The ratio of LTA4H to METRNL in the assessment of polycystic ovary syndrome

Abstract

The present invention relates to a method for diagnosing polycystic ovary syndrome (PCOS) in a subject, the method comprising the steps of: a) determining the amount or concentration of total LTA4H in a sample from the subject; b) determining the amount or concentration of METRNL in a sample from the subject; c) calculating a score of the 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.

Description

[Technical Field]

[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 the sample from the subject; c) calculating a score of the 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. The present invention further relates to a method for selecting a patient for treatment of PCOS, 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 the sample from the subject; c) calculating a score of the amounts or concentrations determined in steps a) and b); d) comparing the calculated score with a reference score; and e) selecting a patient for PCOS treatment. Furthermore, the present invention relates to a method for monitoring PCOS progression in a subject with PCOS or for monitoring the response to treatment in a subject with PCOS, 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 a score of the amounts or concentrations determined in steps a) and b), d) comparing the calculated score with a reference score, and e) monitoring progression in a subject suffering from or being treated for PCOS.Finally, the present invention relates to a computer-implemented method for diagnosing PCOS in a subject. [Background technology]

[0002] Background of the Invention Polycystic ovary syndrome (PCOS) is a heterogeneous gynecological condition defined by the combination of androgen excess and ovarian dysfunction. Patients with PCOS may have a variety of clinical manifestations, which can be reproductive and / or metabolic. Reproductive manifestations include irregular menstrual cycles, infertility, pregnancy complications, and hirsutism. 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 guidelines for the assessment and management of polycystic ovary syndrome 2018).

[0003] Symptoms are not specific for PCOS, and patients are often diagnosed only after a lengthy infertility evaluation. For a final diagnosis of PCOS, other conditions or diseases, such as pregnancy, nonclassical adrenal hyperplasia (NCAH), congenital adrenal hyperplasia, androgen-secreting tumors, Cushing's syndrome, thyroid disorders, 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 include, for example: 17α-hydroxyprogesterone (17-OHP) to rule out NCAH (Nordenstrom and Falhammar 2018) Prolactin to rule out hyperprolactinemia Cortisol to rule out patients with Cushing's syndrome Thyroid-stimulating hormone (TSH) to rule out thyroid disorders is.

[0004] PCOS can be caused by a combination of genetic, epigenetic and environmental factors, including heredity.

[0005] Despite the fact that PCOS is one of the most common endocrine disorders in women, affecting 10% of women during their reproductive years, 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).

[0006] The most widely used criteria for diagnosing PCOS are the so-called Rotterdam criteria. PCOS is indicated by at least two of the following criteria: (i) irregular cycles (hypomenorrhea) and / or ovulatory dysfunction (ovoovulation, 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 menstrual cycles with a cycle length of less than 21 days or more than 35 days, or fewer than eight cycles per year. Clinical and / or biochemical signs of hyperandrogenism (clinical hyperandrogenism is defined as hirsutism (excessive male-pattern hair growth) and / or acne, and biochemical hyperandrogenism is defined as higher levels of free androgen compared to healthy controls). Clinical hyperandrogenism is also defined as a modified Ferriman-Gallwey score of >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 typically determined according to the "International Evidence-based Guideline for PCOS 2018" using an intravaginal ultrasound transducer with a frequency bandwidth that includes 8 MHz. The threshold for PCOM is considered to be a follicle count of >20 per ovary and / or an ovarian volume of ≥10 ml on either ovary, ensuring the absence of corpora lutea, cysts, or dominant follicles. When older ultrasound techniques are used, the threshold for PCOM may be an ovarian volume of ≥10 ml or a follicle count of >12 on either ovary.

[0007] Another method for detecting PCOM is to measure the subject's anti-Müllerian hormone (AMH). AMH is a glycoprotein hormone whose expression is important for sex differentiation at specific times during fetal development. Furthermore, AMH, produced by granulosa cells of growing follicles, typically correlates with the number of follicles in the ovary. Therefore, serum AMH levels can be a surrogate biomarker for follicle count / number (AFC), determined by transvaginal ultrasound. Several studies have suggested serum AMH as a biochemical marker for PCOM. Several studies have proposed AMH thresholds for PCOM in women with PCOS (Nicholas et al. 2014; Pigny et al. 2016; Dietz de Loos et al., Fertil Steril, 2021). However, according to the "International Evidence-Based Guideline for the Assessment and Management of Polycystic Ovary Syndrome 2018," serum AMH levels should not be used as a substitute for the detection of PCOM or the diagnosis of PCOS.

[0008] An additional method for detecting PCOS is the three-item PCOS criteria system (Indran et al., 2018). This system proposes that a diagnosis of PCOS is made if two of three items are present: (i) hypomenorrhea (defined as a mean menstrual cycle length >35 days); (ii) suprathreshold AMH; and (iii) hyperandrogenism, defined as either suprathreshold testosterone and / or the presence of hirsutism (mFG score ≥5). Alternatively, AMH has been suggested in combination with hyperandrogenism and hypomenorrhea (Sahmay et al., 2014) or in combination with SHBG (Calzada et al., 2019).

[0009] Another method for detecting PCOS is to measure other hormones, such as luteinizing hormone (LH) and follicle-stimulating hormone (FSH). However, the diagnostic utility of the LH:FSH ratio for diagnosing PCOS appears low, as only a small proportion of women with PCOS have significantly elevated LH:FSH ratios (Cho et al. 2005). In fact, there is a wide range of LH:FSH ratios found in women diagnosed with PCOS (Malini and George 2018).

[0010] The need to consider the results of multiple diagnostic tests and laboratory tests requires specific expertise, making it extremely difficult for less specialized physicians (such as general practitioners) to diagnose PCOS in clinical routine. For example, determining PCOM by transvaginal ultrasound requires appropriate ultrasound equipment and subjective analysis of ultrasound images by the physician. Furthermore, results may also depend on the specific ultrasound device used to assess PCOM. As a result, a diagnosis of PCOS based on the Rotterdam criteria always involves at least one subjective, device- and operator-dependent measurement that is prone to error.

[0011] To assess biochemical hyperandrogenism, well-established normal ranges for measured androgens must be established. Testosterone is the most abundant androgen measured in its total, bound, and free forms. Techniques for measuring free testosterone have limitations. Direct measurement of free testosterone using radioimmunoassays is highly inaccurate and does not reflect true values. Assays have high intra- and interassay variability. Alternatively, greater accuracy can be achieved by measuring total testosterone concentrations using extraction and chromatography, or gas (GC-MS) or liquid (LC-MS) chromatography-mass spectrometry, particularly for clinical studies. The diagnostic performance of measuring serum testosterone can be enhanced by simultaneous measurement of SHBG; thus, calculation of free T concentration from total testosterone and SHBG levels requires only solving a quadratic equation (Azziz R, Carmina E, Dewailly D, et al. Task Force on the Phenotype of the Polycystic Ovary Syndrome of The Androgen Excess and PCOS Society. The Androgen Excess and PCOS Society criteria for the polycystic ovary syndrome: the complete task force report. Fertil Steril. 2009;91(2):456-88). The definition of hirsutism may vary depending on ethnicity. An mFG score of >8 to diagnose hirsutism in women with PCOS may not be appropriate for diagnosis in all ethnicities. East Asian women have a lower prevalence of hirsutism compared with Caucasians, and a score of >5 has been proposed to define hirsutism in Chinese women.There are also suggestions that blood androgen levels differ between ethnic groups, with the Japanese population having a lower prevalence of elevated androgens, and testosterone is only recommended as a complementary factor in 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).

[0012] Patients with PCOS can be classified into four distinct phenotypes, designated 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 Jan;36(1):5-12). Phenotype A is characterized by patients exhibiting hyperandrogenism, ovulatory dysfunction, and / or irregular cycles and polycystic ovarian morphology. Phenotype B is characterized by hyperandrogenism, ovulatory dysfunction, and / or irregular cycles. Phenotype C is characterized by hyperandrogenism and polycystic ovarian morphology. Phenotype D is characterized by ovulatory dysfunction, ovarian dysfunction, and / or irregular cycles and polycystic ovarian morphology.

[0013] Currently, there are no specific medications available for PCOS. Treatment is symptom-directed and tailored to individual needs. Therapeutic approaches target hyperandrogenism, irregular cycles and / or ovulatory dysfunction, and associated metabolic disorders such as diabetes. The 2018 International Evidence-Based Guidelines for the Evaluation and Management of Polycystic Ovary Syndrome provide information to support clinical decision-making and patient management.

[0014] Inconsistent diagnostic criteria, variable provider knowledge, and lack of consensus pose particular challenges to the diagnosis and care of women with PCOS. These factors contribute to inaccurate diagnosis, both underdiagnosis and overdiagnosis. This unfavorable diagnostic experience worsens 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, Pena AS. Curtailing PCOS. Pediatr Res. 2020;87(2):353-361). Furthermore, timely diagnosis is crucial for preventing further metabolic complications in affected women, such as type 2 diabetes.

[0015] In the largest study of PCOS diagnostic experiences, many women reported delayed diagnosis and inadequate information. More than one-third of women reported more than two years (33.6%) and more than three healthcare professionals (47.1%) before a diagnosis was established. Few women were satisfied with their diagnostic experience (35.2%) or the information they received (15.6%). These gaps in early diagnosis, education, and support represent clear opportunities to improve 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).

[0016] A particularly interesting area for diagnosing PCOS is young women, i.e., adolescents and young women under 25 years of age, whose characteristics of normal pubertal development overlap with the adult diagnostic criteria. This makes the diagnosis controversial and challenging. Many of the signs used to diagnose PCOS evolve over time and can change during the first few years after menarche. Normal pubertal physiological changes, such as irregular menstrual cycles, acne, and PCOM, overlap with the adult PCOS diagnostic criteria. In adolescent and young adult women, PCOS is diagnosed when both OA and HA criteria are met. Pelvic ultrasound is not recommended in adolescents less than 8 years after menarche due to the high incidence of polycystic ovaries at this life stage (Pena 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). Evaluating irregular menstrual cycles in adolescents can be challenging. Menstrual cycles are often irregular in adolescents. Immaturity of the hypothalamic-pituitary-ovarian axis during the early years after menarche often results in anovulation, and cycles may be somewhat longer. However, although 90% of cycles fall within the 21-45 day range, cycles shorter than 20 days and longer than 45 days can occur. By 3 years after menarche, 60–80% of menstrual cycles are 21–34 days long, as is typical for adults. Young women 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 about irregular or missed periods.Patients are also 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, establishing reliable biomarkers as an aid for diagnosing PCOS or identifying patients at risk for developing PCOS is paramount, especially for this patient population. Delayed diagnosis in adolescents and young women is often due to a lack of willingness to diagnose at-risk adolescents due to puberty and fears of overdiagnosis or underdiagnosis. 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 adolescent and young women define oligoovulation, irregular menstrual cycles, and hyperandrogenism as criteria to improve diagnostic accuracy in this patient population (Pena 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 guidelines also recommend diagnostic reevaluation at 3-year intervals and lifestyle changes to minimize symptoms and comorbidities associated with PCOS, such as anxiety and depression.

[0017] To date, there are no universal biomarkers available to be used alone or in combination with another biomarker, or in combination with the above-mentioned symptoms or the aforementioned hormone levels, to assess whether a subject has or is at risk of developing PCOS, and / or to determine response to therapy in subjects with PCOS, and / or to monitor the progression of PCOS in a subject, and / or to monitor response to treatment in subjects with PCOS.

[0018] Therefore, there is an unmet need to establish better diagnostic assays for diagnosing young women and adolescents. Summary of the Invention

[0019] Summary of the Invention In a first aspect, the present invention relates to a method for diagnosing PCOS in a subject, the method comprising: a) determining the amount or concentration of LTA4H in a sample from a subject; b) determining the amount or concentration of METRNL in a sample from the subject; c) calculating a score for the amount or concentration determined in steps a) and b); and d) Comparing the calculated score with a reference score. e) diagnosing PCOS in a subject; Includes.

[0020] Preferably, step e) is based on the result of the comparison step d). Thus, step e) may be as follows: e) diagnosing PCOS in the subject based on the results of the comparison in step d).

[0021] In a second aspect, the present invention relates to a method for selecting a patient for treatment of PCOS, comprising: a) determining the amount or concentration of LTA4H in a sample from a subject; b) determining the amount or concentration of METRNL in a sample from the subject; c) calculating a score for the amount or concentration determined in steps a) and b); d) comparing the calculated score to a reference score; and e) selecting a patient for PCOS treatment; Includes.

[0022] Preferably, step e) is based on the result of the comparison step d). Thus, step e) may be as follows: e) selecting patients for PCOS treatment based on the results of the comparison in step d).

[0023] In a third aspect, the present invention relates to a method for monitoring PCOS progression in a subject with PCOS or for monitoring response to treatment in a subject with PCOS, the method comprising: a) determining the amount or concentration of LTA4H in a sample from a subject; b) determining the amount or concentration of METRNL in a sample from the subject; c) calculating a score for the amount or concentration determined in steps a) and b); d) comparing the calculated score to a reference score; and e) monitoring the progression in a subject suffering from or being treated for PCOS; Includes.

[0024] Preferably, step e) is based on the result of the comparison step d). Thus, step e) may be as follows: e) monitoring the progression in the subject suffering from or being treated for PCOS based on the results of step d).

[0025] In a fourth aspect, the present invention relates to a computer-implemented method for diagnosing PCOS in a subject, the method comprising: (a) in a processing unit, (a1) the amount or concentration value of LTA4H in a sample from a subject; and (a2) the amount or concentration of METRNL in a sample from the subject; receiving the (b) processing, by a processing unit, the values received in step (a), said processing comprising: (b1) calculating a score of the values (a1) and (a2) received in step a); (b2) comparing the calculated score with a reference score; a processing step comprising: (c) optionally providing a diagnosis via an output device, the diagnosis being based on the results of step b); Includes.

[0026] In a fifth aspect, the present invention relates to the use of LTA4H and METRNL as biomarkers for the diagnosis of PCOS. Furthermore, 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 the diagnosis of PCOS.

[0027] 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 explanation of the drawings]

[0028] [Figure 1] ROC curve analysis of the ratio between the two serum biomarkers LTA4H and METRNL for PCOS cases for all phenotypes combined (phenotypes A-D). Ratios were performed between biomarker concentrations obtained by ELISA immunoassay. [Figure 2] Ratio values of two serum biomarkers, LTA4H and METRNL, in controls versus PCOS cases for all phenotypes combined (phenotypes A–D). Ratios were performed between biomarker concentrations obtained by ELISA immunoassay. [Figure 3] ROC curve analysis of the ratio between two serum biomarkers, LTA4H and METRNL, for PCOS phenotypes A-D compared with controls. Ratios were performed between biomarker concentrations obtained by ELISA immunoassay. [Figure 4] Ratio values between two serum biomarkers, LTA4H and METRNL, in healthy controls and PCOS phenotypes A to D. Ratios were performed between biomarker concentrations obtained by ELISA immunoassay. [Figure 5]ROC curve analysis of the ratio between two serum biomarkers, LTA4H and METRNL, for young PCOS cases (age ≤25 years) for all phenotypes A–D combined. Ratios were performed between biomarker concentrations obtained by ELISA immunoassay. [Figure 6] Ratio values between two serum biomarkers, LTA4H and METRNL, in young PCOS cases (age 25 or younger) versus young controls in combined phenotypes A–D. Ratios were performed between biomarker concentrations obtained by ELISA immunoassay. [Figure 7] ROC curve analysis of the ratio between the two serum biomarkers LTA4H and METRNL for young PCOS cases (age ≤25 years) divided by phenotypes A-D relative to young healthy controls (age ≤25 years). Ratios were performed between biomarker concentrations obtained by ELISA immunoassay. [Figure 8] Ratio values between two serum biomarkers, LTA4H and METRNL, in young healthy controls (age ≤25 years) and in patients with PCOS phenotypes A–D (age ≤25 years). Ratios were performed between biomarker concentrations obtained by ELISA immunoassay. [Figure 9] ROC curve analysis of the ratio between the two serum biomarkers LTA4H and METRNL for PCOS cases for all phenotypes combined (phenotypes A-D). Ratios were performed between biomarker concentrations obtained by ELISA immunoassay. [Figure 10] Ratio values of two serum biomarkers, LTA4H and METRNL, in controls versus PCOS cases for all phenotypes combined (phenotypes A–D). Ratios were performed between biomarker concentrations obtained by ELISA immunoassay. [Figure 11] ROC curve analysis of the ratio between two serum biomarkers, LTA4H and METRNL, for PCOS phenotypes A-D compared with controls. Ratios were performed between biomarker concentrations obtained by ELISA immunoassay. [Figure 12]Ratio values between two serum biomarkers, LTA4H and METRNL, in healthy controls and PCOS phenotypes A to D. Ratios were performed between biomarker concentrations obtained by ELISA immunoassay. [Figure 13] ROC curve analysis of the ratios between the two serum biomarkers LTA4H and METRNL for different PCOS age groups (15–20, 20–25, 25–45) compared with controls. Ratios were performed between biomarker concentrations obtained by ELISA immunoassay. [Figure 14] Ratio values between two serum biomarkers, LTA4H and METRNL, in healthy controls and PCOS age groups (15–20, 20–25, 25–45). Ratios were performed between biomarker concentrations obtained by ELISA immunoassay. [Figure 15] ROC curve analysis of the ratio between two serum biomarkers, LTA4H and METRNL, for young PCOS cases (15-25 years old) for all phenotypes A-D combined. Ratios were performed between biomarker concentrations obtained by ELISA immunoassay. [Figure 16] Ratio values between two serum biomarkers, LTA4H and METRNL, in young PCOS cases (15-25 years old) versus young healthy controls, combined phenotypes A-D. Ratios were performed between biomarker concentrations obtained by ELISA immunoassay. [Figure 17] ROC curve analysis of the ratio between two serum biomarkers, LTA4H and METRNL, for young PCOS cases (15-25 years old) divided by phenotypes A-D relative to young healthy controls (15-25 years old). Ratios were performed between biomarker concentrations obtained by ELISA immunoassay. [Figure 18] Ratio values between two serum biomarkers, LTA4H and METRNL, in young healthy controls (15–25 years old) and in patients with PCOS phenotypes A–D (15–25 years old). Ratios were performed between biomarker concentrations obtained by ELISA immunoassay. DETAILED DESCRIPTION OF THE INVENTION

[0029] Detailed Description of the Invention The methods referred to in accordance with the first, second, or third aspects of the present invention include methods consisting essentially of the aforementioned steps or methods including additional steps. Furthermore, the methods of the present invention are preferably ex vivo, more preferably in vitro. Furthermore, the methods of the first, second, or third aspects of the present invention may include steps in addition to those explicitly mentioned above. For example, the additional steps may involve determining additional markers and / or taking pre-treatment samples or evaluating the results obtained by the above-mentioned methods. The methods may 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, for example, by suitable robots and sensory devices for the determination in steps (a) and (b), the computer-implemented calculation in step (c), or the computer-implemented comparison in step (d). Thus, the methods of the present invention may be computer-implemented.

[0030] 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 (Haeggstrom JZ. Leukotriene A4 hydrolase and the committed step in leukotriene B4 biosynthesis. Clin Rev Allergy Immunol. 1999 Spring-Summer;17(1-2):111-31). According to the Human Protein Atlas, moderate expression of LTA4H is detected in normal ovarian tissue (http: / / www.proteinatlas.org). The expression pattern of LTA4H has been investigated in human ovarian tissue, revealing weak and moderate expression in preovulatory follicles on granulosa and intrathecal cells, respectively. After ovulation, the intensity of LTA4H on large luteal cells increases and is highest during the mid-luteal phase. High expression was also observed in the corpus luteum during 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 Aug;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, committed step in the biosynthesis of leukotriene B4 (LTB4), an eicosanoid with potent 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 function as potent chemical mediators in a variety of allergic and inflammatory responses. Emerging data suggest that leukotrienes may have an important role in carcinogenesis.LTB4 levels are elevated 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 reticulum molecular chaperones GRP94 and GRP78 in human lung cancer tissues and its significance. Cancer Detect Prev. 2005;29(6):544-51; Larre 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).Expression of the LTB4 receptor is increased in human pancreatic cancer (Hennig R, Ding XZ, Tong WG, et al. 5-Lipoxygenase and leukotriene B(4) receptors are expressed in human pancreatic cancers but not in pancreatic ducts in normal tissue. Am J Pathol. 2002 Aug;161(2):421-8). LTB4 expression 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 B(4) receptor, in Ras transformation. Oncogene. 2004 Dec 9;23(57):9259-68). Inhibition of LTB4 synthesis by treatment with the LTA4H inhibitor bestatin reduced tumor formation 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).Furthermore, topical application of LTB4 to the skin resulted in not only inflammation but also substantial hyperplasia of the epidermis (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; Ruzicka 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 the hypoxia marker HIF1α and 5-lipoxygenase, a macrophage marker, CD68, was positively correlated in ovarian cancer tissues. Furthermore, hypoxic ovarian cancer cell lines showed increased production of LTA4H and 5-LOX metabolites, which are involved in enhanced tumor-associated macrophage infiltration (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 Mar 5;34(10):1241-52).LTA4H may also counteract inflammation through its aminopeptidase activity, which inactivates the tripeptide Pro-Gly-Pro (PGP) by cleaving it (Stsiapanava A, Olsson U, Wan M, et al. Binding of Pro-Gly-Pro at the active site of leukotriene A4 hydrolase / aminopeptidase and development of an epoxide hydrolase selective inhibitor. Proc Natl Acad Sci USA. 2014 Mar 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 efficacy 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 variants was associated with increased survival during glucocorticoid treatment in patients with tuberculous meningitis with moderate disease (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).

[0031] Meteorin-like protein (METRNL) is a hormone (28 kDa secreted protein) induced in skeletal muscle and adipose tissue after exercise and cold exposure, respectively. Increased METRNL expression in the circulation or adipose tissue resulted in the "browning" of white adipose tissue (WAT). Intraperitoneal injection of Metrnl-Fc protein into mice for 7 days induced significant weight loss, increased O2 consumption, and glucose tolerance. Metrnl did not directly affect thermogenesis in white adipocytes in vitro, indicating the involvement of non-adipocyte types in the induction of beige fat. Rather, Metrnl appeared to stimulate several immune cell subtypes to enter adipose tissue and activate their thermogenic effects. METRNL-treated mice showed increased numbers of macrophages and eosinophils in WAT and increased expression of genes related to alternative macrophage activation (Rao RR, Long JZ, White JP, et al. Meteorin-like is a hormone that regulates immune-adipose interactions to increase beige fat thermogenesis. Cell. 2014 Jun 5;157(6):1279-1291). METRNL is associated with innate and possibly adaptive immunity. High METRNL expression was identified in activated monocytes (M2-polarized macrophages), skin, and mucosal tissues. In the skin, METRNL is expressed by resting fibroblasts and IFNγ-treated keratinocytes. 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 a cytokine associated with barrier tissues and alternatively activated macrophages. Clin Immunol. 2015 Feb;156(2):119-27). Recently, Baht and colleagues described a role for METRNL in regulating skeletal muscle repair through macrophage adhesion and phenotypic conversion.The results suggested that METRNL is primarily secreted by macrophages in response to local injury. Furthermore, METRNL promoted anti-inflammatory functions through a STAT3-dependent auto / paracrine mechanism, inducing insulin-like growth factor 1 (IGF-1), which activates muscle precursors and supports 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 Mar;2(3):278-289. Erratum in: Nat Metab. 2020 Aug;2(8):794). Interestingly, METRNL has been suggested to act as a neurotrophic factor with therapeutic potential in neurogenesis. METRNL can actually cross the blood-brain barrier (BBB), and increased blood-brain barrier dysfunction caused increased cerebrospinal fluid METRNL concentrations (Berghoff M, Hopfinger A, Rajendran R, et al. Evidence of a Muscle-Brain Axis by Quantification of the Neurotrophic Myokine METRNL (Meteorin-Like Protein) in Human Cerebrospinal Fluid and Serum. Journal of Clinical Medicine. 2021;10(15):3271).

[0032] Serum METRNL levels have been studied in relation to type 2 diabetes mellitus (T2DM), 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 glucose tolerance,impaired endothelial function and atherosclerosis.Diabetes Res Clin Pract.2019 Apr;150:57-63;Wang C,Pan Y,Song J,et al.Serum Metrnl Level is Correlated with Insulin Resistance,But Not with β-Cell Function in Type 2 Diabetics.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 serum 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 levels of metrnl compared to controls. Furthermore, METRNL was negatively correlated with IL-6 and TNF-α in both CAD patients, and also with 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 2 diabetes mellitus are negatively associated with insulin resistance and inflammatory cytokines. PLoS One. 2018 Sep 13;13(9):e0204180). Furthermore, a case-control study of CAD patients showed a significant association between the presence and severity of CAD and serum METRNL (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). Recently, two studies have examined circulating levels of METRNL in PCOS patients compared with controls. The study by Fouani et al. was conducted in a cohort of PCOS-recurrent pregnancy loss (PCOS-RPL, n=60) and infertile PCOS (n=60) patients and 60 healthy controls. The women ranged in age from 20 to 40 years (mean age of controls: 30.02 ± 4.60 years; mean age of PCOS cases: 29.88 ± 4.22 years). The authors found lower serum METRNL levels in PCOS patients compared with controls. Furthermore, serum METRNL correlated with BMI, adiponectin, and homocysteine in controls and inversely correlated with FBG, fasting insulin, and HOMA-IR in PCOS groups and subgroups. Furthermore, it inversely correlated with hs-CRP in controls and PCOS groups 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 (subfascin) and asprosin levels in plasma samples from 30 PCOS cases and 30 healthy controls (mean age of controls: 28.22 ± 2.6 years; mean age of PCOS cases: 27.14 ± 3.21 years old). Asprosin levels were significantly higher in women with PCOS compared to healthy controls, whereas METRNL levels were significantly lower compared to controls, consistent with the results shown by Fouani et al. Both asprosin and METRNL levels showed a significant correlation with HOMA-IR in the PCOS subgroup (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).

[0033] In patients with inflammatory bowel disease (IBD), serum levels of METRNL were decreased and negative correlations with TNF-α, IL-6, and BMI levels were identified (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 in metabolic and inflammatory diseases, serum METRNL was lower and synovial fluid METRNL was higher in osteoarthritis patients compared to 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 in obesity-osteoarthritis interplay. Cytokine. 2021 Feb;138:155368).

[0034] There is an unmet medical need for an accurate test for the reliable diagnosis of PCOS. Measuring the ratio between LTA4H and METRNL in a sample has the advantage of being a reliable, biofluid-based test for identifying women with PCOS, which is currently not possible. This method can be reliably used for the diagnosis of PCOS in adolescent subjects and young women under the age of 25, particularly under the age of 20, particularly between the ages of 15 and 25, and particularly between the ages of 15 and 20. Diagnosis of PCOS in adolescent patients is difficult for the reasons mentioned above. Therefore, for the first time, the inventors provide an accurate test for the diagnosis of PCOS in adolescent and young female populations. Furthermore, measuring the ratio between LTA4H and METRNL in a sample has the advantage of identifying whether a patient will respond to treatment. An additional benefit of measuring the ratio between LTA4H and METRNL in patient samples is monitoring the progression of PCOS. Furthermore, the inventors include a computer-implemented method for assessing a subject suffering from PCOS by measuring the ratio between LTA4H levels and METRNL levels in a sample, optionally using further criteria such as values for oligoovulation and / or irregular cycles, hyperandrogenism and / or polycystic ovarian morphology, or using further biomarkers or hormones, to assess said subject based on comparison and / or calculation of the above data.

[0035] As mentioned above, patients with PCOS can exhibit two types of characteristics: reproductive and metabolic. The metabolic form of PCOS includes obesity, insulin resistance, metabolic syndrome, prediabetes, type 2 diabetes, nonalcoholic fatty liver disease (NAFLD), and cardiovascular factors. The term "phenotype" can be used instead of "reproductive." The term "reproductive" (or "phenotype") refers to any phenotypic characteristics of a woman known to exhibit PCOS. For example, these reproductive characteristics include polycystic ovarian morphology (PCOM) and / or clinical hyperandrogenism, such as acne, seborrhea, alopecia, and / or hirsutism. Preferably, these reproductive characteristics include polycystic ovarian morphology (PCOM) and / or clinical hyperandrogenism, more preferably acne, seborrhea, alopecia, deep voice, and / or hirsutism. These reproductive characteristics of clinical hyperandrogenism can be diagnosed simply by questioning the woman or are evident after a brief physical examination of the woman's body. Typically, the reference population exhibits none or more than one of these phenotypic characteristics known to be indicative of PCOS.

[0036] In embodiments, the PCOS is assessed from the group consisting of metabolic or phenotypic PCOS. In further embodiments, the PCOS is assessed from the group consisting of phenotype A, phenotype B, phenotype C, and phenotype D PCOS.

[0037] In an embodiment, the method according to the invention is an in vitro method.

[0038] As used herein, the term "diagnose" preferably means assessing whether the subject referred to in accordance with the method of the present invention suffers from PCOS.Preferably, the expression "diagnose PCOS" used herein should be understood as "assisting" or "supporting" the diagnosis of PCOS.For example, a doctor can be supported in diagnosing PCOS by additional information and / or devices.Therefore, the actual diagnosis can be performed by a doctor.

[0039] As will be understood by those skilled in the art, the diagnosis of the present invention is not intended to be accurate for 100% of the subjects tested. The term "diagnose" preferably requires that a correct diagnosis can be made for a statistically significant portion of the subjects. Whether a portion is statistically significant can be easily determined by those skilled in the art using various well-known statistical evaluation tools, such as determining confidence intervals, p-values, Student's t-tests, and Mann-Whitney tests. Details can be found in 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.

[0040] The term "sample" refers to a sample of a body fluid, a sample of separated cells, or a sample from a tissue or organ. Body fluid samples can be obtained by well-known techniques and preferably include blood, plasma, serum, capillary blood, interstitial fluid, peritoneal fluid, menstrual fluid, more preferably blood, plasma or serum samples. Tissue or organ samples can be obtained from any tissue or organ, for example by biopsy. Separated cells may be obtained from body fluids or tissues or organs by separating techniques such as centrifugation or cell sorting. Preferably, cell, tissue or organ samples are obtained from cells, tissues or organs which express or produce the peptides referred to herein.

[0041] 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.

[0042] The term "subject" as referred to herein preferably refers to a mammal. Mammals include, but are not limited to, livestock animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, e.g., 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 certain embodiments, the patient is a female human patient under the age of 25. In certain embodiments, the patient is a female human patient under the age of 20. In certain embodiments, the patient is a female human patient between the ages of 15 and 25. In certain embodiments, the patient is a female human patient between the ages of 15 and 20. In certain embodiments, the patient is a female human patient under the age of 25 and three years postmenarche. In certain embodiments, the patient is a female human patient under the age of 20 and three years postmenarche. In certain embodiments, the patient is a female human patient between the ages of 15 and 25 and three years postmenarche. In certain embodiments, the patient is a female human patient under the age of 15-20 and 3 years post-menarche.

[0043] According to step a) of the method of the present invention, the amount or concentration of LTA4H in a sample from a subject is determined, i.e., measured. According to step b), the amount or concentration of METRNL in a sample from a subject is determined. It should be understood that steps a) and b) can be performed in any order. Also, the steps can be performed simultaneously.

[0044] In embodiments, the amount or concentration of LTA4H and METRNL is determined using an antibody, particularly a monoclonal antibody. In embodiments, steps a) and b) of determining the amount or concentration of LTA4H and METRNL in a patient sample comprise performing an immunoassay. In embodiments, the immunoassay is performed in either a direct or indirect format. In embodiments, such immunoassay is selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), or immunoassays based on luminescence, fluorescence, chemiluminescence, or electrochemiluminescence detection.

[0045] In certain embodiments, step a) of determining the amount or concentration of LTA4H in a patient sample comprises: i) incubating a patient sample with one or more antibodies that specifically bind to LTA4H, thereby producing a complex between the antibody and LTA4H; and ii) quantifying the complex formed in step i) and thereby quantifying the amount or concentration of LTA4H in the patient sample; Includes.

[0046] In certain embodiments, in step i), the sample is incubated with two antibodies that specifically bind to LTA4H. As will be apparent to those skilled in the art, the sample can be contacted with the first and second antibodies in any desired order, for example, the first antibody first, then the second antibody, or the second antibody first, then the first antibody, or simultaneously, for a time and under conditions sufficient to form a first anti-LTA4H antibody / LTA4H / second anti-LTA4H antibody complex. As will be readily apparent to those skilled in the art, it is no more than routine experimentation to determine the appropriate 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 for the formation of a secondary 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).

[0047] The detection of the anti-LTA4H antibody / LTA4H complex can be carried out by any suitable means. The detection of the first anti-LTA4H antibody / LTA4H / second anti-LTA4H antibody complex can be carried out by any suitable means. Those skilled in the art are fully familiar with such means / methods.

[0048] In certain embodiments, a sandwich is formed comprising a first antibody to LTA4H, LTA4H (the analyte), and a second antibody to LTA4H, wherein the second antibody is detectably labeled.

[0049] In one embodiment, a sandwich is formed comprising a first antibody against LTA4H, LTA4H (analyte) and a second antibody against LTA4H, the second antibody being detectably labeled, and the first anti-LTA4H antibody being capable of binding to a solid phase or is bound to a solid phase.

[0050] 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 its antigen-binding fragment that specifically detects METRNL.Preferably, the antibody or its antigen-binding fragment that specifically detects METRNL specifically binds to an epitope of METRNL.The complex formed between the antibody (or fragment) and the biomarker is proportional to the amount or concentration of METRNL.

[0051] 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 is proportional to the amount or concentration of LTA4H.

[0052] The term "antibody" is known in the art. As used herein, this term refers to any immunoglobulin (Ig) molecule composed of four polypeptide chains, two heavy (H) chains and two light (L) chains. As used herein, the term "antibody" also includes antigen-binding fragments of antibodies. As used herein, an antigen-binding fragment of an antibody is one that can specifically bind to an antigen. Thus, 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 (e.g., METRNL or LTA4H). An 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 a Fab fragment, a Fab' fragment, a Facb fragment, a F(ab')2 fragment, an scFv fragment, and an Fv fragment. For example, the antigen-binding fragment is a F(ab')2 fragment. Methods for producing antigen-binding fragments are well known in the art. For example, fragments can be produced by enzymatic cleavage of the antibodies of the present invention. Furthermore, fragments can be generated synthetically or by recombinant techniques. Fab fragments are preferably produced by papain digestion of the antibody, Fab' fragments by pepsin digestion and partial reduction, F(ab')2 fragments by pepsin digestion, and facb fragments by plasmin digestion. Fv or scFv fragments are preferably produced by molecular biology techniques.

[0053] The antibody according to the method of the present invention 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 population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, but excludes possible variants that may arise during the production of the monoclonal antibody, which variants are generally present in minor amounts. The monoclonal antibodies of the present invention can be produced by the well-known hybridoma method described in Kohler and Milstein, Nature, 256:495 (1975), or by recombinant DNA methods. In some embodiments, the monoclonal antibody is selected from the group consisting of a sheep monoclonal antibody, a mouse monoclonal antibody, a rabbit monoclonal antibody, a goat monoclonal antibody, a horse monoclonal antibody, and a chicken monoclonal antibody. In some embodiments, the monoclonal antibody is a mouse monoclonal antibody.

[0054] The antibodies (or fragments) used in steps a) and b) of the method of the invention can be used in a sandwich assay as capture antibodies in combination with at least one other antibody that binds to a different epitope.

[0055] Antibodies can be used in sandwich assays. Sandwich assays are among the most useful and commonly used assays and encompass several variations of sandwich assay technology. For example, in a typical assay, an unlabeled (capture) binding agent is immobilized or can be immobilized on a solid substrate, and the sample to be tested is contacted with the capture binding agent. After a suitable incubation period sufficient to allow the formation of a binding agent-biomarker complex, a second (detection) binding agent labeled with a reporter molecule capable of producing a detectable signal is added and incubated for a period sufficient to allow the formation of another binding agent-biomarker-labeled binding agent complex. Unreacted material can be washed away, and the presence of the biomarker is determined by observation of a signal produced by the reporter molecule bound to the detection binding agent. Results can be qualitative, by simple observation of the visible signal, or quantitated, for example, by comparison with a control sample containing known amounts of the biomarker to be determined (as a standard or calibrator, as described elsewhere herein).

[0056] The incubation step of typical sandwich assay can be modified as needed and suitable.This modification includes, for example, simultaneous incubation, 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 simultaneously added to the immobilized capture binding agent.Also, the sample to be analyzed and the labeled binding agent can be first incubated, and then the antibody that is bound to solid phase or can be bound to solid phase is added.

[0057] The complex formed between the specific binding agent and the biomarker is proportional to the amount or concentration of the biomarker present in the sample. It will be understood that the specificity and / or sensitivity of the applied binding agent defines the extent to which the proportion of at least one marker contained in the sample can be specifically bound. Details of how to perform the measurement are described elsewhere in this specification. The amount or concentration of the complex formed is converted into the amount or concentration of the biomarker, which reflects the amount or concentration actually present in the sample.

[0058] The term "amount" as used herein encompasses the absolute amount of LTA4H or METRNL, the relative amount or concentration of said LTA4H or METRNL, and any value or parameter that correlates thereto or can be derived therefrom. Such values or parameters include intensity signal values derived from any specific physical or chemical property obtained from the peptide by direct measurement, e.g., intensity values in a mass spectrum or NMR spectrum. Furthermore, all values or parameters obtained by indirect measurement as specified elsewhere herein are encompassed, e.g., response levels determined by a biological readout system in response to a peptide or intensity signal obtained from a specifically bound ligand. It should be understood that values that correlate with the above-mentioned amounts or parameters can also be obtained by any standard mathematical operation. According to a preferred embodiment of the present invention, the determination of the "amount" is performed by the disclosed system, whereby a computing device determines the "amount" based on the contacting and measuring steps performed by one or more analyzer units of the system.

[0059] In step c) of the method according to the first, second or third aspect of the invention, a score of the amounts or concentrations determined in steps a) and b), i.e. the amount or concentration of LTA4H and the amount or concentration of METRNL, is calculated.

[0060] The term "calculating" as used herein refers to evaluating a score based on the amount or concentration of LTA4H and the amount or concentration of METRNL determined in a subject's sample(s). For example, it is assumed 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 this 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. Furthermore, biomarkers can be weighted according to their contribution to establishing a diagnosis. The score can be considered as a classification parameter for diagnosing PCOS. In particular, the score should enable the diagnosis of PCOS based on comparison with the reference score. The reference score is preferably a value, particularly a cutoff value, that allows for the distinction between subjects with PCOS and those without PCOS.

[0061] Preferably, the score 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 present 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 another embodiment, the ratio is the ratio of the amount or concentration of METRNL to the amount or concentration of LTA4H.

[0062] In step d) according to the first, second or third embodiment of the method of the present invention, the score calculated in step c) is compared to a reference score, for example, the calculated ratio is compared to a reference ratio.

[0063] The term "comparing" as used herein includes comparing the calculated scores of samples from test subjects with appropriate reference sources specified elsewhere in this specification.Comparing is preferably assisted by automation.For example, a suitable computer program containing an algorithm for comparing the calculated scores of subjects with reference scores can be used.Such computer programs and algorithms are well known in the art.Notwithstanding the above, comparison can also be performed manually.The computer program may further evaluate the results of the comparison, i.e., automatically provide the desired evaluation, i.e., diagnostic result, in an appropriate output format.The diagnostic result can preferably serve as an aid for, for example, medical professionals to establish a final diagnosis of PCOS.

[0064] The calculating and / or comparing steps may be performed using a computer comprising a processing unit.

[0065] Based on the comparison of the calculated score with the reference score, it is possible to assess whether the test subject suffers from PCOS. For example, the result of the comparison may be presented as raw data, or in some cases as an indicator in the form of a word, phrase, symbol, or number that may indicate a specific diagnosis. Therefore, the reference score is selected so that either the difference or identity of the calculated score with respect to the reference score allows for the identification of the test subject as belonging to a group of subjects suffering from PCOS or not suffering from PCOS. This method allows for either the exclusion (rule-out) or identification (rule-in) of subjects suffering from PCOS. The difference in score, i.e., increase or decrease, as used herein, is preferably a statistically significant difference. This method may also use other diagnostic criteria, such as oligoovulation or hyperandrogenism, to assess whether a patient suffers from PCOS. (Page 26, paragraph 3)

[0066] Preferably, reference score, such as reference ratio, can distinguish whether subject suffers from PCOS or not.Preferably, diagnosis is made by assessing whether test subject's score is above or below reference score.It is not necessary to provide an accurate reference score.Relevant reference score can be obtained by correlating sensitivity and specificity and the sensitivity / specificity for any score.The reference score that brings about high sensitivity brings about lower specificity, and vice versa.

[0067] In some embodiments, the reference score is derived from a sample from a subject (or a group of subjects) known to have PCOS.

[0068] In some embodiments, the reference score is derived from a sample from a subject (or a sample group of subjects) known not to have PCOS.

[0069] As mentioned above, the score calculated in step c) according to the first, second or third aspect of the present invention may 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 (i.e., calculated ratio) greater than the reference ratio is indicated for a subject suffering from PCOS. A ratio lower than the reference ratio is indicated for a subject not suffering from PCOS. In another 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 (i.e., calculated ratio) lower than the reference ratio is indicated for a subject suffering from PCOS. A ratio higher than the reference ratio is indicated for a subject not suffering from PCOS.

[0070] In a preferred embodiment, the method of the present invention further comprises the step of recommending an appropriate treatment if PCOS is diagnosed. Alternatively, the method further comprises the step of initiating an appropriate treatment if PCOS is diagnosed.

[0071] The term "recommend" as used herein means to establish a suggested treatment that can be applied to a subject. However, it should be understood that this term does not include any actual application of treatment. The recommended treatment depends on the diagnostic result provided by the method of the present invention. The above recommendation step can also be preferably automated. Preferably, the diagnosis obtained by the method of the present invention, i.e., the diagnostic result of the method, is used to search a database containing recommended treatment measures for each possible diagnostic result.

[0072] In one embodiment, the recommended or initiated treatment is selected for a drug-based treatment of PCOS or lifestyle changes to control metabolic symptoms. In an embodiment, the drug-based treatment of PCOS is selected from the group consisting of drugs to regulate periods, particularly oral contraceptives or progestin therapy, drugs to prevent or control diabetes, particularly type 2 diabetes, drugs to prevent or control high cholesterol, hormones or drugs to enhance fertility, drugs, hormones or procedures to remove excess hair, and drugs or procedures to control acne.

[0073] The method of the present invention may also be implemented as a computer-implemented invention. In one embodiment, one or more steps, such as the comparing step and / or the calculating step, are performed by a computer (i.e., a computer) including a processing unit. In another embodiment, all steps are performed by a computer with a processing unit.

[0074] Accordingly, a fourth aspect of the present invention provides a computer-implemented method for diagnosing PCOS in a subject, comprising: (a) in a processing unit, (a1) the amount or concentration value of LTA4H in a sample from a subject; and (a2) the amount or concentration of METRNL in a sample from the subject; receiving the (b) processing, by a processing unit, the values received in step (a), said processing comprising: (b1) calculating a score of the values (a1) and (a2) received in step a); (b2) comparing the calculated score with a reference score; a processing step comprising: (c) optionally providing a diagnosis via an output device, the diagnosis being based on the results of step b); The present invention relates to a computer-implemented method, including:

[0075] In some embodiments, the processing unit is included in a computer.

[0076] In some embodiments, step b) according to the fourth aspect of the invention further comprises, in the processing unit, retrieving a reference score from memory, i.e. a standard score suitable for diagnosing PCOS.

[0077] In one embodiment of the method of the present invention, information about the diagnosis (by the final step of the method of the present invention) is provided via a display configured to present the evaluation. Thus, as described elsewhere herein, information can be provided regardless of whether the subject suffers from PCOS. Furthermore, a recommendation for an appropriate treatment can be displayed. As described elsewhere herein, various treatment measures can be recommended. In this case, the treatment option(s) can be displayed on the display.

[0078] In one embodiment of the method of the present invention, the method may comprise the further step of transferring information relating to the evaluation of the method of the present invention to the subject's electronic medical record.

[0079] Alternatively, the assessment performed in the final step of the method of the present invention can be printed by a printer, with the printout containing information on whether the patient is at risk or not at risk and / or a recommendation for appropriate treatment measures.

[0080] The present invention further relates to the use of i) LTA4H and METRNL as biomarkers, or ii) at least one agent that specifically binds to METRNL and at least one agent that specifically binds to LTA4H for diagnosing PCOS.Preferably, this use is carried out in vitro, i.e., 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.

[0081] 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.

[0082] The term "kit" as used herein refers to a collection of the aforementioned means, provided, for example, separately or in a single container. The container may contain instructions for carrying out the method of the present invention. In one embodiment, the kit includes reagents for diagnosing PCOS. The reagents of the kit may include an antibody or antibody fragment. Preferably, the antibody or antibody fragment recognizes an epitope or antigen of LTA4H and / or METRLN. The kit may further include other reagents that recognize other biomarkers. Thus, the kit may also include a combination of at least three reagents. According to the present invention, the biomarker may also include a hormone such as anti-Müllerian hormone (AMH). The kit can be used in any diagnostic assay. [Example]

[0083] The present invention is illustrated solely by the following examples, which should not be construed in any way as limiting the scope of the invention.

[0084] Example 1: Diagnostic performance of the ratio between the two biomarkers LTA4H and METRNL in women with PCOS (phenotypes A, B, C and D) and controls, determined by ELISA technique Performance validation was performed on a sample set of 82 cases (serum samples from women with PCOS) and 44 controls (serum samples from healthy women).

[0085] The concentrations of 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.

[0086] The concentration of LTA4H in human serum was determined using the Human LTA4H ELISA Kit Ver. 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 kits are solid-phase sandwich enzyme-linked immunosorbent assays (ELISAs) designed to detect and quantitate human LTA4H and METRNL levels in cell culture supernatants, plasma, and serum, respectively. The two analytes were measured on separate plates, and only one analyte was measured at a time.

[0087] For LTA4H quantification, the kit was provided with a human LTA4H antibody-precoated plate. Samples were measured at two-fold dilutions. After all reagents were brought to room temperature, 100 μL of each sample and standard was added. Samples and standards were measured in duplicate. Any LTA4H present was allowed to bind to the immobilized capture antibody on the microtiter plate during a 2.5-hour incubation at room temperature on a microplate shaker set at 650 rpm. Unbound material was removed from the plate during a washing step (4 × 300 μL), after which 100 μL of diluted anti-LTA4H biotin conjugate was added to the wells. After a 1-hour incubation on the shaker and another washing step (4 × 300 μL) to remove unbound detection antibody, 100 μL of the prepared streptavidin-HRP solution was added to the plate. This was followed by a 45-minute incubation at room temperature and four washing steps (4 × 300 μL). After the final wash, 100 µL of TMB substrate was added to the plate. The plate was incubated for 30 minutes at room temperature in the dark with gentle shaking. During incubation, the substrate turned blue. Color developed in proportion to the amount of LTA4H bound in the first step. Color development was stopped by adding 50 µL of stop solution, and the solution in the wells changed from blue to yellow. The color intensity was measured using a plate reader at 450 nm for detection and 570 nm for background subtraction. To generate a calibration curve, the lyophilized recombinant LTA4H delivered with the kit was reconstituted and diluted with calibrator diluent. The calibration range of the assay is 2.048 ng / mL to 500 ng / mL. Calibrator 1 (500 ng / mL) corresponds to the reconstituted stock solution, and calibrators 2 to 7 (2.048 ng / mL) were prepared by serial 2.5-fold dilution steps in calibrator diluent. Pure calibrator dilutions served as blanks (0 ng / mL). Calibration curves were fitted using unweighted four-parameter nonlinear regression (Newton / Raphson).

[0088] The capture antibody against 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 three times with 400 μL of wash buffer per well. After the final wash, the wash buffer was completely removed, and the plate was blocked by adding 300 μL of reagent diluent to each well and incubated at room temperature for a minimum of 1 hour. The plate was washed three times with 400 μL of wash buffer per well and was ready for use. Samples were measured at four-fold dilutions. After all reagents were brought to 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 allowed to bind to the immobilized capture antibody on the microtiter plate. During washing steps (3 × 400 μL), unbound material was removed from the plate before 100 μL of anti-METRNL detection antibody diluted in reagent diluent was added to the wells. After a 2-hour incubation and another washing step (3 × 400 μL) to remove unbound detection antibody, 100 μL of the prepared streptavidin-HRP solution was added to the plate. This was followed by a 20-minute incubation at room temperature and washing steps (3 × 400 μL) avoiding direct exposure to light. After the final wash, 100 μL of substrate solution was added to the plate. The plate was incubated for 20 minutes at room temperature avoiding direct exposure to light. During the incubation, the substrate turned blue. The color developed was proportional to the amount of METRNL bound in the first step. Color development was stopped by adding 50 µL of stop solution. The color of the solution in the wells changed from blue to yellow, and the color intensity was measured using a plate reader at 450 nm for detection and 540 or 570 nm for background subtraction. This subtraction compensates for optical imperfections in the plate. To create a standard curve, the lyophilized recombinant METRNL delivered with the kit was reconstituted and diluted in reagent diluent. The calibration range for the METRNL assay is 15.6 pg / mL to 1000 pg / mL. Two-fold serial dilutions of recombinant METRNL in reagent diluent were used to obtain a seven-point standard curve.The calibration curve was fitted using a four-parameter logistic (4-PL, Newton / Raphson) curve fit.

[0089] Once the concentrations of the two analytes were obtained by ELISA immunoassay, the LTA4H:METRNL ratio was calculated for each sample.

[0090] A receiver operating characteristic (ROC) curve was constructed for the LTA4H:METRNL ratio (Figure 1). Model performance is determined by examining the area under the curve (AUC). The best possible AUC is 1, and the worst possible AUC is 0.5. ROC curve analysis of the LTA4H:METRNL ratio for PCOS cases when all phenotypes were combined (phenotypes A-D) 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 for discriminating between women with PCOS (cases, PCOS phenotypes A-D) and healthy control subjects using ROC analysis is shown in Table 1, which lists the AUC and associated 95% confidence intervals for the ROC curve analysis. [Table 1]

[0091] Using the data obtained by running the ratio of LTA4H to METRNL concentrations, box plots were generated for controls and PCOS cases for all phenotypes combined (phenotypes A-D). The LTA4H:METRNL ratio is increased in women with PCOS compared to healthy controls (Figure 2).

[0092] Table 2 shows the diagnostic performance of the LTA4H:METRNL ratio for discriminating women with PCOS relative to healthy control subjects when separated by the different phenotypes A, B, C and D. The AUC for each phenotype is reported in the table. [Table 2]

[0093] ROC curve analysis of the LTA4H:METRNL ratio for PCOS cases divided by different phenotypes relative to healthy controls showed AUCs of 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-D, respectively (Figure 3). The results confirm the high diagnostic accuracy of the LTA4H:METRNL ratio for PCOS. The LTA4H:METRNL ratio in all different PCOS phenotypes (phenotypes A-D) showed increased levels compared to healthy controls (Figure 4).

[0094] Table 3 shows the diagnostic performance of the LTA4H:METRNL ratio in young women (age 25 and under) for distinguishing young women with PCOS from young healthy control subjects for all phenotypes combined (phenotypes A-D). [Table 3]

[0095] ROC curve analysis of the LTA4H:METRNL ratio for young PCOS cases (age 25 or younger) for all phenotypes combined (phenotypes A-D) showed an AUC of 1.00, confirming high diagnostic accuracy for women aged 25 or younger in distinguishing PCOS cases from controls (95% CI 1-1, Figure 5). When only young women (age 25 or younger) were included in the analysis, PCOS cases (all phenotypes A-D combined) showed an increased LTA4H:METRNL ratio compared to young controls (age 25 or younger, Figure 6).

[0096] The diagnostic performance of the LTA4H:METRNL ratio for discriminating young women (aged 25 years and under) with PCOS when separated by the different phenotypes A, B, C, and D relative to healthy control subjects (aged 25 years and under) was evaluated, and the results are reported in Table 4 (AUC of PCOS phenotypes relative to controls). [Table 4]

[0097] ROC curve analysis of the LTA4H:METRNL ratio for young PCOS cases (age 25 or younger, phenotypes A–D) 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 or younger (Figure 7). The LTA4H:METRNL ratio was increased in all different PCOS phenotypes (phenotypes A–D, age 25 or younger) when compared with the LTA4H:METRNL ratio in young healthy controls (age 25 or younger, Figure 8).

[0098] Example 2: Diagnostic performance of the ratio between the two biomarkers LTA4H and METRNL in women with PCOS (phenotypes A, B, C and D) and controls, determined by ELISA technique, in different age groups Performance validation was performed on an additional sample set of 240 cases (serum samples from women with PCOS) and 48 controls (serum samples from healthy women).

[0099] The concentrations of 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) and belonged to three different age groups: 15-20 (n = 70), 20-25 (n = 99), and 25-40 (n = 71). The control group included healthy women without PCOS.

[0100] The concentration of LTA4H in human serum was determined using the Human LTA4H ELISA Kit Ver. 1 manufactured by Invitrogen (Cat. No.: EH308RB). The concentration of METRNL in human serum was determined using the Human METRNL ELISA Kit manufactured by R&D Systems (Cat. No.: DY7867-05). Measurements were performed as described in Example 1.

[0101] A receiver operating characteristic (ROC) curve was constructed for the LTA4H:METRNL ratio (Figure 9). Model performance is determined by examining the area under the curve (AUC). ROC curve analysis of the LTA4H:METRNL ratio for PCOS cases when all phenotypes were combined (phenotypes A-D) showed an AUC of 0.93 (95% CI 0.90-0.96), confirming the high diagnostic accuracy of the LTA4H:METRNL ratio for PCOS (Figure 9).

[0102] The diagnostic performance of the LTA4H:METRNL ratio to distinguish between women with PCOS (cases, PCOS phenotypes A-D) and healthy control subjects using ROC analysis is shown in Table 5, which lists the AUC of the ROC curve analysis and the associated 95% confidence interval. Results were obtained using an ELISA immunoassay. [Table 5]

[0103] Using data obtained by ELISA immunoassay, box plots were generated for controls and PCOS cases for all phenotypes combined (phenotypes A-D). The LTA4H:METRNL ratio is increased in women with PCOS compared to healthy controls (Figure 10).

[0104] Table 6 shows the diagnostic performance of the LTA4H:METRNL ratio for discriminating women with PCOS relative to healthy control subjects when separated by the different phenotypes A, B, C and D. The AUC for each phenotype is reported in the table. [Table 6]

[0105] ROC curve analysis of the LTA4H:METRNL ratio for PCOS cases separated by different phenotypes versus healthy controls showed AUCs of 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-D, respectively (Figure 11). The results confirm the high diagnostic accuracy of the LTA4H:METRNL ratio for PCOS.

[0106] The LTA4H:METRNL ratio in all different PCOS phenotypes (phenotypes AD) was increased compared to healthy controls (Figure 12).

[0107] Table 7 shows the diagnostic performance of the LTA4H:METRNL ratio in different age groups (15 to <20 years, 20 to <25 years, 25 to <40 years) for discriminating women with PCOS from healthy control subjects when all phenotypes are combined (phenotypes A to D). [Table 7]

[0108] ROC curve analysis of the LTA4H:METRNL ratio for PCOS cases divided by different age groups versus healthy controls showed AUCs of 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 (Figure 13). The results confirm the high diagnostic accuracy of the LTA4H:METRNL ratio for PCOS in all different age groups.

[0109] An increased LTA4H:METRNL ratio in women with PCOS compared to controls was observed across all different age groups (Figure 14).

[0110] Given the lack of reliable biomarkers for diagnosing PCOS, especially in younger women (<25 years), a separate analysis was performed for the age group ≥15 and <25 years.

[0111] Table 8 shows the diagnostic performance of the LTA4H:METRNL ratio in young women (aged 15 to under 25 years) for distinguishing young women with PCOS from young healthy control subjects for all phenotypes combined (phenotypes A to D). [Table 8]

[0112] ROC curve analysis of the LTA4H:METRNL ratio for young PCOS cases (ages 15 to 25) for all phenotypes combined (phenotypes A-D) showed an AUC of 0.92, confirming the high diagnostic accuracy of 15-25 year old women in distinguishing PCOS cases from controls (95% CI 0.87-0.97, Figure 15). When only young women (ages 15-25) were included in the analysis, PCOS cases (all phenotypes A-D combined) showed an increased LTA4H:METRNL ratio compared to young controls (ages 15 to 25, Figure 16).

[0113] The diagnostic performance of the LTA4H:METRNL ratio for discriminating young women (aged ≥ 15 and < 25 years) with PCOS when separated by the different phenotypes A, B, C and D relative to healthy control subjects (aged ≥ 15 and < 25 years) was evaluated and the results are reported in Table 9 (AUC of PCOS phenotypes relative to controls). [Table 9]

[0114] Receiver operating characteristic curve analysis of the LTA4H:METRNL ratio for young PCOS cases (ages 15 to <25 years, phenotypes A to D) showed AUCs of 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) for each phenotype, respectively, confirming the high diagnostic accuracy of the LTA4H:METRNL ratio for PCOS in the subgroup of women aged 15 to 25 years (Figure 17). The LTA4H:METRNL ratio was increased in all different PCOS phenotypes (phenotypes A to D, ages 15 to <25 years) when compared with the LTA4H:METRNL ratio in young healthy controls (ages 15 to <25 years, Figure 18).

Claims

1. 1. A method for diagnosing PCOS in a subject, 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 a score for said amounts or concentrations determined in steps a) and b); d) comparing the calculated score with a reference score; and e) diagnosing PCOS in a subject; A method comprising:

2. 1. A method of selecting a patient for treatment of PCOS, 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 a score for 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 treatment; A method comprising:

3. 1. A method for monitoring PCOS progression in a subject with PCOS or for monitoring response to treatment in a subject with PCOS, 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 a score for said amounts or concentrations determined in steps a) and b); d) comparing the calculated score with a reference score; and e) monitoring the progression in said subject suffering from or being treated for PCOS; A method comprising:

4. The method of any one of 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 the subject is a human subject.

5. 5. The method of any one of claims 1 to 4, wherein the subject is suspected of suffering from PCOS, such as 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 the amount or concentration of LTA4H comprises contacting said sample with an antibody or antigen-binding fragment thereof that specifically detects LTA4H.

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

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

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

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

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

12. i) the use of LTA4H and METRNL as biomarkers, 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.

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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