Diagnostic test

Abstract

The present invention relates to a non-invasive test for the detection of deep endometriosis. Particularly, although not exclusively, aspects of the present invention relate to one or more glycolysis-associated biomarkers (e.g., phosphoglycerate mutase 1 (PGAM1), Enolase-1 (ENO1), phosphofructokinase-1 (PFKP) and / or Hexokinase) and / or cytokines associated with aerobic glycolysis (e.g., IL-6 and / or TGF-β) which can be used to determine the likelihood of a subject having deep endometriosis based on a sample obtained from the subject.

Description

[0001] DIAGNOSTIC TEST

[0002] Field of the Invention

[0003] Aspects of the present invention relate to a non-invasive test for the detection of deep endometriosis. Particularly, although not exclusively, aspects of the present invention relate to one or more glycolysis-associated biomarkers (e.g., phosphoglycerate mutase 1 (PGAM1), Enolase-1 (ENO1), phosphofructokinase- 1 (PFKP) and / or Hexokinase) and / or cytokines associated with aerobic glycolysis (e.g., IL-6 and / or TGF-P) which can be used to determine the likelihood of a subject having deep endometriosis based on a sample obtained from the subject.

[0004] Background to the Invention

[0005] Endometriosis is a chronic inflammatory disease associated with debilitating pain and subfertility affecting more than 196 million women worldwide. Despite affecting at least 1 in 10 women worldwide there are no accurate non-invasive tests or biomarkers for endometriosis, leading to an average 8 year wait for diagnosis. Treatment is currently limited to surgical excision, hormonal treatments, and analgesics, which are associated with many adverse effects and do not always provide long-term relief.

[0006] Endometriosis is typically defined as the presence of endometrial-like tissue outside the uterus. The symptoms of endometriosis are classically described as chronic pelvic pain, dysmenorrhoea, dyschezia, dyspareunia and fatigue. These symptoms can be anywhere on a spectrum from mild to severely debilitating. Endometriosis is a heterogeneous condition and can be subdivided into five main types; superficial peritoneal endometriosis, ovarian endometriosis, deep endometriosis, extra-abdominal endometriosis and iatrogenic endometriosis. Deep endometriosis is a severe subtype of the disease whereby endometrial tissue invades more than 5mm into peritoneal surfaces or directly into other organs such as bladder or bowel.

[0007] Deep endometriosis can have the appearance of so-called “implants", "nodules", or "lesions”, which are areas of endometrial-like tissue present outside the uterus. All types of endometriosis can be associated with fibrosis and adhesions. The diagnosis of endometriosis has long been recognised as a clinical challenge. There is no international consensus on the best approach and local policies often diverge from national guidance. Clinical experience and training are therefore critical for accurate surgical diagnosis. Diagnosis is reliably established only through surgical visualization with histological verification, although ovarian endometrioma and deep nodular forms of disease can be detected through Transvaginal ultrasonography (TVLIS) and magnetic resonance imaging (MRI). TVLIS and MRI have both been extensively investigated for the pre-operative diagnosis of endometriosis, these methods can accurately diagnose, but cannot be used to exclude endometriosis.

[0008] Currently in the UK, the diagnosis of endometriosis is instigated through clinical suspicion of symptoms such as cyclical pelvic pain or subfertility. NICE guidelines encourage clinicians to consider endometriosis in the presence of chronic pelvic pain, dysmenorrhoea, deep dyspareunia, cyclical gastrointestinal symptoms, cyclical urinary symptoms, or infertility in association with one of these symptoms. There are not yet any diagnostic biomarkers that can be reliably used in clinical practice. The lack of a non-invasive diagnostic test may lead to the severe under diagnosis of the disease, particularly within adolescence, which may cost the UK economy £8.2 billion annually.

[0009] It is an aim of certain embodiments to at least partially overcome the technical problems associated with the prior art.

[0010] It is an aim of certain embodiments of the present invention to provide a method of determining the likelihood of a subject having deep endometriosis.

[0011] It is an aim of certain embodiments of the present invention to provide protein biomarkers that can be used to determine the likelihood of a subject having deep endometriosis based on a sample obtained from the subject.

[0012] Summary of Certain Embodiments of the Invention

[0013] The present invention relates to the unexpected finding that one or more glycolysis-associated biomarkers are associated with deep endometriosis. Advantageously, the one or more biomarkers are capable of distinguishing deep endometriosis from the other types of endometriosis, e.g., superficial endometriosis (e.g., superficial peritoneal), ovarian endometriosis, extra-abdominal endometriosis and / or iatrogenic endometriosis. In addition, the one or more glycolysis-associated biomarkers of the present invention are capable of differentiating deep endometriosis from symptomatic subjects and / or healthy subjects. As used herein, “symptomatic subjects” relate to subjects with symptoms of endometriosis but no clinical signs of disease. As used herein, “healthy subjects” relate to subjects with no clinical signs or symptoms of endometriosis.

[0014] Without being bound by theory, the present invention relates, at least in part, to the inventor’s insight that subjects with deep endometriosis may display cells having altered metabolic pathways resulting in altered glycolysis pathways in the presence of oxygen (“aerobic glycolysis”) and thus display altered levels of glycolysis-associated enzymes such as PGAM1 , ENO1 , PFKP and / or Hexokinase. As such, the invention relates to the development of a non- invasive test for identifying deep endometriosis utilising one or more of such biomarkers with high levels of specificity and sensitivity.

[0015] Advantageously, the non-invasive test is capable of being used on samples obtained from female subjects at any stage of the menstrual cycle. The test is shown herein to be especially specific and sensitive when performed on samples obtained from female subjects without any one or more of (i) an endometrioma, (ii) fibromyalgia, (iii) a thyroid condition, (iv) menstrual migraines, (v) hormonal therapy and / or (vi) prior treatment of endometriotic lesions. In certain embodiments, the non-invasive test of the present invention is especially specific and sensitive when performed on samples obtained from female subjects without an endometrioma and / or without a thyroid condition.

[0016] Accordingly, the invention provides a method of determining the likelihood of a subject having deep endometriosis, the method comprising detecting levels of one or more glycolysis- associated biomarkers in a sample obtained from the subject.

[0017] Typically, the subject is a female human (e.g., aged between about 18 to 50).

[0018] In exemplary embodiments, the subject does not have an endometrioma as further described herein.

[0019] In exemplary embodiments, the subject does not have fibromyalgia as further described herein.

[0020] In exemplary embodiments, the subject does not have a thyroid condition as further described herein.

[0021] In exemplary embodiments, the subject is not experiencing migraines as further described herein. In exemplary embodiments, the subject is not undergoing hormonal therapy as further described herein.

[0022] In exemplary embodiments, the subject does not have a past medical history of endometriosis as further described herein. Aptly the subject has not had a previous laparoscopy (PL) to treat endometriosis as further described herein.

[0023] In exemplary embodiments, the subject has not had a prior surgical treatment (often laparoscopy) to remove endometriotic lesions as further described herein.

[0024] In exemplary embodiments, the sample is urine. The sample may be frozen and / or stored at a low temperature prior to testing. The sample may be normalised by any suitable technique. For example, the sample may be normalised by specific gravity (SG) as further described herein.

[0025] Without being bound by theory, the invention further relates, at least in part, to the unexpected finding that glycolysis-associated biomarkers of the invention (e.g., PGAM1) are stable in urine at 4°C for at least one week. In particular glycolysis-associated biomarkers of the invention (e.g., PGAM1) are stable in urine at 4°C for at least two weeks. Moreover glycolysis- associated biomarkers of the invention (e.g., PGAM1) are stable in urine at 4°C for three weeks. Advantageously, urine samples from subjects therefore do not require freezing prior to testing but rather can be stored in a refrigerator.

[0026] Creatinine (2-amino-methyl-5H-imadazol-4-one) is a metabolite of creatinine and phosphocreatinine (p-creatinine). It is converted through a non-enzymatic process, diffused into the blood, and is excreted by the kidneys. The conversion appears to be irreversible in vivo. Creatinine forms spontaneously from p-creatine at a rate that is relatively constant and with an intra-individual variation of <15% from day to day making it a useful tool for normalising the levels of other molecules found in urine. Urinary creatinine levels have previously been used as a proxy for hydration status; but without being bound by theory, urine creatinine levels can also be influenced by factors such as muscle mass, diet, urinary tract obstruction or damage, infection, NSAIDs use, and ureteric or bladder involvement of endometriosis. In the context of deep endometriosis, these factors may pose key confounding factors and preclude the use of urine creatinine to accurately normalise urine samples in the context of deep endometriosis. Specific gravity (SG) in samples has a good correlation with urine osmolality, and thus can be used as an adequate proxy. The invention further relates, at least in part, to the inventor’s insight that SG can be used to normalise urine samples and improve the sensitivity and specificity of the methods as described herein.

[0027] In certain embodiments, the method comprises comparing the levels of the one or more glycolysis-associated biomarkers with one or more reference levels. For example, a difference in levels of the one or more biomarkers, compared to the one or more reference levels, may be indicative of an increased likelihood of deep endometriosis.

[0028] In some embodiments, the one or more reference levels are levels of the glycolysis-associated biomarker that are characteristic of a subject having superficial endometriosis and / or a healthy subject. In such embodiments, a decrease in levels of the one or more biomarkers, compared to the one or more reference levels, is indicative of an increased likelihood of deep endometriosis.

[0029] In some embodiments, the one or more reference levels are levels of the glycolysis-associated biomarker that are characteristic of a subject having deep endometriosis. In such embodiments, no difference in levels of the one or more biomarkers, compared to the one or more reference levels, is indicative of an increased likelihood of deep endometriosis.

[0030] In certain embodiments, the glycolysis-associated biomarker is a protein. For example, the biomarker may be selected from any one or more of phosphoglycerate mutase 1 (PGAM1), Enolase-1 (ENO1), phosphofructokinase- 1 (PFKP) and / or Hexokinase. Typically, glycolysis- associated biomarker comprises PGAM1.

[0031] In some embodiments, the method further comprises detecting levels of one or more cytokines associated with aerobic glycolysis, e.g., IL-6, TGF-p or the like.

[0032] Typically, the levels of the biomarker(s) are detected by immunoassay (e.g., ELISA).

[0033] The invention further provides a method of treating or preventing deep endometriosis in the subject, wherein the subject is identified as having an increased likelihood of deep endometriosis according to any method described herein.

[0034] The invention further provides ex vivo assays for diagnosing deep endometriosis in the subject, wherein the assay comprises contacting the sample obtained from the subject with one or more ligand(s) specific for one or more of the glycolysis-associated biomarker(s), wherein the presence of the biomarker(s) creates one or more biomarker-ligand complexes; and detecting and / or quantifying the one or more biomarker-ligand complexes.

[0035] The invention further provides kits for determining whether the subject has deep endometriosis comprising one or more ligand(s) specific for the glycolysis-associated biomarker(s).

[0036] Detailed Description of Certain Embodiments of the Invention

[0037] Brief Description of the Figures

[0038] Certain embodiments of the present invention will be described in more detail below, with reference to the accompanying Figures in which:

[0039] Figure 1 shows a schematic illustrating that endometriosis can be divided into superficial peritoneal, deep, ovarian, extra-abdominal or iatrogenic subtypes.

[0040] Figure 2 shows a schematic illustrating the glycolysis pathway along with glycolytic-associated enzymes and products.

[0041] Figure 3 shows a graph illustrating that a glycolysis-associated biomarker, PGAM1 , was not normally distributed in all study groups.

[0042] Figure 4 shows graphs illustrating PGAM1 levels in all participants. PGAM1 levels are significantly decreased in endometriosis patients compared to controls - healthy donors and symptomatic controls (A) and increased in healthy volunteers compared to participants with deep or superficial endometriosis or symptomatic controls (B). Dark blue = deep endometriosis, light blue = superficial endometriosis, green = symptomatic control, orange = healthy volunteers.

[0043] Figure 5 shows graphs illustrating PGAM1 levels in participants not receiving hormonal medications. PGAM1 levels are significantly decreased in participants with endometriosis compared to (healthy and symptomatic) controls (A) and increased in healthy volunteers and symptomatic controls compared to participants with deep or superficial endometriosis (B). Dark blue = deep endometriosis, light blue = superficial endometriosis, green = symptomatic controls, orange = healthy volunteers. Figure 6 shows graphs illustrating the role of hormonal medications on urinary PGAM1 levels in A) deep endometriosis and B) superficial endometriosis. COCP = combined oral contraceptive pill. GnRH = gonadotropin hormone-releasing hormone analogues. HRT = hormone replacement therapy.

[0044] Figure 7 shows graphs illustrating associations of PGAM1 with various phases of the menstrual cycle in A) deep endometriosis B) superficial endometriosis and C) controls. D) There is an equal representation of all phases of the menstrual cycle in the cohort. There is no change to the pattern of PGAM1 levels when only samples from the E) proliferative and F) secretory phases are assessed. Blue = proliferative phase, purple = secretory phase, black = unknown menstrual phase.

[0045] Figure 8 shows a graph illustrating that PGAM1 levels remain stable at 4°C for at least 7 days when compared to fresh samples on day 0. PGAM1 levels (ng / ml SGnorm - shown on the y- axis) in three endometriosis patients and five healthy volunteers in fresh samples on the day they were received (day 0) and on days 4 and 7 following storage at 4°C. There was no significant difference in PGAM1 levels between days 0 and 4, day 0 and 7, or day 4 and 7 (Shapiro- Wilk normality testing and paired T-test).

[0046] Figure 9A-H show graphs illustrating gene ontology analysis of the GEO dataset GSE141549 using the GEOexplorer package. (A) Gene ontology analysis of biological processes showed the genes that were significantly downregulated in endometrium and peritoneum from endometriosis patients (>±0.5 FC and p<0.001) when compared to endometrium and peritoneum from controls were enriched for genes involved in mitochondrial gene translation, gene expression and regulation of mitochondrial translation. The x-axis shows -p-value, the y- axis shows the pathways affected and colour denotes enrichment score. (B) The gene ontology analysis of biological processes showed that the genes that were significantly upregulated in the endometrium and peritoneum from endometriosis patients (>±0.5 FC and p<0.001) compared to endometrium and peritoneum from controls were enriched for genes involved in inflammatory responses. The x-axis shows -p-value, the y-axis shows the pathways affected and colour denotes enrichment score. (C) Gene ontology analysis of cellular components showed that the genes that were significantly downregulated endometrium and peritoneum from endometriosis patients (>±0.5 FC and p<0.001) compared to endometrium and peritoneum from controls were enriched for genes involved in respiratory chain complex I and mitochondrial ribosome. The x-axis shows -p-value, the y-axis shows the cellular location and colour denotes enrichment score. (D) Gene ontology analysis of cellular components showed that the genes that were significantly upregulated endometrium and peritoneum from endometriosis patients (>±0.5 FC and p<0.001) compared to endometrium and peritoneum from controls were enriched for genes involved in PTW / PP1 phosphatase complex. The x-axis shows -p-value, the y-axis shows the cellular location and colour denotes enrichment score. (E) Gene ontology analysis of molecular function showed that the genes that were significantly downregulated endometrium and peritoneum from endometriosis patients (>±0.5 FC and p<0.001) compared to endometrium and peritoneum from controls were enriched for genes involved in oxidoreduction-driven active transmembrane transporter activity and NADH dehydrogenase activity. The x-axis shows -p-value, the y-axis shows the gene product function and colour denotes enrichment score. (F) Gene ontology analysis of molecular function showed that the genes that were significantly upregulated endometrium and peritoneum from endometriosis patients (>±0.5 FC and p<0.001) compared to endometrium and peritoneum from controls were enriched for genes involved in actin binding and nuclear glucocorticoid receptor binding. The x-axis shows -p-value, the y-axis shows the gene product function and colour denotes enrichment score. (G) Reactome analysis showed that the genes that were significantly downregulated endometrium and peritoneum from endometriosis patients (>±0.5 FC and p<0.001) compared to healthy endometrium and peritoneum from controls were enriched for genes involved in mitochondrial gene expression and translation. The x-axis shows -p-value, the y-axis shows the pathways affected and colour denotes enrichment score. (H) Reactome analysis showed that the genes that were significantly upregulated in endometrium and peritoneum from endometriosis patients (>±0.5 FC and p<0.001) compared to healthy endometrium and peritoneum from controls, were enriched for genes involved in extracellular matrix reorganization, signal transduction and RHO GTPase kinase activity. The x-axis shows -p-value, the y-axis shows the pathways affected and colour denotes enrichment score.

[0047] Figure 10 shows graphs illustrating that patients with endometriosis who have previously had a laparoscopy to treat endometriosis (PL) and patients who have deep endometriosis and the presence of endometriomas have higher levels of urinary PGAM1 compared to patients with deep endometriosis alone. (A) Patients with deep endometriosis who have been surgically treated for endometriosis previously have significantly higher levels of PGAM1 compared to those patients who have not been treated for the disease previously (p=0.0003). (B) Similarly, patients with deep endometriosis with a concurrent diagnosis of endometrioma(s) have significantly higher PGAM1 than those without endometriomas (p<0.0001). (C) Concurrent diagnoses of adenomyosis does not impact PGAM1 levels in any participant pool. SC = Symptomatic Control. HV = Healthy Volunteer. The Mann-Whitney significance test was used in all cases. Figure 11 shows graphs illustrating the urinary PGAM1 can robustly differentiate all participant subgroups from healthy volunteers. (A) When all participants are included, both endometriosis groups and symptomatic controls have significantly lower urinary PGAM1 than healthy volunteers (HV-Deep p=0.001 , HV-Superficial p=0.0004, HV-Symptomatic control p<0.0001). (B) Upon removal of patients who have had a laparoscopy previously and those with a concurrent diagnosis of endometrioma(s), urinary PGAM1 is even lower in all participant subgroups compared to healthy volunteers. Additionally, urinary PGAM1 is significantly lower in patients with deep endometriosis compared to those with superficial endometriosis (HV-SC p<0.0001 , HV-Superficial p<0.0001 , HV-Deep p<0.0001 , Deep-Superficial p=0.0024, Deep- SC p=0.087). The Mann-Whitney significance test was used in all cases.

[0048] Figure 12 shows graphs illustrating that the hormonal therapies typically given to endometriosis patients do not impact urinary PGAM1 levels. In patients with deep endometriosis (A) and superficial endometriosis (B) there was no significant difference in urinary PGAM1 levels between participants on various hormonal therapies, nil = no hormonal therapy. GnRH = Gonadotropin-Releasing Hormone. HRT = Hormone Replacement Therapy. COCP = Combined Oral Contraceptive Pill. Only patients who have not had a laparoscopy to treat their endometriosis previously were included in this analysis. 2-way ANOVA statistical test was used in both cases.

[0049] Figure 13 shows graphs illustrating that the phase of the menstrual cycle does not impact urinary PGAM1 levels. In patients with deep endometriosis (A), superficial endometriosis (B) and symptomatic controls and healthy volunteers (C), no significant difference was seen in PGAM1 levels between individuals who experience amenorrhea, those with regular periods, or between the phases of the menstrual cycle. Only patients with no previous history of a laparoscopy to treat their endometriosis were included in this analysis. 2-way ANOVA statistical test was used in all cases.

[0050] Figure 14 shows a graph illustrating that the location of endometriotic tissue does not impact urinary PGAM1 concentration. There is no significant difference in PGAM1 levels between patients who do and do not have endometriosis to the rectovaginal (RV) nodule, pelvic side wall (PSW), bowel, or pararectal region. Only patients who have not had a previous history of a laparoscopy for endometriosis were included in this analysis. The Mann-Whitney significance test was used in all cases.

[0051] Figure 15 shows a graph illustrating that urinary PGAM1 is significantly higher in endometriosis patients who also have thyroid conditions. Urinary PGAM1 was unaffected by the presence or absence of fibromyalgia, mental health conditions (MHC), migraines, asthma and anaemia, however PGAM1 was significantly higher for endometriosis patients with thyroid disease (p=0.0008). The Mann-Whitney significance test was used in all cases. In this instance, patients who have a previous history of a laparoscopy to treat their endometriosis and those with a concurrent diagnosis of endometrioma(s) were included in this analysis.

[0052] Figure 16 shows a graph illustrating PGAM1 levels (ng / ml SGnorm) remain stable at 4°C for at least 21 days when compared to fresh or freeze-thawed samples on the day they were received (day 0). There was no significant difference in PGAM1 levels between fresh or freeze- thawed samples on day 0 and on days 7, 14, or 21 .

[0053] Figure 17 Receive Operating Characteristics (ROC) is a probability model that we have used to test the ability of the PGAM1 biomarker to distinguish patients with deep (red line) or superficial endometriosis (green line) or symptomatic controls (blue line) from healthy volunteers. Data shown in (A) includes all patients; (B) includes all patients except those with endometrioma and (C) showed all patients except those with endometrioma, thyroid disease and / or migraines. Area Under the Curve (AUC) is an overall measure of the model’s ability to distinguish patients from healthy volunteers. AUC ranges from 0.5-1 , indicates the model cannot distinguish patient from healthy volunteer, whilst a value of 1 indicates a perfect classification model capable of distinguishing all patients from healthy donors.

[0054] Sequence listing

[0055] SEQ ID NO: 1 shows the full-length sequence of phosphoglycerate mutase 1 (PGAM1 , Uniprot accession number: P18669): MAAYKLVLIRHGESAWNLENRFSGWYDADLSPAGHEEAKRGGQALRDAGYEFDICFTSVQ KRAIRTLWTVLDAIDQMWLPWRTWRLNERHYGGLTGLNKAETAAKHGEAQVKIWRRSYD VPPPPMEPDHPFYSNISKDRRYADLTEDQLPSCESLKDTIARALPFWNEEIVPQIKEGKR VLIAAHGNSLRGIVKHLEGLSEEAIMELNLPTGIPIVYELDKNLKPIKPMQFLGDEETVR KAMEAVAAQGKAKK

[0056] SEQ ID NO: 2 shows the full-length sequence of Enolase-1 (ENO1 , Uniprot accession number: P06733): MSILKIHAREIFDSRGNPTVEVDLFTSKGLFRAAVPSGASTGIYEALELRDNDKTRYMGK GVSKAVEH I N KTIAPALVSKKLNVTEQEKI DKLM I EM DGTENKSKFGANAI LGVSLAVCK AGAVEKGVPLYRHIADLAGNSEVILPVPAFNVINGGSHAGNKLAMQEFMILPVGAANFRE AMRIGAEVYHNLKNVIKEKYGKDATNVGDEGGFAPNILENKEGLELLKTAIGKAGYTDKV VIGMDVAASEFFRSGKYDLDFKSPDDPSRYISPDQLADLYKSFIKDYPWSIEDPFDQDD WGAWQKFTASAGIQVVGDDLTVTNPKRIAKAVNEKSCNCLLLKVNQIGSVTESLQACKLA QANGWGVMVSHRSGETEDTFIADLVVGLCTGQIKTGAPCRSERLAKYNQLLRIEEELGSK AKFAGRNFRNPLAK SEQ ID NO: 3 shows the full-length sequence of phosphofructokinase- 1 (PFKP, Uniprot accession number: Q01813):

[0057] MDADDSRAPKGSLRKFLEHLSGAGKAIGVLTSGGDAQGMNAAVRAVVRMGIYVGAKVYFI

[0058] YEGYQGMVDGGSNIAEADWESVSSILQVGGTIIGSARCQAFRTREGRLKAACNLLQRGIT NLCVIGGDGSLTGANLFRKEWSGLLEELARNGQIDKEAVQKYAYLNWGMVGSIDNDFCG

[0059] TDMTIGTDSALHRIIEWDAIMTTAQSHQRTFVLEVMGRHCGYLALVSALACGADWVFLP ESPPEEGWEEQMCVKLSENRARKKRLNIIIVAEGAIDTQNKPITSEKIKELWTQLGYDT

[0060] RVTILGHVQRGGTPSAFDRILASRMGVEAVIALLEATPDTPACWSLNGNHAVRLPLMEC

[0061] VQMTQDVQKAMDERRFQDAVRLRGRSFAGNLNTYKRLAIKLPDDQIPKTNCNVAVINVGA PAAGMNAAVRSAVRVGIADGHRMLAIYDGFDGFAKGQIKEIGWTDVGGWTGQGGSILGTK RVLPGKYLEEIATQMRTHSINALLIIGGFEAYLGLLELSAAREKHEEFCVPMVMVPATVS NNVPGSDFSIGADTALNTITDTCDRIKQSASGTKRRVFIIETMGGYCGYLANMGGLAAGA DAAYIFEEPFDIRDLQSNVEHLTEKMKTTIQRGLVLRNESCSENYTTDFIYQLYSEEGKG VFDCRKNVLGHMQQGGAPSPFDRNFGTKISARAMEWITAKLKEARGRGKKFTTDDSICVL GISKRNVIFQPVAELKKQTDFEHRIPKEQWWLKLRPLMKILAKYKASYDVSDSGQLEHVQ PWSV

[0062] SEQ ID NO: 4 shows the full-length sequence of Hexokinase (Uniprot accession number:

[0063] P19367):

[0064] M IAAQLLAYYFTELKDDQVKKI DKYLYAM RLSDETLI DI MTRFRKEM KNGLSRDFN PTAT VKMLPTFVRSIPDGSEKGDFIALDLGGSSFRILRVQVNHEKNQNVHMESEVYDTPENIVH GSGSQLFDHVAECLGDFMEKRKIKDKKLPVGFTFSFPCQQSKIDEAILITWTKRFKASGV EG ADVVKLLN KAI KKRGDYDAN I VAVVN DTVGTM MTCGYD DQ HCEVGLI I GTGTN ACYM E ELRHIDLVEGDEGRMCINTEWGAFGDDGSLEDIRTEFDREIDRGSLNPGKQLFEKMVSGM YLGELVRLILVKMAKEGLLFEGRITPELLTRGKFNTSDVSAIEKNKEGLHNAKEILTRLG VEPSDDDCVSVQHVCTIVSFRSANLVAATLGAILNRLRDNKGTPRLRTTVGVDGSLYKTH PQYSRRFHKTLRRLVPDSDVRFLLSESGSGKGAAMVTAVAYRLAEQHRQIEETLAHFHLT KDMLLEVKKRMRAEMELGLRKQTHNNAWKMLPSFVRRTPDGTENGDFLALDLGGTNFRV LLVKIRSGKKRTVEMHNKIYAIPIEIMQGTGEELFDHIVSCISDFLDYMGIKGPRMPLGF TFSFPCQQTSLDAGILITWTKGFKATDCVGHDVVTLLRDAIKRREEFDLDVVAWNDTVG TMMTCAYEEPTCEVGLIVGTGSNACYMEEMKNVEMVEGDQGQMCINMEWGAFGDNGCL DDIRTHYDRLVDEYSLNAGKQRYEKMISGMYLGEIVRNILIDFTKKGFLFRGQISETLKTRG IFETKFLSQIESDRLALLQVRAILQQLGLNSTCDDSILVKTVCGVVSRRAAQLCGAGMAA WDKIRENRGLDRLNVTVGVDGTLYKLHPHFSRIMHQTVKELSPKCNVSFLLSEDGSGKG AALITAVGVRLRTEASS

[0065] SEQ ID NO: 5 shows the full-length sequence of interleukin-6 (IL-6, Uniprot accession number: P05231):

[0066] MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLTSSERIDKQIRYI LDGISALRKETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEETCLVKIITGLL EFEVYLEYLQNRFESSEEQARAVQMSTKVLIQFLQKKAKNLDAITTPDPTTNASLLTKLQ AQNQWLQDMTTHLILRSFKEFLQSSLRALRQM

[0067] SEQ ID NO: 6 shows the full-length sequence of Transforming growth factor beta (TGF-p,

[0068] Uniprot accession number: P01137):

[0069] MPPSGLRLLPLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLA

[0070] SPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEI

[0071] YDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWR

[0072] YLSNRLLAPSDSPEWLSFDVTGWRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFT TGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYI DFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQA LEPLPIVYYVGRKPKVEQLSNMIVRSCKCS

[0073] SEQ ID NO: 7 shows a full-length sequence of phosphoglycerate mutase-M (PGAM-M, Uniprot accession number: P15259): MATHRLVMVRHGESTWNQENRFCGWFDAELSEKGTEEAKRGAKAIKDAKMEFDICYTSVL KRAIRTLWAILDGTDQMWLPWRTWRLNERHYGGLTGLNKAETAAKHGEEQVKIWRRSFD IPPPPMDEKHPYYNSISKERRYAGLKPGELPTCESLKDTIARALPFWNEEIVPQIKAGKR VLIAAHGNSLRGIVKHLEGMSDQAIMELNLPTGIPIVYELNKELKPTKPMQFLGDEETVR KAMEAVAAQGKAK

[0074] Detailed Description

[0075] Further features of certain embodiments of the present invention are described below.

[0076] The practice of embodiments of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, protein biology, bioinformatics, microbiology, recombinant DNA technology and immunology, which are within the skill of those working in the art.

[0077] Most general molecular biology, protein biology, microbiology, recombinant DNA technology and immunological techniques can be found in Sambrook et al, Molecular Cloning, A Laboratory Manual (2001) Cold Harbor-Laboratory Press, Cold Spring Harbor, N.Y. or Ausubel et al., Current protocols in molecular biology (1990) John Wiley and Sons, N.Y. Immunohistochemical techniques are described, for example, in Antibodies: A Laboratory Manual by Ed Harlow (Editor), David Lane (Editor) (1988, Cold Spring Harbor Laboratory Press, ISBN 0-87969-314-2), 1855; Price and Newman, “Principles and Practice of Immunoassay,” 2ndEdition, Grove's Dictionaries (1997); and Gosling, “Immunoassays: A Practical Approach,” Oxford University Press (2000). Bioinformatics can be performed using programmes such as R and details of these are freely available through the web (https: / / www.r-project.org / about.html).

[0078] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei- Show, 2nded., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rded., Academic Press; and the Oxford University Press, provide a person skilled in the art with a general dictionary of many of the terms used in this disclosure. Units, prefixes, and symbols are denoted in their Systeme International de Unitese (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation and nucleic acid sequences are written left to right in 5’ to 3’ orientation.

[0079] Deep Endometriosis

[0080] In certain embodiments, the invention provides a method of determining the likelihood of a subject having deep endometriosis.

[0081] Endometriosis is classically defined as a chronic, gynaecological disease characterised by endometrial-like tissue present outside of the uterus and one way it is thought to arise is by retrograde menstruation (see Figure 1). In particular, endometriosis is a condition characterised by ectopic stroma and endometrial glands present outside of the uterus, particularly within the peritoneal cavity and often accompanied by fibrosis. It is one of the most common conditions that affect women of reproductive age, with an estimated 1 in 10 affected worldwide. Endometriosis is a benign disease that exhibits malignant-like features such as invasion, metastasis, and angiogenesis.

[0082] There are three localisations of the inflammatory disease, the most common type is superficial peritoneal endometriosis where flat, shallow lesions invade the peritoneum and no further. Cystic ovarian endometriosis is less common and involves the formation of ‘chocolate cysts’ due to their colour and the inability of old blood to seep away and deep endometriosis, a highly invasive form of the disease, penetrating >5 mm under the peritoneal surface.

[0083] Deep endometriosis is a disease characterized by the presence of endometrium-like epithelium and / or stroma outside the endometrium and myometrium, usually with an associated inflammatory process. For example, deep endometriosis is a severe subtype of the disease where endometrial tissue invades more than 5mm into peritoneal surfaces or directly into other organs such as bladder or bowel. Vascularisation is a key element of the pathogenesis of endometriosis as the new vascular supply is vital to allow proliferation of newly established endometriotic nodules. In comparison with other forms of endometriosis, deep endometriosis lesions are highly vascularised.

[0084] There is no cure for endometriosis, however treatment for managing pain is often provided in the forms of surgery, hormone therapy and analgesia. The route of treatment, whether it be surgical or medication, solely depends on the priority and preferences of the subject in regard to fertility and pain management. Due to the diagnosis of endometriosis only being possible through invasive methods, the identification of biomarkers herein allows the earlier and more efficient diagnosis of the disease. Advantageously, the non-invasive method of detecting deep endometriosis described herein may reduce the cost associated with underdiagnosis of the disease using conventional techniques, as well as reducing the prolonged suffering of subjects before diagnosis and the start of pain management treatments.

[0085] In certain embodiments, the subject having deep endometriosis is characterised by endometriotic lesions about 5mm, about 6mm, about 7mm, about 8mm, about 9mm, about 10mm or more in depth under the peritoneal surface.

[0086] In certain embodiments, the deep endometriosis is characterised by adenomyosis externa with lesions that are more than about 1 cm in diameter (type II and type III lesions). Adenomyosis is endometrial tissue (lining of the uterus) that is found to have grown into the myometrium (muscular wall of the uterus). Adenomyosis can be distinguished into four subtypes based on the myometrial region involved: type I — intrinsic (inner myometrium), type II — extrinsic (outer myometrium), type III — intramural (surrounded by normal outer myometrium), and type IV — indeterminate (not fit into any of the other types).

[0087] In certain embodiments, the deep endometriosis is characterised by infiltration of fibrous and muscular tissue in endometrial tissue.

[0088] In certain embodiments, the deep endometriosis is characterised by one or more of:

[0089] (a) endometriotic lesions about 5mm or more in depth under the peritoneal surface;

[0090] (b) adenomyosis externa with lesions that are more than about 1 cm in diameter (type II and type III lesions); and / or

[0091] (c) infiltration of fibrous and muscular tissue in endometrial tissue.

[0092] In certain embodiments, the subject is identified as having (or not having) one or more endometriotic lesions. For example, the subject may not have ovarian endometrioma.

[0093] Endometriotic lesions (endometriomas) are cystic lesions that result from the disease process of endometriosis. Three major forms of endometriosis are found in the pelvic region: ovarian lesions, peritoneal lesions, and infiltrating endometriotic lesions. The peritoneum is a membrane that lines the inside of the abdomen and pelvis (parietal layer). The peritoneum is comprised of two layers: the superficial parietal layer and the deep visceral layer. The peritoneal cavity contains the omentum, ligaments, mesentery, and intraperitoneal organs. Intraperitoneal organs include but are not limited to the stomach, spleen, liver, first and fourth parts of the duodenum, jejunum, ileum, transverse, and sigmoid colon. Endometriomas are often called "chocolate cysts" due to the thick, dark brown fluid they contain, which is a result of the old blood and menstrual debris accumulating within the cyst.

[0094] Morphologically, there are three types of endometriotic lesions: white, red, and black lesions. In particular, red lesions represent activity with a high level of vascularization, while the white lesions are later phases of red lesions that have undergone a process of inflammation and fibrosis. The classical black lesions are attributable to cyclic tissue decomposition and healing with the subsequent formation of scar tissue.

[0095] Non-invasive test

[0096] In certain embodiments, the method of determining the likelihood of a subject having deep endometriosis is performed ex vivo. Typically, the test is an immunoassay (e.g., ELISA) using a urine sample, as further described herein.

[0097] The subject may be a human or animal (e.g., mammal) suffering (or suspected of suffering) from deep endometriosis. Typically, the subject is a female human. Typically, the female is of reproductive age. In exemplary embodiments, the female is between about 18 and 50 years old.

[0098] In some embodiments, the subject has already been diagnosed as having endometriosis. Advantageously, the methods described herein enable the likelihood of the subject having deep endometriosis to be determined as compared to other types of endometriosis (e.g., superficial endometriosis).

[0099] In some exemplary embodiments, the subject does not have an endometrioma. For example, the subject may be identified as not having an endometrioma prior to being selected for the test. Alternatively, the subject may be identified as not having an endometrioma during, and or after, the test is performed. Any suitable technique for determining whether or not a subject may have an endometrioma may be used, including clinical or biochemical tests within a primary care setting.

[0100] In certain embodiments, the subject may or may not be experiencing migraines. Typically, the subject does not experience migraines (e.g., has not experienced any migraine for at least one week, 1 month, 2 months, 3 months or more). This may be determined, for example, by asking the subject about their symptoms and / or checking their medical records before, during and / or after the method is performed.

[0101] In certain embodiments, the subject may or may not have a thyroid condition. In certain embodiments, the subject does not have a thyroid condition. As used herein “a thyroid condition” refers to any condition or disease that affects how the thyroid functions (for example, hypothyroidism and hyperthyroidism). This may be determined, for example, by asking the subject about their symptoms and / or checking their medical records before, during and / or after the method is performed.

[0102] In certain embodiments, the subject may or may not have a past medical history of endometriosis. As used herein a past medical history refers to a subject having a record of endometriosis prior to the current presenting of symptoms, illness or condition. Aptly the subject may or may not have a had a previous laparoscopy to treat their endometriosis. In certain embodiments, the subject has not had a previous laparoscopy to treat their endometriosis. As used herein “a previous laparoscopy” refers to a subject having had a laparoscopy as a form of treatment for endometriosis prior to the current point in time at which a sample of urine is taken and / or received for testing. Aptly a subject may have had a previous laparoscopy due to a suspicion or diagnosis of endometriosis. This may be determined, for example, by checking their medical records before, during and / or after the method is performed but more often is identified in the first instance by the patient as the World Endometriosis Research Foundation (WERF) questionnaire is completed. Without being bound by theory, diagnostic biomarkers (e.g., PGAM1) levels in a patient may increase the more laparoscopies have been performed as a form of treatment for endometriosis.

[0103] In certain embodiments, the subject may or may not have fibromyalgia. In certain embodiments, the subject does not have fibromyalgia. As used herein “fibromyalgia” refers to a chronic condition that causes symptoms such as musculoskeletal pain. This may be determined, for example, by asking the subject about their symptoms and / or checking their medical records before, during and / or after the method is performed.

[0104] In certain embodiments, the subject may or may not be undergoing hormonal therapy and / or contraception (e.g., COCP, GnRH or HRT as described elsewhere herein). In some exemplary embodiments, the subject may not be undergoing hormonal therapy and / or contraception. This may be determined, for example, by asking the subject about their symptoms and / or checking their medical records before, during and / or after the method is performed. In certain embodiments, the subject exhibits one or more of the following:

[0105] (i) does not have an endometrioma;

[0106] (ii) is not undergoing hormonal therapy;

[0107] (iii) does not have fibromyalgia;

[0108] (iv) does not have a thyroid condition;

[0109] (v) is not experiencing migraines; and / or

[0110] (vi) has not had a previous laparoscopy.

[0111] For example, the subject may be identified as (i) not having an endometrioma, (ii) not undergoing hormonal therapy, (iii) not having fibromyalgia, (iv) not having a thyroid condition and / or (v) not experiencing migraines prior to being selected for the test. Alternatively, the subject may be identified as (i) not having an endometrioma, (ii) not undergoing hormonal therapy, (iii) not having fibromyalgia, (iv) not having a thyroid condition and / or (v) not experiencing migraines during, and or after, the test is performed. Any suitable technique for determining whether or not a subject may or may not have (i) to (v) may be used, including clinical or biochemical tests within a primary care setting and / or asking the subject about their symptoms and / or checking their medical records as already discussed herein.

[0112] In certain embodiments, the subject may be identified as not having:

[0113] (i) an endometrioma;

[0114] (ii) a thyroid condition; and / or

[0115] (iii) a previous laparoscopy during which time an endometriosis diagnosis was given and some diseased tissue was removed (excised or ablated).

[0116] In certain embodiments, the sample comprises saliva, urine, blood, peritoneal fluid and / or endometriotic tissue. In certain embodiments, the sample comprises menstrual or peripheral blood. Typically, the sample comprises urine.

[0117] In some embodiments, the sample has previously been obtained from the subject such that the sampling itself does not form a part of the methods of the invention. The sample may have been obtained immediately prior to the method, or hours, days or weeks prior to the method. In other embodiments, a method of the invention may additionally comprise the step of obtaining the tissue sample from the subject.

[0118] Advantageously, the sample may be obtained from the subject during any menstrual phase. For example, the sample may have been obtained from the subject during the proliferative, secretory and / or menstrual phase of the cycle. In some embodiments, the sample is obtained during the proliferative phase.

[0119] Advantageously, urine samples obtained from the subject may be stored at 4°C for at least one week prior to detecting levels of one or more glycolysis-associated biomarkers (e.g., PGAM1) in the sample. Unexpectedly, enzymes such as PGAM1 are shown herein to be stable at 4°C for at least one week, improving ease of handling samples prior to conducting the methods of the invention. Aptly urine samples obtained from the subject may be stored at 4°C for at least one week, at least two weeks or at least three weeks prior to testing.

[0120] In certain embodiments, one or more glycolysis-associated biomarkers (e.g., PGAM1) is stable in urine at room temperature (about 25°C) for at least 4 days. Aptly urine samples obtained from the subject may be stored at room temperature (about 25°C) for at least 1 day, at least 2 days, at least 3 days, or at least 4 days prior to testing.

[0121] In certain embodiments the sample comprises urine and is normalised by specific gravity (SG) as described herein.

[0122] In certain embodiments the urine sample is fresh. For example, the sample may have been obtained within 5 days, 4 days, 3 days, 2 days, 1 days or less prior to testing. Aptly the method is performed before freezing.

[0123] In certain embodiments the urine sample is frozen. Aptly specific gravity is measured before the sample is frozen. Aptly the method is performed on a thawed sample.

[0124] In certain embodiments, the method of determining the likelihood of a subject having deep endometriosis comprises detecting levels of one or more glycolysis-associated biomarkers in a sample obtained from the subject.

[0125] In certain embodiments the method of determining likelihood of a subject having deep endometriosis comprises comparing the levels of one or more glycolysis-associated biomarkers with one or more reference levels. As used herein, a “difference” is understood to mean a significant increase or decrease in the levels of the target molecule as compared to the reference level (s).

[0126] In certain embodiments, a difference in levels of the one or more biomarkers, compared to the one or more reference levels, is indicative of an increased likelihood of deep endometriosis. For example, the subject may be identified as having deep endometriosis. In certain embodiments, no difference in levels of the one or more biomarkers, compared to the one or more reference levels, is indicative of a decreased likelihood of deep endometriosis. For example, the subject may be identified as not having deep endometriosis.

[0127] In certain embodiments, the method of determining the likelihood of a subject having deep endometriosis comprises:

[0128] (i) detecting levels of one or more glycolysis-associated biomarkers in a sample obtained from the subject; and

[0129] (ii) comparing the levels of the one or more glycolysis-associated biomarkers with one or more reference levels; wherein a difference in levels of the one or more biomarkers, compared to the one or more reference levels, is indicative of an increased likelihood of deep endometriosis.

[0130] In certain embodiments, the one or more reference levels are levels of the glycolysis- associated biomarker(s) that are characteristic of one or more healthy subjects (e.g., control subjects not having endometriosis).

[0131] In addition, or alternatively, the one or more reference levels are levels of the glycolysis- associated biomarker(s) that are characteristic of superficial endometriosis (e.g., superficial peritoneal), ovarian endometriosis, extra-abdominal endometriosis and / or iatrogenic endometriosis. Typically, the one or more reference levels are levels of the glycolysis- associated biomarker are characteristic of superficial endometriosis.

[0132] In certain embodiments, the one or more reference levels may be obtained by detecting levels of the glycolysis-associated biomarker(s) in a sample from one or more healthy subjects (e.g., control subjects not having endometriosis).

[0133] In addition, or alternatively, the one or more reference levels may be obtained by detecting levels of the glycolysis-associated biomarker(s) in a sample from one or more subjects having superficial endometriosis (e.g., superficial peritoneal), ovarian endometriosis, extra-abdominal endometriosis and / or iatrogenic endometriosis. Typically, the one or more reference levels may be obtained by detecting levels of the glycolysis-associated biomarker(s) in a sample from one or more subjects having superficial endometriosis.

[0134] In certain embodiments, a decrease in the level of the glycolysis associated biomarker(s), compared to the one or more reference levels, is indicative of an increased likelihood of deep endometriosis. Typically, the levels of PGAM1 are decreased by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 500% or more as compared to the one or more reference levels. Aptly the one or more reference levels are from a healthy subject.

[0135] In certain embodiments, a level of the glycolysis associated biomarker(s) below a cut-off value is indicative of an increased likelihood of deep endometriosis. Aptly, PGAM1 levels below about 30 ng / ml as standardised to specific gravity is indicative of an increased likelihood of deep endometriosis. Aptly, PGAM1 levels below about 25 ng / ml as standardised to specific gravity is indicative of an increased likelihood of deep endometriosis. Aptly, PGAM1 levels below about 20 ng / ml as standardised to specific gravity is indicative of an increased likelihood of deep endometriosis. Aptly, PGAM1 levels below about 15 ng / ml as standardised to specific gravity is indicative of an increased likelihood of deep endometriosis. Aptly, PGAM1 levels below about 8 ng / ml as standardised to specific gravity is indicative of an increased likelihood of deep endometriosis. Aptly, PGAM1 levels below about 7 ng / ml as standardised to specific gravity is indicative of an increased likelihood of deep endometriosis. Aptly, PGAM1 levels below about 6 ng / ml as standardised to specific gravity is indicative of an increased likelihood of deep endometriosis. Aptly, PGAM1 levels below about 5 ng / ml as standardised to specific gravity is indicative of an increased likelihood of deep endometriosis.

[0136] In certain embodiments, the one or more reference levels are levels of the glycolysis associated biomarker(s) that are characteristic of one or more subjects having deep endometriosis. In addition, or alternatively, the one or more reference levels may be obtained by detecting levels of the glycolysis associated biomarker(s) that are characteristic of one or more subjects having deep endometriosis. In such embodiments, no difference (or an increase) in levels of the one or more biomarkers, compared to the one or more reference levels, is indicative of an increased likelihood of deep endometriosis.

[0137] In certain embodiments, the reference level is less than about 30 ng / ml, less than about 25 ng / ml, less than about 20 ng / ml, less than about 15 ng / ml, less than about 10 ng / ml, less than about 8 ng / ml, less than about 5 ng / ml or less, as standardised to specific gravity (sg). In certain embodiments, the reference level is less than 10 ng / ml as standardised to specific gravity. In certain embodiments the level of one or more glycolysis-associated biomarkers detects deep endometriosis with a likelihood ratio (LR) of about 1 or more, about 5 or more, about 10 or more or about 17 or more.

[0138] Advantageously, the methods, assays and kits of the invention are capable of differentiating between subjects having deep endometriosis vs. superficial endometriosis and / or other types of endometriosis as described herein. In certain embodiments, a difference in levels of the one or more glycolysis-associated biomarkers, compared to the one or more reference levels, is indicative of the subject having a decreased likelihood of superficial endometriosis. In certain embodiments, a difference in levels of the one or more glycolysis-associated biomarkers, compared to the one or more reference levels, is indicative of the subject having an increased likelihood of superficial endometriosis.

[0139] In certain embodiments, superficial endometriosis is characterised by presence of endometrium-like epithelium and / or stroma extending up to 5mm under the peritoneal pelvic surface and / or the serosa of pelvic viscera.

[0140] In certain embodiments, detecting the levels of the glycolysis-associated biomarker comprises outputting, optionally on a computer, (i) an indication of the levels of the glycolysis-associated biomarker, and (ii) this indicates whether the subject is likely to have the deep endometriosis.

[0141] Glycolysis-associated biomarkers

[0142] In certain embodiments, glycolysis-associated biomarkers are used as diagnostic biomarkers to enable a non-invasive diagnosis of deep endometriosis. The glycolysis pathway and glycolysis-associated enzymes are shown in Figure 2.

[0143] In certain embodiments the glycolysis-associated biomarker is a protein. In certain embodiments, the glycolysis-associated biomarker is phosphoglycerate mutase 1 (PGAM1 , Uniprot accession number: P18669 as set forth in SEQ ID NO: 1). In certain embodiments, the protein comprises a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to SEQ ID NO: 1 based on amino acid identity over the entire length of the sequence.

[0144] In certain embodiments, the glycolysis-associated biomarker comprises a sequence having at least about 80% or more homology to SEQ ID NO: 1 based on amino acid identity over the entire length of the sequence. For example, the glycolysis-associated biomarker may comprise SEQ ID NO: 7 (PGAM-M, Uniprot accession number P15259).

[0145] Without being bound by theory, the human genome contains two PGAM genes thereby two iso-types of dPGM genes exist, which includes PGAM-M. PGAM1 may be present in different body parts such as liver, brain, kidney, red blood cells (RBCs), and early fetal skeletal muscle. PGAM-M can be found in adult skeletal muscles and myocardium (cardiac muscles).

[0146] In certain embodiments, reference to “PGAM1” as used herein includes PGAM-M. In alternative embodiments, reference to “PGAM1” as used herein excludes PGAM-M.

[0147] In certain embodiments, the glycolysis-associated biomarker is Enolase-1 (ENO1 , Uniprot accession number: P06733 as set forth in SEQ ID NO: 2). In certain embodiments, the protein comprises a sequence having at least about 90%, 95%, 96%, 97%, 98%, 99% or more homology to SEQ ID NO: 2 based on amino acid identity over the entire length of the sequence.

[0148] In certain embodiments, the glycolysis-associated biomarker is phosphofructokinase- 1 (PFKP, Uniprot accession number: Q01813 as set forth in SEQ ID NO: 3). In certain embodiments, the protein comprises a sequence having at least about 90%, 95%, 96%, 97%, 98%, 99% or more homology to SEQ ID NO: 3 based on amino acid identity over the entire length of the sequence.

[0149] In certain embodiments, the glycolysis-associated biomarker is Hexokinase (Uniprot accession number: P19367 as set forth in SEQ ID NO: 4). In certain embodiments, the protein comprises a sequence having at least about 90%, 95%, 96%, 97%, 98%, 99% or more homology to SEQ ID NO: 4 based on amino acid identity over the entire length of the sequence.

[0150] In certain embodiments the glycolysis-associated biomarker is OX-2 membrane glycoprotein (CD200, Uniprot accession number: P41217). In certain embodiments the glycolysis- associated biomarker is Carboxypeptidase A4 (CPA4, Uniprot accession number: Q9UI42). In certain embodiments the glycolysis-associated biomarker is Inhibin beta B chain (INHBB, Uniprot accession number: P09529). In certain embodiments the glycolysis-associated biomarker is Microfibrillar-associated protein 5 (MFAP5, Uniprot accession number: Q13361). In certain embodiments the glycolysis-associated biomarker is Transcription factor JunD (JunD, Uniprot accession number: P17535). In certain embodiments the glycolysis-associated biomarker is CCAAT / enhancer-binding protein delta (CEBPD, Uniprot accession number: P49716). In certain embodiments the glycolysis-associated biomarker is soluble fms-like tyrosine kinase 1 (sFItl or sVEGFR-1 , Uniprot accession number: P17948).

[0151] In certain embodiments, the protein comprises a sequence having at least about 90%, 95%, 96%, 97%, 98%, 99% or more homology to any one the sequences of P41217, Q9UI42, P09529, Q13361 , P17535, P49716 and / or P17948 based on amino acid identity over the entire length of the sequence.

[0152] In certain embodiments, the glycolysis-associated biomarker is selected from at least two of phosphoglycerate mutase 1 (PGAM1), Enolase-1 (ENO1), phosphofructokinase- 1 (PFKP) and / or Hexokinase. In certain embodiments, the glycolysis-associated biomarkers are selected from at least three of phosphoglycerate mutase 1 (PGAM1), Enolase-1 (ENO1), phosphofructokinase-1 (PFKP) and / or Hexokinase. In certain embodiments, the glycolysis- associated biomarkers are phosphoglycerate mutase 1 (PGAM1), Enolase-1 (ENO1), phosphofructokinase-1 (PFKP) and Hexokinase.

[0153] The levels of glycolysis-associated biomarkers in the sample obtained from a subject can be detected by any appropriate method known in the art. In certain embodiments, levels of glycolysis-associated biomarkers are detected using enzyme-linked immunosorbent assay (ELISA). For example, PGAM1 levels in a sample can be analysed using Human PGAM1 ELISA kit (Abx252944), ENO1 levels in a sample can be analysed using Human Anti-Enolase 1 Antibody (Anti-ENO1) ELISA Kit (abx585629), PFKP levels in a sample can be analysed using Human Phosphofructokinase, Platelet ELISA Kit (abx252942), and / or Hexokinase levels in a sample can be analysed using Human Hexokinase 1 (HK1) ELISA Kit (abx151800). Other kits for detecting biomarkers as described herein are commercially available.

[0154] In certain embodiments, the method further comprises detecting levels of one or more cytokines associated with aerobic glycolysis. For example, the method may further comprise detecting levels of interleukin 6 in the sample obtained from the subject (IL-6, Uniprot accession number: P05231 as set forth in SQ ID NO: 5). In certain embodiments, the protein comprises a sequence having at least about 90%, 95%, 96%, 97%, 98%, 99% or more homology to SEQ ID NO: 5 based on amino acid identity over the entire length of the sequence.

[0155] The levels of IL-6 in the sample obtained from the subject can be detected by any appropriate method known in the art. In certain embodiments, the level of IL-6 is detected using ELISA. For example, IL-6 levels in a sample can be analysed using Human Interleukin 6 (IL6) ELISA Kit (abx050124). In certain embodiments the method further comprises detecting levels of Transforming growth factor beta (TGF-p, Uniprot accession number: P01137 as set forth in SEQ ID NO: 6) in the sample obtained from the subject. In certain embodiments, the protein comprises a sequence having at least about 90%, 95%, 96%, 97%, 98%, 99% or more homology to SEQ ID NO: 6 based on amino acid identity over the entire length of the sequence.

[0156] The above-mentioned homology is calculated on the basis of amino acid sequence identity (sometimes referred to as "hard homology"). The UWGCG Package provides programs including GAP, BESTFIT, COMPARE, ALIGN and PILEUP that can be used to calculate homology or line up sequences (for example used on their default settings). The BLAST algorithm can also be used to compare or line up two sequences, typically on its default settings. Software for performing a BLAST comparison of two sequences is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.njm, iiih.gov / ). This algorithm is further described below. Similar publicly available tools for the alignment and comparison of sequences may be found on the European Bioinformatics Institute website (http: / / www.ebi.ac.uk), for example the ALIGN and CLUSTALW programs.

[0157] The levels of TGF-p in the sample obtained from the subject can be detected by any appropriate method known in the art. In certain embodiments, the level of TGF-p is detected using ELISA. For example, TGF-p levels in a sample can be analysed using TGF beta-1 Human ELISA Kit (Thermo Fisher Scientific, Catalog # BMS249-4). In certain embodiments the method further comprises detecting levels of IL-6 and / or TGF-p in the sample obtained from the subject. IL-6 and TGF-p do not belong to enzymes in the glycolysis pathway, but are cytokines that act on the genes involved in aerobic glycolysis, upregulating their activity.

[0158] In certain embodiments, the method further comprises comparing the levels of IL-6 with one or more reference levels. Aptly a difference in levels of IL-6, compared to the one or more reference levels, is further indicative of an increased likelihood of deep endometriosis. In certain embodiments, the method further comprises comparing the levels of TGF-p with one or more reference levels. Aptly a difference in levels of TGF-p, compared to the one or more reference levels, is further indicative of an increased likelihood of deep endometriosis. In certain embodiments, the method further comprises comparing the levels of IL-6 and / or TGF-p with one or more reference levels. Aptly a difference in levels of IL-6 and / or TGF-p, compared to the one or more respective reference levels, is further indicative of an increased likelihood of deep endometriosis. The one or more reference levels may be levels of the cytokine associated with aerobic glycolysis that are characteristic of (and / or obtained from) a subject having superficial endometriosis and / or a healthy subject.

[0159] In certain embodiments the method further comprises:

[0160] (iii) detecting levels of IL-6 and / or TGF-p in the sample obtained from the subject; and

[0161] (iv) comparing the levels of IL-6 and / or TGF-p with one or more reference levels; wherein a difference in levels of IL-6 and / or TGF-p, compared to the one or more reference levels, is further indicative of an increased likelihood of deep endometriosis.

[0162] In certain embodiments, the glycolysis-associated biomarker is phosphoglycerate mutase 1 (PGAM1). Aptly a difference in the level of PGAM1 in a sample obtained from the subject, compared to one or more reference levels, is indicative of an increased likelihood of deep endometriosis. In certain embodiments, a decrease in the level of PGAM1 in a sample obtained from the subject, compared to one or more reference levels, is indicative of an increased likelihood of deep endometriosis. Aptly, a decrease in the level of PGAM1 of at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 500% as compared to the one or more reference levels is indicative of an increased likelihood of deep endometriosis.

[0163] In certain embodiments, the reference level is less than about 30 ng / ml, less than about 25 ng / ml, less than about 20 ng / ml, less than about 15 ng / ml, less than about 10 ng / ml, less than about 8 ng / ml or less than about 5 ng / ml as standardised to specific gravity (sg). In certain embodiments the level of one or more glycolysis-associated biomarkers detects deep endometriosis with a likelihood ratio (LR) of about 1 or more, about 5 or more, about 10 or more or about 17 or more.

[0164] In certain embodiments, the glycolysis-associated biomarker is Enolase-1 (ENO1). Aptly a difference in the level of ENO1 in a sample obtained from the subject, compared to one or more reference levels, is indicative of an increased likelihood of deep endometriosis. In certain embodiments, a decrease in the level of ENO1 in a sample obtained from the subject, compared to one or more reference levels, is indicative of an increased likelihood of deep endometriosis. Aptly, a decrease in the level of ENO1 of at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 500% as compared to the one or more reference levels is indicative of an increased likelihood of deep endometriosis. In certain embodiments, the glycolysis-associated biomarker is phosphofructokinase- 1 (PFKP). Aptly a difference in the level of PFKP in a sample obtained from the subject, compared to one or more reference levels, is indicative of an increased likelihood of deep endometriosis. In certain embodiments, a decrease in the level of PFKP in a sample obtained from the subject, compared to one or more reference levels, is indicative of an increased likelihood of deep endometriosis. Aptly, a decrease in the level of PFKP of at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 500% as compared to the one or more reference levels is indicative of an increased likelihood of deep endometriosis.

[0165] In certain embodiments, the glycolysis-associated biomarker is Hexokinase. Aptly a difference in the level of Hexokinase in a sample obtained from the subject, compared to one or more reference levels, is indicative of an increased likelihood of deep endometriosis. In certain embodiments, a decrease in the level of Hexokinase in a sample obtained from the subject, compared to one or more reference levels, is indicative of an increased likelihood of deep endometriosis. Aptly, a decrease in the level of Hexokinase of at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 500% as compared to the one or more reference levels is indicative of an increased likelihood of deep endometriosis.

[0166] In certain embodiments, the sample (e.g., the urine sample) is normalised by specific gravity (SG). Typically, the sample is normalised by SG using the equation: wherein Craw is the measured protein level of the one or more glycolysis-associated biomarker (e.g., PGAM1), SGsam is specific gravity of the urine sample, and SGref is 1.02 which represents the mean SG of urine derived from a healthy subject (e.g., female subject not having endometriosis). Aptly, the urine sample is not normalised based on total protein.

[0167] As used herein, the term “specific gravity” (also known as relative density), is a measure of the density of a substance in comparison to the density of water. In particular, specific gravity is a dimensionless quantity defined as the ratio of the density of a substance to the density of a given reference material. Specific gravity for liquids is typically measured with respect to water at its densest. Specific gravity can be calculated directly by methods known in the art. For example, by measuring the density of a sample and dividing it by the (known) density of the reference substance. The density of the sample is simply its mass divided by its volume.

[0168] In certain embodiments, detecting the levels of the one or more biomarkers comprises performing an immunoassay. Aptly the immunoassay is an ELISA as further described herein.

[0169] Methods of treatment

[0170] In certain embodiments, the invention provides a method of treating or preventing deep endometriosis in a subject, wherein the subject has been identified as having an increased likelihood of deep endometriosis according to any method as described herein. In certain embodiments, the subject (e.g., identified as having an increased likelihood of deep endometriosis) is identified as non-suitable for a de-escalated treatment pathway may comprise or consist of hormonal therapy and / or pain medication. Aptly, hormone therapy may treat pain from endometriosis and / or inhibit endometrial proliferation.

[0171] For example, hormone therapies include, but are not limited to: gonadotropin-releasing hormone (GnRH) medicines (such as Buserelin, Goserelin, Leuprorelin, Leuprolide, Histrelin, Naferelin or Triptorelin), oral contraceptives (estrogen-progestin and progestin-only contraceptives), progesterone and progestin (taken as a pill, by injection, or through an intrauterine device), and Danazol.

[0172] For example, pain medications include, but are not limited to nonsteroidal anti-inflammatory drugs (NSAIDS).

[0173] In certain embodiments, the subject is treated with Buserelin, Goserelin, Leuprorelin, Leuprolide, Histrelin, Naferelin or Triptorelin. In certain embodiments, the subject is treated with estrogen-progestin or progestin-only contraceptives. In certain embodiments, the subject is treated with progesterone and progestin. In certain embodiments, the subject is treated with Danazol. In certain embodiments, the subject is treated with nonsteroidal anti-inflammatory drugs.

[0174] In certain embodiments, the subject (e.g., identified as having an increased likelihood of deep endometriosis) is identified as suitable for an escalated treatment pathway. For example, the escalated treatment pathway may comprise (i) surgical treatments; (ii) treatment with a glycolysis-associated inhibitor; and / or (iii) treatment with a drug that can increase mitochondrial fitness.

[0175] For example, surgical treatments include, but are not limited to: laparoscopy (such as laparoscopic excision), laparotomy, surgery to sever pelvic nerves (such as presacral neurectomy or laparoscopic uterine nerve ablation). Aptly the glycolysis-associated inhibitor is dichloroacetate and the drug that can increase mitochondrial fitness is gemcitabine.

[0176] In certain embodiments, the subject is treated by laparoscopic excision.

[0177] In certain embodiments the subject is treated with a glycolysis-associated inhibitor. Aptly the glycolysis-associated inhibitor is dichloroacetate.

[0178] In certain embodiments the invention provides a method of triaging a subject into a suitable treatment group.

[0179] Without being bound by theory, subjects with deep endometriosis may display increased PGAM 1 levels in diseased tissue, resulting in decreased PGAM 1 levels in the urine of patients. In certain embodiments, the subject is treated with therapeutics that reduce PGAM1 levels in the diseased tissue. Aptly such therapeutics may directly or indirectly degrade or break-down PGAM1 in diseased tissue and increase the levels of PGAM1 in the urine.

[0180] In certain embodiments, the invention provides a method of monitoring a subject’s response to treatment of deep endometriosis. In certain embodiments, the method of monitoring a subject’s response to treatment of deep endometriosis comprises detecting levels of one or more glycolysis-associated biomarkers in a sample obtained from the subject. In certain embodiments, the method of monitoring a subject’s response to treatment of deep endometriosis comprises comparing the levels of the one or more glycolysis-associated biomarkers with one or more reference levels.

[0181] In certain embodiments, the method of monitoring a subject’s response to treatment of deep endometriosis comprises:

[0182] (i) detecting levels of one or more glycolysis-associated biomarkers in a sample obtained from the subject; and

[0183] (ii) comparing the levels of the one or more glycolysis-associated biomarkers with one or more reference levels. Aptly the one or more reference levels are predetermined value(s) associated with a healthy subject. Aptly a non-difference in levels of the one or more biomarkers, compared to the one or more reference levels, is indicative of a response to treatment.

[0184] In certain embodiments, the invention provides a method of monitoring a subject’s response to treatment of deep endometriosis before, during and / or after treatment.

[0185] In certain embodiments, the method comprises detecting levels of one or more glycolysis- associated biomarkers in samples obtained from the subject at two or more time points. Aptly a difference in levels of the one or more biomarkers between the two or more time points is indicative of a response to treatment.

[0186] In certain embodiments, the glycolysis-associated biomarker is a protein. Aptly the glycolysis- associated biomarker is selected from any one or more of the glycolysis pathways proteins including, but not limited to: PGAM1 , Enolase-1 , PFKP, and / or Hexokinase.

[0187] In certain embodiments, the glycolysis-associated biomarker is PGAM1. Aptly a difference in levels of PGAM1 between the two or more time points is indicative of a response to treatment. Aptly an increase in levels of PGAM1 between the two or more time points is indicative of a positive response to treatment.

[0188] In certain embodiments, the glycolysis-associated biomarker is Enolase-1 (ENO1). Aptly a difference in levels of ENO1 between the two or more time points is indicative of a response to treatment. Aptly an increase in levels of ENO1 between the two or more time points is indicative of a positive response to treatment.

[0189] In certain embodiments, the glycolysis-associated biomarker is PFKP. Aptly a difference in levels of PFKP between the two or more time points is indicative of a response to treatment. Aptly an increase in levels of PFKP between the two or more time points is indicative of a positive response to treatment.

[0190] In certain embodiments, the glycolysis-associated biomarker is Hexokinase. Aptly a difference in levels of Hexokinase between the two or more time points is indicative of a response to treatment. Aptly an increase in levels of Hexokinase between the two or more time points is indicative of a positive response to treatment.

[0191] In certain embodiments, the method further comprises detecting and comparing levels of one or more cytokines associated with aerobic glycolysis such as IL-6 and / or TGF-p in the sample. In certain embodiments, the glycolysis-associated biomarker is IL-6. Aptly a difference in levels of IL-6 between the two or more time points is indicative of a response to treatment. Aptly an increase in levels of IL-6 between the two or more time points is indicative of a positive response to treatment.

[0192] In certain embodiments, the glycolysis-associated biomarker is TGF-p. Aptly a difference in levels of TGF-p between the two or more time points is indicative of a response to treatment. Aptly an increase in levels of TGF-p between the two or more time points is indicative of a positive response to treatment.

[0193] In certain embodiments, the presence, absence, or level of the glycolysis-associated biomarker may be indicative of a disease, condition, or status of a subject from which the sample (e.g., cell or tissue) is derived.

[0194] In certain embodiments, the level of a glycolysis-associated biomarker is monitored in a subject over time, e.g., monitor the efficacy of one or more treatments. For example, the invention also provides a method of determining a subject’s response to a treatment, wherein the method comprises:

[0195] (i) detecting a glycolysis-associated biomarker in one or more samples from the subject over the course of a treatment, wherein the glycolysis-associated biomarker is detected according to any method as described herein, and

[0196] (ii) comparing the presence, absence and / or level of the glycolysis-associated biomarker with a reference sample or levels obtained therefrom; wherein a difference in the biomarker levels over time (e.g., before treatment commences, at one or more points during treatment, and / or after cessation of treatment) is indicative of a response (or non-response) to the treatment.

[0197] Kits

[0198] In certain embodiments, the invention provides a kit comprising reagents to carry out the method as described herein.

[0199] As used herein the term "binding agent” refers to an agent (e.g., an antibody) comprising a suitable binding moiety enabling binding to a target molecule. Aptly, the binding moiety is specific for a glycolysis-associated biomarker. The antibody that is capable of binding to the target molecule may be a polyclonal or monoclonal antibody. Antibodies that are capable of specifically binding to PGAM1 , Enolase- 1 , PFKP, and / or Hexokinase are commercially available as detailed above.

[0200] Typically, the kit comprises at least one binding agent capable of specifically binding to a glycolysis-associated biomarker. Aptly the glycolysis-associated biomarker(s) is selected from PGAM1 , Enolase-1, PFKP, and / or Hexokinase.

[0201] In certain embodiments, the kit comprises at least two binding agents each capable of specifically binding to a glycolysis-associated biomarker selected from PGAM1, Enolase-1 , PFKP, and / or Hexokinase. In certain embodiments, the kit comprises at least three binding agents each capable of specifically binding to a glycolysis-associated biomarker selected from PGAM1 , Enolase-1 , PFKP, and / or Hexokinase. In certain embodiments, the kit comprises at least four binding agents each capable of specifically binding to a glycolysis-associated biomarker selected from PGAM1 , Enolase-1 , PFKP, and / or Hexokinase.

[0202] In certain embodiments, the kit further comprises a binding agent capable of specifically binding to IL-6 or TGF-p. In certain embodiments, the kit further comprises a binding agent capable of specifically binding to IL-6 and a binding agent capable of specifically binding to TGF-p.

[0203] As used herein the term “detection agent” refers to an agent comprising a suitable detection moiety enabling detection of the binding agent (e.g., an antibody). A suitable detection moiety is selected from a fluorescent moiety, a luminescent moiety, a bioluminescent moiety, a radioactive material, a colorimetric moiety, a nanoparticle having suitable detectable properties, a chromogenic moiety, biotin or an enzyme.

[0204] Examples of detection moieties include but are not limited to, enzymes, e.g. glucose-6- phosphate dehydrogenase ("G6PDH"), alpha-D-galactosidase, glucose oxydase, glucose amylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase and, peroxidase (e.g., horseradish peroxidase); dyes; additional fluorescent labels or fluorescers, such as fluorescein and its derivatives, fluorochrome, GFP ("Green Fluorescent Protein"), dansyl, umbelliferone, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fiuorescamine; fluorophores such as lanthanide cryptates and chelates e.g. Europium etc (Perkin Elmer and Cisbio Assays); radiolabels, e.g., radioisotopes or radionuclides including 3H, 14C, 15N, 35S, 90Y, 99Tc, mln, 1251 , 1311; chemoluminescent labels, e.g. isoluminol, luminol and the dioxetanes; sensitisers; coenzymes; enzyme substrates; particles, such as latex or carbon particles; metal sol; crystallite; liposomes; cells, etc., which may be further labelled with a dye, catalyst or other detectable group; molecules such as biotin, digoxygenin or 5- bromodeoxy uridine; toxin moieties, such as for example a toxin moiety selected from a group of Pseudomonas exotoxin (PE or a cytotoxic fragment or mutant thereof), Diptheria toxin or a cytotoxic fragment or mutant thereof, a botulinum toxin A, B, C, D, E or F, ricin or a cytotoxic fragment thereof e.g. ricin A, abrin or a cytotoxic fragment thereof, saporin or a cytotoxic fragment thereof, pokeweed antiviral toxin or a cytotoxic fragment thereof and bryodin 1 or a cytotoxic fragment thereof.

[0205] The kit may further comprise one or more detection agents (e.g., secondary antibody), wherein each detection agent is capable of specifically binding to a binding agent. Aptly the kit may further comprise two or more detection agents. Aptly the kit may further comprise three or more detection agents.

[0206] The binding agent and / or detection agent may be present in an isolated or substantially purified form. They may be mixed with carriers or diluents that will not interfere with their intended use and still be regarded as substantially isolated. They may also be in a substantially purified form, in which case they will generally comprise at least 90%, e.g. at least 95%, at least 98% or at least 99% of polynucleotides or polypeptides of the kit.

[0207] In some embodiments, the kit further comprises instructions for using the kit to detect the glycolysis-associated biomarkers. In some embodiments, the kit may further comprise one or more additional components such as reagents and / or apparatus necessary for carrying out at immunoassay, e.g., buffers, fixatives, wash solutions, blocking reagents, diluents, chromogens, enzymes, substrates, test tubes, plates, pipettes etc.

[0208] The kit may advantageously be used for carrying out any method described herein and could be employed in a variety of applications, for example in the diagnostic field or as a research tool. It will be appreciated that the parts of the kit may be packaged individually in vials or in combination in containers or multi-container units. Typically, manufacture of the kit follows standard procedures which are known to the person skilled in the art.

[0209] EXAMPLES

[0210] In the following, the invention will be explained in more detail by means of non-limiting examples of specific embodiments. In the example experiments, standard reagents, and buffers free from contamination are used. EXAMPLE 1 - Gene ontology

[0211] The GEO dataset GSE141549, was analyzed using GEOExplorer, an R package and SHINY web application used for DGE. This analysis included 90 patient samples (62 endometrium samples: age 22-48 yrs, and 28 peritoneum samples: age 22-46 yrs) and 62 control samples (38 endometrium samples: age 24-48 yrs, and 24 peritoneum samples: age 33-47 yrs). Batch corrected, normalized data was used and Benjamini and Hochberg p-value adjustment, with a significance cut-off = 0.05. Results were exported in .csv format as downregulated and upregulated genes.

[0212] Gene enrichment analysis was conducted using the GEOexplorer package. Gene Ontology and Reactome databases queried with the results of the previous DGE. Data was exported, for each category and database, in .csv format. The top 30 results from each category were plotted from raw data using Microsoft PowerBI. These were repeated for both upregulated and downregulated genes.

[0213] As depicted in Figure 9A, gene ontology analysis of biological processes showed that the genes that were significantly downregulated endometrium and peritoneum from endometriosis patient (>±0.5 FC and p<0.001) compared to endometrium and peritoneum from controls were enriched for genes involved in mitochondrial gene translation and gene expression.

[0214] As depicted in Figure 9B, gene ontology analysis of biological processes showed that the genes that were significantly upregulated in the endometrium and peritoneum from endometriosis patients (>±0.5 FC and p<0.001) compared to endometrium and peritoneum from controls were enriched for genes involved in inflammatory responses.

[0215] As depicted in Figure 9C, gene ontology analysis of cellular components showed that the genes that were significantly downregulated endometrium and peritoneum from endometriosis patients (>±0.5 FC and p<0.001) compared to endometrium and peritoneum from controls were enriched for genes involved in mitochondrial membrane and matrix as well as the organelle inner membrane.

[0216] As depicted in Figure 9D, gene ontology analysis of cellular components showed that the genes that were significantly upregulated endometrium and peritoneum from endometriosis patients (>±0.5 FC and p<0.001) compared to endometrium and peritoneum from controls were enriched for genes involved in collagen containing extracellular matrix, cell substrate junctions, focal adhesion and cytoskeleton.

[0217] As depicted in Figure 9E, gene ontology analysis of molecular function showed that the genes that were significantly downregulated endometrium and peritoneum from endometriosis patients (>±0.5 FC and p<0.001) compared to endometrium and peritoneum from controls were enriched for genes involved in oxidoreduction-driven active transmembrane transporter activity and RNA binding.

[0218] As depicted in Figure 9F, gene ontology analysis of molecular function showed that the genes that were significantly upregulated endometrium and peritoneum from endometriosis patients (>±0.5 FC and p<0.001) compared to endometrium and peritoneum from controls were enriched for genes involved in actin binding and nuclear glucocorticoid receptor binding.

[0219] As depicted in Figure 9G, reactome analysis showed that the genes that were significantly downregulated endometrium and peritoneum from endometriosis patients (>±0.5 FC and p<0.001) compared to healthy endometrium and peritoneum from controls were enriched for genes involved in mitochondrial gene expression and translation.

[0220] As depicted in Figure 9H, reactome analysis showed that the genes that were significantly upregulated endometrium and peritoneum from endometriosis patients (>±0.5 FC and p<0.001) compared to healthy endometrium and peritoneum from controls were enriched for genes involved in extracellular matrix reorganization, the immune system and signal transduction.

[0221] Following this analysis, glycolysis-associated biomarkers (e.g., phosphoglycerate mutase 1 (PGAM1), Enolase-1 (ENO1), phosphofructokinase- 1 (PFKP) and / or Hexokinase) and / or cytokines associated with aerobic glycolysis (e.g., IL-6 and / or TGF-P) were identified as potential markers to determine the likelihood of a subject having deep endometriosis.

[0222] EXAMPLE 2 - Patient selection

[0223] All patients were identified by reviewing the elective theatre lists at the Hull University Teaching Hospitals NHS Trust (HUTH). Women who matched the inclusion and exclusion criteria (Table 1 and Table 2) were approached and asked if they wished to participate in the study by one of the designated personnel on the NHS staff, or the Principal Investigator who were each trained in Good Clinical Practice (GCP).

[0224] Table 1 : Principal inclusion criteria All participants were asked to provide a clean-catch urine sample into a sterile universal collection tube. For patients at HUTH, this was pre-operative sample taken on the morning of their surgical procedure. The specimens were either stored at 4°C or placed on ice until they could be transported to the laboratory. Healthy volunteers were asked to provide an early- morning first void sample using a Colli-Pee tube (Novosanis, Wijnegem, Belgium) or 100ml specimen pot. They were provided with cold packs to ensure samples were kept cold until they were collected, on the same day, by a member of the research team.

[0225] On arrival at the laboratory, a urine dipstick was performed, and the specific gravity and any abnormalities were documented. The urine was then aliquoted into 2ml cryovials and / or 1 ,5ml Eppendorf and stored at -80°C.

[0226] To generate Figures 3-7, urine samples from participants with deep endometriosis (n=52), superficial endometriosis (n=32) and symptomatic controls (n=18) were defrosted, centrifuged and the supernatant collected. Samples were analysed by ELISA to determine the urine concentration of glycolysis-associated protein (e.g., PGAM1). Eighteen patients who were experiencing uterine bleeding at the time of sample collection were excluded from the primary analysis but including in assessment of menstrual cycle. All results are presented as protein concentration normalised based on specific gravity, which is an accepted method of urine biomarker normalisation.

[0227] Differential expression between the groups was evaluated using the Kruskal-Wallis statistical test. Differences between individual group were appraised using Mann- Whitney statistical test as previously described. Correlations between urinary concentrations and key variables were assessed using Spearman Rank Correlation test. Results were determined to be statistically significant where p<0.05.

[0228] EXAMPLE 3 - urine PGAM1 levels in endometriosis

[0229] PGAM1 is a pivotal glycolytic enzyme which catalyses the reciprocal conversion of 3- phosphoglycerate to 2-phosphoglycerate. Among several key functions, PGAM1 can modulate the rate of glycolysis. PGAM1 is known to promote proliferation and metastasis of cancer cells and is closely associated with clinical metastasis and poor prognosis.

[0230] PGAM 1 levels in urine samples were analysed using Human PGAM1 ELISA kit (Abx252944), which is a sandwich enzyme-linked immune-sorbent assay (Abbexa, Cambridge, UK). Briefly, an antibody is pre-coated onto a 96-well plate to which standards, test samples, biotin- conjugated reagent are added to the wells and incubated. Horseradish peroxidase (HRP)- conjugate reagent is then added, incubated and the unbound conjugates are washed away at each stage. 3, 3'5, 5'- tetramethylbenzidine (TMB) substrate is used to quantify the HRP enzymatic reaction as only wells which contain sufficient PGAM1 will produce a blue coloured product. Once an acidic stop solution is added, this changes to yellow and is proportional to the amount bound to the plate. The OD is measured spectrophotometrically at 450nm in a microplate reader from which a concentration of PGAM1 can be calculated.

[0231] The 96-well plate is loaded with 10OpI of standards and undiluted urine samples in triplicate, covered with an adhesive strip, mixed gently and incubated at 37°C for 90 minutes. All liquid is discarded but the plate is not washed. 10OpI Detection Reagent A working solution to all wells, covered with a new adhesive strip, and incubated at 37°C for 60 minutes. The cover is removed, and the solution discarded. The plate is washed 3 times with 1x wash buffer by adding 350pl to each well for 1-2 minutes before discarding the liquid. Complete removal of liquid at each step is ensured and diligence required to ensure that bubbles do not affect performance. Next 100 pl of Detection Reagent B working solution to each well, the plate sealed and incubated for 30 mins at 37°C. The cover is removed, and the solution discarded. The wash process described above is repeated 5 times. 90pl TMB substrate is added to each well, covered with a plate sealer and mixed gently. The plate is incubated at 37°C for 15 minutes avoiding light exposure before 50pl Stop Solution is added to each well quickly and uniformly to inactive the enzyme. The plate should be read immediately and the OD at 450nm calculated.

[0232] PGAM1 was detected in all samples with a mean concentration 13.8 ng / ml (range 0.25 - 54.22 ng / ml). PGAM1 was normally distributed in both control groups but not endometriosis groups (see Figure 3), so non-parametric tests were used. The concentration of PGAM1 was normalised based on specific gravity, which is an accepted method of biomarker normalisation.

[0233] Urine samples from 152 participants with endometriosis, 19 symptomatic controls and 19 healthy volunteers were collected according to the criteria outlined previously and analysed using ELISA.

[0234] In participants receiving hormonal medications, as shown in Figure 4A, PGAM1 levels were significantly lower with endometriosis compared to control (KW p=0.008). In addition, healthy volunteers receiving hormonal medications exhibited significantly higher PGAM1 levels than deep endometriosis receiving hormonal medications (MW p=0.0005), superficial endometriosis receiving hormonal medications (MW p=0.002) and symptomatic controls receiving hormonal medications (MW p=0.006), as depicted in Figure 4B.

[0235] In participants not taking hormonal medications, as shown in Figure 5A, levels of PGAM1 were significantly higher in controls compared to participants with endometriosis (KW p=0.002). In addition, participants with deep endometriosis not taking hormonal medications exhibited PGAM1 levels significantly lower compared to superficial endometriosis (MW p=0.02), symptomatic controls (MW p=0.05) and healthy volunteers (MW p<0.0001). There was no difference in PGAM1 levels between participants not taking hormonal medications with superficial endometriosis and control participants not taking hormonal medications (MW p=0.06), as depicted in Figure 5B.

[0236] The effect of hormonal medication on urinary PGAM1 levels was evaluated by comparing PGAM1 levels in deep and superficial endometriosis during treatment with either combined oral contraceptive pill (COCP), progestogens, Mirena I US, and gonadotropin hormone- releasing hormone analogues (GnRH), or hormone replacement therapy (HRT). As can be seen from Figure 6, the only statistically significant difference between groups was between Mirena IUS and other progestogens in superficial endometriosis (MW p=0.01). This finding is interesting considering the clinical challenge that progesterone resistance poses in many patients. However, this observation does not appear to be replicated in the deep endometriosis group. In addition, the large distribution of PGAM1 levels in the GnRH group may reflect the extended period which many patients have been receiving add-back HRT whilst awaiting surgery which in many cases is in excess of 24 months.

[0237] The effect of menstrual cycle phase on urinary PGAM1 levels was evaluated by comparing PGAM1 levels in proliferative, secretory, menstrual, and unknown menstrual phase. There is no significant change to the pattern of PGAM1 levels between the proliferative, secretory, menstrual phases in deep endometriosis (Figure 7A), superficial endometriosis (Figure 7B) and control (Figure 7C). There was a representative spread of proliferative, secretory, menstrual, and unknown menstrual phase in each group (Figure 7D).

[0238] As can be seen in Figure 7E-F, when only samples which had been collected in either the secretory phase (Figure 7E) or proliferative phase (Figure 7F) of the menstrual cycle were compared, a simple pattern was seen with significantly higher levels in the healthy volunteers when compared to deep endometriosis (MW p=0.003 and p=0.009 respectively). In summary, PGAM1 was detectable in the urine of all participants with symptoms suggestive of endometriosis. In particular, PGAM1 demonstrated the lowest urinary concentration in the urine of participants with deep endometriosis when compared to either superficial endometriosis, symptomatic controls or healthy volunteers. There is also evidence to support a relationship between exogenous hormone use and menstrual cycle phase with urinary concentration of these glycolysis associated proteins. It therefore follows that this data exemplifies glycolysis associated proteins, and particularly PGAM1 , as diagnostic urine biomarkers for deep endometriosis.

[0239] EXAMPLE 4 - sensitivity and specificity of PGAM1 as a diagnostic marker

[0240] Receiver Operating Characteristic (ROC) analysis was performed to assess the performance of PGAM1 as a diagnostic marker. In particular levels of PGAM1 were measured in the urine of each test group via ELISA (as detailed above), the readings were normalised to the Specific gravity and then compared to identify the sensitivity, specificity and likelihood ratio based on the following equations:

[0241] Sensitivity is the percentage of true positives (e.g. 90% sensitivity = 90% of people who have the target disease will test positive). A high sensitivity indicates that the test is good at detecting people with a disease.

[0242] Specificity is the percentage of true negatives (e.g. 90% specificity = 90% of people who do not have the target disease will test negative) and is calculated by the number of true negatives (TN) / TN + false positives (FP). A high specificity means that the test has a low rate of saying people have a disease when they don’t.

[0243] A positive likelihood ratio (LR+) = sensitivity / (100 - specificity) where sensitivity is = true positives (TP) I (TP + false negatives (FN)).

[0244] There are many ways in which a diagnostic marker for endometriosis could be used clinically, therefore multiple permutations of study groups were assessed (Table 3). For example, a screening test for endometriosis might be used within primary care where both symptomatic and asymptomatic control groups would be the appropriate comparison. Whereas where clinicians require assistance in triaging patients with deep endometriosis directly to specialist endometriosis centres, the appropriate control group would be superficial endometriosis and symptomatic controls. In all comparisons, including participants taking hormonal medications, PGAM1 had a diagnostic capability of AUG <0.65. When participants receiving exogenous hormonal medications were excluded this increased AUC in all groups (Table 3). The best performance seen here was in detecting deep endometriosis where AUC was 0.76 (p<0.001) independent of whether healthy volunteers were included in the control group.

[0245] Table 3: Diagnostic potential of PGAM1 to detect endometriosis.

[0246] Several parameters were seen to have significantly altered urinary PGAM1 levels including the presence of an endometrioma, thyroid disease and migraines. Analysis was performed to see whether excluding participants who reported these possible confounders improved the diagnostic power of PGAM1 (Table 4). The aforementioned parameters are all easily clinically or biochemically verified within a primary care setting.

[0247] When patients with an endometrioma were excluded from analysis, area under the ROC Curve (AUC) improved to 0.68 (p=0.002) in patients taking hormonal treatments and AUC 0.74 (p=0.002) in patients not taking hormonal treatments. The highest AUC was seen in detecting deep endometriosis in patients not taking hormonal treatments and where those with endometrioma had been excluded (AUC 0.91 , p<0.0001).

[0248] Table 4: Prognostic value of PGAM1 in detecting participants with endometriosis from controls.

[0249] The aim of any diagnostic test is for it to be both highly sensitive and specific. The acceptable balance of sensitivity and specificity is dependent on the clinical situation. Table 4 presents the sensitivity, specificity and likelihood ratios have been calculated for various cut-off values for PGAMTs ability to detect (i) participants with endometriosis from controls not taking hormonal medications, (ii) deep endometriosis from superficial endometriosis and controls, and (iii) deep endometriosis from superficial endometriosis and controls in patients without an endometrioma. As can be seen in Table 4, in participants not taking exogenous hormonal medications, PGAM1 levels <5.91 ng / ml standardised to specific gravity could be used to detect deep endometriosis with a likelihood ratio of 9.8, which suggests a useful test. When participants who have had an endometrioma excluded, the utility of PGAM1 in clinical I tests improved with a likelihood ratio of 17.45 and high levels of both sensitivity and specificity.

[0250] Table 5: Sensitivity, specificity and likelihood ratios for PGAM1 to detect endometriosis in participants not taking hormonal medications.

[0251] A likelihood ratio >10 is deemed strong evidence to rule in a disease. This is a positive likelihood ratio. A likelihood ration <0.1 is strong evidence to rule out a disease and is a negative likelihood ratio. <7.81ng / ml PGAM1 normalised to specific gravity has a high positive likelihood ratio in patients without an endometrioma. In patients with deep endometriosis, not taking hormones, <5.9ng / ml PGAM1 normalised to specific gravity has a high positive likelihood ratio (+9.8). Very few diseases can be diagnosed using a single biomarker and with the correct considerations (i.e. ruling out thyroid conditions and endometriomas), PGAM1 alone can be used to diagnose deep endometriosis, which needs further laparoscopic investigation and treatment.

[0252] Although the sample size of participants not taking hormonal therapy restricts further analysis, these data show that PGAM1 levels are a useful diagnostic test in detecting deep endometriosis. Particularly when PGAM1 level is combined with other clinical or biochemical parameters.

[0253] EXAMPLE 5 - Stability of PGAM1 in urine

[0254] PGAM1 levels in urine of three endometriosis patients and five healthy volunteers were analysed via ELISA (as detailed above) on day 0, 4 and 7 following storage at 4°C. As can be seen in Figure 8, there was no statistically significant difference in PGAM1 levels between day 0 (fresh samples) and day 4, day 0 and day 7, or day 4 and day 7. This data demonstrates that PGAM1 levels are stable at 4°C for at least 7 days. It therefore follows that urine samples from subjects do not require freezing prior to testing but rather can be stored in a refrigerator.

[0255] Further, PGAM1 levels in the urine of eight study participants was analysed via ELISA (as detailed above) on the day of sample receipt (i.e. fresh), after one week storage at -80°C (i.e. freeze-thaw), and after 7-days, 14-days or 21-days storage at 4°C. As seen in Figure 16, there was no significant difference in urinary PGAM1 between any of the aforementioned storage conditions, as determined by the analysis of group means using the ANOVA statistical test. This demonstrates that urine for testing can be stored in the refrigerator or -80°C and urinary PGAM1 levels do not significantly change from that present in the fresh sample.

[0256] EXAMPLE 6 - Urine PGAM1 levels in participant subgroups

[0257] Patients were selected and their urine samples collected, dipstick analysed and stored as per Example 2. To generate Figures 10-15, urine samples from the participants detailed in Table 6 were analysed by ELISA (Abx252944) to determine the concentration of glycolysis- associated protein PGAM1 , as described in Example 3. In summary, urine samples from 195 participants with endometriosis, 30 symptomatic controls and 34 healthy volunteers were collected according to the criteria outlined previously and analysed using ELISA.

[0258] Participants diagnosed with deep endometriosis who previously had a laparoscopy to treat endometriosis demonstrated a significantly higher urinary concentration of PGAM1 than participants with deep endometriosis who did not have a previous laparoscopy for endometriosis, Figure 10A. Similarly, participants diagnosed with deep endometriosis and endometrioma(s) had significantly higher urinary PGAM1 compared to participants with deep endometriosis who did not have any endometrioma(s), Figure 10B. In contrast, concurrent diagnosis of adenomyosis had no impact on urinary PGAM1 in participants with endometriosis, Figure 10C. In summary, previous laparoscopy to treat endometriosis and / or concurrent diagnosis of endometrioma(s) significantly increases the urinary PGAM1 concentration in deep endometriosis patients.

[0259] In addition, urinary PGAM1 is significantly lower in patients with deep endometriosis, superficial endometriosis and symptomatic control participants than healthy volunteers, Figure 11 A. When patients diagnosed with endometriosis who also had a previous laparoscopy to treat endometriosis and / or concurrent diagnoses of endometrioma(s) were removed, urinary PGAM1 is further reduced in the endometriosis patients, increasing the significance of this relationship, Figure 11 B. Additionally, when patients who have had a previous laparoscopy to treat endometriosis and those with concurrent diagnoses of endometrioma(s) were removed from the analysis, urinary PGAM1 was significantly lower in deep endometriosis patients than superficial endometriosis, Figure 11 B. As such, urinary PGAM1 can not only detect endometriosis but also differentiate deep from superficial endometriosis. Moreover, urinary PGAM1 concentration was not significantly impacted by the presence or absence of any hormonal treatment in deep (Figure 12A) or symptomatic (Figure 12B) endometriosis patients by analysing group means using the ANOVA statistical test. Further, urinary PGAM1 concentration was not significantly impacted by the menstrual cycle, including phase of the menstrual cycle at the time of sampling or the presence or absence of regular menses, in participants with deep (Figure 13A) or superficial (Figure 13B) endometriosis or healthy volunteers and symptomatic controls (Figure 13C), as determined by analysing group means using the ANOVA statistical test.

[0260] Urinary PGAM1 concentration was not significantly impacted by location of the endometriotic lesions, as defined by the British Society for Gynaecological Endoscopy (BGSE) accredited surgeons who performed the laparoscopic evaluation of these patients, Figure 14. The Mann- Whitney significance test was used in all cases.

[0261] In addition, urinary PGAM1 concentration was not significantly impacted by the presence or absence of the common comorbidities fibromyalgia, mental health conditions (MHC), migraines, asthma and anaemia in any of the endometriosis patients, Figure 15. However, a concurrent diagnosis of endometriosis and thyroid conditions significantly increased urinary PGAM1 compared to endometriosis patients who do not have thyroid conditions. The Mann- Whitney significance test was used in all cases.

[0262] In all patients, urinary PGAM1 was capable of distinguishing both deep and superficial endometriosis from healthy donors and was considered clinically viable in symptomatic controls, Figure 17A. In patients without endometrioma, urinary PGAM1 was capable of distinguishing superficial endometriosis form healthy donors and was considered clinically viable in both deep endometriosis and symptomatic controls, Figure 17B. In patients without endometrioma, thyroid disease, and / or migraine, urinary PGAM1 was capable of distinguishing superficial endometriosis and symptomatic controls from healthy donors and was considered clinically viable in deep endometriosis, Figure 17C.

[0263] In summary, PGAM1 was detectable in the urine of all participants. Urinary PGAM1 concentration was elevated in patients who have had a previous laparoscopy to treat endometriosis and those individuals with a concurrent diagnosis of endometrioma(s) but was unaffected by concurrent diagnoses of adenomyosis. In particular, urinary PGAM1 was significantly lower in endometriosis patients than healthy volunteers. In addition, upon removal of patients who had undergone a previous laparoscopy to treat endometriosis and those individuals with a concurrent diagnosis of endometrioma(s), urinary PGAM1 was significantly lower in deep endometriosis patients compared to superficial endometriosis patients. This not only demonstrates that urinary PGAM1 can detect endometriosis but also that it can differentiate deep from superficial endometriosis. There is evidence to support that, when comparing group means using the ANOVA statistical test, that neither the menstrual cycle (referring to the stage of or presence of menses) nor use of hormonal medications impacts the urinary concentration of PGAM1 . Additionally, there is evidence to support that location of the endometriotic lesion(s) does not impact urinary PGAM1 concentration and only one common comorbidity, thyroid conditions, impacts urinary PGAM1 concentration. Taken together this data exemplifies glycolysis associated proteins, and particularly PGAM1 , as diagnostic urine biomarkers for deep and superficial endometriosis.

[0264] Table 6: Demographics of the participants whose urine PGAM1 concentration was analysed and presented in Figures 10-15. Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to” and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0265] Features, integers, characteristics or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of the features and / or steps are mutually exclusive. The invention is not restricted to any details of any foregoing embodiments. The invention extends to any novel one, or novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0266] The reader’s attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

Claims

CLAIMS1. A method of determining likelihood of a subject having deep endometriosis, the method comprising:(i) detecting levels of one or more glycolysis-associated biomarkers in a sample obtained from the subject; and(ii) comparing the levels of the one or more glycolysis-associated biomarkers with one or more reference levels; wherein a difference in levels of the one or more biomarkers, compared to the one or more reference levels, is indicative of an increased likelihood of deep endometriosis.

2. The method of claim 1 , wherein the subject is identified as having one or more of the following characteristics prior, during, or after detecting levels of the one or more glycolysis- associated biomarkers:(a) does not have an endometrioma;(b) does not have a past medical history of endometriosis, optionally the subject has not had a previous laparoscopy for endometriosis; and / or(c) does not have a thyroid condition.

3. The method of claim 1 or 2, wherein the one or more reference levels are levels of the glycolysis-associated biomarker that are characteristic of a subject having superficial endometriosis and / or a healthy subject.

4. The method of any one of the preceding claims, wherein the glycolysis-associated biomarker is a protein.

5. The method of any one of the preceding claims, wherein the glycolysis-associated biomarker is selected from any one or more of: phosphoglycerate mutase 1 (PGAM1), Enolase-1 (ENO1), phosphofructokinase- 1 (PFKP) and / or Hexokinase.

6. The method of any one of the preceding claims, wherein the method further comprises detecting levels of one or more cytokines associated with aerobic glycolysis, optionally wherein the method further comprises:(iii) detecting levels of IL-6 and / or TGF-p in the sample obtained from the subject; and(iv) comparing the levels of IL-6 and / or TGF-p with one or more reference levels; wherein a difference in levels of IL-6 and / or TGF-p, compared to the one or more reference levels, is further indicative of an increased likelihood of deep endometriosis.

7. The method of any one of the preceding claims, wherein the glycolysis-associated biomarker comprises PGAM1.

8. The method of claim 7, wherein a decrease in the level of PGAM1 , compared to one or more reference levels, is indicative of an increased likelihood of deep endometriosis.

9. The method of claim 8, wherein the subject is human.

10. The method of any preceding claim, wherein the sample comprises urine.11 . The method of claim 10, wherein the glycolysis-associated biomarker is stable in urine at 4°C for at least one week, optionally wherein the glycolysis-associated biomarker is stable in urine at 4°C for three weeks.

12. The method of claim 10 or 11 , wherein the sample is normalised by specific gravity (SG).

13. The method of claim 12, wherein the sample is normalised by SG using the equation:wherein Crawis the level of the one or more glycolysis-associated biomarker, SGsam is specific gravity of the urine sample, and SGref is 1.02 which represents the mean SG of urine derived from a healthy subject.

14. The method of any one of claims 4 to 13, wherein detecting the levels of the protein biomarker comprises performing an immunoassay.

15. The method of claim 14, wherein the immunoassay is an ELISA.

16. A method of treating or preventing deep endometriosis in a subject, wherein the subject has been identified as having an increased likelihood of deep endometriosis according to a method of any one of claims 1 to 15.

17. The method of claim 16, wherein the subject is identified as non-suitable for a deescalated treatment pathway, optionally wherein the de-escalated treatment pathway consists of hormonal therapy and / or pain relief.

18. The method of claim 16 or 17, wherein the subject is identified as suitable for an escalated treatment pathway, optionally wherein the escalated treatment pathway comprises:(i) surgical management, optionally a laparoscopic excision; and / or(iii) treatment with a glycolysis-associated inhibitor, optionally wherein the glycolysis-associated inhibitor is dichloroacetate.

19. A method of monitoring a subject’s response to treatment of deep endometriosis, wherein the method comprises:(i) detecting levels of one or more glycolysis-associated biomarkers in a sample obtained from the subject; and(ii) comparing the levels of the one or more glycolysis-associated biomarkers with one or more reference levels, wherein the one or more reference levels are predetermined value(s) associated with a healthy subject, wherein a non-difference in levels of the one or more biomarkers, compared to the one or more reference levels, is indicative of a response to treatment.

20. A method of monitoring a subject’s response to treatment of deep endometriosis before, during and / or after treatment, wherein the method comprises detecting levels of one or more glycolysis-associated biomarkers in samples obtained from the subject at two or more time points, wherein a difference in levels of the one or more biomarkers between the two or more time points is indicative of a response to treatment.

21. The method of claim 20, wherein the subject is treated by laparoscopic excision and / or with a glycolysis-associated inhibitor, optionally wherein the glycolysis-associated inhibitor is dichloroacetate.

22. The method of any one of claims 19 to 21 , wherein the glycolysis-associated biomarker is a protein.

23. The method of any one of claims 19 to 22, wherein the glycolysis-associated biomarker is selected from one or more of PGAM1 , Enolase, PFKP, IL-6, TGF-p and / or Hexokinase.

24. The method of any one of claims 20 to 23, wherein the method further comprises detecting levels of one or more cytokines associated with aerobic glycolysis, optionally wherein the method comprises detecting and comparing levels of IL-6 and / or TGF-p in the sample.

25. The method of claim 23 or 24, wherein the glycolysis-associated biomarker comprises PGAM1 , optionally wherein an increase in levels of PGAM1 between the two or more time points is indicative of a response to treatment.

26. The method of any one of claims 20 to 25, wherein the subject is human.

27. The method of any of claims 19 to 26, wherein the sample comprises urine.

28. The method of claim 27, wherein the glycolysis-associated biomarker is stable in urine at 4°C for at least one week, optionally wherein the glycolysis-associated biomarker is stable in urine at 4°C for up to three weeks.

29. The method of claim 27 or 28, wherein the sample is normalised by specific gravity (SG).

30. The method of claim 29, wherein the sample is normalised by SG using the equation:wherein Crawis the level of the one or more glycolysis-associated biomarker, SGsam is specific gravity of the urine sample, and SGref is 1.02 which represents the mean SG of urine derived from a healthy subject.

31. The method of any one of claims 22 to 30, wherein detecting the levels of the protein biomarker comprises performing an immunoassay, optionally wherein the immunoassay is an ELISA.

32. An ex vivo assay for diagnosing deep endometriosis in a subject, the assay comprising:(a) contacting a sample obtained from the subject with one or more ligand(s) specific for one or more glycolysis-associated biomarker(s), wherein the presence of the biomarker(s) creates one or more biomarker-ligand complexes; and(b) detecting and / or quantifying the one or more biomarker-ligand complexes.

33. The assay of claim 32, wherein the subject exhibits one or more of the following:(i) does not have an endometrioma;(ii) does not have a past medical history of endometriosis, optionally the subject has not had a previous laparoscopy for endometriosis; and / or(iii) does not have a thyroid condition.

34. The assay of claim 32 or 33, wherein the glycolysis-associated biomarker is a protein.

35. The assay of any one of claims 32 to 34, wherein the glycolysis-associated biomarker is selected from any one or more of PGAM1, Enolase, PFKP, IL-6, TGF-p and / or Hexokinase.

36. The assay of any one of claims 32 to 35, wherein the assay further comprises:(c) detecting levels of one or more cytokines associated with aerobic glycolysis, optionally wherein the assay comprises contacting the sample obtained from the subject with one or more ligand(s) specific for IL-6 and / or TGF-p, wherein the presence of IL-6 and / or TGF-p creates an IL-6 -ligand complex and / or TGF-p-ligand complex; and(d) detecting and / or quantifying the IL-6 -ligand complex and / or TGF-p-ligand complex.

37. The assay of any one of claims 32 to 36, wherein the glycolysis-associated biomarker comprises PGAM1.

38. The assay of claim 37, wherein a decrease in the level of PGAM1 , compared to one or more reference levels, in indicative of deep endometriosis.

39. The assay of any one of claims 32 to 38, wherein the subject is human.

40. The assay of any of claims 32 to 39, wherein the sample comprises urine.41 . The assay of claim 40, wherein the glycolysis-associated biomarker is stable in urine at 4°C for at least one week, optionally wherein the glycolysis-associated biomarker is stable in urine at 4°C for three weeks.

42. The assay of claim 40 or 41 , wherein the sample is normalised by specific gravity (SG).

43. The method of claim 42, wherein the sample is normalised by SG using the equation:wherein Crawis the level of the one or more glycolysis-associated biomarker, SGsam is specific gravity of the urine sample, and SGref is 1.02 which represents the mean SG of urine derived from a healthy subject.

44. The assay of any one of claims 32 to 43, wherein the step of detecting the amount of the at least one protein comprises performing an immunoassay, optionally wherein the immunoassay is an ELISA.

45. A kit for determining whether a subject has deep endometriosis, the kit comprising one or more ligands specific for one or more glycolysis-associated biomarker(s).

46. The kit of claim 45, wherein the glycolysis-associated biomarker is selected from any one or more of PGAM1, Enolase, PFKP, IL-6, TGF-p and / or Hexokinase.

47. The kit of claim 45 or 46, further comprising one or more ligands specific for one or more cytokines associated with aerobic glycolysis, optionally wherein the cytokines are IL-6 and / or TGF-p.

48. The kit of any one of claims 45 to 47, wherein the glycolysis-associated biomarker is PGAM1.

49. The kit of any one of claims 45 to 47, wherein the ligands are antibodies.

50. Use of one or more glycolysis-associated biomarkers in determining the likelihood of a subject having deep endometriosis.

51. Use according to claim 50, wherein the glycolysis-associated biomarker is selected from one or more of PGAM1, ENO1, PFKP and / or Hexokinase.

52. Use according to claim 50 or 51 , wherein the glycolysis-associated biomarker comprises PGAM1.

Images