDAIWY M.A. ET AL.: "Prediction of endometriosis with serum and peritoneal fluid markers: a prospective controlled trial", HUMAN REPRODUCTION, vol. 17, no. 1, 2002, pages 426 - 431, XP002967254 *
KAMADA Y. ET AL.: "GnRH agonist-suppressed expression of nitric oxide synthesis and generation of peroxymitrite in adenomyosis", HUMAN REPRODUCTION, vol. 15, no. 12, 2000, pages 2512 - 2519, XP003009906 *
MARKOWSKA J. ET AL.: "Cytokines and Endometriosis", CLINICAL AND EXPERIMENTAL OBSTETRICS AND GYNAECOLOGY, vol. 31, no. 4, 2004, pages 269 - 270, XP008078728 *
WU M-Y. ET AL.: "Nitric oxide synthesis is increased in the endometrial tissue of women with endometriosis", HUMAN REPRODUCTION, vol. 18, no. 12, 2003, pages 2668 - 2671, XP003009904 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114144674A (zh) * 2019-07-22 2022-03-04 豪夫迈·罗氏有限公司 P物质作为用于子宫内膜异位症的非侵入性诊断的血液生物标志物

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