FGFBP1 to METRNL ratio in the assessment of polycystic ovary syndrome

Abstract

The present invention relates to a method for diagnosing polycystic ovary syndrome (PCOS) in a subject, comprising the steps of: a) determining the amount or concentration of total FGFBP1 in a sample from the subject; b) determining the amount or concentration of METRNL in the sample from the subject; c) calculating a score for 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 FGFBP1 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 amount or concentration determined in steps a) and b); d) comparing the calculated score with a standard score; and e) diagnosing PCOS in the subject. Furthermore, the present invention 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 FGFBP1 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 amount or concentration determined in steps a) and b); d) comparing the calculated score with a standard 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, said method comprising the steps of: a) determining the amount or concentration of FGFBP1 in a sample from the subject; b) determining the amount or concentration of METRNL in a sample from the subject; c) calculating a score for 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 symptoms, which can be reproductive and / or metabolic. Reproductive symptoms include irregular menstrual cycles, infertility, pregnancy complications, and hirsutism. Metabolic symptoms include obesity, insulin resistance, metabolic syndrome, prediabetes, type 2 diabetes, and cardiovascular factors. These clinical symptoms 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 definitive diagnosis of PCOS, other conditions or disorders, 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 disorders include, for example: 17α-hydroxyprogesterone (17-OHP) to rule out NCAH (Nordenstrom and Falhammar 2018) Prolactin to eliminate hyperprolactinemia Cortisol to rule out patients with Cushing's syndrome Thyroid-stimulating hormone (TSH) to rule out thyroid disorders

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

[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 Rotterdam criteria are the most widely used diagnostic criteria for PCOS. PCOS is indicated by the presence of at least two of the following criteria: (i) irregular cycles (oligomenorrhea) and / or ovulatory dysfunction (oligo-anovulation, 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 a menstrual 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 are defined as clinical hyperandrogenism as hirsutism (excessive male-pattern hair growth) and / or acne, and biochemical hyperandrogenism as higher levels of free androgen compared to healthy controls. Clinical hyperandrogenism is also defined as a modified Ferriman-Galway score greater than 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 either: a follicle count of >20 per ovary and / or an ovarian volume of ≥10 ml, which ensures 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 in either ovary or a follicle count of >12.

[0007] Another method for detecting PCOM is to measure anti-Müllerian hormone (AMH) in subjects. AMH is a glycoprotein hormone whose expression is important for sexual differentiation at specific times during fetal development. Furthermore, AMH, produced by granulosa cells of growing follicles, typically correlates with the number of antral follicles in the ovary. Therefore, serum AMH levels can be a surrogate biomarker for antral follicle count (AFC), as 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 the presence of two of the following three criteria constitutes a diagnosis of PCOS: (i) oligomenorrhea (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 oligomenorrhea (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-to-FSH ratio for diagnosing PCOS appears low, as only a small proportion of women with PCOS had significantly elevated LH-to-FSH ratios (Cho et al. 2005). In fact, the range of LH-to-FSH ratios seen in women diagnosed with PCOS is wide (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 an appropriate ultrasound device 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 serum testosterone measurements may be enhanced by simultaneous measurement of SHBG, in which case calculating free T concentrations 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 for diagnosing 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 indications that blood androgen levels differ between ethnic groups, with the Japanese population having a low 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 PCOS medications available. Treatment is symptom-directed and tailored to individual needs. Therapeutic approaches target hyperandrogenism, irregular cycles, and / or ovulatory dysfunction, as well as associated metabolic disorders such as diabetes. The 2018 International Evidence-Based Guidelines for the Evaluation and Management of Polycystic Ovary Syndrome provide information to aid 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 it took more than two years (33.6%) and more than three healthcare professionals (47.1%) to establish a 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 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 particular area of concern in diagnosing PCOS is young women, i.e., adolescents and young women under the age of 25, when characteristics of normal pubertal development overlap with adult diagnostic criteria. This makes the diagnosis controversial and difficult. Many of the symptoms used to diagnose PCOS evolve over time and may change during the first few years after menstruation. Normal pubertal physiological changes, such as irregular menstrual cycles, acne, and PCOM, overlap with 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 for adolescents younger than 8 years after menarche because of 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 during adolescence. During the early years of menstruation, immaturity of the hypothalamic-pituitary-ovarian axis often results in anovulation, and cycles may be somewhat longer. However, 90% of cycles are within the 21-45 day range. However, cycles shorter than 20 days and longer than 45 days can occur. From menarche to 3 years, 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 know what is normal, and patients may not inform their caregivers about irregular or missed periods.Additionally, 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, establishing reliable biomarkers to aid in the diagnosis of PCOS or to identify patients at risk for developing PCOS is of utmost importance, particularly for this patient population. Delayed diagnosis in adolescents and young women is often due to a reluctance to diagnose at-risk adolescents due to adolescence and fear of over- 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 oligo-anovulation, 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 is no universal biomarker 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] Thus, there is an unmet need to establish better diagnostic assays for diagnosing PCOS in 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, said method comprising: a) determining the amount or concentration of FGFBP1 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 with a reference score; e) diagnosing PCOS in the subject.

[0020] Preferably, step e) is based on the result of the comparison step d). Thus, step e) comprises the following steps: 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 FGFBP1 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 with a reference score; e) selecting a patient for PCOS treatment.

[0022] Preferably, step e) is based on the result of the comparison step d). Thus, step e) comprises the following steps: e) selecting a patient 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, said method comprising: a) determining the amount or concentration of FGFBP1 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 with a reference score; e) monitoring progression in a subject suffering from or being treated for PCOS.

[0024] Preferably, step e) is based on the result of the comparison step d). Thus, step e) comprises the following steps: e) monitoring the progression in a 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, said method comprising: (a) in a processing unit, (a1) the amount or concentration value of FGFBP1 in a sample derived from a subject; and (a2) the amount or concentration of METRNL in a sample from the subject; receiving a (b) processing the values received in step (a) in a processing unit, (b1) calculating the scores of the values (a1) and (a2) received in step a); (b2) comparing the calculated score with a reference score; a processing step including: (c) optionally providing a diagnosis via an output device, said diagnosis being based on the results of step b).

[0026] In a fifth aspect, the present invention relates to the use of FGFBP1 and METRNL as biomarkers for diagnosing 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 FGFBP1 for diagnosing 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 FGFBP1. [Brief explanation of the drawings]

[0028] [Figure 1] ROC curve analysis of the ratio between the two serum biomarkers FGFBP1 and METRNL for PCOS cases for all phenotypes combined (phenotypes A–D). The ratio was taken as the performance between the concentrations of the biomarkers obtained by ELISA immunoassay. [Figure 2]Ratio values of two serum biomarkers, FGFBP1 and METRNL, in controls versus PCOS cases for all phenotypes combined (phenotypes A-D). Ratios were calculated as the performance between the concentrations of the biomarkers obtained by ELISA immunoassay. [Figure 3] ROC curve analysis of the ratio between two serum biomarkers, FGFBP1 and METRNL, for PCOS phenotypes A-D compared with controls. The ratio was taken as the performance between the concentrations of the biomarkers obtained by ELISA immunoassay. [Figure 4] Ratio values between two serum biomarkers, FGFBP1 and METRNL, in healthy controls and PCOS phenotypes A to D. The ratios were taken as performance between the concentrations of the biomarkers obtained by ELISA immunoassay. [Figure 5] ROC curve analysis of the ratio between two serum biomarkers, FGFBP1 and METRNL, for young PCOS cases (age 25 or younger) for all phenotypes A-D combined. Ratios were taken as the performance between the concentrations of biomarkers obtained by ELISA immunoassay. [Figure 6] Ratio values between two serum biomarkers, FGFBP1 and METRNL, in young controls versus young PCOS cases (age ≤25 years) for combined phenotypes A–D. Ratios were taken as performance between biomarker concentrations obtained by ELISA immunoassay. [Figure 7] ROC curve analysis of the ratio between two serum biomarkers, FGFBP1 and METRNL, for young PCOS cases (age ≤25 years) versus young healthy controls (age ≤25 years) separated by phenotypes A–D. Ratios were taken as the performance between biomarker concentrations obtained by ELISA immunoassay. [Figure 8]Ratio values between two serum biomarkers, FGFBP1 and METRNL, in young healthy controls (age ≤25 years) and in patients with PCOS phenotypes A-D (age ≤25 years). The ratios were taken as the performance between the concentrations of the biomarkers obtained by ELISA immunoassay. [Figure 9] ROC curve analysis of the ratio between the two serum biomarkers FGFBP1 and METRNL for PCOS cases for all phenotypes combined (phenotypes A–D). The ratio was taken as the performance between the concentrations of the biomarkers obtained by ELISA immunoassay. [Figure 10] Ratio values of two serum biomarkers, FGFBP1 and METRNL, in controls versus PCOS cases for all phenotypes combined (phenotypes A-D). Ratios were calculated as the performance between the concentrations of the biomarkers obtained by ELISA immunoassay. [Figure 11] ROC curve analysis of the ratio between two serum biomarkers, FGFBP1 and METRNL, for PCOS phenotypes A-D compared with controls. The ratio was taken as the performance between the concentrations of the biomarkers obtained by ELISA immunoassay. [Figure 12] Ratio values between two serum biomarkers, FGFBP1 and METRNL, in healthy controls and PCOS phenotypes A to D. The ratios were taken as performance between the concentrations of the biomarkers obtained by ELISA immunoassay. [Figure 13] ROC curve analysis of the ratio between two serum biomarkers, FGFBP1 and METRNL, for different PCOS age groups (15-20, 20-25, 25-45) compared with controls. The ratio was taken as the performance between the concentrations of the biomarkers obtained by ELISA immunoassay. [Figure 14] Ratio values between two serum biomarkers, FGFBP1 and METRNL, in healthy controls and PCOS age groups (15-20, 20-25, 25-45). Ratios were taken as the performance between the concentrations of the biomarkers obtained by ELISA immunoassay. [Figure 15] ROC curve analysis of the ratio between two serum biomarkers, FGFBP1 and METRNL, for young PCOS cases (15-25 years old) for all phenotypes A-D combined. The ratio was taken as the performance between the concentrations of the biomarkers obtained by ELISA immunoassay. [Figure 16] Ratio values between two serum biomarkers, FGFBP1 and METRNL, in young healthy controls versus young PCOS cases (15-25 years old) for combined phenotypes A-D. Ratios were calculated as the performance between biomarker concentrations obtained by ELISA immunoassay. [Figure 17] ROC curve analysis of the ratio between two serum biomarkers, FGFBP1 and METRNL, for young PCOS cases (15-25 years old) versus young healthy controls (15-25 years old) separated by phenotypes A-D. The ratio was taken as the performance between the concentrations of the biomarkers obtained by ELISA immunoassay. [Figure 18] Ratio values between two serum biomarkers, FGFBP1 and METRNL, in young healthy controls (15-25 years old) and in PCOS phenotypes A-D (15-25 years old). The ratios were calculated as the performance between the concentrations of the biomarkers 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 that consist essentially of the steps described above or methods that include 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 further markers and / or collecting pre-treatment samples or evaluating the results obtained by the above 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 determining steps (a) and (b), or for computer-implemented calculation of step (c), or computer-implemented comparison of step (d). Therefore, the methods of the present invention may be computer-implemented.

[0030] Fibroblast growth factor binding protein 1 (FGFBP1, aliases FGF-BP, FGFBP1, FGFBP, FGFBP-1 or HBP17) is a 26.2 kDa protein that belongs to the fibroblast growth factor binding protein family. FGFBP1 binds to FGF1, FGF2, FGF7, FGF10, FGF22, and HSPG2 (perlecan) in a reversible and noncovalent manner (Wu DQ, Kan MK, Sato GH, et al. Characterization and molecular cloning of a putative binding protein for heparin-binding growth factors. J Biol Chem. 1991 Sep 5;266(25):16778-85; Beer HD, Bittner M, Niklaus G, et al. The fibroblast growth factor binding protein is a novel interaction partner of FGF-7, FGF-10, and FGF-22 and regulates FGF activity: implications for epithelial repair. Oncogene. 2005 Aug 11;24(34):5269-77; Abuharbeid S, Czubayko F, Aigner A. The fibroblast growth factor-binding protein FGF-BPs. Int J Biochem Cell Biol. 2006;38(9):1463-8). Many members of the FGF family are anchored in the extracellular matrix (ECM), bind to heparin sulfate proteoglycans (HSPGs), and are released from this storage site by proteases and heparanases. FGFBPs shuttle FGFs from their storage sites to their receptors (Turner N, Grose R. Fibroblast growth factor signaling: from development to cancer.Nat Rev Cancer 2010,10:116-129). Biochemical studies have revealed that FGFBP1 binds to FGF2 in a dose-dependent and specific manner, and that this binding is inhibited by FGF1, heparan sulfate, and heparinoids (Tassi E, Al-Attar A, Aigner A, et al. Enhancement of fibroblast growth factor (FGF) activity by an FGF-binding protein. J Biol Chem 2001,276:40247-40253). This FGFBP1 / FGF2 interaction results in a significant decrease in the affinity of FGF2 for heparin, thus leading to the FGFBP1-mediated release of FGF2 from the ECM (Aigner A, Butscheid M, Kunkel P, et al. An FGF-binding protein (FGF-BP) exerts its biological function by parallel paracrine stimulation of tumor cell and endothelial cell proliferation through FGF-2 release. Int J Cancer 2001, 92:510-517). Several findings from different laboratories indicate that FGFBP1 may contribute to embryonic development, angiogenesis, wound healing, tumor growth and malignant progression, as well as maintenance and reinnervation of neuromuscular junctions and blood-brain barrier development (Czubayko F, Smith RV, Chung HC, Wellstein A. Tumor growth and angiogenesis induced by a secreted binding protein for fibroblast growth factors. J Biol Chem 1994,269:28243-28248; Mongiat M, Otto J, Oldershaw R, et al. Fibroblast growth factor-binding protein is a novel partner for perlecan protein core.J Biol Chem 2001,276:10263-10271;Tassi E,Al-Attar A,Aigner A,et al.Enhancement of fibroblast growth factor(FGF)activity by an FGF-binding protein.J Biol Chem 2001,276:40247-40253;Gibby KA,McDonnell K,Schmidt MO,Wellstein A.A distinct role for secreted fibroblast growth factor-binding proteins in development,Proc Natl Acad Sci USA:2009,106:8585-8590;Czubayko F,Liaudet-Coopman ED,Aigner A,et al.A secreted FGF-binding protein can serve as the angiogenic switch in human cancer.Nat Med 1997,3:1137-1140;Kurtz A,Aigner A,Cabal-Manzano RH,et al.Differential regulation of a fibroblast growth factor-binding protein during skin carcinogenesis and wound healing.Neoplasia 2004,6:595-602;Kurtz A,Wang HL,Darwiche N,et al.Expression of a binding protein for FGF is associated with epithelial development and skin carcinogenesis.Oncogene 1997,14:2671-2681;Tassi E,Henke RT,Bowden ET,et al.Cancer Res 2006,66:1191-1198;Tassi E,Wellstein A.Expression of a fibroblast growth factor-binding protein during the development of adenocarcinoma of the pancreas and colon.Semin Oncol 2006,326:1549-1554;Williams AH,Valdez G,Moresi V,et al.MicroRNA-206 delays ALS progression and promotes regeneration of neuromuscular synapses in mice Am J Pathol.2011 Nov;179(5):2220-32;Cottarelli A,Corada M,Beznoussenko GV,et al.Fgfbp1 promotes blood-brain barrier development by regulating collagen IV deposition and maintaining Wnt / β-catenin signaling.Development.2020 Aug 24;147(16):dev185140) FGFBP1 The mRNA was analyzed, transcribed, and quantified using a fon Tacer (Fon Tacer). K,Bookout AL,Ding X,et al.Research resource: Comprehensive expression atlas of the Fibroblast Growth Factor system in adult mice. Mol Endocrinol 2010,24:2050-2064; Kurtz A, Wang HL, Darwiche N, et al. Expression of a binding protein for FGF is associated with epithelial development and skin carcinogenesis. Oncogene 1997,14:2671-2681). Genetic polymorphisms in the human FGFBP1 gene were associated with higher gene and protein expression in the human kidney and an increased risk of familial hypertension (Tomaszewski M, Charchar FJ, Nelson CP, et al. Pathway analysis shows association between FGFBP1 and hypertension. J Am Soc Nephrol. 2011 May;22(5):947-55). Regulation of FGF signaling by FGFBP1 has been shown to regulate vascular sensitivity to endogenous angiotensin II and thus control steady-state blood pressure (Tassi E, Lai EY, Li L, et al. Blood Pressure Control by a Secreted FGFBP1 (Fibroblast Growth Factor-Binding Protein). Hypertension. 2018 Jan;71(1):160-167). FGF2 signaling has proven central to maintaining cellular plasticity in epithelial ovarian cancer, already at the very early stages of carcinogenesis (De Cecco L, Marchionni L, Gariboldi M, et al. Gene expression profiling of advanced ovarian cancer: characterization of a molecular signature involving fibroblast growth factor 2).Oncogene. 2004 Oct 21;23(49):8171-83). Higher serum FGF2 levels have been found in patients with epithelial ovarian cancer (both benign and malignant subtypes) compared with healthy controls (Barton DP, Cai A, Wendt K, et al. Angiogenic protein expression in advanced epithelial ovarian cancer. Clin Cancer Res 3:1579-1586, 1997; Le Page C, Ouellet V, Madore J, et al. From gene profiling to diagnostic markers: IL-18 and FGF-2 complement CA125 as serum-based markers in epithelial ovarian cancer. Int J Cancer 118:1750-1758, 2006; Madsen CV, Steffensen KD, Olsen DA, et al. Serum platelet-derived growth factor and fibroblast growth factor in patients with benign and malignant ovarian tumors. Anticancer Res. 2012 Sep;32(9):3817-25). In rats, FGF2 treatment of ovarian cultures inhibited primordial follicle assembly. Furthermore, many differentially expressed genes identified in treated ovaries correlated with previously known genes associated with PCOS, suggesting that FGF2-dependent aberrant follicle assembly may be a component of later-life PCOS (Nilsson E, Zhang B, Skinner MK. Gene bionetworks that regulate ovarian primordial follicle assembly. BMC Genomics. 2013 Jul 23;14:496). In cows, FGF2 mRNA expression is high in preovulatory follicles and the early luteal phase, then it decreases in the late luteal phase and remains at low levels throughout pregnancy (Berisha B, Schams D, Rodler D, Pfaffl MW. Angiogenesis in the Ovary - The Most Important Regulatory Event for Follicle and Corpus Luteum Development and Function in Cow - An Overview. Anat Histol Embryol. 2016 Apr;45(2):124-30).Intraperitoneal injection of FGF2 in a mouse model of estradiol valerate-induced PCOS had protective and ameliorative effects (Moayeri A, Rostamzadeh A, Raoofi A, et al. Retinoic acid and fibroblast growth factor-2 play a key role on modulation of sex hormones and apoptosis in a mouse model of polycystic ovary syndrome induced by estradiol valerate. Taiwan J Obstet Gynecol. 2020 Nov;59(6):882-890). Conflicting results have been published regarding serum FGF2 levels in patients with PCOS. Artini et al. reported that there was no difference in serum FGF2 levels between untreated PCOS patients and control patients, but that FGF2 levels increased in PCOS patients after FSH stimulation (Artini PG, Monti M, Matteucci C, et al. Vascular endothelial growth factor and basic fibroblast growth factor in polycystic ovary syndrome during controlled ovarian hyperstimulation. Gynecol Endocrinol. 2006 Aug;22(8):465-70). Instead, Patil et al. reported lower serum FGF2 levels in women with PCOS undergoing controlled ovarian hyperstimulation for IVF compared with age- and BMI-matched controls (Patil K, Hinduja I, Mukherjee S. Alteration in angiogenic potential of granulosa-lutein cells and follicular fluid contributes to luteal defects in polycystic ovary syndrome. Hum Reprod. 2021 Mar 18;36(4):1052-1064).Although the role of FGF2 signaling in PCOS is unclear, its metabolic function has been investigated in recent years. Depending on its concentration, FGF2 can function as a positive or negative factor for adipogenesis in vitro (Kim S, Ahn C, Bong N, et al. Biphasic effects of FGF2 on adipogenesis. PLoS One. 2015 Mar 19;10(3):e0120073. doi:10.1371 / journal.pone.0120073). Mathes et al. demonstrated that FGF2-dependent signaling enhances the differentiation of fibrinogenic / adipogenic progenitors, which promote the formation of intramuscular adipose tissue (Mathes S, Fahrner A, Ghoshdastider U, et al. FGF-2-dependent signaling activated in aged human skeletal muscle promotes intramuscular adipogenesis. Proc Natl Acad Sci U S A. 2021 Sep 14;118(37):e2021013118.doi:10.1073 / pnas.2021013118). The appearance of adipose tissue between skeletal muscle fibers is a unique feature of aging, obesity, and type 2 diabetes and has been linked to insulin resistance. Furthermore, FGF2 has been shown to be a negative regulator of thermogenesis in both brown and beige fat. Disruption of FGF2 strongly enhanced the thermogenic activity of brown and beige fat, resulting in increased energy expenditure and improved lipid homeostasis. Furthermore, FGF2 deficiency protected mice from the development of adiposity and fatty liver induced by high fat levels (Li H, Zhang X, Huang C, et al. FGF2 disruption enhances thermogenesis in brown and beige fat to protect against adiposity and hepatic steatosis. Mol Metab. 2021 Dec;54:101358. doi:10.1016 / j.molmet.2021).

[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 quiescent 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 et al. 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 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 METRNL 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 METRNL levels 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). 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 PCOS subgroups (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 a negative correlation was identified 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 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 FGFBP1 and METRNL in a sample has the advantage of being a reliable, biofluid-based test for identifying women with PCOS, which is not currently possible. This method can also be reliably used to diagnose 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. Diagnosing PCOS in adolescent patients is difficult for the reasons mentioned above. Therefore, the present inventors have, for the first time, realized an accurate test for the diagnosis of PCOS in adolescent and young female populations. Furthermore, measuring the ratio between FGFBP1 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 FGFBP1 and METRNL in a patient's sample 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 the levels of FGFBP1 and METRNL in a sample, and optionally using further criteria such as values for oligo-anovulation and / or irregular cycles, hyperandrogenism and / or polycystic ovarian morphology, or further biomarkers or hormones, to assess said subject based on the above data comparison and / or calculation.

[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, a deep voice, and / or hirsutism. These reproductive characteristics of clinical hyperandrogenism can be diagnosed simply by questioning the woman or are apparent after a brief physical examination of the woman's body. Typically, the reference population exhibits none or none of these phenotypic characteristics known to be indicative of PCOS.

[0036] In an embodiment, the PCOS is assessed from the group consisting of metabolic or phenotypic PCOS. In a further embodiment, 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" refers to assessing whether the subject referred to in accordance with the method of the present invention is suffering from PCOS.Preferably, the expression "diagnose PCOS" used herein should be understood as "supporting" or "assisting" the diagnosis of PCOS.For example, a doctor can be assisted in the diagnosis of 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 to be examined. The term "diagnose" preferably requires that a correct diagnosis can be made for a statistically significant portion of subjects. Whether a portion is statistically significant can be determined by those skilled in the art without further ado using various well-known statistical evaluation tools, such as determining confidence intervals, determining p-values, Student's t-test, Mann-Whitney test, etc. 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 an 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 samples, 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 separation techniques such as centrifugation or cell sorting. Preferably, cell, tissue, or organ samples are obtained from cells, tissues, or organs that 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] As used herein, the term "subject" 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 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 "subject" and "patient" 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 postmenstrual. In certain embodiments, the patient is a female human patient under the age of 20 and three years postmenstrual. In certain embodiments, the patient is a female human patient between the ages of 15 and 25 and three years postmenstrual. In a particular embodiment, the patient is a female human patient between the ages of 15 and under 20 years old, and 3 years postmenstrual.

[0043] According to step a) of the method of the present invention, the amount or concentration of FGFBP1 is determined, i.e., measured, in a sample from a subject. 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, each step can be performed simultaneously.

[0044] In embodiments, the amount or concentration of FGFBP1 and METRNL is determined using antibodies, particularly monoclonal antibodies. In embodiments, steps a) and b) of determining the amount or concentration of FGFBP1 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, steps a) and b) of determining the amount or concentration of FGFBP1 and METRNL in a patient sample comprise: i) incubating a patient sample with one or more antibodies that specifically bind to FGFBP1, thereby generating a complex between the antibody and FGFBP1; ii) incubating the patient sample with one or more antibodies that specifically bind to METRNL, thereby generating a complex between the antibody and METRNL; and iii) quantifying the complexes formed in steps i) and ii), thereby quantifying the amount or concentration of FGFBP1 and METRNL in the patient sample.

[0046] In certain embodiments, in step i), the sample is incubated with two antibodies that specifically bind to FGFBP1. 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, i.e., 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-FGFBP1 antibody / FGFBP1 / second anti-FGFBP1 antibody complex. As will be readily apparent to those skilled in the art, it will only take routine experimentation to determine the time and conditions that are appropriate or sufficient for the formation of a complex between a specific anti-FGFBP1 antibody and an FGFBP1 antigen / analyte (=anti-FGFBP1 complex), or for the formation of a secondary or sandwich complex (=first anti-FGFBP1 antibody / FGFBP1 / second anti-FGFBP1 antibody complex) comprising a first antibody against FGFBP1, FGFBP1 (analyte), and a second anti-FGFBP1 antibody.

[0047] Detection of the anti-FGFBP1 antibody / FGFBP1 complex can be carried out by any suitable means. Detection of the first anti-FGFBP1 antibody / FGFBP1 / second anti-FGFBP1 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 FGFBP1, FGFBP1 (the analyte), and a second antibody to FGFBP1, wherein the second antibody is detectably labeled.

[0049] In one embodiment, a sandwich is formed comprising a first antibody against FGFBP1, FGFBP1 (analyte) and a second antibody against FGFBP1, wherein the second antibody is detectably labeled and the first anti-FGFBP1 antibody is capable of binding to a solid phase or is bound to a solid phase.

[0050] In a specific embodiment, in step i), the sample is incubated with two antibodies that specifically bind to METRNL. 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, i.e., 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-METRNL antibody / METRNL / second anti-METRNL antibody complex. As will be readily apparent to those skilled in the art, it is merely a matter of routine experimentation to determine the time and conditions that are appropriate or sufficient for the formation of a complex between a specific anti-METRNL antibody and a METRNL antigen / analyte (= anti-METRNL complex), or for the formation of a secondary complex or sandwich complex (= first anti-METRNL antibody / METRNL / second anti-METRNL antibody complex) comprising a first antibody against METRNL, METRNL (analyte), and a second anti-METRNL antibody.

[0051] Detection of the anti-METRNL antibody / METRNL complex can be carried out by any suitable means. Detection of the first anti-METRNL antibody / METRNL / second anti-METRNL antibody complex can be carried out by any suitable means. Those skilled in the art are fully familiar with such means / methods.

[0052] In certain embodiments, a sandwich is formed comprising a first antibody to METRNL, METRNL (the analyte), and a second antibody to METRNL, wherein the second antibody is detectably labeled.

[0053] In one embodiment, a sandwich is formed comprising a first antibody against METRNL, METRNL (analyte) and a second antibody against METRNL, wherein the second antibody is detectably labeled and the first anti-METRNL antibody is capable of binding to a solid phase or is bound to a solid phase.

[0054] In embodiments, the second antibody is detectably labeled, either directly or indirectly. In certain embodiments, the second antibody is detectably labeled with a luminescent dye, particularly a chemiluminescent or electrochemiluminescent dye.

[0055] It will be appreciated by those skilled in the art that steps i) and ii) can be performed simultaneously or sequentially in any order.

[0056] 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 capable of specifically binding 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 FGFBP1). An antibody fragment preferably comprises a portion of a full-length antibody, preferably its variable domain, or at least its antigen-binding site. In embodiments, 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 produced synthetically or by recombinant techniques. Fab fragments are preferably produced by papain digestion of an antibody, pepsin digestion and partial reduction to produce Fab' fragments, pepsin digestion to produce F(ab')2 fragments, and plasmin digestion to produce facb fragments. Fv or scFv fragments are preferably produced by molecular biology techniques.

[0057] 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, excluding possible variants that may arise during the production of the monoclonal antibody, although such variants are generally present in minor amounts. The monoclonal antibodies of the present invention can be produced by the well-known hybridoma method described by Kohler and Milstein, Nature, 256:495 (1975), or can also be produced by recombinant DNA technology. In some embodiments, the monoclonal antibody is selected from the group consisting of sheep monoclonal antibodies, mouse monoclonal antibodies, rabbit monoclonal antibodies, goat monoclonal antibodies, horse monoclonal antibodies, and chicken monoclonal antibodies. In some embodiments, the monoclonal antibody is a mouse monoclonal antibody.

[0058] 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.

[0059] 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 observing the signal produced by the reporter molecule bound to the detection binding agent. Results can be qualitative, by simple observation of the visible signal, or quantified, for example, by comparison with a control sample containing a known amount of the biomarker to be determined (as a standard or calibrator, as described elsewhere herein).

[0060] The incubation step of typical sandwich assay can be modified as needed and appropriate.This modification includes, for example, simultaneous incubation, in which two or more binding agents and biomarkers are incubated together.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.

[0061] 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 of the proportion of at least one marker contained in the sample that can be specifically bound. Details of how to perform the measurement are also described elsewhere in this specification. The amount or concentration of the complex formed is converted into the amount or concentration of the biomarker that reflects the amount actually present in the sample.

[0062] The term "amount" as used herein encompasses the absolute amount of FGFBP1 or METRNL, the relative amount or concentration of said FGFBP1 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 preferred embodiments of the subject 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.

[0063] In step c) of the method according to the first, second or third aspect of the present invention, a score of the amounts or concentrations determined in steps a) and b), i.e. the amount or concentration of FGFBP1 and the amount or concentration of METRNL, is calculated.

[0064] The term "calculating" as used herein refers to assessing a score based on the amount or concentration of FGFBP1 and the amount or concentration of METRNL determined in a subject's sample.For example, it is assumed that a score, i.e., a single score, is calculated based on the amount or concentration of FGFBP1 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 FGFBP1 and the amount or concentration of METRNL.Furthermore, biomarkers can be weighted in the score according to their contribution to establishing a diagnosis.The score can be considered as a classifier parameter for diagnosing PCOS.In particular, the score allows for 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.

[0065] Preferably, the score is a ratio, i.e., the ratio of the amount or concentration of FGFBP1 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 FGFBP1 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 FGFBP1.

[0066] In step d) according to the first, second or third aspect 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.

[0067] As used herein, the term "comparing" includes comparing the calculated score for a sample from a test subject with a suitable standard source as specified elsewhere in this specification. The comparison is preferably assisted by automation. For example, a suitable computer program containing an algorithm for comparing the calculated score of a subject with a standard score may be used. Such computer programs and algorithms are well known in the art. As described above, the comparison can also be performed manually. The computer program may further evaluate the result of the comparison, i.e., automatically present the desired evaluation, i.e., diagnostic result, in a suitable output format. The diagnostic result can preferably serve as an aid for, for example, medical professionals to establish a final diagnosis of PCOS.

[0068] The calculating and / or comparing steps may be performed by using a computer comprising a processing unit.

[0069] 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 comparison result may be presented as raw data, or in some cases as an indicator in the form of a word, phrase, symbol, or number, which may indicate a specific diagnosis.Therefore, the reference score is selected so that the difference or the sameness of the calculated score with respect to the reference score allows the test subject to be identified as belonging to a group of subjects suffering from PCOS or not.The method allows either the exclusion (exclusion) or identification (inclusion) of subjects suffering from PCOS.The difference in score, i.e., increase or decrease, as used herein, is preferably a statistically significant difference.The method can also use other diagnostic criteria, such as oligo-anovulation or hyperandrogenism, to assess whether a patient suffers from PCOS.

[0070] Preferably, the reference score, such as the reference ratio, can distinguish whether the subject suffers from PCOS or not.Preferably, diagnosis is made by assessing whether the test subject's score is above or below the reference score.It is not necessary to set an exact reference score.Relevant reference score can be obtained by correlating the sensitivity and specificity of any score and sensitivity / specificity.The reference score that brings about high sensitivity brings about lower specificity, and vice versa.

[0071] In some embodiments, the normative score is derived from a sample from a subject (or a sample group of subjects) known to have PCOS.

[0072] In some embodiments, the reference score is not derived from samples from subjects (or sample groups of subjects) known to be afflicted with PCOS.

[0073] As described above, the score calculated in step c) according to the first, second or third aspect of the present invention may be a ratio. In an embodiment, the ratio is the ratio of the amount or concentration of FGFBP1 to the amount or concentration of METRNL. In this case, a ratio (i.e., the 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 FGFBP1. In this case, a ratio (i.e., the 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.

[0074] 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.

[0075] 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 the application of any actual treatment is not included in this term. The recommended treatment depends on the diagnosis result provided by the method of the present invention. The recommendation step mentioned above can also preferably be 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, including a recommended treatment measure for each possible diagnosis result.

[0076] In embodiments, the recommended or initiated treatment is selected for a drug-based treatment of PCOS or lifestyle changes to control metabolic symptoms. In embodiments, the drug-based therapy for PCOS is selected from the group consisting of drugs for regulating menstruation, particularly oral contraceptives or progestin therapy, drugs for preventing or controlling diabetes, particularly type 2 diabetes, drugs for preventing or controlling high cholesterol, hormones or drugs for enhancing fertility, drugs, hormones, or treatments for removing excess hair, and drugs or treatments for controlling acne.

[0077] The method of the present invention may also be implemented as a computer-implemented invention. In an embodiment, one or more steps, such as the comparing step and / or the calculating step, are performed by a computer including a processing unit (i.e., a computer). In another embodiment, all steps are performed by a computer with a processing unit.

[0078] 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 FGFBP1 in a sample derived from a subject; and (a2) the amount or concentration of METRNL in a sample from the subject; receiving the (b) processing the values received in step (a) in a processing unit, (b1) calculating the scores of the values (a1) and (a2) received in step a); (b2) comparing the calculated score with a reference score; a processing step including: (c) optionally performing said diagnosis via an output device, said diagnosis being based on the results of step b).

[0079] In some embodiments, the processing unit is included in a computer.

[0080] In some embodiments, step b) according to the fourth aspect of the invention further comprises, in the processing unit, retrieving from memory a reference score, i.e. a reference score suitable for diagnosing PCOS.

[0081] In an embodiment of the method of the present invention, information regarding the diagnosis (according to the final step of the method of the present invention) is presented 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, treatment options can be displayed on the display.

[0082] In one embodiment of the method of the present invention, the method may include the further step of transferring information from the evaluation of the method of the present invention to the subject's electronic medical record.

[0083] 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.

[0084] The present invention further relates to the use of i) FGFBP1 and METRNL as biomarkers, or ii) at least one agent that specifically binds to METRNL and at least one agent that specifically binds to FGFBP1 for diagnosing PCOS.Preferably, the use is carried out in vitro, i.e., with a sample from a subject.Preferably, the agent that specifically binds to FGFBP1 is an antibody or an antigen-binding fragment.Preferably, the agent that specifically binds to METRNL is an antibody or an antigen-binding fragment.

[0085] 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 FGFBP1.

[0086] The term "kit" as used herein refers to a collection of the aforementioned means, for example, supplied 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 FGFBP1 and / or METRNL. 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]

[0087] 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.

[0088] Example 1: Diagnostic performance of the ratio between the two biomarkers FGFBP1 and METRNL in women with PCOS (phenotypes A, B, C and D) and controls as determined by ELISA technique Performance validation was performed on a sample set of 85 cases (serum samples from women with PCOS) and 44 controls (serum samples from healthy women).

[0089] The concentrations of two analytes, FGFBP1 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.

[0090] The concentration of FGFBP1 in human serum was determined using a Human FGFBP1 ELISA kit from Sigma-Aldrich (catalog number: RAB1460). The concentration of METRNL in human serum was determined using a Human METRNL ELISA kit from R&D Systems (catalog number: DY7867-05). The kits are solid-phase sandwich enzyme-linked immunosorbent assays (ELISAs) designed to detect and quantitate human FGFBP1 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.

[0091] For FGFBP1 quantification, the kit was equipped with a human FGFBP1 antibody-precoated plate. Samples were measured at 75-fold dilutions. After all reagents were brought to room temperature, 100 μL of each sample and standard was added to the plate. Samples and standards were measured in duplicate. During a 2.5-hour incubation at room temperature with gentle shaking, any FGFBP1 present bound to the immobilized capture antibody on the microtiter plate. During a wash step (4 × 300 μL), unbound material was removed from the plate before 100 μL of diluted detection antibody was added to the wells. After a 1-hour incubation with gentle shaking and another wash step (4 × 300 μL) to remove any unbound detection antibody, 100 μL of prepared streptavidin-HRP solution was added to the plate. This was followed by a 45-minute incubation at room temperature with gentle shaking and a wash step (4 × 300 μL). After the final wash, 100 μL of TMB One-Step Substrate Reagent 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 development was proportional to the amount of FGFBP1 bound in the first step. The color development was stopped by adding 50 μL of stop solution, causing the solution in the wells to change from blue to yellow. The color intensity was measured using a plate reader at 450 nm for detection and 570 nm for background subtraction. A 7-point standard curve was obtained using 2.5-fold serial dilutions of recombinant FGFBP1 in assay diluent. The standard curve was fitted using an unweighted, four-parameter nonlinear regression (Newton / Raphson).

[0092] The METRNL capture antibody 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 4-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 any unbound detection antibody, 100 μL of prepared streptavidin-HRP solution was added to the plate. This was followed by a 20-minute incubation at room temperature and three 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 light exposure. During the incubation, the substrate turned blue. Color development was proportional to the amount of METRNL bound in the first step. Color development was stopped by adding 50 µL of stop solution, causing the color of the solution in the wells to change from blue to yellow. 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 generate a calibration curve, the lyophilized recombinant METRNL delivered with the kit was reconstituted and diluted with reagent diluent. The calibration range of the METRNL assay is 15.6 pg / mL to 1000 pg / mL.A seven-point standard curve was obtained using two-fold serial dilutions of recombinant METRNL in reagent diluent, and the standard curve was fitted using a four-parameter logistic (4-PL, Newton / Raphson) curve fit.

[0093] Once the concentrations of the two analytes were obtained by ELISA immunoassay, the ratio of FGFBP1 to METRNL was calculated for each sample.

[0094] A receiver operating characteristic (ROC) curve was constructed for the FGFBP1 to METRNL ratio (Figure 1). Model performance is judged 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 FGFBP1 to METRNL ratio for PCOS cases when all phenotypes were combined (phenotypes A–D) showed an AUC of 0.98 (95% CI, 0.96–1.00), confirming the high diagnostic accuracy of the FGFBP1 to METRNL ratio for PCOS (Figure 1). The diagnostic performance of the FGFBP1 to METRNL ratio in distinguishing women with PCOS (cases, PCOS phenotypes A–D) from healthy controls using ROC analysis is shown in Table 1, which lists the AUC and associated 95% confidence intervals for the ROC curve analysis. [Table 1]

[0095] Data obtained by running the ratio of FGFBP1 to METRNL concentrations were used to generate box plots of PCOS cases for controls and all phenotypes combined (phenotypes A-D). The ratio of FGFBP1 to METRNL is increased in women with PCOS when compared to healthy controls (Figure 2).

[0096] Table 2 shows the diagnostic performance of the FGFBP1 to METRNL ratio in distinguishing women with PCOS relative to healthy controls when separated by the different phenotypes A, B, C, and D. The AUC for each phenotype is reported in the table. [Table 2]

[0097] Receiver operating characteristic curve analysis of the FGFBP1 to METRNL ratio for PCOS cases versus healthy controls separated by different phenotypes showed AUCs of 0.95 (95% CI, 0.87-1.00), 0.99 (95% CI, 0.97-1.00), 1.00 (95% CI, 0.99-1.00), and 0.99 (95% CI, 0.98-1.00) for phenotypes A through D, respectively (Figure 3). The results confirm the high diagnostic accuracy of the FGFBP1 to METRNL ratio for PCOS. The FGFBP1 to METRNL ratio in all different PCOS phenotypes (phenotypes A through D) showed increased levels compared to healthy controls (Figure 4).

[0098] Table 3 shows the diagnostic performance of the FGFBP1 to 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]

[0099] ROC curve analysis of the FGFBP1 to METRNL ratio in young PCOS cases (age 25 or younger) for all phenotypes combined (phenotypes A-D) showed an AUC of 0.99, confirming high diagnostic accuracy for women 25 or younger in distinguishing PCOS cases from controls (95% CI, 0.97-1; 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 FGFBP1 to METRNL ratio compared to young controls (age 25 or younger; Figure 6).

[0100] The diagnostic performance of the FGFBP1 to METRNL ratio in differentiating young women (aged 25 years and younger) with PCOS when separated by the different phenotypes A, B, C, and D relative to young healthy control subjects (aged 25 years and younger) was evaluated, and the results are reported in Table 4 (AUC for PCOS phenotypes vs. controls). [Table 4]

[0101] ROC curve analysis of the FGFBP1-to-METRNL ratio for young PCOS cases (age 25 or younger, phenotypes A–D) showed AUCs of 1.00 (95% CI, 1–1), 0.93 (95% CI, 0.75–1), 1.00 (95% CI, 1–1), and 1.00 (95% CI, 1–1) for each phenotype, respectively, confirming the high diagnostic accuracy of the FGFBP1-to-METRNL ratio for PCOS in the subgroup of women aged 25 or younger (Figure 7). The FGFBP1-to-METRNL ratio was increased in all different PCOS phenotypes (phenotypes A–D, age 25 or younger) when compared with the FGFBP1-to-METRNL ratio in young healthy controls (age 25 or younger, Figure 8).

[0102] Example 2: Diagnostic performance of the ratio between the two biomarkers FGFBP1 and METRNL in women with PCOS (phenotypes A, B, C and D) and controls in different age groups, as determined by the technique ELISA 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).

[0103] The concentrations of two analytes, FGFBP1 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), and 25-40 (n = 71). The control group included healthy women (20-40 years old) without PCOS.

[0104] The concentration of FGFBP1 in human serum was determined using a Human FGFBP1 ELISA kit from Sigma-Aldrich (catalog number: RAB1460). The concentration of METRNL in human serum was determined using a Human METRNL ELISA kit from R&D Systems (catalog number: DY7867-05). Measurements were performed as described in Example 1.

[0105] A receiver operating characteristic (ROC) curve was constructed for the FGFBP1 to METRNL ratio (Figure 9). Model performance was determined by examining the area under the curve (AUC). ROC curve analysis of the FGFBP1 to METRNL ratio for PCOS cases when all phenotypes were combined (phenotypes A to D) showed an AUC of 0.95 (95% CI, 0.93 to 0.97), confirming the high diagnostic accuracy of the FGFBP1 to METRNL ratio for PCOS (Figure 9).

[0106] The diagnostic performance of the FGFBP1 to METRNL ratio in distinguishing women with PCOS (cases, PCOS phenotypes A-D) from healthy controls using ROC analysis is shown in Table 5, which describes the AUC of the ROC curve analysis and the associated 95% confidence interval. Results were obtained using an ELISA immunoassay. [Table 5]

[0107] Using data obtained by ELISA immunoassay, boxplots were generated for controls and PCOS cases for all phenotypes combined (phenotypes A-D). The ratio of FGFBP1 to METRNL is increased in women with PCOS compared to healthy controls (Figure 10).

[0108] Table 6 shows the diagnostic performance of the FGFBP1 to METRNL ratio in distinguishing women with PCOS relative to healthy controls when separated by the different phenotypes A, B, C, and D. The AUC for each phenotype is reported in the table. [Table 6]

[0109] ROC curve analysis of the FGFBP1-to-METRNL ratio for PCOS cases versus healthy controls separated by different phenotypes showed AUCs of 0.95 (95% CI, 0.92-0.98), 1.00 (95% CI, 1.00-1.00), 1.00 (95% CI, 1.00-1.00), and 0.94 (95% CI, 0.90-0.99) for phenotypes A to D, respectively (Figure 11). The results confirm the high diagnostic accuracy of the FGFBP1-to-METRNL ratio for PCOS.

[0110] The ratio of FGFBP1 to METRNL in all different PCOS phenotypes (phenotypes A to D) was increased compared to healthy controls (Figure 12).

[0111] Table 7 shows the diagnostic performance of the FGFBP1 to METRNL ratio in different age groups (15–20, 20–25, 25–40) in distinguishing women with PCOS from healthy controls when all phenotypes are combined (phenotypes A–D). [Table 7]

[0112] ROC curve analysis of the FGFBP1-to-METRNL ratio for PCOS cases segregated by different age groups versus healthy controls showed AUCs of 0.91 (95% CI, 0.85-0.97), 0.96 (95% CI, 0.93-0.99), and 0.98 (95% CI, 0.95-1.00) for the age groups 15-20, 20-25, and 25-40, respectively (Figure 13). The results confirm the high diagnostic accuracy of the FGFBP1-to-METRNL ratio for PCOS in all different age groups.

[0113] An increased FGFBP1 to METRNL ratio in women with PCOS compared to controls was observed across all different age groups (Figure 14).

[0114] 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.

[0115] Table 8 shows the diagnostic performance of the FGFBP1 to 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]

[0116] ROC curve analysis of the FGFBP1 to METRNL ratio in young PCOS cases (ages 15 to 25) for all phenotypes combined (phenotypes A-D) showed an AUC of 0.94, confirming the high diagnostic accuracy of women aged 15 to 25 in distinguishing PCOS cases from controls (95% CI, 0.90-0.97, Figure 15). When only young women (ages 15 to 25) were included in the analysis, PCOS cases (all phenotypes A-D combined) showed an increased FGFBP1 to METRNL ratio compared to young controls (ages 15 to 25, Figure 16).

[0117] The diagnostic performance of the FGFBP1 to METRNL ratio in distinguishing young women (aged ≥15 and <25 years) with PCOS when separated by the different phenotypes A, B, C, and D relative to young healthy control subjects (aged ≥15 and <25 years) was evaluated, and the results are reported in Table 9 (AUC for PCOS phenotypes vs. controls). [Table 9]

[0118] ROC curve analysis of the FGFBP1:METRNL ratio for young PCOS cases (ages 15 to <25 years, phenotypes A to D) showed AUCs of 0.93 (95% CI, 0.88 to 0.97), 1.00 (95% CI, 1.00 to 1.00), 1.00 (95% CI, 1.00 to 1.00), and 0.94 (95% CI, 0.88 to 1.00) for each phenotype, respectively, confirming the high diagnostic accuracy of the FGFBP1:METRNL ratio for PCOS in the subgroup of women aged 15 to 25 years (Figure 17). When compared with the FGFBP1:METRNL ratio in young healthy controls, the FGFBP1:METRNL ratio was increased in all different PCOS phenotypes (phenotypes A to D, ages 15 to <25 years) (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 FGFBP1 in a sample from a subject; b) determining the amount or concentration of METRNL in a sample from said subject; c) calculating a score for the amount or concentration determined in steps a) and b); d) comparing said calculated score with a reference score; 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 FGFBP1 in a sample from a subject; b) determining the amount or concentration of METRNL in a sample from said subject; c) calculating a score for the amount or concentration determined in steps a) and b); d) comparing said calculated score with a reference score; 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 FGFBP1 in a sample from a subject; b) determining the amount or concentration of METRNL in a sample from said subject; c) calculating a score for the amount or concentration determined in steps a) and b); d) comparing said calculated score with a reference score; e) monitoring the progression in said subject suffering from or being treated for PCOS; A method comprising:

4. 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, e.g., the subject suspected of suffering from PCOS has a history of PCOS symptoms.

6. 6. The method of claim 1, wherein the determining the amount or concentration of FGFBP1 comprises contacting the sample with an antibody or antigen-binding fragment thereof that specifically detects FGFBP1.

7. 6. The method of claim 1, wherein the determining the amount or concentration of METRNL comprises contacting the sample with an antibody or antigen-binding fragment thereof that specifically detects METRNL.

8. The score is a ratio, in particular a) the ratio is the ratio of the amount or concentration of the FGFBP1 to the amount or concentration of the METRNL, preferably a ratio higher than the reference ratio is indicative of the subject suffering from PCOS, or b) the ratio is the ratio of the amount or concentration of the METRNL to the amount or concentration of the FGFBP1, preferably a ratio lower than the reference ratio indicates a subject suffering from PCOS; 8. The method according to any one of claims 1 to 7.

9. 9. The method of any one of claims 1 to 8, wherein the PCOS is a PCOS 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) the amount or concentration of FGFBP1 in a sample derived from a subject; and (a2) the amount or concentration of METRNL in a sample derived from the subject; receiving the (b) processing the values received in step (a) in the processing unit, (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 including: (c) optionally performing said diagnosis via an output device, said diagnosis being based on the results of step b); 11. A computer-implemented method comprising:

12. i) the use of FGFBP1 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 FGFBP1 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 FGFBP1.

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