Ratio between LTA4H and METRNL in assessment of polycystic ovarian syndrome

Abstract

The present invention relates to a method for diagnosing polycystic ovarian syndrome (PCOS) in a subject, said method comprising the steps of: a) determining the amount or concentration of total LTA4H in a sample from said subject, b) determining the amount or concentration of METRNL in a sample from said subject, c) calculating the fraction of said amounts or concentrations determined in steps a) and b), d) comparing the calculated score to a reference score, and e) diagnosing the PCOS of the subject.

Description

[0001] The present invention relates to a method for diagnosing PCOS in a subject, the method comprising the steps of: a) determining the amount or concentration of LTA4H in a sample from the subject, b) determining the amount or concentration of METRNL in a sample from the subject, c) calculating the fraction of the amounts or concentrations determined in steps a) and b), d) comparing the calculated fraction with a reference fraction, and e) diagnosing PCOS in the subject. In addition, it relates to a method for selecting a patient for treatment of PCOS, the method comprising: a) determining the amount or concentration of LTA4H in a sample from the subject, b) determining the amount or concentration of METRNL in a sample from the subject, c) calculating the fraction of the amounts or concentrations determined in steps a) and b), d) comparing the calculated fraction with a reference fraction, and e) selecting a patient for treatment of PCOS. Furthermore, it relates to a method for monitoring the progression of PCOS in a subject suffering from PCOS or for monitoring the response to treatment in a subject suffering from PCOS, the method comprising: a) determining the amount or concentration of LTA4H in a sample from the subject, b) determining the amount or concentration of METRNL in a sample from the subject, c) calculating a fraction of the amounts or concentrations determined in steps a) and b), d) comparing the calculated fraction to a reference fraction, and e) monitoring the progression of a subject suffering from PCOS or being treated for PCOS. Finally, the invention relates to a computer-implemented method for diagnosing PCOS in a subject. Background Art

[0002] Polycystic ovary syndrome (PCOS) is a heterogeneous gynecological condition defined by a combination of signs of androgen excess and ovarian dysfunction. Patients with PCOS may have a range of clinical manifestations that can be reproductive and / or metabolic. Reproductive manifestations include irregular menstrual cycles, infertility, pregnancy complications, and hirsutism, while metabolic manifestations include obesity, insulin resistance, metabolic syndrome, prediabetes, type 2 diabetes, and cardiovascular factors. These clinical manifestations are also associated with psychological disorders such as anxiety and depression (Escobar-Morreale, HF 2018; International evidence-based guideline for the assessment and management of polycysticovary syndrome 2018).

[0003] These symptoms are not specific to PCOS, and patients are often diagnosed only after a longer evaluation for infertility. For the final diagnosis of PCOS, other conditions or diseases (such as pregnancy, atypical adrenal hyperplasia (NCAH), congenital adrenal hyperplasia, androgen-secreting tumors, Cushing's syndrome, thyroid disease, or hyperprolactinemia) should be excluded (Escobar-Morreale HF. Polycystic ovary syndrome: definition, aetiology, diagnosis and treatment. Nat Rev Endocrinol. 2018; 14 (5): 270-284; Teede HJ, Misso ML, Costello MF et al. International PCOS Network. Recommendations from the international evidence-based guideline for the assessment and management of polycystic ovary syndrome. Hum Reprod. 2018; 33 (9): 1602-1618). Diagnostic tests that can be used to exclude other diseases, such as:

[0004] 17α-Hydroxyprogesterone (17-OHP) to exclude NCAH (Nordenstrom and Falhammar 2018)

[0006] Prolactin to exclude hyperprolactinemia

[0007] Cortisol to exclude patients with Cushing's syndrome

[0008] Thyroid stimulating hormone (TSH) to rule out thyroid disease

[0009] PCOS can be caused by a combination of genetic, epigenetic, and environmental factors, such as inherited traits.

[0010] Although PCOS is one of the most common endocrine disorders in women, affecting 10% of women of reproductive age, up to 70% of affected women remain undiagnosed (March WA, Moore VM, Willson KJ et al. The prevalence of polycystic ovary syndrome in a community sample assessed under contrasting diagnostic criteria. Hum Reprod. 2010; 25(2):544-51).

[0011] The most widely used criteria for the diagnosis of PCOS are the so-called Rotterdam criteria. PCOS is indicated if at least 2 of the following criteria apply: (i) irregular cycles (oligomenorrhea) and ovulatory dysfunction (oligoovulation, OA), (ii) clinical and / or biochemical hyperandrogenism (HA) and (iii) polycystic ovarian morphology (PCOM) (PCOS Consensus Workshop Group, Fertil Steril 2004; 81: 19-25). The first criterion is defined as the following menstrual cycles: cycle length less than 21 days or more than 35 days or less than 8 cycles per year. Clinical and / or biochemical features of hyperandrogenism, where clinical hyperandrogenism is defined as hirsutism (excessive and male-pattern hair growth) and / or acne, and biochemical hyperandrogenism is defined as higher levels of free androgens compared to healthy controls. Clinical hyperandrogenism is also defined as a modified Ferriman-Gallwey score>8. Biochemical hyperandrogenism can be assessed using free testosterone or the free androgen index (FAI), which can be calculated by measuring total testosterone and sex hormone binding globulin (SHBG). PCOM is usually determined according to the "PCOS 2018 International Evidence-Based Clinical Guidelines" using an intravaginal ultrasound transducer with a bandwidth of up to 8 MHz. The threshold for PCOM is considered to be in either ovary: ensuring the absence of a corpus luteum, cysts, or dominant follicles, and the number of follicles per ovary is >20 and / or the ovarian volume is ≥10 ml. If using older ultrasound technology, the threshold for PCOM can be an ovarian volume ≥10 ml or a follicle count >12 in either ovary.

[0012] Another method for detecting PCOM is to measure anti-Mullerian hormone (AMH) in the subject. AMH is a glycoprotein hormone whose expression is essential for sex differentiation at a specific time during fetal development. In addition, AMH produced by granulosa cells of growing follicles is generally correlated with the number of antral follicles in the ovaries. Therefore, serum AMH levels can be an alternative biomarker to the number / number of antral follicles (AFC) determined by transvaginal ultrasound. Some studies have suggested serum AMH as a biochemical marker for PCOM. In some studies, AMH thresholds for PCOM in women with PCOS have been proposed (Nicholas et al. 2014; Pigny et al. 2016; Dietz de Loos et al., Fertil Steril, 2021). However, according to the 2018 International Evidence-Based Clinical Guidelines for the Assessment and Management of Polycystic Ovary Syndrome, serum AMH levels should not be used as an alternative to detecting PCOM or diagnosing PCOS.

[0013] A further method used for the detection of PCOS is the 3-item PCOS criteria system (Indran et al., 2018). In this system, a diagnosis of PCOS is suggested if two out of three items are present: (i) oligomenorrhea (defined as mean menstrual cycle length >35 days); (ii) AMH above the threshold; and (iii) hyperandrogenemia defined as testosterone above the threshold and / or the presence of hirsutism (mFG score ≥5). Alternatively, AMH is suggested to be combined with hyperandrogenemia and oligomenorrhea (Sahmay et al., 2014) or with SHBG (Calzada et al., 2019).

[0014] Another method for detecting PCOS is to measure other hormones, such as, for example, luteinizing hormone (LH) and follicle stimulating hormone (FSH). However, the diagnostic utility of the LH:FSH ratio for PCOS appears to be low, as only a small fraction of women with PCOS have significantly elevated LH:FSH ratios (Cho et al., 2005). In fact, a wide range of LH:FSH ratios have been found in women diagnosed with PCOS (Malini and George 2018).

[0015] The necessity to consider multiple diagnostic test results and clinical examination results requires specific expertise, which makes it difficult for less specialized physicians (such as general practitioners) to diagnose PCOS in clinical routine. For example, the determination of PCOM by transvaginal ultrasound requires adequate ultrasound equipment and the physician's subjective analysis of the ultrasound images. In addition, the results may also depend on the specific ultrasound equipment used to assess PCOM. Therefore, the diagnosis of PCOS based on the Rotterdam criteria always includes at least one subjective, equipment- and operator-dependent, and error-prone measurement.

[0016] In order to assess biochemical hyperandrogenism, the androgen measured must have a clear normal range. Testosterone is the most abundant androgen measured, whether in total, combined or free form. The practice of measuring free testosterone has its limitations. Using radioimmunoassay to directly measure free testosterone is very inaccurate and cannot reflect the true value. The assay has high intra-assay and inter-assay variability. Alternatively, higher accuracy can be obtained by using extraction and chromatography, or gas phase (GC-MS) or liquid phase (LC-MS) chromatography-mass spectrometry to measure total testosterone concentration, particularly for clinical research. The diagnostic performance of measuring serum testosterone can be enhanced by measuring SHBG at the same time, so that the calculation of free T concentration from total testosterone and SHBG levels only requires solving a second-order equation (Azziz R, Carmina E, Dewailly D et al. Task Force on the Phenotype of the Polycystic Ovary Syndrome of The Androgen Excess and PCOSociety. The Androgen Excess and PCOS Society criteria for the polycysticovary syndrome: the complete task force report. Fertil Steril. 2009; 91 (2): 456-88). The definition of HA may vary by race. An mFG score of >8 used to diagnose hirsutism in women with PCOS may not be appropriate for diagnosis in all races. The prevalence of hirsutism in East Asian women is lower than that in Caucasians, and a score of >5 has been suggested to define hirsutism in Chinese women. There are also indications that there are differences in blood androgen levels between different ethnic groups, with the Japanese population having a lower incidence of elevated androgens, and testosterone is recommended only as a supplementary factor for the diagnosis of PCOS in this population (Huang Z, Yong EL. Ethnic differences: Is there an Asian phenotype for polycystic ovarian syndrome? Best Pract Res Clin Obstet Gynaecol. 2016; 37: 46-55; Kubota T. Update in polycystic ovary syndrome: new criteria of diagnosis and treatment in Japan. Reprod Med Biol. 2013; 12(3): 71-77).

[0017] Patients suffering from PCOS can be divided into four different phenotypes, called A, B, C or D (Neven ACH, Laven J, Teede HJ, Boyle JA. A Summary on Polycystic Ovary Syndrome: Diagnostic Criteria, Prevalence, Clinical Manifestations, and Management According to the Latest International Guidelines. Semin Reprod Med. 2018 January; 36 (1): 5-12). Phenotype A is characterized by patients showing hyperandrogenemia, ovulatory dysfunction and / or irregular cycles and polycystic ovarian morphology. Phenotype B is characterized by hyperandrogenemia, ovulatory dysfunction and / or irregular cycles. Phenotype C is characterized by hyperandrogenemia and polycystic ovarian morphology. Phenotype D is characterized by ovulatory dysfunction and / or irregular cycles and polycystic ovarian morphology.

[0018] Currently, no specific PCOS medications are available. Treatment is symptom-directed and tailored to individual needs. Treatment targets hyperandrogenism, irregular cycles and / or ovulatory dysfunction, and associated metabolic disorders, such as diabetes. The 2018 International Evidence-Based Clinical Guidelines for the Assessment and Management of Polycystic Ovary Syndrome provide information to support clinical decision making and patient management.

[0019] Inconsistent diagnostic criteria, variable provider knowledge, and lack of consensus present unique challenges to the diagnosis and care of women with PCOS. These factors result in inaccurate diagnosis, both underdiagnosis and overdiagnosis. This adverse diagnostic experience deteriorates for affected women and limits opportunities for timely intervention to minimize associated comorbidities, particularly during the transition from pediatric to adult care (Witchel SF, Teede HJ, AS. Curtailing PCOS. Pediatr Res. 2020; 87(2): 353-361). In addition, timely diagnosis is crucial to prevent further metabolic complications (such as, for example, type 2 diabetes) in affected women.

[0020] In the largest study of the experience with PCOS diagnosis, many women reported delayed diagnosis and inadequate information. One-third or more of women reported seeing >2 years (33.6%) and >3 healthcare professionals (47.1%) before diagnosis. Few were satisfied with their diagnostic experience (35.2%) or the information they received (15.6%). These gaps in early diagnosis, education, and support are clear opportunities to improve the patient experience (Gibson-Helm M, Teede H, Dunaif A, Dokras A. Delayed Diagnosis and a Lack of Information Associated With Dissatisfaction in Women With Polycystic Ovary Syndrome. J Clin Endocrinol Metab. 2017;102(2):604-612).

[0021] An area of ​​particular interest in the diagnosis of PCOS is in younger women, namely adolescents and young women younger than 25 years of age, when features of normal pubertal development overlap with adult diagnostic criteria. This makes the diagnosis controversial and challenging. Many of the findings used to diagnose PCOS may evolve over time and change during the first few years after menarche. Normal physiologic changes of puberty, such as irregular menstrual cycles, acne, and PCOM, overlap with adult PCOS diagnostic criteria. In adolescent and young adult women, PCOS is diagnosed when criteria for both OA and HA are met. Pelvic ultrasound is not recommended in adolescents < 8 years from menarche because of the high incidence of polyfollicular ovaries at this stage of life ( AS, Witchel SF, Hoeger KM, Oberfield SE, Vogiatzi MG, Misso M, Garad R, Dabadghao P, Teede H. Adolescent polycystic ovary syndrome according to the international evidence-based guideline. BMC Med. 2020; 18(1): 72). Assessing adolescents for menstrual cycle irregularity can be difficult. Menstrual cycles are often irregular during adolescence. During the first few years after menarche, immaturity of the hypothalamic-pituitary-ovarian axis often results in anovulation, and cycles may be somewhat long. However, 90% of cycles will be in the range of 21 to 45 days, although short cycles of less than 20 days and long cycles of more than 45 days may occur. By the third year after menarche, 60% to 80% of menstrual cycles are 21 to 34 days in length, which is typical of adults. Young girls and their caregivers (e.g., parents or guardians) often have difficulty assessing what constitutes a normal menstrual cycle or bleeding pattern. Patients and their caregivers may not be familiar with what is normal, and patients may not inform their caregivers of irregular or missed periods. In addition, patients are often reluctant to discuss this topic with their caregivers (ACOG Committee Opinion No. 651: Menstruation in Girls and Adolescents: Using the Menstrual Cycle as a Vital Sign. Obstet Gynecol. 2015; 126(6): e143-e146). Therefore, it is critical to establish reliable biomarkers to assist in the diagnosis of PCOS or to identify patients at risk for developing PCOS, especially for this group of patients. Delays in diagnosis in adolescents and young women are often due to reluctance to diagnose adolescents who are at risk due to puberty and concerns about over- or under-diagnosis. This can lead to long-term complications such as obesity and insulin resistance, as well as anxiety or depression. The latest guidelines for the diagnosis of PCOS in adolescents and young women define oligoovulation, irregular menstrual cycles, and hyperandrogenism as criteria to improve diagnostic accuracy in this patient group ( AS, Witchel SF, Hoeger KM et al. Adolescent polycystic ovary syndrome according to the international evidence-based guideline. BMC Med. 2020; 18(1): 72). The guideline also recommends re-evaluation of the diagnosis every 3 years and lifestyle changes to minimize symptoms and comorbidities associated with PCOS, such as anxiety and depression.

[0022] To date, there are no universal biomarkers that can be used to assess whether a subject has PCOS or is at risk of developing PCOS, and / or to determine the response of a subject with PCOS to therapy, and / or to monitor the progression of PCOS in a subject, and / or to monitor the response of a subject with PCOS to therapy, either alone or in combination with another biomarker, or in conjunction with the above-mentioned symptoms or hormone levels previously described.

[0023] Therefore, there is an unmet need to develop better diagnostic assays to diagnose young women and adolescents. Summary of the invention

[0024] In a first aspect, the present invention relates to a method for diagnosing PCOS in a subject, the method comprising the steps of:

[0025] a) determining the amount or concentration of LTA4H in a sample from a subject,

[0026] b) determining the amount or concentration of METRNL in a sample from the subject,

[0027] c) calculating the fractions of the amounts or concentrations determined in steps a) and b), and

[0028] d) Comparing the calculated score with a reference score.

[0029] e) Diagnosing PCOS in the subject.

[0030] Preferably, step (e) is based on the results of comparing step (d). Therefore, step e) can be as follows:

[0031] e) diagnosing PCOS in the subject based on the result of the comparison in step d).

[0032] In a second aspect, the invention relates to a method of selecting a patient for therapy for PCOS, the method comprising:

[0033] a) determining the amount or concentration of LTA4H in a sample from a subject,

[0034] b) determining the amount or concentration of METRNL in a sample from the subject,

[0035] c) calculating the fractions of the amounts or concentrations determined in steps a) and b),

[0036] d) comparing the calculated score with a reference score, and

[0037] e) Selection of patients for PCOS therapy.

[0038] Preferably, step (e) is based on the results of comparing step (d). Therefore, step e) can be as follows:

[0039] e) selecting a patient for PCOS therapy based on the results of the comparison in step d).

[0040] In a third aspect, the present invention relates to a method for monitoring the progression of PCOS in a subject with PCOS or for monitoring the response to treatment in a subject with PCOS, the method comprising:

[0041] a) determining the amount or concentration of LTA4H in a sample from a subject,

[0042] b) determining the amount or concentration of METRNL in a sample from the subject,

[0043] c) calculating the fractions of the amounts or concentrations determined in steps a) and b),

[0044] d) comparing the calculated score with a reference score, and

[0045] e) Monitoring the progress of subjects suffering from or being treated for PCOS.

[0046] Preferably, step (e) is based on the results of comparing step (d). Therefore, step e) can be as follows:

[0047] e) monitoring the progress of the subject suffering from or being treated for PCOS based on the results of step d).

[0048] In a fourth aspect, the present invention relates to a computer-implemented method for diagnosing PCOS in a subject, the method comprising:

[0049] (a) Receiving at the processing unit

[0050] (a1) a value for the amount or concentration of LTA4H in a sample from a subject, and

[0051] (a2) a value for the amount or concentration of METRNL in a sample from a subject,

[0052] (b) processing the value received in step (a) with a processing unit, wherein the processing comprises:

[0053] (b1) calculating the fraction of said values ​​(a1) and (a2) received in step a),

[0054] (b2) comparing the calculated score with a reference score, and

[0055] (c) optionally providing a diagnosis via an output device, wherein the diagnosis is based on the results of step b).

[0056] In a fifth aspect, the present invention relates to the use of LTA4H and METRNL as biomarkers for diagnosing PCOS. In addition, the present invention relates to the use of at least one agent that specifically binds to METRNL and at least one agent that specifically binds to LTA4H for diagnosing PCOS.

[0057] In a sixth aspect, the present invention relates to a kit comprising at least one agent that specifically binds to METRNL and at least one agent that specifically binds to LTA4H. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 ROC curve analysis of the ratio between the two serum biomarkers LTA4H and METRNL in PCOS cases when all phenotypes were combined (phenotype AD). The ratio between the biomarker concentrations obtained by ELISA immunoassay was calculated.

[0059] Figure 2 . Ratio values ​​between the two serum biomarkers LTA4H and METRNL in controls and PCOS cases when all phenotypes were combined (phenotype AD). The ratios between the biomarker concentrations obtained by ELISA immunoassay were calculated.

[0060] Figure 3 ROC curve analysis of the ratio between the two serum biomarkers LTA4H and METRNL in PCOS phenotype AD when compared to controls. The ratio between the biomarker concentrations obtained by ELISA immunoassay was calculated.

[0061] Figure 4 Ratio values ​​between the two serum biomarkers LTA4H and METRNL in healthy controls and PCOS phenotype AD. Ratios between the biomarker concentrations obtained by ELISA immunoassay were calculated.

[0062] Figure 5ROC curve analysis of the ratio between the two serum biomarkers LTA4H and METRNL in young PCOS cases (age ≤ 25 years) when all phenotype AD were combined. The ratio between the biomarker concentrations obtained by ELISA immunoassay was calculated.

[0063] Figure 6 . Ratio values ​​between the two serum biomarkers LTA4H and METRNL in young controls and young PCOS cases (age ≤ 25 years) with combined phenotype AD. The ratio between the biomarker concentrations obtained by ELISA immunoassay was calculated.

[0064] Figure 7 ROC curve analysis of the ratio between the two serum biomarkers LTA4H and METRNL in young PCOS cases (age ≤ 25 years) and young healthy controls (age ≤ 25 years) separated by phenotype AD. The ratio between the biomarker concentrations obtained by ELISA immunoassay was calculated.

[0065] Figure 8 Ratio values ​​between the two serum biomarkers LTA4H and METRNL in young healthy controls (age ≤ 25 years) and PCOS phenotype AD (age ≤ 25 years). The ratio between the biomarker concentrations obtained by ELISA immunoassay was calculated.

[0066] Fig. 9 ROC curve analysis of the ratio between the two serum biomarkers LTA4H and METRNL in PCOS cases when all phenotypes were combined (phenotype AD). The ratio between the biomarker concentrations obtained by ELISA immunoassay was calculated.

[0067] Fig.10 . Ratio values ​​between the two serum biomarkers LTA4H and METRNL in controls and PCOS cases when all phenotypes were combined (phenotype AD). The ratios between the biomarker concentrations obtained by ELISA immunoassay were calculated.

[0068] Fig.11 ROC curve analysis of the ratio between the two serum biomarkers LTA4H and METRNL in PCOS phenotype AD when compared to controls. The ratio between the biomarker concentrations obtained by ELISA immunoassay was calculated.

[0069] Fig.12 Ratio values ​​between the two serum biomarkers LTA4H and METRNL in healthy controls and PCOS phenotype AD. Ratios between the biomarker concentrations obtained by ELISA immunoassay were calculated.

[0070] Fig.13 ROC curve analysis of the ratio between the two serum biomarkers LTA4H and METRNL in different PCOS age groups (15-20, 20-25, 25-45) when compared to controls. The ratio between the biomarker concentrations obtained by ELISA immunoassay was calculated.

[0071] Fig.14 . Ratio values ​​between two serum biomarkers LTA4H and METRNL in healthy controls and PCOS age groups (15-20, 20-25, 25-45). The ratio between the biomarker concentrations obtained by ELISA immunoassay was calculated.

[0072] Fig.15 ROC curve analysis of the ratio between the two serum biomarkers LTA4H and METRNL in young PCOS cases (age 15-25 years) when all phenotype AD were combined. The ratio between the biomarker concentrations obtained by ELISA immunoassay was calculated.

[0073] Fig.16 . Ratio values ​​between the two serum biomarkers LTA4H and METRNL in young healthy controls and young PCOS cases (age 15-25 years) with combined phenotype AD. The ratio between the biomarker concentrations obtained by ELISA immunoassay was calculated.

[0074] Fig.17 ROC curve analysis of the ratio between the two serum biomarkers LTA4H and METRNL in young PCOS cases (age 15-25 years) and young healthy controls (age 15-25 years) separated by phenotype AD. The ratio between the biomarker concentrations obtained by ELISA immunoassay was calculated.

[0075] Fig.18 Ratio values ​​between the two serum biomarkers LTA4H and METRNL in young healthy controls (age 15-25 years) and PCOS phenotype AD (age 15-25 years). The ratio between the biomarker concentrations obtained by ELISA immunoassay was calculated. DETAILED DESCRIPTION

[0076] The method mentioned according to the first, second or third aspect of the present invention includes a method consisting essentially of the above steps or a method including other steps. In addition, the inventive method is preferably an in vitro method, and more preferably an in vitro method. In addition, the method according to the first, second or third aspect of the present invention may include steps other than those explicitly mentioned above. For example, other steps may involve determining other markers and / or sample pretreatment or evaluating the results obtained by the method. The method can be performed manually or assisted by automation. Preferably, steps (a), (b), (c), (d) and / or (e) may be assisted in whole or in part by automation, such as by suitable robots and sensory equipment to carry out determination in steps (a) and (b) or calculation implemented by a computer in step (c) or comparison implemented by a computer in step (d). Therefore, the method of the inventive method can be implemented by a computer.

[0077] Leukotriene A4 hydrolase (LTA4H) is part of the 5-lipoxygenase (5-LO) pathway, which converts arachidonic acid (AA) into pro-inflammatory leukotrienes and anti-inflammatory lipoxins. LTA4H is widely expressed in various organs, tissues, and individual cell types ( JZ.Leukotriene A4hydrolase and the committed step inleukotriene B4 biosynthesis.Clin Rev Allergy Immunol.1999 Spring-Summer;17(1-2):111-31). According to the human protein atlas, moderate expression of LTA4H was detected in normal ovarian tissue (http: / / www.proteinatlas.org). The expression pattern of LTA4H has been studied in human ovarian tissue, revealing weak expression and moderate expression on granulosa cells and intrathecal cells in preovulatory follicles, respectively. After ovulation, the intensity of LTA4H on large luteal cells increases and is highest in the mid-luteal phase. High expression is also observed in the corpus luteum in early pregnancy (Hattori N, Fujiwara H, Maeda M et al. Human large luteal cells in the menstrual cycle and early pregnancy express leukotriene A4 hydrolase.Mol Hum Reprod.1998 August;4(8):803-10). LTA4H is a unique bifunctional zinc metalloenzyme with both epoxide hydrolase (intracellular) and aminopeptidase (extracellular) activities. As an epoxide hydrolase, LTA4H catalyzes the final and key step in the biosynthesis of leukotriene B4 (LTB4), which is an eicosanoid with effective chemoattractant and proinflammatory properties (Serhan CN. Resolution phase of inflammation: novel endogenous anti-inflammatory and proresolving lipid mediators and pathways. Annual review of immunology. 2007; 25: 101–137). Leukotrienes are a family of eicosanoids that act as effective chemical mediators in various allergic and inflammatory reactions. New data suggest that leukotrienes may play an important role in carcinogenesis.LTB4 levels are increased in several human cancers, including skin, lung, colon and prostate cancers (Dreyling KW, Hoppe U, Peskar BA et al. Leukotriene synthesis by human gastrointestinal tissues. Biochim Biophys Acta. 1986 Sep 12;878(2):184-93; Chen X, Wang S, Wu N, Yang CS. Leukotriene A4 hydrolase as a target for cancer prevention and therapy. Curr Cancer Drug Targets. 2004 May;4(3):267-83; Wang Q, He Z, Zhang J et al. Overexpression of endoplasmic reticulummolecular chaperone GRP94 and GRP78 in human lung cancer tissues and its significance. Cancer Detect Prev. 2005;29(6):544-51; Larré S, Tran N, Fan C et al. PGE2 and LTB4 tissue levels in benign and cancerous prostates. Prostaglandins Other Lipid Mediat. 2008 Dec; 87(1-4): 14-9; Oi N, Yamamoto H, Langfald A et al. LTA4H regulates cell cycle and skin carcinogenesis. Carcinogenesis. 2017 Jul 1; 38(7): 728-737). The expression of LTB4 receptor is increased in human pancreatic cancer (Hennig R, Ding XZ, Tong WG et al. 5-Lipoxygenase and leukotriene B(4) receptor are expressed in human pancreatic cancers but not in pancreatic ducts in normal tissue. Am J Pathol. 2002 Aug; 161(2): 421-8).The expression of LTB4 is also increased in HRAS-v12 transformed cells, and the receptor BLT2 is required for Ras-induced transformation in vivo (Yoo MH, Song H, Woo CH et al. Role of the BLT2, a leukotriene B4 receptor, in Ras transformation. Oncogene. 2004 Dec 9;23(57):9259-68). Inhibition of LTB4 synthesis by treatment with the LTA4H inhibitor bestatin reduced tumorigenesis in in vivo models of esophageal adenocarcinoma and colorectal cancer (Chen X, Li N, Wang S et al. Leukotriene A4 hydrolase in rat and human esophageal adenocarcinomas and inhibitory effects of bestatin. J Natl Cancer Inst. 2003 Jul 16; 95(14): 1053-61; Zhao S, Yao K, Li D et al. Inhibition of LTA4H by bestatin in human and mouse colorectal cancer. EBioMedicine. 2019 Jun; 44: 361-374). In addition, topical application of LTB4 to the skin causes not only inflammation but also massive epidermal hyperplasia (Bauer RW, van der Kerhof PC, de Grood RM. Epidermal hyperproliferation following the induction of microabscesses by leukotriene B4. Br J Dermatol 1986; 114: 409–12; Ruzika T, Burg G. Effects of chronic intracutaneous administration of arachidonic acid and its metabolites. Induction of leukocytoclastic vasculitis by leukotriene B4 and 12-hydroxyeicosatetraenoic acid and its prevention by prostaglandin E2. J Invest Dermatol 1987; 88: 120–3).The expression of 5-lipoxygenase, hypoxia marker HIF1α and macrophage marker CD68 is positively correlated in ovarian cancer tissues. In addition, hypoxic ovarian cancer cell lines show increased production of LTA4H and 5-LOX metabolites, which are responsible for enhancing the infiltration of tumor-associated macrophages (Wen Z, Liu H, Li M et al. Increased metabolites of 5-lipoxygenase from hypoxic ovarian cancer cells promote tumor-associated macrophage infiltration. Oncogene. 2015 March 5; 34 (10): 1241-52). LTA4H can also fight inflammation through its aminopeptidase activity, which inactivates the tripeptide Pro-Gly-Pro (PGP) by cleavage (Stsiapanava A, Olsson U, Wan M et al. Binding of Pro-Gly-Pro at the active site of leukotriene A4hydrolase / aminopeptidase and development of an epoxidehydrolase selective inhibitor. Proc Natl Acad Sci USA. 2014 March 18; 111(11): 4227-32). PGP is a biomarker for chronic obstructive pulmonary disease (COPD) and promotes neutrophil accumulation (Snelgrove RJ, Jackson PL, Hardison MT et al. A critical role for LTA4H in limiting chronic pulmonary neutrophilic inflammation. Science. 2010 Oct 1; 330(6000): 90-4). Serum LTA4H has recently been suggested as a potential biomarker for predicting the efficiency of allergen immunotherapy for the treatment of allergic rhinitis (Ma TT, Cao MD, Yu RL, et al. Leukotriene A4 Hydrolase Is a Candidate Predictive Biomarker for Successful Allergen Immunotherapy. Front Immunol. 2020 Nov 24;11:559746).Higher expression of LTA4H due to LTA4H gene variation is associated with increased survival after glucocorticoid therapy in patients with moderate tuberculous meningitis (Whitworth L, Coxon J, van Laarhoven A, et al. A Bayesian analysis of the association between Leukotriene A4 Hydrolase genotype and survival in tuberculous meningitis. Elife. 2021 Jan 8;10:e61722).

[0078] Meteorin-like protein (METRNL) is a hormone (28KDa secreted protein) induced in skeletal muscle and adipose tissue after exercise and cold exposure, respectively. Increased expression of METRNL in the circulation or adipose tissue causes 'browning' of white adipose tissue (WAT). Intraperitoneal injection of Metrnl-Fc protein into mice for 7 days induced significant weight loss and increased O2 consumption and glucose tolerance. Metrnl did not show a direct effect on thermogenesis of white adipocytes in vitro, suggesting that non-adipocyte cell types are involved in the induction of beige fat. Metrnl appears to stimulate several immune cell subtypes to enter adipose tissue and activate their pro-thermogenic effects. The number of macrophages and eosinophils in WAT of mice treated with METRNL increased, and the expression of genes associated with alternative macrophage activation increased (Rao RR, Long JZ, White JP et al. Meteorin-like is a hormone that regulates immune-adipose interactions to increasebeige fat thermogenesis. Cell. 2014 June 5; 157(6): 1279-1291). METRNL has been associated with innate and possible acquired immunity. High expression of METRNL has been identified in activated monocytes (M2 polarized macrophages), skin and mucosal tissues. In the skin, METRNL is expressed by resting fibroblasts and keratinocytes treated with IFNγ. Overexpression of METRNL has been described in several human skin diseases including psoriasis. METRNL is also upregulated in the synovium of human rheumatoid arthritis (Ushach I, Burkhardt AM, Martinez C et al. METEORIN-LIKE is acytokine associated with barrier tissues and alternatively activated macrophages. Clin Immunol. 2015 February; 156 (2): 119-27). Recently, Baht and colleagues described the role of METRNL in coordinating skeletal muscle repair through macrophage accumulation and phenotypic conversion. The results showed that in response to local injury, METRNL is mainly secreted by macrophages. In addition, METRNL promotes anti-inflammatory function by inducing insulin-like growth factor 1 (IGF-1) through a STAT3-dependent autocrine / paracrine mechanism, thereby activating muscle progenitor cells to help myogenesis.Finally, METRNL has been shown to be a key regulator of muscle regeneration, acting directly on immune cells to promote an anti-inflammatory / pro-regenerative environment and myogenesis (Baht GS, Bareja A, Lee DE et al. Meteorin-like facilitates skeletal muscle repair through a Stat3 / IGF-1 mechanism. Nat Metab. 2020 March; 2(3): 278-289. Erratumin: Nat Metab. 2020 August; 2(8): 794). Interestingly, METRNL has been suggested to act as a neurotrophic factor with therapeutic potential in neural development. METRNL is indeed able to cross the blood-brain barrier (BBB), and increases in blood-brain barrier dysfunction lead to increased cerebrospinal fluid METRNL concentrations (Berghoff M,. A, Rajendran R et al. Evidence of a Muscle–Brain Axis by Quantification of the NeurotrophicMyokine METRNL (Meteorin-Like Protein) in Human Cerebrospinal Fluid and Serum. Journal of Clinical Medicine. 2021; 10(15): 3271).

[0079] The association of serum METRNL levels with type 2 diabetes mellitus (T2DM) has been investigated with conflicting results (Lee JH, Kang YE, Kim JM, et al. Serum Meteorin-like protein levels decreased in patients newly diagnosed with type 2 diabetes. Diabetes Res Clin Pract. 2018 Jan;135:7-10; Chung HS, Hwang SY, Choi JH, et al. Implications of circulating Meteorin-like (Metrnl) levels in human subjects with type 2 diabetes. Diabetes Res Clin Pract. 2018 Feb;136:100-107; Wang K, Li F, et al. Serum Levels of Meteorin-Like (Metrnl) Are Increased in Patients with Newly Diagnosed Type 2 Diabetes Mellitus and Are Associated with Insulin Resistance. Med Sci Monit. 2019 Mar 31; 25:2337-2343; El-Ashmawy HM, Selim FO, Hosny TAM, Almassry HN. Association of low serum Meteorin like (Metrnl) concentrations with worsening of glucosetolerance, impaired endothelial function and atherosclerosis. Diabetes Res ClinPract. 2019 April; 150:57-63; Wang C, Pan Y, Song J, et al. Serum Metrnl Level is Correlated with Insulin Resistance, But Not withβ-Cell Function in Type2Diabetics. Med Sci Monit.2019 Nov 25;25:8968-8974; Ferns GA, Fekri K, Shahini Shams Abadi M et al. A meta-analysis of the relationship between serums metrnl-like protein / subfatin and risk of type 2 diabetes mellitus and coronary artery disease. Arch Physiol Biochem. 2021 May 5:1-7; Lappas M. Maternal obesity and gestational diabetes decrease Metrnl concentrations in cord plasma. J Matern Fetal Neonatal Med. 2021 Sep;34(18):2991-2995). Patients with T2DM and coronary artery disease (CAD) showed lower serum Metrnl levels compared with controls. In addition, it was shown that METRNL was negatively correlated with IL-6 and TNF-α in CAD patients and BMI, insulin resistance, IL-6 and TNF-α in T2DM patients (Dadmanesh M, Aghajani H, Fadaei R, Ghorban K. Lower serum levels of Meteorin-like / Subfatin in patients with coronary artery disease and type 2diabetes mellitus arenegatively associated with insulin resistance and inflammatory cytokines. PLoS One. 2018 September 13; 13 (9): e0204180). In addition, a case-control study of CAD patients showed that serum METRNL is now associated with the presence and severity of CAD (Liu ZX, Ji HH, Yao MP et al. Serum Metrnl is associated with the presence and severity of coronary artery disease. J Cell Mol Med.2019 Jan;23(1):271-280). Obese patients undergoing bariatric surgery showed reduced circulating levels of METRLN and improved glucose and lipid homeostasis compared with normal weight controls (Pellitero S, Piquer-Garcia I, Ferrer-Curriu G et al. Opposite changes in meteorin-like and oncostatin m levels are associated with metabolic improvements after bariatric surgery. Int J Obes (Lond). 2018 Apr;42(4):919-922). Two recent studies have investigated circulating levels of METRNL in PCOS patients compared with controls. The study by Fouani et al. was conducted on the following cohorts: PCOS recurrent pregnancy loss (PCOS-RPL, n=60) and infertile PCOS patients (n=60) as well as 60 healthy controls. The women were aged 20 to 40 years (mean age of controls: 30.02±4.60; mean age of PCOS cases: 29.88±4.22). The authors found that serum METRNL levels were lower in PCOS patients when compared to controls. Moreover, serum METRNL was correlated with BMI, adiponectin, and homocysteine ​​in controls, and negatively correlated with FBG, fasting insulin, and HOMA-IR in the PCOS group and subgroups. Moreover, it was negatively correlated with hs-CRP in controls, PCOS group, and subgroups (Fouani FZ, Fadaei R, Moradi N, et al. Circulating levels of Meteorin-like protein in polycystic ovary syndrome: A case-control study. PLoS One. 2020 Apr 24;15(4):e0231943). Deniz et al. measured METRNL (subfatin) and asprosin levels in plasma samples from 30 PCOS cases and 30 healthy controls (mean age of controls: 28.22 ± 2.6; mean age of PCOS cases: 27.14 ± 3.21). Although asprosin levels were significantly higher in women with PCOS compared with healthy controls, METRNL levels were significantly lower than those in controls, which is consistent with the results shown by Fouani et al. In the PCOS subgroup, asprosin and METRNL levels were significantly correlated with HOMA-IR (Deniz R, Yavuzkir S, Ugur K et al. Subfatin and asprosin, two new metabolic players of polycystic ovary syndrome. J Obstet Gynaecol. 2021 Feb; 41(2): 279-284. doi: 10.1080 / 01443615.2020.1758926). .

[0080] In patients with inflammatory bowel disease (IBD), serum levels of METRNL are reduced and have been identified to be negatively correlated with TNF-α, IL-6, and BMI levels (Gholamrezayi A, Mohamadinarab M, Rahbarinejad P, et al. Characterization of the serum levels of Meteorin-like in patients with inflammatory bowel disease and its association with inflammatory cytokines. Lipids Health Dis. 2020 Oct 30;19(1):230). Consistent with other studies on metabolic and inflammatory diseases, serum METRNL was lower, whereas synovial fluid METRNL was higher, in patients with osteoarthritis compared with non-osteoarthritis subjects (Sobieh BH, Kassem DH, Zakaria ZM, El-Mesallamy HO. Potential emerging roles of the novel adipokines adipolin / CTRP12 and meteorin-like / METRNL inobesity-osteoarthritis interplay. Cytokine. 2021 Feb;138:155368).

[0081] There is an unmet medical need for an accurate test for the reliable diagnosis of PCOS. The ratio between LTA4H and METRNL in a measuring sample has the advantage of a reliable test based on biological fluids, which identifies women suffering from PCOS, which is currently impossible. The method can also be reliably used for adolescent subjects and young women aged less than 25 years old, particularly less than 20 years old, particularly 15 years old to less than 25 years old, particularly 15 years old to less than 20 years old, for diagnosing PCOS. Due to the above reasons, the diagnosis of PCOS in adolescent patients is difficult, and therefore, we provide an accurate test for the diagnosis of PCOS in adolescent and young female populations for the first time. In addition, the ratio between LTA4H and METRNL in a measuring sample has the advantage of identifying whether a patient responds to treatment. Another advantage of measuring the ratio between LTA4H and METRNL in a patient sample is to monitor the progress of PCOS. In addition, we attach a computer-implemented method for assessing a subject suffering from PCOS by measuring the ratio between LTA4H and METRNL levels in a sample, and optionally utilizing further criteria, such as values ​​for oligoovulation and / or irregular cycles, hyperandrogenemia and / or polycystic ovarian morphology, or utilizing further biomarkers or hormones based on comparison and / or calculation of the above data to assess the subject.

[0082] As mentioned above, patients suffering from PCOS may show two types of characteristics-reproductive or metabolic. The metabolic types of PCOS include obesity, insulin resistance, metabolic syndrome, prediabetes, type 2 diabetes, non-alcoholic fatty liver disease (NAFLD) and cardiovascular factors. The term "phenotype" can be used instead of "reproductive". The term "reproductive" (or "phenotype") describes any feature of the female phenotype known to indicate PCOS. For example, these reproductive characteristics include polycystic ovary morphology (PCOM) and / or clinical hyperandrogenism, such as acne, seborrhea, alopecia and / or hirsutism. Preferably, these reproductive characteristics include polycystic ovary morphology (PCOM) and / or clinical hyperandrogenism, more preferably acne, seborrhea, alopecia, deep voice and / or hirsutism. These reproductive characteristics of clinical hyperandrogenism can be simply diagnosed by asking females, or it is obvious after a brief physical examination of the female body. Typically, the reference population does not show any or no more than one of these phenotypic characteristics known to indicate PCOS.

[0083] In an embodiment, PCOS is assessed from the group consisting of metabolic or phenotypic PCOS. In a further embodiment, PCOS is assessed from the group consisting of phenotype A, phenotype B, phenotype C, and phenotype D PCOS.

[0084] In an embodiment, the method according to the invention is an in vitro method.

[0085] As used herein, the term "diagnosis" preferably refers to assessing whether a subject referred to in the method of the present invention suffers from PCOS. Preferably, as used herein, the expression "diagnosing PCOS" should be understood as "helping" or "assisting" the diagnosis of PCOS. For example, additional information and / or devices can be used to assist a physician in diagnosing PCOS. Therefore, the actual diagnosis may be performed by a physician.

[0086] As will be appreciated by those skilled in the art, the diagnosis of the present invention is generally not intended to be correct for 100% of the subjects to be tested. The term "diagnosis" preferably requires that a correct diagnosis can be made for a portion of subjects with statistical significance. Various well-known statistical evaluation tools (e.g., determination of confidence intervals, determination of p-values, Student's t-tests, Mann-Whitney tests, etc.) can be used by those skilled in the art to determine whether a certain portion is statistically significant without further effort. For details, see Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. The p-value is preferably 0.4, 0.1, 0.05, 0.01, 0.005 or 0.0001.

[0087] The term "sample" refers to a body fluid sample, a separated cell sample or a sample from a tissue or organ. The body fluid sample can be obtained by well-known techniques, and preferably includes a sample of blood, plasma, serum, capillary blood, interstitial fluid, peritoneal fluid, menstrual fluid, more preferably a sample of blood, plasma or serum. Tissue or organ samples can be obtained from any tissue or organ by, for example, biopsy. Separated cells can be obtained from body fluids or tissues or organs by separation techniques such as centrifugation or cell sorting. Preferably, cells, tissues or organ samples are obtained from those cells, tissues or organs expressing or producing the peptides mentioned herein.

[0088] In some embodiments of the methods of the present invention, the sample is a blood sample (ie, a whole blood sample), a serum sample, or a plasma sample.

[0089] The term "subject" mentioned herein is preferably a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits and rodents (e.g., mice and rats). In some embodiments, the subject is a human. The subject can be male or female. The terms "patient" and "subject" are used interchangeably herein. In a particular embodiment, the patient is a female human patient less than 25 years old. In a particular embodiment, the patient is a female human patient less than 20 years old. In a particular embodiment, the patient is a female human patient from 15 years old to less than 25 years old. In a particular embodiment, the patient is a female human patient from 15 years old to less than 20 years old. In a particular embodiment, the patient is a female human patient from 25 years old and three years after menarche. In a particular embodiment, the patient is a female human patient from 20 years old and three years after menarche. In a particular embodiment, the patient is a female human patient from 15 years old to less than 25 years old and three years after menarche. In a particular embodiment, the patient is a female human patient from 15 years old to less than 20 years old and three years after menarche.

[0090] According to step a) of the method of the present invention, the amount or concentration of LTA4H is determined (i.e. measured) in a sample from a subject. According to step b), the amount or concentration of METRNL is determined in a sample from a subject. It should be understood that steps a) and b) can be performed in any order. Further, the steps can be performed simultaneously.

[0091] In an embodiment, the amount or concentration of LTA4H and METRNL is determined using antibodies, in particular monoclonal antibodies. In an embodiment, steps a) and b) of determining the amount or concentration of LTA4H and METRNL in a patient sample comprise performing an immunoassay. In an embodiment, the immunoassay is performed in a direct or indirect form. In an embodiment, such an immunoassay is selected from the group consisting of: an enzyme-linked immunosorbent assay (ELISA), an enzyme immunoassay (EIA), a radioimmunoassay (RIA), or an immunoassay based on luminescence, fluorescence, chemiluminescence, or electrochemiluminescence detection.

[0092] In certain embodiments, step a) of determining the amount or concentration of LTA4H in a sample from a patient comprises the following steps:

[0093] i) incubating the patient's sample with one or more antibodies that specifically bind to LTA4H, thereby generating a complex between the antibody and LTA4H, and

[0094] ii) quantifying the complex formed in step i), thereby quantifying the amount or concentration of LTA4H in the patient sample.

[0095] In a specific embodiment, in step i), the sample is incubated with two antibodies that specifically bind to LTA4H. As will be apparent to the skilled person, the sample may be contacted with the first and second antibodies in any desired order, e.g., first with the first antibody and then with the second antibody; or first with the second antibody and then with the first antibody; or simultaneously with the first and second antibodies, the contact being carried out for a time and under conditions sufficient to form a first anti-LTA4H antibody / LTA4H / second anti-LTA4H antibody complex. As will be readily appreciated by the skilled person, this is merely a matter of routine experimentation to set suitable or sufficient time and conditions for the formation of a complex between a specific anti-LTA4H antibody and an LTA4H antigen / analyte (=anti-LTA4H complex), or a secondary complex or sandwich complex comprising a first antibody against LTA4H, LTA4H (analyte) and a second anti-LTA4H antibody (=first anti-LTA4H antibody / LTA4H / second anti-LTA4H antibody complex).

[0096] The detection of the anti-LTA4H antibody / LTA4H complex may be performed by any suitable means. The detection of the first anti-LTA4H antibody / LTA4H / second anti-LTA4H antibody complex may be performed by any suitable means. Those skilled in the art are fully familiar with such means / methods.

[0097] In certain embodiments, a sandwich will be formed comprising a first antibody to LTA4H, LTA4H (analyte), and a second antibody to LTA4H, wherein the second antibody is detectably labeled.

[0098] In one embodiment, a sandwich will be formed comprising a first antibody to LTA4H, LTA4H (analyte) and a second antibody to LTA4H, wherein the second antibody is detectably labeled and wherein the first anti-LTA4H antibody is capable of binding to or associated with a solid phase.

[0099] In a preferred embodiment, determining the amount or concentration of METRNL in a sample from a subject comprises contacting the sample with an antibody or antigen-binding fragment thereof that specifically detects METRNL. Preferably, the antibody or antigen-binding fragment thereof that specifically detects METRNL specifically binds to an epitope of METRNL. The complex formed between the antibody (or fragment) and the biomarker should be proportional to the amount or concentration of METRNL.

[0100] Preferably, determining the amount or concentration of LTA4H comprises contacting the sample with an antibody or antigen binding fragment thereof that specifically detects LTA4H. Preferably, the antibody or antigen binding fragment thereof that specifically detects LTA4H specifically binds to an epitope of LTA4H. The complex formed between the antibody (or fragment) and the biomarker should be proportional to the amount or concentration of LTA4H.

[0101] The term "antibody" is known in the art. As used herein, the term refers to any immunoglobulin (Ig) molecule consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains. As used herein, the term "antibody" also includes an antigen-binding fragment of an antibody. As used herein, an antigen-binding fragment of an antibody should be able to specifically bind to an antigen. Therefore, an antigen-binding fragment of an antibody is a fragment that retains the ability of a (full-length) antibody to specifically bind to an antigen (such as METRNL or LTA4H). The antibody fragment preferably comprises a portion of a full-length antibody, preferably its variable domain, or at least its antigen-binding site. In one embodiment, the antigen-binding fragment is selected from the group consisting of the following items: Fab fragment, Fab' fragment, Facb fragment, F(ab')2 fragment, scFv fragment, Fv fragment. For example, the antigen-binding fragment is a F(ab')2 fragment. How to produce an antigen-binding fragment is well known in the art. For example, fragments can be produced by enzymatic cleavage of the antibodies of the present invention. In addition, fragments can be produced by synthetic or recombinant techniques. Fab fragments are preferably produced by papain digestion of antibodies, Fab' fragments are produced by pepsin digestion and partial reduction, F(ab')2 fragments are produced by pepsin digestion, and facb are produced by plasmin digestion. Fv or scFv fragments are preferably produced by molecular biology techniques.

[0102] The antibody according to the inventive method can be a polyclonal antibody or a monoclonal antibody. In a preferred embodiment, the antibody is a monoclonal antibody. The term "monoclonal antibody" is well known in the art. As used herein, the term preferably refers to an antibody obtained from a substantially homogeneous antibody population, that is, the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variants that may be produced during the production of the monoclonal antibody, which variants are generally present in small amounts. The monoclonal antibody of the present invention can be prepared by the well-known hybridoma method described by Kohler and Milstein, Nature, 256:495 (1975), or can be prepared by a recombinant DNA method. In certain embodiments, the monoclonal antibody is selected from the group consisting of: sheep monoclonal antibody, mouse monoclonal antibody, rabbit monoclonal antibody, goat monoclonal antibody, horse monoclonal antibody, chicken monoclonal antibody. In certain embodiments, the monoclonal antibody is a mouse monoclonal antibody.

[0103] The antibodies (or fragments) used in steps a) and b) of the method of the invention may be used in a sandwich assay as capture antibody in combination with at least one other antibody binding to a different epitope.

[0104] Antibodies can be used in sandwich assays. Sandwich assays are one of the most useful and commonly used assays, including many variations of sandwich assay techniques. For example, in a typical assay, an unlabeled (capture) binding agent is fixed or can be fixed on a solid substrate, and the sample to be tested is contacted with the capture binding agent. After an appropriate incubation period, a second (detection) binding agent labeled with a reporter molecule capable of producing a detectable signal is added and incubated for a period of time sufficient to allow the formation of a binding agent-biomarker complex, thereby allowing time to form another complex of a binding agent-biomarker-labeled binding agent. Any unreacted material can be washed away, and the presence of a biomarker can be determined by observing the signal generated by the reporter molecule bound to the detection binding agent. The result can be qualitatively characterized by simply observing a visible signal, or can be quantified by comparing with a control sample containing, for example, a known amount of a biomarker to be determined (such as a standard or calibrator described elsewhere herein).

[0105] The incubation steps of a typical sandwich assay can be varied as needed and as appropriate. Such variations include, for example, simultaneous incubations, in which two or more binding agents and biomarkers are co-incubated. For example, the sample to be analyzed and the labeled binding agent are added to the fixed capture binding agent simultaneously. It is also possible to first incubate the sample to be analyzed and the labeled binding agent, and then add the antibody that is bound to the solid phase or capable of binding to the solid phase.

[0106] The complex formed between the specific binding agent and the biomarker should be proportional to the amount or concentration of the biomarker present in the sample. It should be understood that the specificity and / or sensitivity of the binding agent to be applied defines the degree of proportion of at least one marker contained in the sample that can be specifically bound. Further details on how the measurement can be implemented can also be found elsewhere herein. The amount or concentration of the complex formed should be converted into the amount or concentration of the biomarker, thereby reflecting the amount or concentration actually present in the sample.

[0107] As used herein, the term "amount" encompasses the absolute amount of LTA4H or METRNL, the relative amount or concentration of said LTA4H or METRNL, and any value or parameter associated therewith or derivable therefrom. Such values ​​or parameters include intensity signal values ​​from all specific physical or chemical properties obtained from said peptide by direct measurement, such as intensity values ​​in a mass spectrometry or NMR spectrum. In addition, included are all values ​​or parameters obtained by indirect measurements specified elsewhere in this specification, for example, the reaction level determined from a biological readout system in response to an intensity signal obtained from a peptide or from a specifically bound ligand. It should be understood that the values ​​associated with the above-mentioned amounts or parameters can also be obtained by all standard mathematical operations. According to a preferred embodiment of the present invention, the determination of the "amount" is performed by the disclosed system, wherein the computing device determines the "amount" based on contact and measurement steps performed by one or more analyzer units of the system.

[0108] In step c) of the method according to the first, second or third aspect of the invention, the fractions of the amounts or concentrations determined in steps a) and b), ie the amount or concentration of LTA4H and the amount or concentration of METRNL, are calculated.

[0109] As used herein, the term "calculation" refers to the assessment of a score based on the amount or concentration of LTA4H and the amount or concentration of METRNL determined in the sample of the subject. For example, it is envisioned that a score, i.e., a single score, is calculated based on the amount or concentration of LTA4H and the amount or concentration of METRNL, and the score is compared with a reference score. The calculated score combines information about the amount or concentration of LTA4H and the amount or concentration of METRNL. In addition, the biomarker can be weighted in the score according to its contribution to the establishment of the diagnosis. The score can be regarded as a classifier parameter for diagnosing PCOS. In particular, the score should be able to diagnose PCOS based on a comparison with a reference score. Preferably, the reference score is able to distinguish between a subject with PCOS and a subject without PCOS, in particular a critical value.

[0110] Preferably, the fraction is a ratio, i.e. the ratio of the amount or concentration of LTA4H to the amount or concentration of METRNL. Thus, the ratio calculated in step c) according to the first, second or third aspect of the invention is compared with a reference ratio. In one embodiment, the ratio is the ratio of the amount or concentration of LTA4H to the amount or concentration of METRNL. In an alternative embodiment, the ratio is the ratio of the amount or concentration of METRNL to the amount or concentration of LTA4H.

[0111] In step d) of the first, second or third aspect of the method according to the invention, the score calculated in step c) is compared with a reference score. For example, the calculated ratio should be compared with a reference ratio.

[0112] As used herein, the term "comparison" encompasses comparison of scores calculated for samples from test subjects, which are suitable reference sources specified elsewhere in this specification. Preferably, the comparison is assisted by automation. For example, a suitable computer program comprising an algorithm for comparing the calculated scores and reference scores of the subject can be used. Such computer programs and algorithms are well known in the art. Nevertheless, the comparison can also be implemented manually. The computer program can further evaluate the results of the comparison, i.e., automatically provide the required assessment in a suitable output format (i.e., diagnosis). Preferably, the diagnosis can be used as, for example, an auxiliary for a professional physician to establish the final diagnosis of PCOS.

[0113] The calculating step and / or the comparing step may be performed by using a computer comprising a processing unit.

[0114] Based on the comparison of the calculated score with the reference score, it should be possible to assess whether the test subject suffers from PCOS. For example, the result of the comparison can be given as raw data, and in some cases as an indicator in the form of words, phrases, symbols or numerical values ​​that can indicate a specific diagnosis. Therefore, the reference score to be selected allows the difference or identity of the calculated score and the calculated score to identify those test subjects who belong to or do not belong to the subject group suffering from PCOS. The method allows the exclusion (crossing out) or identification (crossing in) of subjects suffering from PCOS. As used herein, the difference in the score, i.e., increase or decrease, is preferably a statistically significant difference. The method can also use other diagnostic criteria, such as oligoovulation or hyperandrogenism to assess whether the patient suffers from PCOS. (Page 26, third paragraph)

[0115] Preferably, a reference score, such as a reference ratio, should allow for differentiation of whether a subject suffers from PCOS. Preferably, the diagnosis is made by assessing whether the test subject's score is above or below the reference score. It is not necessary to provide an exact reference score. A relevant reference score can be obtained by correlating sensitivity and specificity with the sensitivity / specificity of any score. A reference score that results in a high sensitivity results in a lower specificity, and vice versa.

[0116] In some embodiments, the reference score is derived from a sample from a subject (or a group of samples from a subject) known to suffer from PCOS.

[0117] In some embodiments, the reference score is derived from a sample from a subject (or a group of samples from a subject) known not to have PCOS.

[0118] As described above, the score calculated in step c) according to the first, second or third aspect of the present invention can be a ratio. In one embodiment, the ratio is the ratio of the amount or concentration of LTA4H to the amount or concentration of METRNL. In this case, a ratio greater than the reference ratio (i.e., the calculated ratio) indicates a subject suffering from PCOS. A ratio lower than the reference ratio indicates a subject not suffering from PCOS. In an alternative embodiment, the calculated ratio is the ratio of the amount or concentration of METRNL to the amount or concentration of LTA4H. In this case, a ratio lower than the reference ratio (i.e., the calculated ratio) indicates a subject suffering from PCOS. A ratio greater than the reference ratio indicates a subject not suffering from PCOS.

[0119] In a preferred embodiment, the method of the present invention further comprises the step of suggesting an appropriate therapy if PCOS has been diagnosed. Alternatively, the method further comprises the step of initiating an appropriate therapy if PCOS has been diagnosed.

[0120] As used herein, the term "recommendation" means a proposal to establish a therapy that can be applied to a subject. However, it should be understood that the term does not include the application of actual treatment. The therapy to be recommended depends on the diagnosis provided by the method of the present invention. Preferably, the above-mentioned recommendation step can also be automated. Preferably, the diagnosis obtained by the method of the present invention, i.e. the diagnosis of the method, will be used to search a database of recommendations for treatment measures including possible diagnosis results for individuals.

[0121] In one embodiment, the therapy to be suggested or initiated is selected from a drug-based therapy for PCOS or a lifestyle change for controlling metabolic symptoms. In an embodiment, the drug-based therapy for PCOS is selected from the group consisting of: drugs for regulating the cycle, in particular oral contraceptives or progestin therapy, drugs for preventing or controlling diabetes, in particular type 2 diabetes, drugs for preventing or controlling high cholesterol, hormones or drugs to increase fertility, drugs, hormones or surgical procedures to remove excess hair, drugs or surgical procedures to control acne.

[0122] The method of the present invention can also be implemented as a computer-implemented invention. In one embodiment, one or more steps, such as the comparison step and / or the calculation step, are implemented by a computer (i.e., a computer) comprising a processing unit. In another embodiment, all steps are implemented by a computer comprising a processing unit.

[0123] Accordingly, a fourth aspect of the present invention relates to a computer-implemented method for diagnosing PCOS in a subject, the method comprising:

[0124] (a) Receiving at the processing unit

[0125] (a1) a value for the amount or concentration of LTA4H in a sample from a subject, and

[0126] (a2) a value for the amount or concentration of METRNL in a sample from a subject,

[0127] (b) processing the value received in step (a) with a processing unit, wherein the processing comprises:

[0128] (b1) calculating the fraction of said values ​​(a1) and (a2) received in step a),

[0129] (b2) comparing the calculated score with a reference score, and

[0130] (c) optionally providing a diagnosis via an output device, wherein the diagnosis is based on the results of step b).

[0131] In some embodiments, the processing unit is embodied in a computer.

[0132] In some embodiments, step b) according to the fourth aspect of the present invention further comprises retrieving, at the processing unit, a reference score from the memory, ie a reference score suitable for diagnosing PCOS.

[0133] In one embodiment of the method of the present invention, information about the diagnosis (according to the last step of the method of the present invention) is provided by a display, which is configured to present the assessment. Therefore, as described elsewhere herein, information on whether the subject suffers from PCOS can be provided. Further, suggestions for suitable treatment can be displayed. As described elsewhere herein, various treatment measures can be suggested. In this case, one or more treatment options can be displayed in the display.

[0134] In one embodiment of the method of the invention, the method may comprise the further step of transferring information regarding the assessment of the method of the invention to the electronic medical record of the subject.

[0135] Alternatively, the assessment performed in the last step of the method of the invention can be printed out by a printer. The printout should contain information on whether the patient is at risk and / or recommendations for suitable treatment measures.

[0136] The present invention further relates to i) the use of LTA4H and METRNL as biomarkers for diagnosing PCOS, or ii) the use of at least one agent that specifically binds to METRNL and at least one agent that specifically binds to LTA4H for diagnosing PCOS. Preferably, the use is in vitro, i.e. carried out in a sample from a subject. Preferably, one agent that specifically binds to LTA4H is an antibody or an antigen-binding fragment. Preferably, one agent that specifically binds to METRNL is an antibody or an antigen-binding fragment.

[0137] Finally, the present invention relates to a kit comprising at least one agent that specifically binds to METRNL and at least one agent that specifically binds to LTA4H.

[0138] As used herein, the term "kit" refers to a collection of the above-mentioned means, for example, provided separately or in a single container. The container may contain instructions for implementing the method of the present invention. In one embodiment, the kit includes reagents for diagnosing PCOS. The reagents of the kit may include antibodies or antibody fragments. Preferably, the antibody or antibody fragment recognizes an epitope or antigen of LTA4H and / or METRLN. The kit may further contain other reagents that recognize other biomarkers. Therefore, the kit may also include a combination of at least three reagents. According to the present invention, biomarkers may also include hormones, such as anti-Mullerian hormone AMH. The kit can be used for any diagnostic assay.

[0139] Examples

[0140] The present invention is illustrated by the following examples only. The examples are not to be construed in any way as limiting the scope of the present invention.

[0141] Example 1: Diagnostic performance of the ratio between the two biomarkers LTA4H and METRNL determined by ELISA technique in women with PCOS (phenotypes A, B, C and D) and controls

[0142] Performance validation has been performed on a sample set of 82 cases (serum samples from women with PCOS) and 44 controls (serum samples from healthy women).

[0143] The concentrations of the two analytes, LTA4H and METRNL, were determined by ELISA (enzyme-linked immunosorbent assay). The case group consisted of patients diagnosed with PCOS according to the Rotterdam criteria (26 phenotype A, 19 phenotype B, 20 phenotype C, and 20 phenotype D). The control group included healthy women without PCOS.

[0144] The concentration of LTA4H in human serum was determined using the Human LTA4H ELISA Kit Version 1 from Invitrogen (Catalog Number: EH308RB). The concentration of METRNL in human serum was determined using the Human METRNL ELISA Kit from R&D Systems (Catalog Number: DY7867-05). The kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) designed to detect and quantify the levels of human LTA4H and METRNL in cell culture supernatants, plasma, and serum, respectively. The two analytes were measured in separate plates, and only one analyte was measured at a time.

[0145] To quantify LTA4H, the kit provides human LTA4H antibody pre-coated plates. Samples were measured at 2-fold dilution. After all reagents were placed at room temperature, 100 μL of each sample and standard was added. Samples and standards were measured in duplicate. During a 2.5-hour incubation at room temperature on a microplate oscillator set to 650 rpm, any LTA4H present was bound to the capture antibody fixed on the microtiter plate. During the washing step (4x 300 μL), unbound material was removed from the plate, and then 100 μL of diluted anti-LTA4H biotin conjugate was added to the well. After incubation on the oscillator for 1 hour and another washing step (4x 300 μL) to remove any unbound detection antibody, 100 μL of prepared streptavidin-HRP solution was added to the plate. Subsequently, incubated at room temperature for 45 minutes and a washing step (4x 300 μL) was performed. After the last wash, 100 μL of TMB substrate was added to the plate. The plate was incubated at room temperature, in the dark for 30 minutes, while gently shaking. During the incubation period, the substrate turned blue. The color development was proportional to the amount of LTA4H combined in the initial step. The color development was stopped by adding 50 μL of stop solution, the solution in the well changed from blue to yellow, and the color intensity was measured at 450nm using a plate reader for detection, and the color intensity was measured at 570nm for background subtraction. In order to generate a calibration curve, the lyophilized recombinant LTA4H provided with the kit was reconstructed and diluted in a calibration diluent. The calibration range of this assay is 2.048ng / mL to 500ng / mL. Calibrator 1 (500ng / mL) corresponds to the reconstructed stock solution, and calibrators 2 to calibrators 7 (2.048ng / mL) are prepared by continuous 2.5-fold dilution steps in a calibration diluent. Pure calibration diluent is used as blank (0ng / mL). The calibration curve is fitted using a non-weighted 4-parameter nonlinear regression (Newton / Raphson).

[0146] The capture antibody of METRNL was diluted to a working concentration in PBS without carrier protein. A 96-well microplate was incubated with 100 μL of diluted capture antibody per well. The plate was sealed and incubated overnight at room temperature. The plate was then washed 3 times with wash buffer, 400 μL per well each time. After the last wash, the wash buffer was completely removed, the plate was closed by adding 300 μL of reagent buffer to each well, and incubated at room temperature for at least 1 hour. The plate was washed 3 times with wash buffer, 400 μL per well each time, and was set aside. The sample was measured at a 4-fold dilution. After all reagents were placed at room temperature, 100 μL of each sample and standard was added. Samples and standards were measured in duplicate. During the 2-hour incubation at room temperature, any METRNL present was bound to the capture antibody fixed on the microtiter plate. In the washing step (3x 400 μL), unbound material was removed from the plate, and then 100 μL of anti-METRNL detection antibody diluted in reagent diluent was added to the well. After 2 hours incubation and another washing step (3x 400 μL) to remove any unbound detection antibody, 100 μL prepared streptavidin-HRP solution is added to the plate. Subsequently incubated at room temperature for 20 minutes and washed (3x 400 μL), avoid direct exposure to light. After the last wash, 100 μL substrate solution is added to the plate. The plate is incubated at room temperature for 20 minutes, avoiding direct light exposure. During the incubation, the substrate turns blue. Color development is proportional to the amount of METRNL combined in the initial step. Color development is stopped by adding 50 μL stop solution, and the solution color in the hole changes from blue to yellow, and the color intensity is measured at 450nm using a plate reader for detection, and the color intensity is measured at 540nm or 570nm for background subtraction. This deduction corrects the optical defects in the plate. In order to generate a calibration curve, the lyophilized recombinant METRNL provided with the kit is reconstructed and diluted in a reagent diluent. The calibration range of the METRNL assay was 15.6 pg / mL to 1000 pg / mL. A seven-point standard curve was obtained using a 2-fold serial dilution of recombinant METRNL in reagent diluent. The calibration curve was fitted using a four-parameter logistic (4-PL, Newton / Raphson) curve fit.

[0147] Once the concentrations of both analytes were obtained by ELISA immunoassay, the LTA4H:METRNL ratio was calculated for each sample.

[0148] A receiver operating characteristic (ROC) curve was generated for the LTA4H:METRNL ratio ( Figure 1). Model performance was determined by looking at the area under the curve (AUC). The best possible AUC was 1, while the lowest possible AUC was 0.5. ROC curve analysis of the LTA4H:METRNL ratio for PCOS cases when all phenotypes were combined (phenotype AD) showed an AUC of 0.98 (95% CI 0.95-1.00), confirming the high diagnostic accuracy of the LTA4H:METRNL ratio for PCOS ( Figure 1 The diagnostic performance of the LTA4H:METRNL ratio in distinguishing women with PCOS (cases, PCOS phenotype AD) from healthy control subjects using ROC analysis is shown in Table 1 , which depicts the AUC and associated 95% confidence intervals of the ROC curve analysis.

[0149] Table 1

[0150]

[0151] The data obtained by calculating the ratio between LTA4H and METRNL concentrations were used to generate box and whisker plots for controls and PCOS cases when all phenotypes were combined (phenotype AD). When compared to healthy controls, women with PCOS had an increased LTA4H:METRNL ratio ( Figure 2 ).

[0152] Table 2 shows the diagnostic performance of the LTA4H:METRNL ratio for distinguishing women with PCOS from healthy control subjects when separated by different phenotypes A, B, C and D. The AUC for each phenotype is reported in the table.

[0153] Table 2

[0154]

[0155] ROC curve analysis of the LTA4H:METRNL ratio of PCOS cases and healthy controls separated by different phenotypes showed that the AUCs were 0.99 (95% CI 0.97-1.00), 0.99 (95% CI 0.97-1.00), 0.97 (95% CI 0.93-1.00), and 0.97 (95% CI 0.93-1.00) for phenotypes A to D, respectively ( Figure 3 The results confirmed the high diagnostic accuracy of the LTA4H:METRNL ratio for PCOS. The LTA4H:METRNL ratio showed increased levels in all different PCOS phenotypes (phenotype AD) compared to healthy controls ( Figure 4 ).

[0156] Table 3 shows the diagnostic performance of the LTA4H:METRNL ratio in young women (age ≤ 25 years) for distinguishing young women with PCOS from young healthy control subjects when all phenotypes were combined (phenotype AD).

[0157] Table 3

[0158]

[0159] ROC curve analysis of the LTA4H:METRNL ratio in young PCOS cases (age ≤ 25 years) when all phenotypes were combined (phenotype AD) showed an AUC of 1.00, confirming the high diagnostic accuracy (95% CI 1-1, Figure 5 When only young women (age ≤ 25 years) were included in the analysis, PCOS cases (combining all phenotypes AD) showed an increased LTA4H:METRNL ratio compared with young controls (age ≤ 25 years, Figure 6 ).

[0160] The diagnostic performance of the LTA4H:METRNL ratio for distinguishing young women (age ≤ 25 years) with PCOS from young healthy control subjects (age ≤ 25 years) when separated by different phenotypes A, B, C and D has been evaluated and the results are reported in Table 4 (AUC for PCOS phenotypes vs. controls).

[0161] Table 4

[0162]

[0163] ROC curve analysis of the LTA4H:METRNL ratio in young PCOS cases (age ≤25 years, phenotype AD) showed an AUC of 1.00 (95% CI 1-1) for all phenotypes, confirming the high diagnostic accuracy of the LTA4H:METRNL ratio for PCOS in the subgroup of women aged 25 years or younger ( Figure 7 When compared to the LTA4H:METRNL ratio in young healthy controls, the LTA4H:METRNL ratio was increased in all different PCOS phenotypes (phenotype AD, age ≤ 25 years, Figure 8 ).

[0164] Example 2: Diagnostic performance of the ratio between the two biomarkers LTA4H and METRNL in different age groups determined by ELISA technique in women with PCOS (phenotypes A, B, C and D) and controls

[0165] Performance validation has been performed in an additional sample set of 240 cases (serum samples from women with PCOS) and 48 controls (serum samples from healthy women).

[0166] The concentrations of the two analytes, LTA4H and METRNL, were determined by ELISA (enzyme-linked immunosorbent assay). The case group consisted of patients diagnosed with PCOS according to the Rotterdam criteria (155 phenotype A, 5 phenotype B, 8 phenotype C and 72 phenotype D), belonging to three different age groups: 15-20 (n=70), 20-25 (n=99), 25-40 (n=71). The control group included healthy women who did not suffer from PCOS.

[0167] The concentration of LTA4H in human serum was determined using the Human LTA4H ELISA Kit Version 1 from Invitrogen (Cat. No. EH308RB). The concentration of METRNL in human serum was determined using the Human METRNL ELISA Kit from R&D Systems (Cat. No. DY7867-05). The measurements were performed as described in Example 1.

[0168] A receiver operating characteristic (ROC) curve was generated for the LTA4H:METRNL ratio ( Fig. 9 Model performance was determined by looking at the area under the curve (AUC). ROC curve analysis of the LTA4H:METRNL ratio for PCOS cases when all phenotypes were combined (phenotype AD) showed an AUC of 0.93 (95% CI 0.90-0.96), confirming the high diagnostic accuracy of the LTA4H:METRNL ratio for PCOS ( Fig. 9 ).

[0169] The diagnostic performance of the LTA4H:METRNL ratio in distinguishing women with PCOS (cases, PCOS phenotype AD) from healthy control subjects using ROC analysis is shown in Table 5, which depicts the AUC and associated 95% confidence intervals of the ROC curve analysis. The results were obtained using an ELISA immunoassay.

[0170] Table 5

[0171]

[0172] The data obtained by ELISA immunoassay were used to generate box and whisker plots for controls and PCOS cases when all phenotypes were combined (phenotypes AD). When compared to healthy controls, women with PCOS had an increased LTA4H:METRNL ratio ( Fig.10 ).

[0173] Table 6 shows the diagnostic performance of the LTA4H:METRNL ratio for distinguishing women with PCOS from healthy control subjects when separated by different phenotypes A, B, C and D. The AUC for each phenotype is reported in the table.

[0174] Table 6

[0175]

[0176] ROC curve analysis of the LTA4H:METRNL ratio of PCOS cases and healthy controls separated by different phenotypes showed that the AUCs were 0.93 (95% CI 0.90-0.96), 1.00 (95% CI 0.98-1.00), 0.93 (95% CI 0.85-1.00), and 0.92 (95% CI 0.87-0.96) for phenotypes A to D, respectively ( Fig.11 ). The results confirmed the high diagnostic accuracy of the LTA4H:METRNL ratio for PCOS.

[0177] Compared with healthy controls, the LTA4H:METRNL ratio was increased in all the different PCOS phenotypes (phenotype AD) ( Fig.12 ).

[0178] Table 7 shows the diagnostic performance of the LTA4H:METRNL ratio in different age groups (15 ≤ age < 20 years, 20 ≤ age < 25 years, 25 ≤ age < 40 years) for distinguishing women with PCOS from healthy control subjects when all phenotypes were combined (phenotype AD).

[0179] Table 7

[0180]

[0181] ROC curve analysis of the LTA4H:METRNL ratio of PCOS cases and healthy controls separated by different age groups showed that the AUCs were 0.91 (95% CI 0.86-0.96), 0.91 (95% CI 0.86-0.95), and 0.97 (95% CI 0.94-1.00) for age groups 15-20, 20-25, and 25-40, respectively ( Fig.13 ). The results confirmed the high diagnostic accuracy of the LTA4H:METRNL ratio for PCOS in all different age groups.

[0182] The LTA4H:METRNL ratio was confirmed to be increased in women with PCOS compared with controls in all age groups ( Fig.14 ).

[0183] Considering the lack of reliable biomarkers for the diagnosis of PCOS, especially in younger women (age <25 years), separate analyses were performed for the age groups ≥15 years and <25 years.

[0184] Table 8 shows the diagnostic performance of the LTA4H:METRNL ratio in young women (15 years ≤ age < 25 years) for distinguishing young women with PCOS from young healthy control subjects when all phenotypes were combined (phenotype AD).

[0185] Table 8

[0186]

[0187] ROC curve analysis of the LTA4H:METRNL ratio in young PCOS cases (15 years ≤ age < 25 years) when all phenotypes were combined (phenotype AD) showed an AUC of 0.92, confirming the high diagnostic accuracy (95% CI 0.87-0.97, Fig.15 When only young women (age 15-25 years) were included in the analysis, PCOS cases (combining all phenotypes AD) showed an increased LTA4H:METRNL ratio compared with young controls (15 ≤ age < 25 years, Fig.16 ).

[0188] The diagnostic performance of the LTA4H:METRNL ratio for distinguishing young women with PCOS (15 years ≤ age < 25 years) from young healthy control subjects (15 years ≤ age < 25 years) when separated by different phenotypes A, B, C and D has been evaluated and the results are reported in Table 9 (AUC for PCOS phenotypes vs. controls).

[0189] Table 9

[0190]

[0191] ROC curve analysis of the LTA4H:METRNL ratio in young PCOS cases (15 years ≤ age < 25 years, phenotype AD) showed that for each phenotype, the AUC was 0.92 (95% CI 0.88-0.97), 1.00 (95% CI 1.00-1.00), 0.94 (95% CI 0.85-1.00), and 0.91 (95% CI 0.84-0.98), respectively, confirming the high diagnostic accuracy of the LTA4H:METRNL ratio for PCOS in the subgroup of women aged 15-25 years ( Fig.17When compared with the LTA4H:METRNL ratio in young healthy controls, the LTA4H:METRNL ratio was increased in all different PCOS phenotypes (phenotype AD, 15 ≤ age < 25 years, Fig.18 ).

Claims

1. A method for diagnosing PCOS in a subject, the method comprising the following steps: a) determining the amount or concentration of LTA4H in a sample from said subject, b) determining the amount or concentration of METRNL in a sample from said subject, c) calculating the fractions of said amounts or concentrations determined in steps a) and b), d) comparing the calculated score with a reference score, and e) Diagnosing PCOS in the subject.

2. A method of selecting a patient for therapy for PCOS, the method comprising: a) determining the amount or concentration of LTA4H in a sample from said subject, b) determining the amount or concentration of METRNL in a sample from said subject, c) calculating the fractions of said amounts or concentrations determined in steps a) and b), d) comparing the calculated score with a reference score, and e) selecting said patient for PCOS therapy.

3. A method for monitoring the progression of PCOS in a subject with PCOS or for monitoring the response of a subject with PCOS to treatment, the method comprising: a) determining the amount or concentration of LTA4H in a sample from said subject, b) determining the amount or concentration of METRNL in a sample from said subject, c) calculating the fractions of said amounts or concentrations determined in steps a) and b), d) comparing the calculated score with a reference score, and e) monitoring the progress of said subject suffering from or being treated for PCOS.

4. The method according to claims 1 to 3, wherein the sample is blood, serum, plasma, capillary blood, interstitial fluid, peritoneal fluid or menstrual fluid, a menstrual fluid sample, and / or wherein the subject is a human subject.

5. The method of claims 1 to 4, wherein the subject is suspected of suffering from PCOS, for example wherein the subject suspected of suffering from PCOS has a history of PCOS symptoms.

6. The method of any one of claims 1 to 5, wherein said determining of said amount or concentration of LTA4H comprises contacting said sample with an antibody or antigen-binding fragment thereof that specifically detects LTA4H.

7. The method according to any one of claims 1 to 5, wherein said determining of said amount or concentration of METRNL comprises contacting said sample with an antibody or antigen-binding fragment thereof that specifically detects METRNL.

8. The method according to any one of claims 1 to 7, wherein the fraction is a ratio, in particular a) wherein said ratio is the ratio of said amount or concentration of LTA4H to said amount or concentration of METRNL, wherein, Preferably, a ratio higher than the reference ratio is indicative of a subject suffering from PCOS, or b) wherein said ratio is the ratio of said amount or concentration of METRNL to said amount or concentration of LTA4H, wherein, preferably, a ratio below the reference ratio is indicative for a subject suffering from PCOS.

9. The method according to any one of claims 1 to 8, wherein PCOS is selected from the group consisting of: metabolic or phenotypic PCOS.

10. The method according to any one of claims 1 to 9, wherein PCOS is selected from the group consisting of: phenotype A, phenotype B, phenotype C and phenotype D.

11. A computer-implemented method for diagnosing PCOS in a subject, the computer-implemented method comprising: (a) Receiving at the processing unit (a1) a value for the amount or concentration of LTA4H in a sample from the subject, and (a2) a value for the amount or concentration of the amount of METRNL in a sample from the subject, (b) processing the value received in step (a) with the processing unit, wherein the processing comprises: (b1) calculating the fraction of said values ​​(a1) and (a2) received in step a), (b2) comparing the calculated score with a reference score, and (c) optionally providing a diagnosis via an output device, wherein the diagnosis is based on the results of step b).

12. i) Use of LTA4H and METRNL as biomarkers for diagnosing PCOS, or ii) Use of at least one agent that specifically binds to METRNL and at least one agent that specifically binds to LTA4H for diagnosing PCOS.

13. A kit comprising at least one agent that specifically binds to METRNL and at least one agent that specifically binds to LTA4H.

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