Endometriosis markers and methods of diagnosing endometriosis

Abstract

The present disclosure relates to methods and kits for detecting levels of androgens, androgen precursors and androgen metabolites. In particular, the present disclosure relates to methods and kits for diagnosing and / or prognosing endometriosis based on the levels of these androgens, androgen precursors and androgen metabolites.

Description

Endometriosis markers and methods of diagnosing endometriosisTECHNICAL FIELDThe present disclosure relates to methods and kits for detecting levels of androgens, androgen precursors and androgen metabolites. The present disclosure further relates to methods and kits for diagnosing and / or prognosing endometriosis based on the levels of these androgens, androgen precursors and androgen metabolites.BACKGROUNDEndometriosis is a chronic condition affecting the reproductive system of females. It is characterized by the presence of endometrial-like tissue outside the uterus. Endometriosis is associated with various symptoms including dysmenorrhoea, dyspareunia, pelvic pain and infertility.Endometriosis is understood to be an estrogen-dependent disease in which endometriotic tissue proliferates in response to estrogen. Consequently, many of the clinical treatments of endometriosis rely on hormone-based therapies which look to reduce levels of estrogen.Huhtinen et al describe a study in which local estradiol (E2) and estrone (E1) concentrations in the endometrium and different types of endometriotic lesions were measured in endometriosis patients, and also mRNA expression of the estrogen- metabolizing enzymes. The study indicated that endometrial or endometriotic E2 concentrations are actively regulated by local estrogen metabolism in the tissue (“Endometrial and Endometriotic Concentrations of Estrone and Estradiol are determined by local metabolism rather than circulating levels”, J. Clin. Endocrinol. Metab. November 2012, 97(11), pages 4228-4235).Huhtinen et al further describe a study which sought to evaluate whether the tissue steroid hormone concentrations in endometriosis differ from the endometrium or serum (“Intra-tissue steroid profiling indicates differential progesterone and testosterone metabolism in the endometrium and endometriosis lesions”, J. Clin. Endocrinol. Metab. November 2014, 99(11), pages E2188 to E2197). In the described study, steroid analysis of serum and tissue specimens of women with endometriosis and healthy controls were measured. The authors concluded that endometriosis lesions present with progestin and androgen metabolism which are different from that of the endometrium, and the lesions are characterized by high levels of testosterone (T) in the tissue and a loss of cyclical changes in the concentration of progesterone (P4) in the tissue.Currently, the only reliable method to diagnose endometriosis is by performing a laparoscopy. This is an expensive and invasive procedure and so endometriosis has significant diagnostic delays for patients, typically an average of eight years from onset of symptoms.Some attempts to use various biomarkers for diagnosing endometriosis have been described in US11001892B1, US7879562B2, US20150132295A1 and US8541173B2. However, it has proved difficult to identify biomarkers that can reliably identify the presence of the disease and / or distinguish patients from healthy controls.Therefore, there remains a need for quick and / or minimally invasive methods of diagnosing and / or prognosing endometriosis.SUMMARYThe present disclosure is based on the finding that certain steroids and their metabolites can provide useful biomarkers of endometriosis. In particular, the present inventors have identified that certain androgens, androgen precursors and androgen metabolites may be elevated in females suffering or at risk of developing endometriosis. Indeed, the present inventors have observed that the hormone metabolism signature (particularly the androgen metabolism signature) is different in patients suffering from endometriosis compared to healthy controls. Accordingly, the present disclosure provides methods and kits for detecting levels of certain androgens, androgen precursors and androgen metabolites, and also methods and kits for diagnosing and / or prognosing endometriosis based on these insights.Described herein is a method for detecting one or more steroid(s) in a test sample, the method comprising: probing a test sample for the presence of the one or more steroid(s) to determine a level of the one or more steroid(s) in the test sample; wherein the one or more steroid(s) is selected from an androgen, an androgen precursor, an androgen metabolite, and combinations thereof.In some examples, the method may comprise a step of comparing the level of the one or more steroid(s) detected for the test sample with a reference or baseline level of the same one or more steroid(s) in a healthy subject (e.g. one that is not suffering from endometriosis). This reference or baseline level may be the level of the one or more steroid(s) detected in a sample obtained from the healthy subject.Where the level of the one or more steroid(s) is different for the test sample to the reference or baseline level, the test sample is determined to have been derived froma subject suffering from endometriosis. In particular, in many cases, where the level of the one or more steroid(s) is higher than the reference or baseline level, the test sample is determined to have been derived from a subject suffering from endometriosis. In some examples, the level of the one or more steroid(s) may be at least 1.5-fold, 2-fold, 2.5- fold, 3-fold, 3.5 fold, 4-fold, 5-fold, 6-fold, 7-fold or 8-fold higher in a test sample derived from a patient suffering from endometriosis than the reference or baseline level.In some examples, the method may find use in a method for diagnosing and / or prognosing endometriosis. In particular, the level of the one or more steroid(s) may be indicative of the likelihood and / or severity of endometriosis.Accordingly, in a first aspect of the disclosure, there is provided a method for diagnosing and / or prognosing endometriosis, the method comprising: probing a test sample for the presence of one or more steroid(s) to determine a level of the one or more steroid(s) in the test sample; wherein the one or more steroid(s) is selected from 11 -ketotestosterone (11 KT), 11 -ketoandrostenedione (11 KA4), 11 -hydroxyandrostenedione (11OHA4), 11- hydroxytestosterone (11OHT), 11 -hydroxydihydrotestosterone, 11- ketodihydrotestosterone, 11 -hydroxyandrosterone (HOHAndro), 17- hydroxypregnanolone (VHP), and combinations thereof; wherein the level of the one or more steroid(s) in the test sample is indicative of the likelihood and / or severity of endometriosis.More particularly, the present inventors have identified that the levels of certain steroid hormones in the body may be determined to provide an indication of the likelihood of endometriosis and / or may provide an indication of the severity of the condition.In particular, the present inventors analysed the levels of a number of steroid hormones in biological fluid samples obtained from both healthy subjects and subjects suffering from endometriosis. They surprisingly identified that levels of a number of these steroid hormones were altered in those subjects suffering from endometriosis compared to the healthy subjects - meaning that each of these steroid hormones (or combinations of these) may be used to form the basis of a reliable diagnostic test for endometriosis. Thus, the described methods offer the possibility of diagnosing and / or prognosing endometriosis for many subjects without the need for surgery.It was observed that levels of certain androgens, androgen precursors and androgen metabolites were different (in some cases, elevated) in patients suffering from endometriosis compared to levels observed in a healthy control. In particular, it was observed that classic and 11 -oxygenated androgens could find particular application asbiomarkers for endometriosis. Without being bound by theory, the present inventors believe that 11 -oxygenated androgens might be particularly useful as their levels are not as prone to fluctuations, e.g. fluctuations due to age of the subject and / or due to a phase of the menstrual cycle.By way of further example, the method may comprise probing the test sample for one or more steroids selected from 11 -ketotestosterone (11 KT), 11- ketoandrostenedione (11 KA4), 11 -hydroxyandrostenedione (11OHA4), 11- hydroxytestosterone (sometimes referred to herein as 11OH-testosterone (11OHT)), and combinations thereof.In some embodiments, the method comprises additionally probing the test sample for the presence of one or more additional steroid(s) selected from:17-hydroxypregnenolone (sometimes referred to herein as 17OH-pregnenolone (17OHP5)), 17-hydroxyprogesterone (sometimes referred to herein as 17OH- progesterone (17OHP4)), dehydroepiandrosterone (sometimes referred to herein as androstenolone (DHEA)), androstenedione (A4), testosterone (T), 11 -deoxycortisol (S), 11 -deoxycorticosterone (DOC), androstanedione (5-adione), androsterone (An), etiocholanolone (Et), 11 -hydroxyandrosterone (HOHAndro), 5-pregnenediol (5-PD), 5- pregnenetriol (5-PT), pregnanediol (PD), pregnanetriol (PT), pregnanetriolone (PTONE), 17-hydroxypregnanolone (sometimes referred to herein as 17OH-pregnanolone (17HP)), 3a5a-17-OH-pregnanolone (3a5a17HP), tetrahydro-11 -deoxycortisol (THS), and combinations thereof.As would be appreciated by the skilled person, many of the steroids described herein comprise multiple stereocentres and exist in different stereoisomeric forms (e.g. in different diastereomeric and / or enantiomeric forms). In particular, in some steroids certain substituents may be designated as alpha or beta to indicate a particular orientation of the substituent. Both orientations are encompassed within the scope of the present disclosure. By way of example, as used herein, references to 11- hydroxytestosterone may encompass both alpha and beta forms. In some examples, the 11 -hydroxytestosterone may be 11-p-hydroxytestosterone. By way of further example, as used herein, references to 11 -hydroxyandrosterone (HOHAndro), may encompass both alpha and beta forms. In some examples, the 11 -hydroxyandrosterone (HOHAndro) may be 11-p-hydroxyandrosterone (1 I bOHAndro). In some examples of the disclosure, the beta form of the steroids may be identified using the methods described herein.The present disclosure additionally provides a method for detecting one or more steroid(s) in a test sample, the method comprising: probing a test sample for the presence of the one or more steroid(s) to determine a level of the one or more steroid(s) in the test sample; wherein the one or more steroid(s) is selected from 11 -ketotestosterone (11 KT), 11 -ketoandrostenedione (11 KA4), 11 -hydroxyandrostenedione (11OHA4), 11- hydroxytestosterone (11OHT), and combinations thereof.It was surprisingly observed that certain steroids showed markedly increased levels in subjects suffering from endometriosis compared to the healthy subjects. Indeed, certain 11 -oxygenated androgens (such as 110H-testosterone (11OHT) and 11- ketotestosterone (11 KT)) were significantly elevated in the bodily fluids of female subjects suffering from endometriosis. In some examples, 11 -hydroxyandrosterone (HOHAndro) and 17-hydroxypregnanolone (17HP) were significantly elevated in the bodily fluids of female subjects suffering from endometriosis. Thus, where a test sample is determined to comprise a higher level of one or more of 11OHT, 11 KT, HOHAndro and / or 17HP, when compared to a level observed in a healthy subject, the subject may be suffering from endometriosis and / or may be diagnosed accordingly.In some examples, the level of certain 11 -oxygenated androgens (such as 11OH- testosterone (11OHT)) and 11 -ketotestosterone (11 KT) may be at least 1.5-fold, 2-fold,2.5-fold, 3-fold, 3.5 fold, 4-fold, 5-fold, 6-fold, 7-fold or 8-fold higher in a test sample derived from a patient suffering from endometriosis than the reference or baseline level. By way of yet further example, the level of 11OH-testosterone (11OHT) may be at least1.5-fold, 2-fold, 2.5-fold, or 3-fold higher in a test sample derived from a patient suffering from endometriosis than the reference or baseline level (e.g. about 1.5-fold higher). By way of yet further example, the level of 11 -ketotestosterone (11 KT) may be at least 1.5- fold, 2-fold, or 2.5-fold higher in a test sample derived from a patient suffering from endometriosis than the reference or baseline level (e.g. about 2-fold higher).It was also observed that certain steroids may show decreased levels in subjects suffering from endometriosis compared to the healthy subjects. Indeed, concentrations of certain 11 -oxygenated androgens (such 11 -hydroxyandrostenedione (11OHA4) and 11 -ketoandrostenedione (11 KA4)) were found to be lower in the bodily fluids of female subjects suffering from endometriosis. Thus, where a test sample is determined to comprise a lower level of one or more of 11OHA4 and / or 11 KA4, when compared to a level observed in a healthy subject, the subject may be suffering from endometriosis and / or may be diagnosed accordingly.In some examples, the level of certain 11-oxygenated androgens (such as 11OHA4 and 11 KA4) may be at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5 fold, 4-fold, 5- fold, 6-fold, 7-fold or 8-fold lower in a test sample derived from a patient suffering from endometriosis than the reference or baseline level.In some examples, the method may comprise probing a test sample for the presence of 11 -ketotestosterone (11 KT) to determine a level of 11 KT in the test sample. This steroid in particular was surprisingly observed to show highly increased levels in patients suffering from endometriosis. Thus, methods and kits based on determining the level of this particular 11 -oxygenated androgen may provide improved sensitivity, reliability and / or accuracy. In such examples, the method may further comprise determining a level of one or more other androgen, androgen precursors or androgen metabolites as described herein.By way of particular example, the method may comprise probing a test sample to determine a level of at least 11 KT and additionally probing the test sample for the presence of one or more additional steroid(s) selected from:11 -ketoandrostenedione (11 KA4), 11 -hydroxyandrostenedione (11OHA4), 11- hydroxytestosterone (11OHT), and combinations thereof.In some further examples, the method may comprise probing a test sample to determine a level of at least 11 KT, and additionally probing the test sample for the presence of one or more steroids selected from:17-hydroxypregnenolone (17OHP5), 11 -hydroxyandrostenedione (11OHA4), 11- ketoandrostenedione (11 KA4), 17-hydroxyprogesterone (17OHP4), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), 11- hydroxytestosterone (11OHT), 11 -hydroxydihydrotestosterone (11OHDHT), 11- ketodihydrotestosterone (11 KDHT), 11 -deoxycortisol (S), 11 -deoxycorticosterone (DOC), androstanedione (5-adione), androsterone (An), etiocholanolone (Et), 11- hydroxyandrosterone (HOHAndro), 5-pregnenediol (5-PD), 5-pregnenetriol (5-PT), pregnanediol (PD), pregnanetriol (PT), pregnanetriolone (PTONE), 17-OH- pregnanolone (17HP), 3a5a-17-OH-pregnanolone (3a5a17HP), tetrahydro-11- deoxycortisol (THS), and combinations thereof.In some examples and without being bound by theory, the determination of levels of 11 KT (optionally in combination with the determination of levels of one or more other 11-oxygenated androgens, or other steroids as described herein) may show the greatest fold-change difference between healthy controls and those subjects suffering fromendometriosis and / or they may be relatively abundant androgen metabolites. Thus, they may find particular utility in the described methods. As stated previously, and without being bound by theory, these particular steroids may be particularly useful as they may provide a more consistent and / or reliable indicator of the likelihood and / or severity of endometriosis. In particular, these steroids are less prone to decline with age (meaning that they can be useful across a range of age groups), and / or they are less prone to menstrual cycle fluctuation.In some examples, the method may comprise probing the test sample for the presence of at least the following steroids:(i) 11 KT and 11 KA4;(ii) 11OHT and 11OHA4;(iii) 11OHA4 and A4;(iv) 11OHT and T;(v) 11 KA4 and 11OHA4; or(vi) 11 KT and 11OHT.In other words, the method may comprise probing the test sample for the presence of at least a pair of steroids as outlined in (i), (ii), (iii), (iv), (v) or (vi) above. The combinations shown in (i), (ii), (iii), (iv), (v) and (vi) above may also find particular utility in methods of diagnosing and / or prognosing endometriosis.As stated previously, through their investigations, the present inventors have identified that the hormone metabolism signature (and in particular the androgen metabolism signature) is different in subjects suffering from endometriosis compared to healthy subjects. Without being bound by theory, the present inventors believe that the different androgen metabolism signature may be the result of and / or associated with a difference in enzyme activity between subjects suffering from endometriosis and healthy subjects.A general overview of the androgen metabolism pathway (starting from cholesterol) is provided in Figure 1 . A schematic representation of an altered androgen metabolism signature (based on the inventor’s observations) in a subject suffering from endometriosis is shown in Figure 2. In Figure 2, the block arrows superimposed on the structures indicate that these androgen precursors, androgens and androgen metabolites have been particularly observed to be altered in a subject suffering from endometriosis (although it is noted that this figure does not provide an exhaustive list). As explained previously, certain steroids have shown particularly elevated levels, as indicated by the upwards facing arrows on Figure 2.Without being bound by theory, the data obtained by the inventors have allowed them to infer a level of enzyme activity, in particular of certain enzymes involved in the androgen metabolic pathway. By way of particular example, and again without being bound by theory, the inventors believe that the activity of the enzyme Aldo-keto reductase family 1 member C3 (AKR1C3) is multifunctional enzyme that may be particularly important as it plays a pivotal role in all pathways to androgen production including 11OHT and 11 KT (steroids observed to show the most increased levels in subjects suffering from endometriosis). The activity of these enzymes is illustrated on Figure 1. In particular, and without being bound by theory, AKR1C3 catalyses the conversion of 11 KA4 to 11 KT, is further involved in the production of testosterone (T) from androstenedione (A4) and may catalyse conversion of 11OHA4 to 11OHT. Without being bound by theory, the present inventors also believe the activity of enzymes CYP11 B1 and HSD11 B2 may also be of use in distinguishing subjects suffering from endometriosis.Thus, the inventors have further identified that methods based on probing a test sample for levels of enzyme activity associated with the androgen metabolic pathway may provide particularly useful diagnostic tools.Accordingly, the disclosure further provides a method for detecting a level of enzyme activity in a test sample, the method comprising:(i) probing a test sample to determine a level of enzyme activity of an enzyme, wherein the enzyme is selected from the group consisting of: Aldo-keto reductase family 1 member C3 (AKR1C3), CYP11 B1 , and HSD11 B2.By way of further example, the disclosure further provides a method for diagnosing and / or prognosing endometriosis, the method comprising:(i) probing a test sample to determine a level of enzyme activity of an enzyme, wherein the enzyme is selected from the group consisting of: Aldo-keto reductase family 1 member C3 (AKR1C3), CYP11 B1 , and HSD11 B2; wherein the level of enzyme activity is indicative of the likelihood and / or severity of endometriosis.In some examples, the enzyme is AKR1C3.In particular, the method may comprise determining the level of enzyme activity in a test sample and comparing the level with a baseline or reference level. The baseline or reference level may be a level of enzyme activity of the enzyme (e.g. AKR1C3)observed or determined in a healthy subject (i.e. a subject not suffering from endometriosis).The method may further comprising detecting a difference in the level of enzyme activity when compared to the baseline or reference level of enzyme activity, wherein a difference in the levels and / or an altered level of enzyme activity is indicative of the likelihood and / or severity of endometriosis in the subject. By way of further example, in those examples where an altered level of enzyme activity is observed in the subject, the altered level of enzyme activity may indicate that the subject is suffering from endometriosis. In some examples, the altered level of activity of the enzyme may be observed as a different substrate preference. By way of example, AKR1C3 may convert more 11 KA4 to 11 KT than A4 to T in a subject suffering from endometriosis (in comparison to a healthy subject). In a further example, AKR1C3 may convert more 11OHA4 to 11OHT in a subject suffering from endometriosis (in comparison to a healthy subject).Thus, for some specific substrate / product combinations (e.g. substrate steroid and product steroid), the altered level of enzyme activity may manifest as an increased level of enzyme activity (e.g. for the conversion of that specific substrate to that specific product). For other specific substrate / product combinations, the altered level of enzyme activity may manifest as a decreased level of enzyme activity (e.g. for the conversion of that specific substrate to that specific product).By way of further example, where an increased level of enzyme activity is observed in the subject (e.g. an increased level of AKR1C3 activity in a specific reaction in comparison to a reference level), the increased level of enzyme activity may indicate that the subject is suffering from endometriosis.By way of further example, where a decreased level of enzyme activity is observed in the subject (e.g. a decreased level of AKR1C3 activity in a specific reaction in comparison to a reference level), the decreased level of enzyme activity may indicate that the subject is suffering from endometriosis.The levels of enzyme activity may be determined using a variety of different methods as known in the art. In some cases, the level of enzyme activity (e.g. the level of AKR1C3 activity) may be determined indirectly by determining relative levels of a substrate and product. In other examples, the level of enzyme activity may be determined directly by determining a rate of substrate to product conversion e.g. by using labelled substrates. In some examples, the level of enzyme activity may be determined bymeasuring the abundance of products (e.g. unlabelled products) by an analytical techniques, such as mass spectrometry.As described above, in some examples, the levels of enzyme activity may be determined (e.g. indirectly determined) by determining and comparing levels of substrate and product steroid pairs in enzyme-catalysed reactions associated with the androgen metabolic pathway. The relative comparison of levels of such steroid pairs may offer an advantage over the determination of absolute levels of one or more steroids by removing any variability that might be observed between batches of sample or in the analytical technique used.By way of further example, a useful method may involve the determination of levels of a substrate steroid and a product steroid in a reaction step associated with the androgen metabolic pathway, optionally a reaction step catalysed by AKR1C3 CYP11 B1 , or HSD11 B2. The method may then comprise determining the relative levels of the substrate steroid and the product steroid.In some examples, the relative levels of the substrate steroid and the product steroid may be determining by calculating a product steroid to substrate steroid ratio. In examples, where a product steroid to substrate steroid ratio is about 1 , the levels of the product steroid and substrate steroid may be substantially the same. In examples where a product steroid to substrate steroid ratio is greater than about 1 , the level of the product steroid may be higher than the level of the substrate steroid. In examples where a product steroid to substrate steroid ratio is less than about 1 , the level of the product steroid may be lower than the level of the substrate steroid.In some examples, the method may involve the determination of levels of a substrate steroid and a product steroid in a reaction step modulated and / or catalysed by AKR1C3. In such cases, the substrate steroid may be 11OHA4 and the product steroid may be 11OHT. In other examples, the substrate steroid may be 11 KA4 and the product steroid may be 11 KT. In some examples, the substrate steroid may be A4 and the product steroid may be T.In some examples, the method may involve the determination of levels of a substrate steroid and a product steroid in a reaction step modulated and / or catalysed by CYP11 B1 . In such cases, the substrate steroid may be androstenedione and the product steroid may be 1 i p-hydroxyandrostenedione (11OHA4). In some examples, the substrate steroid may be testosterone (T) and the product steroid may be 11 (3- hydroxytestosterone (11OHT).In some examples, the method may involve the determination of levels of a substrate steroid and a product steroid in a reaction step modulated and / or catalysed by HSD11 B2. In such cases, the substrate steroid may be 11OHA4 and the product steroid may be 11 KA4. In some examples, the substrate steroid may be 11OHT and the product steroid may be 11 KT.Accordingly, the disclosure further provides a method for detecting at least two steroids in a test sample, the method comprising:(i) probing a test sample obtained from a subject to determine the relative levels of a steroid pair selected from one of the following:(a) 11 KT and 11 KA4;(b) 11OHT and 11OHA4;(c) androstenedione (A4) and 11OHA4;(d) 11OHT and T;(e) 11 KA4 and 11OHA4; and(f) 11 KT and 11OHT.As stated above, the method may provide a method (e.g. an indirect method) for detecting a level of enzyme activity, optionally wherein the enzyme is AKR1C3 Aldo-keto reductase family 1 member C3 (AKR1C3), CYP11 B1 , or HSD11 B2.In particular, the methods described above might find application in a method for diagnosing and / or prognosing endometriosis. In particular examples, the relative levels of the steroid pair defined in any one of (a), (b), (c), (d), (e), or (f) (i.e. (a) 11 KT and 11 KA4; (b) 11OHT and 11OHA4; (c) 11OHA4 and androstenedione (A4); (d) 11OHT and T; (e) 11 KA4 and 11OHA4; or (f) 11 KT and 11OHT) may be indicative of the likelihood and / or severity of endometriosis.In particular, the relative levels of the steroid pairs defined in any one of (a) to (f) determined in the test sample (and as defined above) may be compared to the relative levels of the same steroid pairs defined in any one of (a) to (f) observed in a healthy subject. In other words, the relative levels of the steroid pairs defined in any one of (a) to (f) observed in a healthy subject may be used as a baseline or reference level.In some examples, the method may comprise probing a test sample to determine the relative levels of 11 KA4 (an example of a substrate steroid) and 11 KT (an example of a product steroid), e.g. the method may comprise determining a ratio of 11 KT to 11 KA4. The method may further comprise comparing the ratio of 11 KT to 11 KA4 with the ratio of 11 KT to 11 KA4 observed or determined in a healthy subject. In some examples, the ratio of 11 KT to 11 KA4 may be less than or equal to: about 1 , about 0.98,or about 0.5. In some examples, the ratio of 11 KT to 11 KA4 may be greater than or equal to: about 0.25, about 0.5, about 0.75 or about 0.85. In some examples, the ratio of 11 KT to 11 KA4 may be about 0.87. In some examples, the ratio of 11 KT to 11 KA4 may be increased in a subject suffering from endometriosis (e.g. in comparison to the ratio of 11 KT to 11 KA4 in a healthy subject).In some examples, the method may comprise probing a test sample to determine the relative levels of 11OHA4 (an example in this case of a substrate steroid) and 11OHT (an example in this case of a product steroid), e.g. the method may comprise determining a ratio of 11OHT to 11OHA4. The method may further comprise comparing the ratio of 11OHT to 11OHA4 with the ratio of 11OHT to 11OHA4 observed or determined in a healthy subject. In some examples, the ratio of 11OHT to 11OHA4 may be greater than or equal to: about 0.05, about 0.075, orabout O.10. In some examples, the ratio of 11 OHT to 11OHA4 may be increased in a subject suffering from endometriosis (e.g. in comparison to the ratio of 11 OHT to 11OHA4 in a healthy subject).In some examples, the method may comprise probing a test sample to determine the relative levels of androstenedione (A4) (an example in this case of a substrate steroid) and 11OHA4 (an example in this case of a product steroid), e.g. the method may comprise determining a ratio of 11OHA4 to androstenedione. The method may further comprise comparing the ratio of 11OHA4 to androstenedione with the ratio of 11OHA4 to androstenedione observed or determined in a healthy subject. In some examples, the ratio of 11OHA4 to androstenedione may be greater than or equal to: about 0.5, about 1 , about 2, about 3 or about 5. In some examples, the ratio of 11OHA4 to androstenedione may be less than or equal to: about 4, about 3 or about 2. In some examples, the ratio of 11OHA4 to androstenedione may be decreased in a subject suffering from endometriosis (e.g. in comparison to the ratio of 11OHA4 to androstenedione in a healthy subject).In some examples, the method may comprise probing a test sample to determine the relative levels of testosterone (T) (an example of a substrate steroid) and 11OHT (an example of a product steroid), e.g. the method may comprise determining a ratio of 11OHT to T. The method may further comprise comparing the ratio of 11OHT to T with the ratio of 11OHT to T observed or determined in a healthy subject. In some examples, the ratio of 11OHT to T may be less than or equal to: about 1 , 0.75, or about 0.5. In some examples, the ratio of 11OHT to T may be about 0.49. In some examples, the ratio of 11OHT to T may be decreased in a subject suffering from endometriosis (e.g. in comparison to the ratio of 11OHT to T in a healthy subject).In some examples, the method may comprise probing a test sample to determine the relative levels of 11OHA4 (an example of a substrate steroid) and 11 KA4 (an example of a product steroid), e.g. the method may comprise determining a ratio of 11 KA4 to 110HA4. The method may further comprise comparing the ratio of 11 KA4 to 11OHA4 with the ratio of 11 KA4 to 11OHA4 observed or determined in a healthy subject. In some examples, the ratio of 11 KA4 to 11OHA4 may be greater than or equal to: about 0.5, about 0.9, about 1 , about 1.1 , or about 1.2. In some examples, the ratio of 11 KA4 to 11OHA4 may be about 1.3. In some examples, the ratio of 11 KA4 to 11OHA4 may be increased in a subject suffering from endometriosis (e.g. in comparison to the ratio of 11 KA4 to 110HA4 in a healthy subject).In some examples, the method may comprise probing a test sample to determine the relative levels of 11OHT (an example of a substrate steroid) and 11 KT (an example of a product steroid), e.g. the method may comprise determining a ratio of 11 KT to 11OHT. The method may further comprise comparing the ratio of 11 KT to 11OHT with the ratio of 11 KT to 11OHT observed or determined in a healthy subject. In some examples, the ratio of 11 KT to 110HT may be less than or equal to: about 5, or about 4. In some examples, the ratio of 11 KT to 11OHT may be greater than or equal to: about2, about 2.25, or about 2.5. In some examples, the ratio of 11 KT to 11OHT may be about3, or about 2.96. In some examples, the ratio of 11 KT to 11OHT may be increased in a subject suffering from endometriosis (e.g. in comparison to the ratio of 11 KT to 11OHT in a healthy subject).As described above, the various methods may be used to provide an indication of a likelihood of endometriosis. In some examples, the methods may be useful in providing an indication of the severity of the disease.In particular, endometriosis is typically graded in the clinic by reference to four stages: stage I (minimal disease, 1 to 5 points) to IV (severe disease, >40 points). The points are assigned according to several parameters such as location, size, and depth (superficial versus deep) of the lesions (T. P. Canavan and L. Radosh

[2000] Postgrad. Med. 107:213, the contents of which are incorporated herein by reference).The larger the disparity or difference between the level of the one or more steroids (e.g. 11 KT) detected in a test sample (and in accordance with the methods described herein), when compared to the reference or baseline level of the corresponding one or more steroids in a healthy subject, may provide an indication of a greater likelihood that the endometriosis is more severe and / or is graded at a higher stage.Similarly, the larger the disparity or difference between the ratio of a pair of steroids (as described above), when compared to the reference or baseline ratio of the corresponding pair of steroids in a healthy subject, may provide an indication of a greater likelihood that the endometriosis is more severe and / or is graded at a higher stage.Each of the methods described herein rely on the provision of a test sample obtained from a subject or patient. Thus, each of the described methods may further comprise a step of providing a test sample from a subject and / or obtaining a test sample from a subject.The term “test sample” may embrace any biological samples, such as biological fluids and / or tissues. A test sample may comprise a biopsy, blood (or a fraction thereof (serum / plasma)), secretions, scrapings, tissue or organ washes or cells.In particular, the test sample probed in the described methods may be obtained from a biological fluid, such as a bodily fluid. By way of example, the test sample may be obtained from blood (e.g. serum or plasma), urine, saliva, peritoneal fluid or the like. In some examples, the test sample may be obtained from blood (e.g. serum) or urine.The one or more steroid(s) that are probed in the test sample may depend upon the nature of the test sample. By way of example only, detecting a steroid metabolome in a serum sample may provide a picture of part of the steroid metabolome, whereas detecting a steroid metabolome in a urine sample may provide an indication of total steroid metabolism throughout the body. The present inventors have identified that detecting one or more steroid(s) present in each of these types of test sample may assist in providing a diagnosis and / or classification of the severity of endometriosis. Thus, the methods described herein may be carried out in either or both of a serum sample and a urine sample.By way of further example, where the test sample is obtained from blood (e.g. serum or plasma), the one or more steroid(s) that are probed in the test sample may be selected from 11 -ketotestosterone (11 KT), 11 -ketoandrostenedione (11 KA4), 11- hydroxyandrostenedione (11OHA4), 11 -hydroxytestosterone (11OHT), 11- hydroxydihydrotestosterone, 11 -ketodihydrotestosterone, and combinations thereof. Additional steroids that may be probed may include: 17-hydroxypregnenolone (17OHP5), 17-hydroxyprogesterone (17OHP4), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), 11 -deoxycortisol (S), 11 -deoxycorticosterone (DOC), androstanedione (5-adione), and combinations thereof.In examples where the test sample is obtained from urine, the method may comprise probing the test sample for metabolites of the 11-oxygenated androgens thatare present in the urine. Accordingly, where the test sample is a urine sample, the present inventors have identified that the described methods should focus on certain androgen metabolites derived from the 11 -oxygenated androgens that have been found to be discriminatory for endometriosis.Accordingly, in a further aspect there is provided a method of diagnosing and / or prognosing endometriosis comprising detecting one or more steroid(s) in a urine sample, the method comprising: probing the urine sample for the presence of the one or more steroid(s) to determine a level of the one or more steroid(s) in the urine sample; wherein the one or more steroid(s) is selected from androsterone (An), etiocholanolone (Et), 11 -hydroxyandrosterone (HOHAndro), 17-hydroxypregnanolone (17HP), and combinations thereof; wherein the level of the one or more steroid(s) in the test sample is indicative of the likelihood or severity of endometriosis.In some examples, the method may comprise probing the urine sample for at least 11 -hydroxyandrosterone (e.g. 11-beta-hydroxyandrosterone, HbOHAndro), which is the main urinary metabolite of 11 -oxygenated androgens.In some examples, the method may further comprise probing the urine sample for additional steroids, in particular any of the one or more steroid(s) as defined herein. Such additional steroids may be selected from 5-pregnenediol (5-PD), 5-pregnenetriol (5-PT), pregnanediol (PD), pregnanetriol (PT), pregnanetriolone (PTONE), 17-OH- pregnanolone (VHP), 3a5a-17-OH-pregnanolone (3a5a17HP), tetrahydro-11- deoxycortisol (THS), and combinations thereof.In some examples, the method may comprise probing the urine sample for at least 17-OH-pregnanolone (17HP), optionally in combination with any one or more other steroid(s) as defined herein.Test samples may be provided by, or obtained from, subjects suffering from endometriosis, subjects suspected to be suffering from endometriosis, or subjects susceptible or predisposed to endometriosis. The subjects may be asymptomatic and / or symptomatic.The sample may be obtained from or provided by subjects between recognised and / or diagnosed instances of endometriosis. Subjects from whom samples may be provided or obtained include, for example, healthy subjects showing no detectable signs of endometriosis.A test sample may be a stored or preserved sample. For example, the test sample may be obtained from a subject and stored or preserved for a period of time prior to the probing step.The methods of the present disclosure comprise a step of probing a test sample to determine a level of at least one steroid selected from an androgen, an androgen precursor and an androgen metabolite. As used herein, the level of at least one steroid may refer to a concentration or an amount of the steroid in the test sample.Methods of determining levels of steroids may involve the use of any standard analytical technique and suitable techniques would be known to those working in the field. Useful methods for determining the level of an androgen, an androgen precursor or an androgen metabolite may include, but are not limited to, mass spectroscopy, infrared spectroscopy, nuclear magnetic resonance spectroscopy, raman spectroscopy, electrochemical analysis and the like.In some examples, determining a level of the steroid in question may comprise the use of liquid chromatography-mass spectroscopy (LC-MS) or gas chromatographymass spectroscopy (GC-MS).In some examples, the step of detecting the relevant androgen, androgen precursor or androgen metabolite, or combination thereof (e.g. one or more of 11- ketotestosterone (11 KT), 11 -ketoandrostenedione (11 KA4), 11- hydroxyandrostenedione (11OHA4), and 11 -hydroxytestosterone (11OHT)) in a test sample may involve the use of antibodies with affinity and / or specificity for the relevant molecule. For example, the disclosed methods may use anti-11 KT antibodies, anti- 11 KA4 antibodies, anti-11OHA4 antibodies and anti-11OHT antibodies.Useful antibodies may be conjugated to or labelled with detectable moieties, for example optically delectable moieties. Examples of useful and detectable moieties may include, fluorescent, chemiluminescent and / or coloured particles - such as colloidal gold, alkaline phosphatase, horseradish peroxidase and the like. One of skill will appreciate that binding a labelled and (optically) detectable antibody to a particular molecule (for example an 11 KT or one of the other androgen precursors, androgens, or androgen metabolites as described herein), allows expression of that molecule to be detected and / or quantified.It should be noted that the term antibody includes any target molecule binding fragment thereof. Within the context of this disclosure, the term “target molecule” may include any of the androgen precursors, androgens, or androgen metabolites as described herein.Immunological techniques such as ELISA, immunohistochemistry and FACS may be used to detect or visualise bound antibody and subsequently to report levels of target molecule expression. Moreover, the amount of antibody bound (and the corresponding amount of any detectable label present on the bound antibodies) may be used as a means by which the amount of any given target molecule may be quantified.According to a further aspect of the present disclosure, there is provided a method of treating and / or preventing endometriosis in a subject in need thereof, comprising the steps of:(i) diagnosing that a subject is suffering from endometriosis in accordance with any one of the methods described in the present disclosure; and(ii) administering an endometriosis therapy to the subject.In other words, there is provided a method of treating endometriosis in a subject in need thereof, comprising the steps of:(i) determining a level of one or more steroid(s) selected from 11- ketotestosterone (11 KT), 11 -ketoandrostenedione (11 KA4), 11- hydroxyandrostenedione (11OHA4), 11 -hydroxytestosterone (11OHT), 11- hydroxydihydrotestosterone (11OHDHT), 11 -ketodihydrotestosterone (11 KDHT), 11- hydroxyandrosterone (HOHAndro), 17-hydroxypregnanolone (VHP), and combinations thereof, in a test sample obtained from a subject;(ii) comparing the level with a reference or baseline level of the one or more steroids in a healthy subject; and(iiii) administering an endometriosis therapy if the determined level of the one or more steroid(s) is different to the reference or baseline level.In other examples, there is provided a method of treating endometriosis in a subject in need thereof, comprising the steps of:(i) determining a ratio of two steroids each independently selected from an androgen, an androgen precursor and an androgen metabolite (as described herein, e.g. (a) 11 KT to 11 KA4; (b) 11OHT to 11OHA4; (c) 11OHA4 to androstenedione (A4); (d) 11OHT to T; (e) 11 KA4 to 11OHA4; or (f) 11 KT to 11OHT) in a test sample obtained from a subject;(ii) comparing the ratio with a reference or baseline ratio of the two steroids in a healthy subject; and(iiii) administering an endometriosis therapy if the determined ratio in the test sample is different to the reference or baseline ratio.As referred to above, an endometriosis therapy may refer to any treatment method suitable to treat and / or prevent endometriosis and / or symptoms thereof. Suitable endometriosis therapies are known in the art (see, for example, “Treatment of endometriosis: a review with comparison of 8 guidelines”, Kalaitzopoulos et al, BMC Womens Health, 2021 , 21 , 397, the contents of which are incorporated by reference).Representative endometriosis therapies include, but are not limited to, painkillers, hormonal treatments, chemotherapy, and surgical treatments.Thus, the method may further comprise administering a painkiller to the subject in a therapeutically effective amount. Painkillers used for the treatment of endometriosis include both simple analgesics, such as paracetamol, COX-2 inhibitors, aspirin, and other non-steroidal anti-inflammatory drugs well known in the art, and narcotic analgesics, such as morphine, codine, oxycodone, and others well known in the art.In some examples, the method may further comprise administering a hormonal treatment to the subject in a therapeutically effective amount. Hormonal treatments include, but are not limited to, oral contraceptives, progestins, such as Dydrogesterone, Medroxyprogesterone acetate, Depot medroxyprogesterone acetate, Norethisterone, Levonorgestrel, and others well known in the art, progesterone and progesterone-like substances, GnRH agonists, such as leuprorelin, buserelin, goserelin, histrelin, deslorelin, nafarelin, and triptorelin, androgens and synthetic androgens like Danazol, and aromatase inhibitors.In some examples, the method may further comprise a surgical treatment, in particular surgery to remove, destroy and / or ablate areas of endometriotic tissue. Surgical treatments include, but are not limited to, laparoscopic surgery, hysterectomy, and oophorectomy.According to a further aspect of the disclosure, there is provided a kit for detecting levels of one or more steroids selected from androgens, androgen precursors, and androgen metabolites (e.g. those steroids described herein).The kit may find particular utility in a method for diagnosing endometriosis.The kit may comprise a probe for detecting levels of one or more steroids selected from androgens, androgen precursors, and androgen metabolites (e.g. those steroids described herein).In particular, there is provided a kit for detecting levels of one or more steroids, wherein the kit comprises a probe for detecting levels of one or more (or all of) steroids selected from:(i) 11 -ketotestosterone (11 KT);(ii) 11 -ketoandrostenedione (11KA4);(iii) 11 -hydroxyandrostenedione (11OHA4);(iv) 11 -hydroxytestosterone (11OHT);(v) 11 -hydroxydihydrotestosterone;(vi) 11 -ketodihydrotestosterone;(vii) 11 -hydroxyandrosterone (HOHAndro); and(viii) 17-OH-pregnanolone (17HP).The kit may additionally comprise one or more probe(s) for detecting levels of one or more (or all of) additional steroids selected from:(ix) androsterone (An);(x) etiocholanolone (Et);(xi) 17OH-pregnenolone (17OHP5);(xii) 17-hydroxyprogesterone (17OHP4);(xiii) dehydroepiandrosterone (or androstenolone (DHEA));(xiv) androstenedione (A4);(xv) testosterone (T);(xvi) 11 -deoxycortisol (S);(xvii) 11 -deoxycorticosterone (DOC);(xviii) androstanedione (5-adione);(xix) 5-pregnenediol (5-PD);(xx) 5-pregnenetriol (5-PT);(xxi) pregnanediol (PD);(xxii) pregnanetriol (PT);(xxiii) pregnanetriolone (PTONE);(xxiv) 3a5a-17-OH-pregnanolone (3a5a17HP); and(xxv) tetrahydro-11 -deoxycortisol (THS).The kit may comprise one or more probe(s) for detecting the relative levels of a steroid pair selected from one of the following:(a) 11 KT and 11KA4;(b) 11OHT and 11OHA4;(c) androstenedione (A4) and 11OHA4;(d) 11OHT and T;(e) 11 KA4 and 11OHA4; and(f) 11 KT and 11OHT.In some preferred examples, the kit may comprise a probe for detecting the relative levels of a steroid pair as defined in (a) or (b).In some examples, the kit may comprise one or more antibodies that bind (e.g. specifically bind) to the one or more steroids being detected.By way of example only, the kit may further comprise one or more antibodies selected from the following:(i) an anti- 11 -ketotestosterone (11 KT) antibody;(ii) an anti-11 -ketoandrostenedione (11KA4) antibody;(iii) an anti-11 -hydroxyandrostenedione (11OHA4) antibody;(iv) an anti-11 -hydroxytestosterone (11OHT) antibody;(v) an anti-11 -hydroxydihydrotestosterone antibody;(vi) an anti-11 -ketodihydrotestosterone antibody;(vii) an anti- 11 -hydroxyandrosterone (HOHAndro) antibody; and(viii) an anti-17-OH-pregnanolone (17HP) antibody.In some examples, the kit may comprise additional antibodies selected from one or more (or all) of the following:(ix) an anti-androsterone (An) antibody;(x) an anti-etiocholanolone (Et) antibody;(xi) an anti-17OH-pregnenolone (17OHP5) antibody;(xii) an anti-17-hydroxyprogesterone (17OHP4) antibody;(xiii) an anti-dehydroepiandrosterone (or androstenolone (DHEA)) antibody;(xiv) an anti-androstenedione antibody;(xv) an anti-testosterone antibody;(xvi) an anti- 11 -deoxycortisol (S) antibody;(xvii) an anti- 11 -deoxycorticosterone (DOC) antibody;(xviii) an anti-androstanedione (5-adione) antibody;(xix) an anti-5-pregnenediol (5-PD) antibody;(xx) an anti-5-pregnenetriol (5-PT) antibody;(xxi) an anti-pregnanediol (PD) antibody;(xxii) an anti-pregnanetriol (PT) antibody;(xxiii) an anti-pregnanetriolone (PTONE) antibody;(xxiv) an anti-3a5a-17-OH-pregnanolone (3a5a17HP) antibody; and(xxv) an anti-tetrahydro-11 -deoxycortisol (THS) antibody.The antibodies contained within a kit of this disclosure may comprise or be conjugated, fused or bound to, a detectable label or moiety, for example an optically detectable label or moiety.In some examples, the kit of the disclosure may comprise at least (i), optionally in combination with any one or more antibodies selected from (ii) to (xxv).In other examples, the kit may comprise at least:(a) an anti-11 KT antibody and an anti-11 KA4 antibody;(b) an anti-11OHT antibody and an anti-11OHA4 antibody;(c) an anti-androstenedione (A4) antibody and an anti-11OHA4 antibody;(d) an anti-11OHT antibody and an anti-T antibody;(e) an anti-11 KA4 antibody and an anti-11OHA4 antibody; and / or(f) an anti-11 KT antibody and an anti-11OHT antibody.A kit of this disclosure may further comprise buffers, solutions, tools, receptacles for use in any of the methods described herein. A kit of this disclosure may comprise instructions for use.DefinitionsAs used herein, a “subject” or “patient” may refer to a mammalian subject, such as a female mammalian subject. Representative examples include, but are not limited to, human, mouse, rabbit, horse, dog, sheep and rat subjects.As used herein and unless the context indicates otherwise, a “healthy subject” is a subject which is not suffering from endometriosis.As used herein, the term “endometriosis” may embrace conditions in which endometrial tissue makes its way outside of the uterus and grows on or within other organs or structures including, for example, the ovaries, fallopian tubes, the cavities of the pelvis, the supporting ligaments of the uterus or the peritoneum. The term “endometriosis” embraces all known types of endometriosis including, but not limited to, peritoneal endometriosis, ovarian endometriosis and deep infiltrating endometriosis.As used herein, symptoms of endometriosis may include, but are not limited to, dysmenorrhoea, dyspareunia, pelvic pain, and infertility.Moreover, it should be noted that the terms “comprise”, “comprising” and / or “comprises” is / are used to denote that aspects and embodiments of this invention “comprise” a particular feature or features. It should be understood that this / these terms may also encompass aspects and / or embodiments which “consist essentially of” or “consist of” the relevant feature or features.Detailed descriptionThe disclosure will now be described by way of example only with reference to the following Figures.Figure 1 shows an overview of androgen metabolism;Figure 2 shows a schematic representation of an altered androgen metabolism signature in a subject suffering from endometriosis;Figure 3 shows serum concentrations of classic androgens and alternative androgen pathway metabolites are higher in women with diagnosed endometriosis (Dx), when compared to healthy control subjects (HC). Serum concentrations of DHEA, A4, T (“Test”, testosterone), DHT, An and 5adione were measured in healthy control women and endometriosis patients. DHEA, A4, T, DHT, An and 5adione were significantly elevated in endometriosis patients compared to HC. Error bars represent the standard deviation of the mean. Statistical comparison Mann Whitney II test or unpaired t test. *p<0.05, **p<0.05, ****p<0.0001.Figure 4 shows serum concentrations of certain 11 -oxygenated androgens are altered in women with diagnosed endometriosis (Dx). Serum concentrations of 11OHA4, 11OHT, 11 KA4 and 11 KT were measured in healthy control women and endometriosis patients. 11 -oxygenated androgen precursors, 11OHA4 and 11 KA4, were decreased in Dx, while 11 -oxygenated androgens, 11OHT and 11 KT were significantly elevated in Dx. Error bars represent the standard deviation of the mean. Statistical comparison Mann Whitney II test. *p<0.05, ****p<0.0001.Figure 5 shows serum concentrations of steroid precursors and glucocorticoids in women with endometriosis. Serum concentrations of B, E, F, 17OHP4, S and DOC were measured in HC and Dx. Concentrations of B, 17OHP4, S and DOC did not differ between groups. E and F were modestly elevated in Dx compared to HC. Error bars represent the standard deviation of the mean. Statistical comparison Mann Whitney II test or unpaired t test. *p<0.05, ****p<0.0001 , ns - non significant.Figure 6 shows serum concentrations of classic androgens are highest in women with stage I and stage II endometriosis. Serum concentrations of DHEA, A4, T and DHT were measured in healthy control women and Dx patients stratified rASRM stage (l-IV). T was elevated in all endometriosis stages. DHEA and A4, were significantly elevated in stage I, III and IV endometriosis, and DHT was significantly elevated in stage I endometriosis. Error bars represent the standard deviation of the mean. Statisticalcomparison Kruskal-Wallis test with multiple comparisons. *p<0.05, **p<0.01 , ***p<0.001 ****p<0.0001 , ns - non significant.Figure 7 shows serum concentrations of 11 -oxygenated androgens stratified by endometriosis stage. Serum concentrations of 11OHA4, 11OHT, 11 KA4 and 11 KT were measured in healthy control women and endometriosis patients by rASRM stage (l-IV). 11 KT was elevated in all endometriosis stages while 11 KA4 was decreased. 11OHT was significantly elevated in stage I endometriosis while concentrations of 11OHA4 did not significantly differ between groups. Error bars represent the standard deviation of the mean. Statistical comparison Kruskal-Wallis test with multiple comparisons. **p<0.01 , ***p<0.001 ****p<0.0001 , ns - non significant.Figure 8 shows ROC curve analysis of serum from healthy control women or diagnosed with endometriosis. DHEA AUC of 0.78 (p<0.001 , Cl 0.7102 to 0.8428), A4 AUG of 0.76 (p<0.001 , Cl 0.6873 to 0.8244). T AUC of 0.804 (p<0.001 , Cl 0.7440 to 0.8637) and 11 KT AUC of 0.8063 (p<0.001 , Cl 0.7437 to 0.8689).Figure 9 shows AKR1C3 enzyme activity based on substrate to product ratios (product / substrate). Ratios were calculated based on measured serum concentrations in healthy control women (HC) and patients diagnosed with endometriosis (Dx). The ratio of T / A4 did not differ between HC and Dx. The ratio of 11OHT / 11OHA4 and 11 KT / 11 KA4 was significantly greater in Dx compared to HC. Statistical comparison Mann Whitney U test. ****p<0.0001 , ns - non significant.Figure 10 shows ROC curve analysis of serum ratios from healthy control women or with diagnosed endometriosis. 11 KT / 11 KA4 (AUC 0.9695; p<0.001 , Cl 0.9484 to 0.9905) and 11OHT / 11OHA4 (AUC 0.8224; p<0.001 , Cl 0.7633 to 0.8815).Figure 11 shows CYP11 B1 enzyme activity based on substrate to product ratios (product / substrate). Ratios were calculated based on measured serum concentrations in healthy control women (HC) and patients diagnosed with endometriosis (Dx). The ratios of 11OHA4 / A4 and 11OHT / T were significantly less in Dx compared to HC. ROC curve analysis of serum ratios from healthy control women or with diagnosed endometriosis for 11OHA4 / A4 and 11OHT / T. Statistical comparison Mann Whitney U test. ****p<0.0001 , ns - non significant.Figure 12 shows HSD11 B2 enzyme activity based on substrate to product ratios (product / substrate). Ratios were calculated based on measured serum concentrations in healthy control women (HC) and patients diagnosed with endometriosis (Dx). The ratios of 11 KA4 / 11OHA4 and 11 KT / 11OHT were significantly greater in Dx compared to HC. ROC curve analysis of serum ratios from healthy control women or with diagnosedendometriosis for 11 KA4 / 11OHA4 and 11 KT / 11OHT. Statistical comparison Mann Whitney II test. ****p<0.0001 , ns - non significant.Figure 13 shows urine analysis of androgen metabolites in healthy control (HC) and diagnosed endometriosis patients (Dx). Androgen metabolites - An (androsterone), Etio (etiocholanolone), H BOHAndro, DHEA and 16aOHDHEA were measured by GC / MS. An (androsterone), Etio (etiocholanolone), H BOHAndro were significantly increased in Dx patients compared to HC. Statistical comparison Mann Whitney II test, ns - non significant.Figure 14 shows ROC curve analysis of H BOHAndro concentrations in urine from healthy control women or diagnosed with endometriosis. AUC of 0.8711 (p<0.001 , Cl 0.8039 to 0.9382).Figure 15 shows urine analysis of steroid precursor metabolites in healthy control (HC) and diagnosed endometriosis patients (Dx). Precursor metabolites - PD: pregnanediol, PT: pregnanetriol, PTONE: pregnanetriolone, 17HP: 17- hydroxypregnanolone, 3a5a-17-OH-pregnanolone (3a5a17HP) and THS: tetrahydro-11- deoxycortisol were measured by GC / MS. All analyte concentrations were significantly greater in Dx compared to HC. Statistical comparison Mann Whitney II test, ns - non significant **p<0.01 ***p<0.001. ****p<0.0001 , ns - non significant.Figure 16 shows ROC curve analysis of 17HP (17-hydroxypregnanolone) concentrations in urine from healthy control women or diagnosed with endometriosis. AUC of 0.9507 (p<0.001 , Cl 0.9073 to 0.9940).EXAMPLESSummaryThe study carried out by the inventors aimed to characterise the steroid metabolome profile of patients with endometriosis. Serum samples from patients with suspected endometriosis (n=157, Edinburgh and Liverpool) and healthy controls matched by age and BMI (n=61 , Birmingham) were profiled using an LC-MS / MS assay to simultaneously measure 21 steroid hormones / metabolites. Analysis controlled for menstrual cycle phase and endometriosis disease stage, participants were not receiving hormone treatment. Additionally, urine samples from patients with suspected endometriosis (n=69) and healthy controls (n=38) were profiled by GC / MS to quantify urinary steroid metabolites.The hormone signature in patients with endometriosis was found to be distinct from healthy controls. The concentrations of 15 / 21 analytes measured were significantlyaltered consistent with a disease-specific hormone profile. Dehydroepiandrosterone (DHEA) was elevated 2-fold (P<0.0001), testosterone (T) was elevated 1.8-fold (P<0.0001), 11 -hydroxytestosterone was elevated 1.5-fold (P<0.0001) and 11- ketotestosterone (11 KT) 2-fold (P<0.0001) in endometriosis compared to healthy controls. ROC curve analysis of T (AUC=0.804, P<0.001) and 11 KT (AUC=0.806, P<0.001) demonstrated these analytes had potential as diagnostic classifiers.Steroid metabolising enzyme activities were also assessed by calculating product to substrate ratios for the androgen enzyme AKR1C3. Notably, AKR1C3 activity when measured as the ratio of 11 KT / 11 KA4 was significantly increased in endometriosis compared to healthy controls. ROC curve analysis of the ratio of 11 KT / 11 KA4 (AUC=0.97, P<0.001) proved most robust for providing diagnostic cut-off values which were applied across the cohort. This analysis produced 8% false positives in unstratified healthy controls and correctly identified 95% of the endometriosis patients.Additionally, urinary androgen metabolites were elevated in endometriosis patients compared to healthy controls and this was associated with increased androsterone, etiocholanolone and H BOHAndro, indicative of increased urinary androgen excretion.The present inventors have accordingly identified a disease-specific hormone signature that identifies endometriosis with high specificity and sensitivity. Measuring the steroid metabolome and particularly 11 -oxygenated androgens; 11 KA4, 11 KT, 11OHT and 11 KT as well as 11 KT / 11 KA4 ratios in endometriosis were identified as particularly useful in this context.Materials and MethodsStudy approvalWritten informed consent was obtained from all endometriosis study participants prior to surgery; ethical approval was granted by the Lothian Research Ethics Committee (LREC 11 / AL / 0376). Methods were carried out in accordance with NHS Lothian Tissue Governance guidelines and EPHect guidelines(https: / / endometriosisfoundation.org / ephect / ).The collection of serum samples from healthy volunteers identified through advertisement received ethical approval by the Science, Technology, Engineering and Mathematics Ethical Review Committees of the University Of Birmingham, UK (ERN_17- 0494, ERN_17-0494B).Patient cohortsEligible participants (n=157) were women with chronic pelvic pain (aged 18-50 years) of >3 months duration who were undergoing diagnostic laparoscopy for suspected endometriosis in NHS Lothian, or Liverpool Women’s Hospital, UK. Pelvic pain was defined as pain located within the true pelvis (between and below the anterior iliac crests). Age, BMI, menstrual cycle stage and hormone status were obtained and recorded along with other key clinical data.Inclusion criteria for healthy controls (n=61) were age 18 years or above and provision of written informed consent prior to study participation. A standardized questionnaire was used to record demographic data including age and BMI; for women, the use of hormonal contraceptives and menopausal status were recorded. Exclusion criteria were any acute or chronic disease affecting steroid biosynthesis or metabolism, the intake of any medication known to interfere with steroid biosynthesis or metabolism. Healthy controls were matched to patient cohort by age and BMI and selected from a broader cohort of participants as detailed in Schiffer et al (European Journal of Endocrinology, Volume 188, Issue 1 , January 2023, I vac017 , https: / / doi.Org / 10.1093 / ejendo / ivac017) .Participants from either cohort using hormonal contraceptives (combined oral contraceptives, contraceptive depot injection, or implant) were excluded.Diagnosis of endometriosis was confirmed macroscopically at laparoscopy in 157 women (‘diagnosed’). Women with endometriosis diagnosis were subsequently classified according to the revised scoring system of the American Society for Reproductive Medicine (rASRM) as stage I (n = 65), II (n = 33), III (26) and IV (n = 33) (Table 1).Table 1. Cohort Characteristics for healthy controls or endometriosis patient serum samples. Patients were classified according to rASRM stage indicating those diagnosed with minimal / mild (I, II) or moderate / severe (III, IV) endometriosis.Serum steroid analysisBlood samples were collected in the morning and, in the case of patients with suspected endometriosis, prior to anaesthesia on the morning of surgery. All blood samples were collected in serum-separating tubes. After centrifugation, aspirated serum was aliquoted and stored at -80°C. Serum samples from patients with endometriosis (n=157) and age- and BMI-matched healthy controls (n=61) were profiled using a sensitive Mass Spec (LC-MS / MS) assay to simultaneously measure 21 steroid hormones / metabolites.Unconjugated steroids were quantified using a previously published and validated multi-steroid profiling liquid chromatography-tandem mass spectrometry (LC- MS / MS) assay (Schiffer et al, Journal of Chromatography B, Volume 1209, 15 October 2022, 123413). In brief, 200 pL of serum were mixed with stable isotope-labelled internal standards (Progesterone-d9, 17a-hydroxyprogesterone (17OHP)-d8, cortisol-d4, cortisone-d7, DHEA-d6, A4-d7, T-d3, DHT-d3, 11 K4-d10, 11 KT-d3, 11OHA4-d7, 5a- dihydroprogesterone (5aDHP)-d4, allopregnanolone (alloP)-d4) and following protein precipitation with 50 pL acetonitrile samples were extracted by liquid-liquid extraction with 1 mL methyl terf-butyl ether. The dried organic phase was reconstituted in the mobile phase and steroids were chromatographically separated using a PhenomenexLuna Omega C18 column (1.6 pm, 100A, 2.1 mm x 50 mm) and a water-methanol gradient. Ammonium fluoride was introduced by post-column infusion and steroid quantification was performed on a Waters Xevo TQ-XS mass spectrometer using electrospray ionization in positive ion mode.DHEAS was extracted and quantified using a separate validated assay (Foster et al, The Journal of Clinical Endocrinology & Metabolism, 105(3), March 2020, pages 925-937). Internal standard (DHEAS-d2) was added to 20 pL of serum followed by 20 pL of ZnSCU thenlOO pL of acetonitrile. The samples were then centrifuged and 100 pL of the solution was transferred to a new vial, dried, and reconstituted in 200 pL of methanol / water prior to LC-MS / MS analysis. DHEAS was quantified against a calibration series ranging from 250 to 8000 ng mL-1.Urine AnalysisUrine was collected from healthy controls (n=38) or patients with suspected endometriosis (n=69) and urinary steroid metabolites quantified using Gas chromatography / mass spectrometry (GC / MS).Analyte keySerum analytes were grouped as follows:• Classic Androgens: dehydroepiandrosterone sulfate (DHEAS), dehydroepiandrosterone (DHEA), androstenedione (A4), androstanedione (5adione), testosterone (T) and dihydrostestosterone (DHT).• Alternative androgen pathway metabolites: androsterone (An) and androstanedione (5adione).• 11 -oxygenated androgens: 1 i p-Hydroxyandrostenedione (11OHA4), 11 p- Hydroxytestosterone (11OHT), 11 -ketoandrostenedione (11 KA4) and 11- ketotestosterone (11 KT).• Steroid precursors: 17-hydroxyprogesterone (17OHP4), 11 -deoxycortisol (S) and 11 -deoxycortisone (DOC).• Glucocorticoids: Corticosterone (B), Cortisone (E) and Cortisol (F).Urine metabolites were grouped as follows:• Androgen metabolites: androsterone (An), etiocholanolone (Etio), 11 - hydroxyandrosterone (H BOHAndro), DHEA, 16a-hydroxyDHEA (16aDHEA).• Additional androgen metabolites: 5-pregnenediol (5-PD) and 5-pregnenetriol (5-PT).• Precursor metabolites: pregnanediol (PD), pregnanetriol (PT), pregnanetriolone (PTONE), 17-OH-pregnanolone (17HP), 3a5a-17-OH- pregnanolone (3a-5a-17HP) and tetrahydro-11 -deoxycortisol (THS).Statistical analysisStatistical analysis was performed using GraphPad Prism 9. Data were summarized using means and SEM, presented for healthy controls (N=61) and diagnosed endometriosis patients (N=157).T test was used to compare the difference in the means of the two groups. Two-way ANOVA was used to determine the significance between treatments in grouped data. Non-parametric testing was utilised where sample sizes were insufficient to confirm normality of data distribution; Kruskal-Wallis test was used to assess differences between multiple groups or Mann-Whitney test to assess variance between two groups. Criterion for significance was P < 0.05. Correlation coefficients for androgen metabolites in serum were plotted into a correlation matrix using GraphPad Prism 9.ResultsEndometriosis is associated with an altered androgen metabolism signatureThe concentrations of androgens and their precursors were quantified using LC- MS / MS in serum samples from healthy controls (n=61) and patients with suspected endometriosis (n=157).The concentrations of ‘classic’ androgens; DHEA, A4, T and DHT were determined (Figure 3). Concentrations of DHEA (p<0.0001), A4 (p<0.0001), T (p<0.0001) and DHT (p<0.05), as well as alternative androgen pathway metabolites; An (p<0.0001) and 5adione (p<0.01), were significantly higher in patients with endometriosis compared to healthy controls.The concentrations of 11-oxygenated androgens were also determined (11OHA4, 11OHT, 11 KA4 and 11 KT; Figure 4). 11-oxygenated androgen analytes were altered in patients with endometriosis characterised by decreased 11OHA4 (p<0.05) and 11 KA4 (P0.0001), and increased 11OHT, (p<0.0001), and 11 KT (p<0.0001) compared to healthy controls.Steroid precursors are not elevated in endometriosis but there is modest increase in glucocorticoidsThe concentrations of steroid precursors and glucocorticoids were quantified using LC-MS / MS in serum samples from healthy controls (n=61) and patients with suspected endometriosis (n=41). The concentrations of glucocorticoids B, E, F and steroid precursors 17OHP4, S and DOC were determined (Figure 5). There was a modest increase in cortisone (E) and cortisol (F) in endometriosis patients but corticosteroid (B) did not differ between groups. Similarly, concentrations of steroid precursors 17OHP4, S and DOC did not differ between endometriosis patients and healthy controls.Classic and 11 -oxygenated androgens are highly elevated in Stage l / ll EndometriosisTo further assess the role of altered steroid metabolome in patients with endometriosis, patients were stratified by disease stage according to the rASRM criteria. The concentrations of ‘classic’ androgens; DHEA, A4, T and DHT were determined as before (Figure 6). Concentrations of DHEA (p<0.0001), A4 (p<0.0001) and T (p<0.0001) were greatest in patients with stage I endometriosis and concentrations of these in androgens in patients with Stage I and Stage II endometriosis were significantly higher than healthy controls.The concentrations of 11 -oxygenated androgens (11OHA4, 11OHT, 11 KA4 and 11 KT) were determined for HC, and stage I to stage IV endometriosis patients (Figure 7). Concentrations of 11OHT (p<0.001) and 11 KT (p<0.0001) were highest in patients with stage I endometriosis and significantly higher than healthy controls. Concentrations of 11 KT were significantly elevated in stage I, II, III and IV endometriosis patients compared to HC. Concentrations of 11OHA4 did not differ between groups while 11 KA4 was significantly reduced in stage l-IV endometriosis patients compared to HC.Single analyte ROC curve for HC vs EndometriosisROC analysis of key androgen analytes in serum from healthy control women or patients diagnosed with endometriosis was performed (Figure 8). DHEA and A4 showed good sensitivity and specificity with an AUC of 0.78 (p<0.001 , Cl 0.7102 to 0.8428) and 0.76 (p<0.001 , Cl 0.6873 to 0.8244) respectively. T and 11 KT showed better sensitivity and specificity with an AUC of 0.804 (p<0.001 , Cl 0.7440 to 0.8637) and 0.8063 (p<0.001 , Cl 0.7437 to 0.8689).AKR1C3 enzyme activities are altered in endometriosisGiven the increase in androgen abundance detected in patients with endometriosis, it was next investigated whether this could be the result of changes in enzyme activity. Aldo-keto reductase family 1 member C3 (AKR1C3) is multifunctional enzyme that plays a pivotal role in all pathways to androgen production including T, DHT, 110HT and 11 KT all of which were found to be elevated in endometriosis patients in the current study. AKR1C3 enzyme activity was calculated based on measured serum concentrations in healthy control women (HC) and patients diagnosed with endometriosis (Dx; Figure 9). Ratios were calculated as substrate to product (product / substrate) based on AKR1C3-mediated androgen actions. The ratio of T / A4 did not differ between groups, but the ratios of 11OHT / 11OHA4 and 11 KT / 11 KA4 were significantly higher in Dx groups (p<0.0001), consistent with greater production of 11OHT and 11 KT in endometriosis patients.Concentrations of 11 KA4 and 11 KT can be used to discriminate between healthy controls and endometriosis patientsNotably, calculated enzyme ratios could be used to discriminate between patient groups (Figure 10). We performed ROC analysis of serum ratios from healthy control women or patients diagnosed with endometriosis. 11 KT / 11 KA4 showed high sensitivity and specificity with an AUC of 0.9695 (p<0.001 , Cl 0.9484 to 0.9905). Likewise, 11OHT / 11OHA4 showed high sensitivity and specificity with an AUC of 0.8224 (p<0.001 , Cl 0.7633 to 0.8815). These data suggest that in addition to absolute quantification of analytes the enzyme ratios have potential to support diagnostic classification.CYP11 B1 and HSD11 B2 enzyme activities are altered in endometriosisCYP11 B1 and HSD11 B2 enzymes mediate interconversion and activation of 11- oxygenated androgens which were found to be altered in endometriosis patients in the current study. CYP11 B1 and HSD11 B2 enzyme activities were calculated based on measured serum concentrations in healthy control women (HC) and patients diagnosed with endometriosis (Dx; Figure 11 and Figure 12). Ratios were calculated as substrate to product (product / substrate) based on known / predicted enzyme activities. The ratios of 11OHA4 / A4 and 11OHT / T were significantly less in Dx compared to HC (CYP11 B1 ; Figure 11). ROC curve analysis of suggested 11OHA4 / A4 ratios may be informative in distinguishing between HC and Dx.The ratios of 11 KA4 / 11OHA4 and 11 KT / 11OHT were significantly greater in Dx compared to HC (HSD11 B2; Figure 12).androgen metabolites are altered in endometriosisMeasuring hormone abundance in serum provides only part of the steroid metabolome, whereas the urine steroid metabolome can provide an indication of the total steroid metabolism in the body. To investigate if alterations in serum hormone concentrations could also be detected in urine, the present inventors assessed urinary steroids using GC / MS in similar cohorts of HC and Dx patients.Steroid metabolites related to androgen metabolism; An (androsterone), Etio (etiocholanolone), H BOHAndro, DHEA, 16aOHDHEA were quantified in urine (Figure 13). Concentrations of classic androgen metabolites; An (androsterone) and Etio (etiocholanolone) as well as 11 -oxygenated androgen metabolites, H BOHAndro, were significantly higher in Dx patients compared to HC whereas concentrations of DHEA or 16aDHEA did not differ between groups (Figure 13).Given the altered abundance of androgen metabolites in urine the present inventors assessed if urinary androgen metabolite concentrations could be used to discriminate between HC and Dx groups (Figure 14). Analysis of the main urinary metabolite of 11 -oxygenated androgens, HbOHAndro, produced good sensitivity and specificity with an AUC of 0.8711 (p<0.001 , Cl 0.8039 to 0.9382; Figure 14).Urinary steroid metabolites related to steroid precursor metabolism; PD: pregnanediol, PT: pregnanetriol, PTONE: pregnanetriolone, 17HP: 17- hydroxypregnanolone, 3a5a-17-OH-pregnanolone (3a5a17HP) and THS: tetrahydro-11- deoxycortisol were measured by GC / MS (Figure 15). All analyte concentrations were significantly greater in Dx compared to HC.Given the altered abundance of steroid precursor metabolites in urine the present inventors then assessed if these could be used to discriminate between HC and Dx groups (Figure 16). Analysis of the precursor metabolite 17HP, produced good sensitivity and specificity with an AUC of 0.9507 (p<0.001 , Cl 0.9073 to 0.9940).ConclusionThe present inventors have discovered a disease-specific hormone signature that identifies endometriosis with high specificity and sensitivity. This is characterised by increased concentrations of classic and 11-oxygenated androgens in serum as well as elevated androgen metabolites in urine. In particular, measuring the steroid metabolome and particularly serum concentrations of 11-oxygenated androgens; 11OHA4, 11 KA4, 11OHT and 11 KT, their ratios; 11 KT / 11 KA4 and 11OHT / 11OHA4, and concentrations ofurinary metabolites; An, Etio, H BOHAndro and 17HP, have been particularly identified as having utility as diagnostic biomarkers in endometriosis.Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated herein in their entirety by reference.

Claims

CLAIMS:

1. A method of diagnosing and / or prognosing endometriosis, the method comprising: probing a test sample for the presence of one or more steroid(s) to determine a level of the one or more steroid(s) in the test sample; wherein the one or more steroid(s) is selected from 11 -ketotestosterone (11 KT), 11 -ketoandrostenedione (11 KA4), 11 -hydroxyandrostenedione (11OHA4), 11- hydroxytestosterone (11OHT), 11 -hydroxydihydrotestosterone (11OHDHT), 11- ketodihydrotestosterone (11 KDHT), 11 -hydroxyandrosterone (HOHAndro), 17- hydroxypregnanolone (17HP), and combinations thereof; wherein the level of the one or more steroid(s) in the test sample is indicative of the likelihood and / or severity of endometriosis.

2. The method according to claim 1 , wherein the one or more steroids is selected from11 -ketotestosterone (11 KT), 11 -ketoandrostenedione (11 KA4), 11- hydroxyandrostenedione (11OHA4), 11 -hydroxytestosterone (11OHT), and combinations thereof.

3. The method according to claim 2, wherein the method comprises additionally probing the test sample for the presence of one or more additional steroid(s) selected from:17-hydroxypregnenolone (17OHP5), 17-hydroxyprogesterone (17OHP4), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), 11- deoxycortisol (S), 11 -deoxycorticosterone (DOC), androstanedione (5-adione), androsterone (An), etiocholanolone (Et), 11 -hydroxyandrosterone (HOHAndro), 5- pregnenediol (5-PD), 5-pregnenetriol (5-PT), pregnanediol (PD), pregnanetriol (PT), pregnanetriolone (PTONE), 17-OH-pregnanolone (17HP), 3a5a-17-OH-pregnanolone (3a5a17HP), tetrahydro-11 -deoxycortisol (THS), and combinations thereof.

4. The method according to any one of the preceding claims, wherein the method comprises probing a test sample for the presence of 11 -ketotestosterone (11 KT) to determine a level of 11 KT in the test sample.

5. The method according to claim 4, wherein the method comprises probing a test sample for the presence of 11 -ketotestosterone (11 KT) to determine a level of 11-ketotestosterone (11 KT) in the test sample, and additionally probing the test sample for the presence of one or more additional steroid(s) selected from:17-hydroxypregnenolone (17OHP5), 11 -hydroxyandrostenedione (11OHA4), 11- ketoandrostenedione (11 KA4), 17-hydroxyprogesterone (17OHP4), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), 11- hydroxytestosterone (11OHT), 11 -hydroxydihydrotestosterone (11OHDHT), 11- ketodihydrotestosterone (11 KDHT), 11 -deoxycortisol (S), 11 -deoxycorticosterone (DOC), androstanedione (5-adione), androsterone (An), etiocholanolone (Et), 11- hydroxyandrosterone (HOHAndro), 5-pregnenediol (5-PD), 5-pregnenetriol (5-PT), pregnanediol (PD), pregnanetriol (PT), pregnanetriolone (PTONE), 17-OH- pregnanolone (VHP), 3a5a-17-OH-pregnanolone (3a5a17HP), tetrahydro-11- deoxycortisol (THS), and combinations thereof.

6. A method according to any one of the preceding claims, wherein the method comprises probing a test sample for the presence of 11 -ketotestosterone (11 KT) to determine a level of 11 -ketotestosterone (11 KT) in the test sample, and additionally probing the test sample for the presence of one or more additional steroid(s) selected from:11 -ketoandrostenedione (11 KA4), 11 -hydroxyandrostenedione (11OHA4), 11- hydroxytestosterone (11OHT), and combinations thereof.

7. A method according to any one of claims 1 to 6, wherein the method comprises probing the test sample for the presence of at least the following steroids:(i) 11 KT and 11 KA4;(ii) 11OHT and 11OHA4;(iii) 11OHA4 and A4;(iv) HOHT and T;(v) 11 KA4 and 11OHA4; or(vi) 11 KT and 11OHT.

8. A method according to any one of the preceding claims, wherein the method further comprises comparing the level of the one or more steroid(s) detected for the test sample with a reference or baseline level of the same or corresponding one or more steroid(s) in a healthy subject.

9. A method according to claim 8, wherein when the level of the one or more steroid(s) in the test sample is higher than the reference or baseline level, the test sample is determined to have been derived from a subject suffering from endometriosis, optionally wherein when the level of 11 KT and / or 11OHT is higher than the reference or baseline level, the test sample is determined to have been derived from a subject suffering from endometriosis.

10. The method according to claim 8, wherein when the level of the one or more steroid(s) in the test sample is lower than the reference or baseline level, the test sample is determined to have been derived from a subject suffering from endometriosis; optionally wherein when the level of 11OHA4 and / or 11 KA4 is lower than the reference or baseline level, the test sample is determined to have been derived from a subject suffering from endometriosis.

11. A method according to any one of the preceding claims, wherein the method comprises detecting at least two steroids in a test sample, and wherein the method further comprises:(i) probing the test sample to determine the relative levels of a steroid pair selected from the following:(a) 11 KT and 11 KA4;(b) 11OHT and 11OHA4;(c) androstenedione (A4) and 11OHA4;(d) 11OHT and T;(e) 11 KA4 and 11OHA4; and(f) 11 KT and 11OHT.

12. The method according to claim 11 , wherein the method further comprises comparing the relative levels of the steroid pair in the test sample with a baseline or reference relative level of the same or corresponding steroid pair.

13. The method according to claim 12, wherein when the relative levels of the steroid pair in the test sample is different or altered to the baseline or reference relative level, the test sample is determined to have been derived from a subject suffering from endometriosis.

14. A method according to any one of claims 11 to 13, further comprising determining a ratio of:(a) 11 KT to 11 KA4;(b) 11OHT to 11OHA4;(c) 11OHA4 to androstenedione (A4);(d) 11OHT to T;(e) 11 KA4 to 11OHA4; and(f) 11 KT to 11OHT; in the test sample.

15. A method according to claim 14, further comprising: comparing the ratio of 11 KT to 11 KA4 with a baseline or reference ratio of 11 KT to 11 KA4, optionally wherein when the ratio of 11 KT to 11 KA4 is increased in the test sample relative to the baseline or reference ratio, the sample is determined to have been derived from a subject suffering from endometriosis.

16. A method according to claim 14, further comprising: comparing the ratio of 11OHT to 11OHA4 in the test sample with a baseline or reference ratio of 11OHT to 11OHA4, optionally wherein when the ratio of 11OHT to 11OHA4 is increased in the test sample relative to the baseline or reference ratio, the test sample is determined to have been derived from a subject suffering from endometriosis.

17. A method according to claim 14, further comprising:(i) comparing the ratio of 11OHA4 to androstenedione in the test sample with a baseline or reference ratio of 11OHA4 to androstenedione, optionally wherein when the ratio of 11OHA4 to androstenedione is decreased in the test sample relative to the baseline or reference ratio, the test sample is determined to have been derived from a subject suffering from endometriosis;(ii) comparing the ratio of 11OHT to testosterone in the test sample with a baseline or reference ratio of 11OHT to testosterone, optionally wherein when the ratio of 11OHT to testosterone is decreased in the test sample relative to the baseline or reference ratio, the test sample is determined to have been derived from a subject suffering from endometriosis;(iii) comparing the ratio of 11 KA4 to 11OHA4 in the test sample with a baseline or reference ratio of 11 KA4 to 11OHA4, optionally wherein when the ratio of 11 KA4 to 11OHA4 is increased in the test sample relative to the baseline or reference ratio, the test sample is determined to have been derived from a subject suffering from endometriosis; or(iv) comparing the ratio of 11 KT to 11OHT in the test sample with a baseline or reference ratio of 11 KT to 110HT, optionally wherein when the ratio of 11 KT to 110HT is increased in the test sample relative to the baseline or reference ratio, the test sample is determined to have been derived from a subject suffering from endometriosis.

18. A method according to any one of the preceding claims, wherein the test sample is a biological fluid selected from blood (e.g. serum or plasma), urine, saliva and peritoneal fluid.

19. A method according to claim 1 , wherein the test sample is serum.

20. A method of diagnosing and / or prognosing endometriosis comprising detecting one or more steroid(s) in a urine sample, the method comprising: probing the urine sample for the presence of the one or more steroid(s) to determine a level of the one or more steroid(s) in the urine sample; wherein the one or more steroid(s) is selected from androsterone (An), etiocholanolone (Et), 11 -hydroxyandrosterone (HOHAndro), 17-hydroxypregnanolone (17HP), and combinations thereof; wherein the level of the one or more steroid(s) in the test sample is indicative of the likelihood or severity of endometriosis; optionally wherein the method comprises probing the urine sample for the presence of at least 11 -hydroxyandrosterone (HOHAndro).21 . A method according to any one of the preceding claims, wherein the level of the one or more steroid(s) in the test sample is determined by an analytical technique, optionally by liquid chromatography-mass spectroscopy (LC-MS) or gas chromatography- mass spectroscopy (GC-MS).

22. A method according to any one of claims 1 to 20, wherein the level of the one or more steroid(s) in the test sample is determined using antibodies with affinity and / or specificity for the relevant steroids.

23. A method of treating and / or preventing endometriosis in a subject in need thereof, comprising the steps of:(i) diagnosing that a subject is suffering from endometriosis in accordance with any one of the methods defined in claims 1 to 22; and(ii) administering an endometriosis therapy to the subject.

24. A kit for detecting levels of one or more steroids, wherein the kit comprises a probe for detecting levels of one or more (or all of) steroids selected from:(i) 11 -ketotestosterone (11 KT);(ii) 11 -ketoandrostenedione (11KA4);(iii) 11 -hydroxyandrostenedione (11OHA4);(iv) 11 -hydroxytestosterone (11OHT);(v) 11 -hydroxydihydrotestosterone;(vi) 11 -ketodihydrotestosterone;(vii) 11 -hydroxyandrosterone (HOHAndro); and(viii) 17-OH-pregnanolone (17HP).

25. The kit of claim 24, wherein the kit comprises a probe for additionally detecting levels of one or more (or all of) additional steroids selected from:(ix) androsterone (An);(x) etiocholanolone (Et);(xi) 17OH-pregnenolone (17OHP5);(xii) 17-hydroxyprogesterone (17OHP4);(xiii) dehydroepiandrosterone (or androstenolone (DHEA));(xiv) androstenedione (A4);(xv) testosterone (T);(xvi) 11 -deoxycortisol (S);(xvii) 11 -deoxycorticosterone (DOC);(xviii) androstanedione (5-adione);(xix) 5-pregnenediol (5-PD);(xx) 5-pregnenetriol (5-PT);(xxi) pregnanediol (PD);(xxii) pregnanetriol (PT);(xxiii) pregnanetriolone (PTONE);(xxiv) 3a5a-17-OH-pregnanolone (3a5a17HP); and(xxv) tetrahydro-11 -deoxycortisol (THS).

26. The kit of claim 24, wherein the kit comprises one or more (or all) antibodies selected from the following:(i) an anti- 11 -ketotestosterone (11 KT) antibody;(ii) an anti-11 -ketoandrostenedione (11 KA4) antibody;(iii) an anti-11 -hydroxyandrostenedione (11OHA4) antibody;(iv) an anti-11 -hydroxytestosterone (11OHT) antibody;(v) an anti-11 -hydroxydihydrotestosterone antibody;(vi) an anti-11 -ketodihydrotestosterone antibody;(vii) an anti- 11 -hydroxyandrosterone (HOHAndro) antibody; and(viii) an anti-17-OH-pregnanolone (17HP) antibody; and optionally wherein the kit comprises additional antibodies selected from one or more (or all) of the following:(ix) an anti-androsterone (An) antibody;(x) an anti-etiocholanolone (Et) antibody;(xi) an anti-17OH-pregnenolone (17OHP5) antibody;(xii) an anti-17-hydroxyprogesterone (17OHP4) antibody;(xiii) an anti-dehydroepiandrosterone (or androstenolone (DHEA)) antibody;(xiv) an anti-androstenedione antibody;(xv) an anti-testosterone antibody;(xvi) an anti- 11 -deoxycortisol (S) antibody;(xvii) an anti- 11 -deoxycorticosterone (DOC) antibody;(xviii) an anti-androstanedione (5-adione) antibody;(xix) an anti-5-pregnenediol (5-PD) antibody;(xx) an anti-5-pregnenetriol (5-PT) antibody;(xxi) an anti-pregnanediol (PD) antibody;(xxii) an anti-pregnanetriol (PT) antibody;(xxiii) an anti-pregnanetriolone (PTONE) antibody;(xxiv) an anti-3a5a-17-OH-pregnanolone (3a5a17HP) antibody; and(xxv) an anti-tetrahydro-11 -deoxycortisol (THS) antibody.

27. The kit of any one of claims 24 to 26, further comprising buffers, solutions, tools, receptacles and / or instructions for use.

28. A method for detecting a level of enzyme activity in a test sample, the method comprising:(i) probing a test sample to determine a level of enzyme activity of an enzyme, wherein the enzyme is selected from: Aldo-keto reductase family 1 member C3 (AKR1C3), CYP11B1, and HSD11 B2.

29. A method according to claim 28, further comprising comparing the level of enzyme activity in the test sample with a baseline or reference level.

30. A method according to claim 29, wherein when an altered level of enzyme activity is observed, the test sample is determined to have been derived from a subject suffering from endometriosis.

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