Metrnl as a (blood) biomarker for the diagnosis of polycystic ovary syndrome

Abstract

The present invention relates to a method for assessing whether a subject has polycystic ovary syndrome (PCOS) or is at risk of developing PCOS, a method for selecting patients for the treatment of PCOS, a method for monitoring PCOS progression or monitoring the response to treatment, and a computer-implemented method for assessing a subject suspected of having PCOS by determining the amount or concentration of meteorin-like protein (METRNL) in a sample of the subject.

Description

Technical Field

[0001] The present invention relates to a method for determining the amount or concentration of S100A12 in a sample from a patient and comparing the determined amount or concentration with a reference to evaluate whether the patient has endometriosis or is at risk of developing endometriosis, a method for selecting a patient for treatment, and a method for monitoring a patient suffering from or being treated for endometriosis. Further, the present invention relates to a computer-implemented method for assessing a subject suspected of having PCOS by determining the amount or concentration of METRNL in a sample of the subject, optionally determining the amount or concentration of a second biomarker and / or additional diagnostic criteria, and by comparing the amount or concentration of METRNL, optionally the amount or concentration of the second biomarker and / or the presence of the diagnostic criteria with a reference.

Background Art

[0002] Polycystic ovary syndrome (PCOS) is a heterogeneous gynecological disorder defined by a combination of androgen excess symptoms and ovarian dysfunction. PCOS patients can have various clinical symptoms that can be reproductive and / or metabolic in nature. Reproductive findings include irregular menstrual cycles, infertility, pregnancy complications, and hirsutism, and metabolic findings 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, H.F. 2018; International evidence-based guideline for the assessment and management of polycystic ovary syndrome 2018).

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

[0004] PCOS can be caused by genetic, epigenetic and environmental factors, such as a combination of genes.

[0005] PCOS is one of the most common endocrine disorders in women, and despite the fact that 10% of women of reproductive age are affected, 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 criteria mainly and widely used for PCOS diagnosis are the so-called Rotterdam criteria. PCOS is indicated when at least two of the following criteria are applied: (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 having a cycle length of less than 21 days or more than 35 days or less than 8 cycles per year. In the case of clinical and / or biochemical signs of hyperandrogenism, clinical hyperandrogenism is defined as hirsutism (excessive male-pattern hair growth) and / or acne, and biochemical hyperandrogenism is defined as higher levels of free androgen compared to healthy controls. Clinical hyperandrogenism is also defined as a modified Ferriman-Gallwey score of more than 8. Biochemical hyperandrogenism can be evaluated using free testosterone or free androgen index (FAI) which can be calculated by measuring total testosterone and sex hormone binding globulin (SHBG). PCOM is usually determined according to the "International Evidence-based Guideline for PCOS 2018" using a vaginal ultrasound transducer with a frequency bandwidth including 8 MHz. The threshold for PCOM is considered, for the ovary, as either >20 follicles per ovary and / or ovarian volume ≥10 ml, which ensures the absence of corpus luteum, cysts or dominant follicles. When older ultrasound techniques are used, the threshold for PCOM can be an ovarian volume of ≥10 ml or a follicle count of >12 for either ovary.

[0007] Another way to detect PCOM is to measure the anti-Müllerian hormone (AMH) of the subject. AMH is a glycoprotein hormone whose expression is important for sexual differentiation at a specific time during fetal development. Furthermore, AMH produced by the granulosa cells of growing follicles usually correlates with the number of follicles in the ovary. Therefore, the serum level of AMH can be a surrogate biomarker for the antral follicle count / number (AFC) determined by transvaginal ultrasound. Several studies have suggested serum AMH as a biochemical marker for PCOM. In several studies, AMH thresholds for PCOM in women with PCOS have been proposed (Nicholas et al. 2014; Pigny et al. 2016; Dietz de Loos et al., Fertil Steril, 2021). However, according to the "International evidence-based guideline for the assessment and management of polycystic ovary syndrome 2018", serum AMH levels should not be used as an alternative for the detection of PCOM or the diagnosis of PCOS.

[0008] A further method for detecting PCOS is the 3-item PCOS criteria system (Indran et al. 2018). In this system, it has been proposed to diagnose PCOS if two of the three items are present: (i) oligomenorrhea (defined as an average menstrual cycle length > 35 days); (ii) AMH above the threshold; (iii) hyperandrogenism defined as either testosterone above the threshold 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 way to detect PCOS is to measure other hormones, such as luteinizing hormone (LH) and follicle-stimulating hormone (FSH). However, the diagnostic utility of the LH:FSH ratio for the diagnosis of PCOS seems low, as only a small proportion of women with PCOS had a significantly elevated LH:FSH ratio (Cho et al. 2005). In fact, the range of LH: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 clinical examinations requires specific expertise, which makes it very difficult for less specialized physicians (such as general practitioners) to diagnose PCOS in clinical routine. For example, the determination of PCOM by transvaginal ultrasound requires an appropriate ultrasound device and a subjective analysis of the ultrasound images by a physician. Furthermore, the results can also depend on the specific ultrasound device used for the assessment of PCOM. As a result, the diagnosis of PCOS based on the Rotterdam criteria always includes at least one subjective, device- and operator-dependent, error-prone measurement.

[0011] To evaluate biochemical hyperandrogenemia, there must be a well-established normal range for the measured androgen. Testosterone is the most abundantly measured androgen in its total, bound, and free forms. There are limitations to the methods for measuring free testosterone. Direct measurement of free testosterone using radioimmunoassay is highly inaccurate and does not reflect the true value. The assay has high variability within and between assays. Alternatively, higher accuracy can be obtained, especially for clinical studies, by measuring total testosterone concentration using extraction and chromatography, or gas (GC-MS) or liquid (LC-MS) chromatography-mass spectrometry. The diagnostic performance of measuring serum testosterone can be enhanced by the simultaneous measurement of SHBG such that the calculation of free T concentration from total testosterone and SHBG levels requires only the solution of 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 HA may vary according to ethnicity. An mFG score of more than 8 for diagnosing hirsutism in women with PCOS is not appropriate for diagnosis in all ethnic groups. East Asian women have a lower prevalence of hirsutism compared to white women, and a score of more than 5 has been proposed to define hirsutism in Chinese women.There are also signs that androgen levels in the blood vary among ethnic groups. Here, the Japanese population has a low prevalence of increased androgen production, 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 different phenotypes named 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 characteristic of patients showing hyperandrogenemia, ovulatory dysfunction and / or irregular cycles, and polycystic ovarian morphology. Phenotype B is characterized by hyperandrogenemia, ovulatory dysfunction, and / or irregular cycles. Phenotype C is characterized by hyperandrogenemia and polycystic ovarian morphology. Phenotype D is characterized by ovulatory dysfunction and / or irregular cycles and polycystic ovarian morphology.

[0013] Currently, there are no specific PCOS drugs available. Treatments are symptom-directed and tailored to the individual's needs. Treatment approaches target hyperandrogenism, irregular cycles and / or ovulatory dysfunction, and related metabolic disorders such as diabetes. International evidence-based guidelines for the assessment and management of polycystic ovary syndrome in 2018 provide information to assist clinical decision-making and patient management.

[0014] Inconsistent diagnostic criteria, variable provider knowledge, and lack of consensus pose specific challenges to the diagnosis and care of women with PCOS. These factors contribute to inaccurate diagnoses, both underdiagnosis and overdiagnosis. This unfavorable diagnostic experience worsens the affected women and limits the timely opportunity for intervention to minimize related co-existing conditions, especially 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 to prevent further metabolic complications in affected women, such as type 2 diabetes.

[0015] In the largest study of PCOS diagnostic experiences, many women reported delays in diagnosis and inadequate information. Before a diagnosis was established, more than one-third of women reported more than 2 years (33.6%) and more than 3 healthcare specialists (47.1%). 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 the patient experience (Gibson-Helm M, Teede H, Dunaif A, Dokras A. Delayed Diagnosis and a Lack of Information Associated With Dissatisfaction in Women With Polycystic Ovary Syndrome. J Clin Endocrinol Metab. 2017;102(2):604 - 612).

[0016] Particularly interesting areas in the diagnosis of PCOS are young women, i.e., adolescents and young women under 25 years of age, preferably under 20 years of age, when the characteristics of normal pubertal development overlap with adult diagnostic criteria. This makes the diagnosis controversial and difficult. Many of the manifestations used to diagnose PCOS progress over time and can change during the first few years after menarche. Normal pubertal physiological changes such as irregular menstrual cycles, acne, and PCOM overlap with adult PCOS diagnostic criteria. In adolescents and young adult women, PCOS is diagnosed when both the OA and HA criteria are met. Pelvic ultrasound examination is not recommended in adolescents less than 8 years from menarche because the incidence of polycystic ovaries is high 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). Assessment of irregular menstrual cycles in adolescents can be difficult. Menstrual cycles are often irregular during adolescence. Immaturity of the hypothalamic-pituitary-ovarian axis during the first few years after menarche often results in anovulation and may result in somewhat longer cycles. However, 90% of cycles are in the range of 21 to 45 days, but short cycles less than 20 days and long cycles over 45 days can occur. By 3 years after menarche, 60 to 80% of menstrual cycles are 21 to 34 days in length, as typical in 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 guardians may not know well what is normal, and patients may not inform their guardians about menstrual irregularities or loss of menstruation.In addition, patients are often reluctant to discuss this topic with their guardians (ACOG Committee Opinion No. 651: Menstruation in Girls and Adolescents: Using the Menstrual Cycle as a Vital Sign. Obstet Gynecol. 2015;126(6):e143 - e146). Therefore, it is of utmost importance to establish reliable biomarkers as an aid for the diagnosis of PCOS or the identification of patients at risk of developing PCOS, especially in this patient group. Delayed diagnosis in adolescents and young women is often due to the reluctance to diagnose at - risk adolescents because of puberty and the fear of over - or under - diagnosis. This can lead to obesity and insulin resistance, as well as long - term complications such as anxiety or depression. The latest guidelines for the diagnosis of PCOS in adolescents and young women define oligo - anovulation, irregular menstrual cycles, and hyperandrogenemia as criteria to improve diagnostic accuracy in this patient group (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 re - evaluation of the diagnosis at 3 - year intervals and lifestyle changes to minimize symptoms and comorbidities associated with PCOS such as anxiety and depression.

[0017] To date, there are no available universal biomarkers that are used alone 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 the response to treatment in subjects with PCOS, and / or to monitor PCOS progression in a subject, and / or to monitor the response to treatment in subjects with PCOS.

[0018] Therefore, there is an unmet need to establish better diagnostic assays for diagnosing PCOS in young women and adolescents. SUMMARY OF THE INVENTION

[0019] In one aspect, the invention is a method for assessing whether a patient has PCOS or is at risk of developing PCOS, comprising: (a) determining the amount or concentration of METRNL in a sample from the patient; (b) comparing the determined amount or concentration to a reference; and relates to a method.

[0020] In a second aspect, the invention is a method for selecting a patient for the treatment of PCOS, comprising: (c) determining the amount or concentration of METRNL in a sample from the subject; (d) comparing the determined amount or concentration to a reference; and relates to a method.

[0021] In a third aspect, the invention is a method for monitoring the progression of PCOS in a subject or the response of a subject having PCOS to treatment, comprising: (e) determining the level of METRNL in a first sample from the subject; (f) determining the level of METRNL in a second sample from the subject obtained after the first sample; (g) comparing the level of METRNL in the first sample to the level of METRNL in the second sample; (h) monitoring the progression in the subject suffering from or being treated for PCOS based on the result of step c); and relates to such a method.

[0022] In a fourth aspect, the invention is a computer-implemented method for assessing a subject suspected of having PCOS, comprising: (a) A step of receiving a value for the amount or concentration of a first biomarker in the sample of the subject, wherein the first biomarker is METRNL, and receiving a value for the amount or concentration of the first biomarker in the sample of the subject; (b) Optionally, a step of receiving a value for the amount or concentration of a second biomarker in the sample of the subject; (c) Optionally, a step of receiving a value for the presence or absence of at least an additional diagnostic criterion selected from the group consisting of oligo-anovulation and hyperandrogenemia and polycystic ovarian morphology; (d) A step of comparing the value for the amount or concentration in steps (a) - (b) with the criteria for the biomarker and the value for the presence or absence of the at least one additional diagnostic criterion, and / or calculating a score for assessing the subject in whom PCOS is suspected based on the amount or concentration of the biomarker and the value; (e) A step of assessing the subject based on the comparison and / or the calculation performed in step (d); Relates to a computer-implemented method comprising.

Brief Description of Drawings

[0023]

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DETAILED DESCRIPTION OF THE INVENTION

[0024] The inventors of the present invention have identified meteorin-like protein (METRNL) as a reliable biomarker for diagnosing PCOS in a subject, determining whether the subject is at risk of developing PCOS, selecting PCOS patients for treatment, monitoring the progression of PCOS in a subject having PCOS, or monitoring the response of a subject having PCOS to treatment. METRNL can be used alone or in combination with at least additional criteria such as hyperandrogenemia, oligo-anovulation, PCOM or irregular cycles for diagnosis, risk assessment and / or monitoring of response to treatment in patients. Furthermore, determination of the level of METRNL compared to a control level can be used to monitor the response to treatment and / or monitor the progression of PCOS in the subject.

[0025] The inventors have for the first time shown that the METRNL value measured in a sample, preferably a biological fluid sample (preferably blood, plasma, serum, capillary blood, interstitial fluid, ascites, or menstrual fluid, and more preferably the biological fluid sample is blood, plasma or serum), is decreased in females suffering from PCOS compared to controls. Further, the inventors have for the first time shown that the METRNL value measured in a sample, preferably a biological fluid sample (preferably blood, plasma, serum, capillary blood, interstitial fluid, ascites, or menstrual fluid, and more preferably the biological fluid sample is blood, plasma or serum), is decreased in females suffering from any of the phenotypes A - D of PCOS. The solution provided by the present invention is an immunoassay for detecting meteorin-like protein in a sample, preferably a biological fluid sample (preferably blood, plasma, serum, capillary blood, interstitial fluid, ascites, or menstrual fluid, and more preferably the biological fluid sample is blood, plasma or serum). This immunoassay can be used in combination with other clinical and / or biochemical features, such as oligo-anovulation and / or irregular cycles, hyperandrogenemia or PCOM, to diagnose females with PCOS. Further, the measurement of the METRNL value can be used to monitor the progression of PCOS in the patient and the response to treatment. The inventors have also shown that the measurement of the METRNL value in a sample, preferably a biological fluid sample (preferably blood, plasma, serum, capillary blood, interstitial fluid, ascites, or menstrual fluid, and more preferably the biological fluid sample is blood, plasma or serum), is particularly suitable for the diagnosis of PCOS in adolescents or young women under 25 years old, particularly under 20 years old, particularly under 15 - 25 years old, particularly under 15 - 20 years old, either alone or in combination with the above additional diagnostic criteria.

[0026] There is an unmet medical need for an accurate test for the reliable diagnosis of PCOS. Measurement of METRNL in a sample, preferably a biological fluid sample (preferably blood, plasma, serum, capillary blood, interstitial fluid, ascites, or menstrual fluid, and the biological fluid sample is more preferably blood, plasma or serum), has the advantage of a highly reliable body fluid-based test for identifying females suffering from currently undiagnosable PCOS. Measurement of METRNL in a sample, preferably a biological fluid sample (preferably blood, plasma, serum, capillary blood, interstitial fluid, ascites or menstrual fluid, and the biological fluid sample is more preferably blood, plasma or serum), can surely be used for the diagnosis of PCOS even in young subjects and young females under 25 years old, especially under 20 years old, especially under 15 to 25 years old, especially under 15 to 20 years old. Diagnosis of PCOS in young patients is difficult for the above reasons, and thus, the inventors provide for the first time an accurate test for the diagnosis of PCOS in young and young female populations. Further, measurement of METRNL in a sample, preferably a biological fluid sample (preferably blood, plasma, serum, capillary blood, interstitial fluid, ascites, or menstrual fluid, and the biological fluid sample is more preferably blood, plasma or serum), has the advantage of identifying whether a patient responds to treatment. A further advantage of measurement of METRNL in a patient's sample, preferably a biological fluid sample (preferably blood, plasma, serum, capillary blood, interstitial fluid, ascites, or menstrual fluid, and the biological fluid sample is more preferably blood, plasma or serum), is to monitor the progression of PCOS.Furthermore, the present inventors provide a computer-implemented method for assessing a patient suffering from PCOS, the method comprising measuring the level of METRNL in a sample, preferably a biological fluid sample (preferably blood, plasma, serum, capillary blood, interstitial fluid, ascites, or menstrual fluid, more preferably blood, plasma or serum), optionally together with further criteria such as values for oligo-anovulation and / or irregular cycles, hyperandrogenemia and / or polycystic ovarian morphology, or together with further biomarkers or hormones, and assessing the subject suffering from PCOS based on the comparison and / or calculation of said data.

[0027] As noted above, patients suffering from PCOS can exhibit characteristics of two types, reproductive or metabolic. Metabolic PCOS includes obesity, insulin resistance, metabolic syndrome, prediabetes, type 2 diabetes, non-alcoholic fatty liver disease (NAFLD), and cardiovascular factors. The term "phenotype" can be used in place of "reproductive." The term "reproductive" (or "phenotype") refers to any characteristic of the phenotype of a female known to exhibit PCOS. For example, these reproductive characteristics include polycystic ovarian morphology (PCOM) and / or clinical hyperandrogenemia, such as acne, seborrhea, alopecia, and / or hirsutism. Preferably, these reproductive characteristics include polycystic ovarian morphology (PCOM) and / or clinical hyperandrogenemia, more preferably acne, seborrhea, alopecia, deepening of the voice, and / or hirsutism. These reproductive characteristics of clinical hyperandrogenemia can be simply diagnosed by asking the female or are apparent after a short physical examination of the female's body. Typically, a reference population exhibits none or no more than one of these phenotypic characteristics known to indicate PCOS.

[0028] Meteorin-like protein (METRNL) is a hormone (28KDa secreted protein) induced after exercise and cold exposure in skeletal muscle and adipose tissue, respectively. Increased METRNL expression in circulation or adipose tissue led to 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 improved glucose tolerance. Metrnl did not directly affect the thermogenesis of white adipocytes in vitro, indicating the involvement of non-adipocyte types in the induction of beige fat. Instead, Metrnl seemed to stimulate several immune cell subtypes to enter adipose tissue and activate their thermogenesis-promoting effects. In METRNL-treated mice, the numbers of macrophages and eosinophils in WAT increased, and the expression of genes related to alternative macrophage activation increased (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 immunity and possibly also acquired immunity. High expression of METRNL has been identified in activated monocytes (M2-polarized macrophages), skin, and mucosal tissues. In the skin, METRNL is expressed by 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 and co-workers described the role of METRNL in the regulation of skeletal muscle repair through macrophage adhesion and phenotypic conversion. The results suggested that METRNL is mainly secreted from macrophages in response to local injury. Furthermore, METRNL promoted anti-inflammatory functions through a STAT3-dependent autocrine / paracrine mechanism that induces insulin-like growth factor 1 (IGF-1), which activates muscle precursors to aid myogenesis. Finally, METRNL has been shown to be an important regulator of muscle regeneration that acts directly on immune cells to promote an anti-inflammatory / regeneration-promoting 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 an increase in cerebrospinal fluid METRNL concentration (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).

[0029] Serum METRNL levels have been studied in relation to type 2 diabetes (T2DM) and have yielded 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) level 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.November 25, 2019; 25:8968 - 8974; Ferns GA, Fekri K, Shahini Shams Abadi M, et al. A meta - analysis of the relationship between serums metrnl - like protein / subfatin and risk of type 2 diabetes mellitus and coronary artery disease. Arch Physiol Biochem. 2021 May 5:1 - 7; Lappas M. Maternal obesity and gestational diabetes decrease Metrnl concentrations in cord plasma. J Matern Fetal Neonatal Med. 2021 Sep;34(18):2991 - 2995). Patients with type 2 diabetes mellitus (T2DM) and coronary artery disease (CAD) showed lower serum METRNL levels compared to the control group. Furthermore, METRNL showed a negative correlation with IL - 6 and TNF - α in both CAD patients and also showed a negative correlation 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). Additionally, 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.January 2019; 23(1): 271 - 280). Obese patients who underwent bariatric surgery showed a decrease in the circulating levels of METRLN and an improvement in glucose and lipid homeostasis compared to 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 examined the circulating levels of METRNL in PCOS patients compared to 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 age of the women was between 20 years and 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 to controls. Furthermore, serum METRNL was correlated with BMI, adiponectin, and homocysteine in controls and inversely correlated with FBG, fasting insulin, and HOMA - IR in the PCOS group and subgroups. Additionally, it was inversely correlated with hs - CRP in controls, as well as in the PCOS group and subgroups (Fouani FZ, Fadaei R, Moradi N, et al. Circulating levels of Meteorin - like protein in polycystic ovary syndrome: A case - control study. PLoS One. 2020 Apr 24; 15(4): e0231943). Deniz et al. measured the levels of METRNL (subfatin) and asprosin in plasma samples obtained from 30 PCOS cases and 30 healthy controls (mean age of controls: 28.22 ± 2.6 years; mean age of PCOS cases: 27.14 ± 3.21 years old). Asprosin levels were significantly higher in women with PCOS compared to healthy controls, while METRNL levels were significantly lower compared to controls, consistent with the results shown by Fouani et al. Both asprosin and METRNL levels showed a significant correlation with HOMA-IR in the PCOS subgroup (Deniz R, Yavuzkir S, Ugur K, et al. Subfatin and asprosin, two new metabolic players of polycystic ovary syndrome. J Obstet Gynaecol. 2021 Feb;41(2):279-284. doi:10.1080 / 01443615.2020.1758926).

[0030] 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 on metabolic and inflammatory diseases, in patients with osteoarthritis, serum METRNL was lower and synovial fluid METRNL was higher compared to non-osteoarthritis controls (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).

[0031] In a first aspect of the present invention, a method for assessing whether a subject has PCOS or is at risk of developing PCOS, comprising: (a) determining the amount or concentration of METRNL in a sample of the subject; and (b) comparing the determined amount or concentration to a reference. The present invention relates to such a method.

[0032] A decrease in the amount or concentration of METRNL in a patient's sample indicates the presence, risk, or development of PCOS in the patient. In particular, the amount or concentration of METRNL in a patient's sample indicates the presence or risk of development of PCOS in the patient if the amount or concentration of METRNL in the patient's sample is higher than the amount or concentration of METRNL in a reference or reference sample. In particular, METRNL is detectable in a biological fluid sample from a patient being evaluated for the presence or risk of development of PCOS in a greater amount or concentration than in the same biological fluid sample from an individual not suffering from PCOS or not at risk of developing PCOS. In particular, an amount or concentration of METRNL reduced by 50% or more indicates the presence or risk of development of PCOS. In particular, an amount or concentration of METRNL reduced by 100% or more indicates the presence or risk of development of PCOS. In particular, an amount or concentration of METRNL reduced by 150% or more indicates the presence or risk of development of PCOS. In particular, an amount or concentration of METRNL reduced by 200% or more indicates the presence or risk of development of PCOS.

[0033] In embodiments, the biological fluid sample is whole blood, serum, plasma, capillary blood, interstitial fluid, ascites, or menstrual fluid, preferably the biological fluid sample is serum or whole blood. In embodiments, the sample is an in vitro sample, i.e., it is analyzed in vitro and not returned to the body.

[0034] In certain embodiments, the patient is an experimental animal, a domestic animal or a primate. In certain embodiments, the patient is a human patient. In certain embodiments, the patient is a human female patient. In certain embodiments, the patient is a human female patient under 25 years of age. In certain embodiments, the patient is a human female patient under 20 years of age. In certain embodiments, the patient is a human female patient between 15 and less than 25 years of age. In certain embodiments, the patient is a human female patient between 15 and less than 20 years of age. In certain embodiments, the patient is a human female patient under 25 years of age, preferably under 20 years of age, and within 3 years after menarche. In certain embodiments, the patient is a human female patient under 20 years of age and within 3 years after menarche. In certain embodiments, the patient is a human female patient between 15 and less than 25 years of age and within 3 years after menarche. In certain embodiments, the patient is a human female patient between 15 and less than 20 years of age and within 3 years after menarche.

[0035] In embodiments, PCOS is assessed from the group consisting of metabolic or phenotypic PCOS. In a further aspect, PCOS is assessed from the group consisting of phenotypic A, phenotypic B, phenotypic C and phenotypic D PCOS.

[0036] In embodiments, the first method of the present invention is an in vitro method.

[0037] In embodiments, the amount or concentration of METRNL is determined using an antibody, particularly a monoclonal antibody. In embodiments, step a) of determining the amount or concentration of METRNL in a patient sample comprises performing an immunoassay. In embodiments, the immunoassay is performed in either a direct or indirect format. In embodiments, such immunoassays are 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.

[0038] In certain embodiments, step a) of determining the amount or concentration of METRNL in a patient sample is i) incubating a sample from the patient with one or more antibodies that specifically bind to METRNL, thereby generating a complex of the antibody and METRNL, and ii) quantifying the complex formed in step i), thereby quantifying the amount or concentration of METRNL in the patient's sample comprising.

[0039] In certain embodiments, 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 anti-METRNL antibody, the second anti-METRNL antibody, or both the first and second antibodies simultaneously, in any desired order, for a time and under conditions sufficient to form a first anti-METRNL antibody / METRNL / second anti-METRNL antibody complex. As will be readily appreciated by those skilled in the art, it is routine experimentation to establish the time and conditions appropriate or sufficient for either the formation of a complex between a specific anti-METRNL antibody and the METRNL antigen / analyte (= anti-METRNL complex), or the formation of a secondary or sandwich complex comprising the first anti-METRNL antibody, METRNL (analyte), and the second anti-METRNL antibody (= first anti-METRNL antibody / METRNL / second anti-METRNL antibody complex).

[0040] The detection of the anti-METRNL antibody / METRNL complex can be performed by any suitable means. The 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 conversant with such means / methods.

[0041] In certain embodiments, a sandwich is formed comprising a first antibody to METRNL, METRNL (analyte), and a second antibody to METRNL, and the second antibody is detectably labeled.

[0042] In one embodiment, a sandwich is formed that includes a first antibody against METRNL, METRNL (the analyte), and a second antibody against METRNL, the second antibody being detectably labeled, and the first anti-METRNL antibody being capable of binding to a solid phase or being bound to a solid phase.

[0043] 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 dye or an electrochemiluminescent dye.

[0044] In a second aspect, the invention is a method of selecting a patient for treatment of PCOS, comprising: (c) determining the amount or concentration of METRNL in a sample of the subject; and (d) comparing the determined amount or concentration to a reference. The invention relates to a method comprising the above steps.

[0045] In embodiments, if a decrease in the amount of METRNL in a patient's sample is determined, the patient is selected for treatment of PCOS. In particular, if the amount of METRNL is greater than the amount of METRNL in a reference sample or reference samples, the patient is selected for treatment of PCOS. In particular, if the amount of METRNL is higher in a biological fluid sample of a patient being evaluated for treatment of PCOS than in the same biological fluid sample of an individual not suffering from PCOS, not at risk of developing PCOS, or not selected for treatment of PCOS, the patient is selected for treatment of PCOS. In particular, if the amount of METRNL is decreased by 50% or more, the patient is selected for treatment of PCOS. In particular, if the amount of METRNL is decreased by 100% or more, the patient is selected for treatment of PCOS. In particular, if the amount of METRNL is decreased by 150% or more, the patient is selected for treatment of PCOS. In particular, if the amount of METRNL is decreased by 200% or more, the patient is selected for treatment of PCOS.

[0046] In particular, patients are selected for drug-based treatment of PCOS or lifestyle modifications to control metabolic syndrome. In embodiments, the drug-based therapy for PCOS includes drugs for regulating the menstrual cycle, particularly oral contraceptives or progestin therapy, drugs for preventing or controlling diabetes, particularly type 2 diabetes, drugs for preventing or controlling high cholesterol, drugs for enhancing hormones or fertility, drugs or treatments for removing excessive hair, and drugs or treatments for controlling acne, and is selected from the group consisting of these.

[0047] In embodiments, the biological fluid sample is whole blood, serum, plasma, capillary blood, interstitial fluid, ascites, or menstrual fluid, and preferably the biological fluid sample is serum or whole blood. In embodiments, the sample is an in vitro sample, i.e., it is analyzed in vitro and not returned to the body.

[0048] In certain embodiments, the patient is an experimental animal, livestock, or a primate. In certain embodiments, the patient is a human patient. In certain embodiments, the patient is a human female patient. In certain embodiments, the patient is a human female patient under 25 years old. In certain embodiments, the patient is a human female patient under 20 years old. In certain embodiments, the patient is a human female patient between 15 and less than 25 years old. In certain embodiments, the patient is a human female patient between 15 and less than 20 years old. In certain embodiments, the patient is a human female patient under 25 years old, preferably under 20 years old, and within 3 years after menarche. In certain embodiments, the patient is a human female patient under 20 years old and within 3 years after menarche. In certain embodiments, the patient is a human female patient between 15 and less than 25 years old and within 3 years after menarche. In certain embodiments, the patient is a human female patient between 15 and less than 20 years old and within 3 years after menarche.

[0049] In embodiments, the second method of the present invention is an in vitro method.

[0050] In an embodiment, the amount of METRNL is determined using an antibody, particularly a monoclonal antibody. In an embodiment, step a) of determining the amount of METRNL in a patient sample comprises performing an immunoassay. In an embodiment, the immunoassay is performed in either a direct or indirect format. In an embodiment, such an immunoassay is selected from the group consisting of an enzyme-linked immunosorbent assay (ELISA), an enzyme immunoassay (EIA), a radioimmunoassay (RIA), or an immunoassay based on the detection of luminescence, fluorescence, chemiluminescence or electrochemiluminescence.

[0051] In certain embodiments, step a) of determining the amount of METRNL in a patient sample comprises i) incubating the patient sample with one or more antibodies that specifically bind to METRNL, thereby generating a complex of the antibody and METRNL, and ii) quantifying the complex formed in step i), thereby quantifying the amount of METRNL in the patient sample and includes.

[0052] In certain embodiments, 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 anti-METRNL antibody, the second anti-METRNL antibody, or both the first and second anti-METRNL antibodies simultaneously, in any desired order, for a time and under conditions sufficient to form a first anti-METRNL antibody / METRNL / second anti-METRNL antibody complex, for example, first with the first antibody and then with the second antibody, or first with the second antibody and then with the first antibody. As will be readily understood by those skilled in the art, establishing the time and conditions appropriate or sufficient for either the formation of a complex between a specific anti-METRNL antibody and a METRNL antigen / analyte (= anti-METRNL complex), or the formation of a secondary or sandwich complex comprising a first anti-METRNL antibody, METRNL (analyte) and a second anti-METRNL antibody (= first anti-METRNL antibody / METRNL / second anti-METRNL antibody complex) is routine experimentation.

[0053] The detection of the anti-METRNL antibody / METRNL complex can be performed by any suitable means. The 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 proficient in such means / methods.

[0054] In certain embodiments, a sandwich is formed that includes a first antibody to METRNL, METRNL (analyte), and a second antibody to METRNL, and the second antibody is detectably labeled.

[0055] In one embodiment, a sandwich is formed that includes a first antibody to METRNL, METRNL (analyte), and a second antibody to METRNL, the second antibody is detectably labeled, and the first anti-METRNL antibody can bind to or is bound to a solid phase.

[0056] 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 dye or an electrochemiluminescent dye.

[0057] Furthermore, the present invention also relates to a kit comprising reagents for the diagnosis of PCOS. The reagents of the kit can include an antibody or an antibody fragment. Preferably, the antibody or antibody fragment recognizes an epitope or antigen of METRNL. The kit can further include other reagents that recognize other biomarkers. Thus, the kit can include a combination of at least two reagents. The kit can specifically measure the amount or concentration of METRNL and any other biomarker of interest. According to the present invention, the biomarker can also include hormones, anti-Mullerian hormone (AMH). The kit can be used in any diagnostic assay.

[0058] In an embodiment, the amount of METRNL is determined using an antibody, particularly a monoclonal antibody. In an embodiment, step a) of determining the amount of METRNL in a patient sample comprises performing an immunoassay. In an embodiment, the immunoassay is performed in either a direct or indirect format. In an embodiment, such immunoassays are selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), or immunoassays based on the detection of luminescence, fluorescence, chemiluminescence or electrochemiluminescence.

[0059] In a third aspect, the invention is a method of monitoring the progression of PCOS in a subject or monitoring the response of a subject having PCOS to treatment, comprising: (a) determining the level of METRNL in a first sample of the subject; (b) determining the level of METRNL in a second sample of the subject obtained after the first sample; (c) comparing the level of METRNL in the first sample with the level of METRNL in the second sample; (d) monitoring the progression in the subject suffering from or being treated for PCOS based on the result of step c); The method is related to the above.

[0060] In an embodiment, the progression of PCOS in a subject having PCOS is monitored to determine whether the amount or concentration of METRNL in a patient sample changes over time. In particular, the progression of PCOS is monitored to determine whether the amount or concentration of METRNL increases, decreases, or does not change over time. In an embodiment, when a decrease in the amount or concentration of METRNL in a subject's sample is determined, the progression of PCOS is monitored.

[0061] In an embodiment, a subject undergoing treatment for PCOS is monitored to determine whether the amount or concentration of METRNL in the subject's sample has changed. In particular, a subject undergoing treatment for PCOS is monitored to determine whether the amount or concentration of METRNL has increased, decreased, or remained unchanged. In particular, a subject undergoing treatment for PCOS is monitored to determine whether the amount or concentration of METRNL has increased, decreased, or remained unchanged due to the applied treatment. In an embodiment, a decrease in the amount or concentration of METRNL in a subject undergoing treatment for PCOS indicates that the treatment is effective. In an embodiment, that the amount or concentration of METRNL in a sample of a subject being treated for PCOS has not changed or has decreased indicates that PCOS persists. In particular, if the amount or concentration of METRNL has decreased by 50% or more, the treatment for PCOS is ineffective. In particular, if the amount or concentration of METRNL has decreased by 100% or more, the treatment for PCOS is ineffective. In particular, if the amount or concentration of METRNL has decreased by 150% or more, the treatment for PCOS is ineffective. In particular, if the amount or concentration of METRNL has decreased by 200% or more, the treatment for PCOS is ineffective.

[0062] In certain embodiments, if an unchanged or decreased amount or concentration of METRNL in a sample of a subject being treated for PCOS is determined, the treatment is adapted.

[0063] In an embodiment, the subject is monitored several times at different time points. In an embodiment, the subject is monitored several times within a time frame of weeks, months, or years. In certain embodiments, the subject is monitored once a month or once a year. In an embodiment, a subject suffering from PCOS is monitored once a month or once a year after diagnosis of PCOS. In an embodiment, a subject being treated for PCOS is monitored once after treatment. In particular, a subject being treated for PCOS is monitored once a month or once a year to determine the effectiveness of the treatment.

[0064] In an embodiment, the treatment of PCOS is selected from the group consisting of drug-based treatment of PCOS and lifestyle changes to control metabolic syndromes. In an embodiment, the drug-based therapy for PCOS is a drug for regulating the period, particularly an oral contraceptive or progestin therapy, a drug for preventing or controlling diabetes, particularly type 2 diabetes, a drug for preventing or controlling high cholesterol, a drug for enhancing hormones or fertility, a drug or treatment for removing excessive hair, or a drug or treatment for controlling acne, and is selected from the group consisting of these.

[0065] In an embodiment, the biological fluid sample is whole blood, serum, plasma, capillary blood, interstitial fluid, ascites, or menstrual fluid, and preferably the biological fluid sample is serum or whole blood. In an embodiment, the sample is an in vitro sample, that is, it is analyzed in vitro and not returned to the body.

[0066] In certain embodiments, the patient is an experimental animal, a domestic animal, or a primate. In certain embodiments, the patient is a human patient. In certain embodiments, the patient is a human female patient. In certain embodiments, the patient is a human female patient under 25 years old. In certain embodiments, the patient is a human female patient under 20 years old. In certain embodiments, the patient is a human female patient aged 15 to less than 25 years old. In certain embodiments, the patient is a human female patient aged 15 to less than 20 years old. In certain embodiments, the patient is a human female patient under 25 years old, particularly under 20 years old, and within 3 years after menarche. In certain embodiments, the patient is a human female patient under 20 years old and within 3 years after menarche. In certain embodiments, the patient is a human female patient aged 15 to less than 25 years old and within 3 years after menarche. In certain embodiments, the patient is a human female patient aged 15 to less than 20 years old and within 3 years after menarche.

[0067] In an embodiment, the second method of the present invention is an in vitro method.

[0068] In an embodiment, the amount or concentration of METRNL is determined using an antibody, particularly a monoclonal antibody. In an embodiment, step a) of determining the amount or concentration of METRNL in a patient sample comprises performing an immunoassay. In an embodiment, the immunoassay is performed in either a direct or indirect format. In an embodiment, such immunoassays are selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), or immunoassays based on the detection of luminescence, fluorescence, chemiluminescence or electrochemiluminescence.

[0069] In certain embodiments, step a) of determining the amount or concentration of METRNL in a patient sample i) incubating the patient sample with one or more antibodies that specifically bind to METRNL, thereby generating a complex of the antibody and METRNL, and ii) quantifying the complex formed in step i), thereby quantifying the amount or concentration of METRNL in the patient sample comprises.

[0070] In certain embodiments, 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 anti-METRNL antibody, the second anti-METRNL antibody, or both the first and second antibodies simultaneously, in any desired order, for a time and under conditions sufficient to form a first anti-METRNL antibody / METRNL / second anti-METRNL antibody complex, e.g., first the first antibody and then the second antibody, or first the second antibody and then the first antibody. As will be readily understood by those skilled in the art, establishing the time and conditions appropriate or sufficient for either the formation of a complex between a specific anti-METRNL antibody and a METRNL antigen / analyte (= anti-METRNL complex), or the formation of a secondary or sandwich complex comprising a first anti-METRNL antibody, METRNL (analyte) and a second anti-METRNL antibody (= first anti-METRNL antibody / METRNL / second anti-METRNL antibody complex) is merely routine experimentation.

[0071] The detection of the anti-METRNL antibody / METRNL complex can be performed by any suitable means. The 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 proficient in such means / methods.

[0072] In certain embodiments, a sandwich is formed that includes a first antibody against METRNL, METRNL (analyte), and a second antibody against METRNL, and the second antibody is detectably labeled.

[0073] In one embodiment, a sandwich is formed that includes a first antibody against METRNL, METRNL (analyte), and a second antibody against METRNL, the second antibody is detectably labeled, and the first anti-METRNL antibody can bind to or is bound to a solid phase.

[0074] 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 dye or an electrochemiluminescent dye.

[0075] In a fourth aspect, the invention is a computer-implemented method for assessing a subject suspected of having PCOS, comprising: (a) receiving a value for the amount or concentration of a first biomarker in a sample of the subject, wherein the first biomarker is METRNL; (b) optionally, receiving a value for the amount or concentration of a second biomarker in the sample of the subject; (c) optionally, receiving a value for the presence or absence of at least an additional diagnostic criterion selected from the group consisting of oligo-ovulation and hyperandrogenemia and polycystic ovarian morphology; (d) Comparing the value of the quantity or concentration in steps (a) to (b) with the reference for the biomarker and the value for the presence or absence of the at least one additional diagnostic criterion, and / or calculating a score for assessing the subject in whom PCOS is suspected based on the quantity or concentration of the biomarker and the value, (e) Evaluating the subject based on the comparison and / or the calculation performed in step (d), A computer-implemented method comprising the above is provided.

[0076] In an embodiment, a computer-implemented method for assessing a subject suspected of having PCOS includes a method that consists essentially of the above-described steps or further steps. Further, the method of the present invention is preferably an ex vivo method, more preferably an in vitro method. Further, the method of the present invention may include steps in addition to those explicitly described above. For example, the further steps may relate to further determining markers and / or collecting samples before treatment or evaluating the results obtained by the above method. The method may be performed manually or assisted by automation.

[0077] As used herein, the term "computer-implemented" means that the method is typically executed in an automated manner on a data processing unit included in a computer or similar data processing device. The data processing unit receives a value for the quantity of the biomarker. Such a value can be a quantity, relative quantity or any other calculated value that reflects the quantities described in detail elsewhere in this specification. Thus, it should be understood that the above method does not require determination of the quantity of the biomarker, but rather uses values for quantities that have already been determined in advance.

[0078] The present invention also generally contemplates a computer program, a computer program product, or a computer-readable storage medium having the computer program tangibly incorporated therein, where the computer program includes instructions for performing the method of the present invention as specified above when executed on a data processing device or a computer. Specifically, the present disclosure further includes the following: - a computer or computer network comprising at least one processor, the processor being configured to execute a method according to one of the embodiments described herein, - a computer loadable data structure configured to execute a method according to one of the embodiments described herein when executed on a computer, - a computer script, wherein the computer program is adapted to execute one of the methods of the embodiments described herein while the program is being executed on a computer, - a computer program comprising program means for executing a method according to one of the embodiments described herein when the computer program is executed on a computer or a computer network, - a computer program comprising the program means described in the preceding embodiments stored on a computer-readable storage medium, - a storage medium having a data structure stored thereon and adapted to execute a method according to one of the embodiments described herein after the data structure is loaded into the main memory and / or working memory of a computer or computer network, - a computer program product having program code means that can be stored or stored on a storage medium for executing a method according to one of the embodiments described herein when the program code means are executed on a computer or a computer network, Data streams signals, typically encrypted, containing data on parameters defined elsewhere in this specification, as well as - Data stream signals, typically encrypted, containing the assessments provided by the method of the present invention.

[0079] Definition: In the context of the kit of the present invention, the term "reagent" refers to a substance or compound added to a sample that enables the indication of the amount or concentration of a specific component in the sample.

[0080] In the context of the kit of the present invention, the term "specifically measure" means detecting the exact amount or concentration of a well-defined molecule. For a specific measurement, a sample obtained from a female can be incubated with a reagent under conditions appropriate for the formation of a binder-marker complex. Such appropriate incubation conditions are well known to those skilled in the art, so it is not necessary to specify such conditions.

[0081] In the context of the kit of the present invention, the term "reagent" can refer to a protein molecule (such as an antibody), a nucleic acid molecule (such as any form of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)), or another biochemical, organic or inorganic substance that can interact with the molecule specifically measured in the sample.

[0082] Furthermore, the reagent can be linked to a detectable reporter moiety or label such as an enzyme, a dye, a radionuclide, a luminescent group, a fluorescent group or biotin, for example a fluorescent marker that can be used in immunoassay analysis. The reporter moiety or label can be used with the reagent of the kit according to the second aspect of the invention as long as its signal can be directly related or proportional to the amount of binder remaining on the support after washing. Then, the amount of any second binder remaining bound to the solid support can be determined using a method suitable for the specific detectable reporter moiety or label. In the case of a radionuclide, scintillation counting or autoradiography methods are generally appropriate. Antibody-enzyme conjugates can be prepared using various coupling techniques (for a review, see, for example, Scouten, W.H., Methods in Enzymology 135:30-65, 1987). Dyes (including, for example, colorimetric products of enzyme reactions), luminescent groups and fluorescent groups can be detected using spectroscopy. Biotin can be detected by binding to avidin or streptavidin and then to a different reporter group (usually a radioactive or fluorescent group or an enzyme). Enzyme reporter groups can generally be detected by addition of a substrate (usually for a specific period) followed by spectroscopic analysis, spectrophotometry or other analysis of the reaction product. Standards and standard additions can be used to determine the level of antigen in a sample using techniques well known to those skilled in the art.

[0083] The reagent may also be a substance that can potentially further bind to the matrix of a column used in chromatography for purification and / or further analysis (such as mass spectrometry). Furthermore, the reagent may be linked to a test strip.

[0084] Preferably, the reagent is an antibody. Suitable antibodies for measuring the amount or concentration of one of the specifically measured molecules in a sample obtained from the female as described above are well known to those skilled in the art.

[0085] Preferably, the reagent can be used in an electrochemiluminescent immunoassay, and more preferably, the reagent is an antibody that can be used in an electrochemiluminescent immunoassay.

[0086] Furthermore, the kit can include more than one reagent, such as two different reagents, three different reagents, four different reagents or more different reagents, preferably two different reagents that interact with one molecule specifically measured in the sample. For example, when the molecule to be specifically measured is measured by an electrochemiluminescent immunoassay, the kit can include two different antibodies that bind to the same molecule being measured. Preferably, the two different antibodies that bind to the same molecule do not compete for the binding site on the molecule and bind to this molecule at different positions. Furthermore, both antibodies can be linked to different detectable reporter moieties or labels.

[0087] The kit can further include a buffer and / or a salt for adjusting the pH as well as the reaction conditions and measurement conditions. Furthermore, the kit comprises a stabilizer for supporting the stability of the reagent and / or the hormone during the specific measurement of, for example, (i) the amount or concentration of FT, or (ii) the amount or concentration of TT and the amount or concentration of SHBG, the amount or concentration of AMH, and the amount or concentration of one or more further hormones indicating PCOS. Suitable buffers, salts and stabilizers are well known to those skilled in the art. Furthermore, sodium azide may be added to all liquid solutions of the kit, such as the reagent or the buffer solution.

[0088] The kit may also comprise all the equipment necessary for collecting a blood sample from a female, such as a container for the blood sample, a needle, and a device for connecting the container and the needle. Preferably, the kit can comprise a syringe.

[0089] Generally, a physician or a physician's assistant may collect blood from a female. Thereafter, the blood may be sent to a laboratory where the sample is measured using the kit with a designated analyzer and the data is sent to the physician. However, the kit may also be applied by the physician or the physician's assistant themselves. The kit can be applied during a physician's outpatient, stationary treatment or home visit.

[0090] All components of the kit may be separately packaged in individual containers. However, it is also possible that two or more components of the kit may be packaged together in one or more containers.

[0091] The kit may further comprise, for example, a label containing instructions on how to use the kit or instructions on the contents of the kit. However, this information may also be provided in any other form, such as on a storage medium such as a CD-ROM or a USB stick.

[0092] It is to be understood that the word "comprise", and variations such as "comprises" and "comprising", mean the inclusion of the stated integer or step or group of integers or steps, but do not mean the exclusion of any other integer or step or group of integers or steps.

[0093] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0094] Concentrations, amounts, and other numerical data may be expressed or presented herein in the form of a "range". It should be understood that such a range format is merely used for convenience and brevity, and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the boundaries of the range, but also all of the individual numerical values or sub-ranges subsumed within that range as if each were explicitly recited. By way of illustration, a numerical range of "150 mg to 600 mg" should be interpreted to include not only the explicitly recited values of 150 mg to 600 mg, but also the individual values and sub-ranges within the indicated range. Thus, this numerical range includes individual values such as 150, 160, 170, 180, 190, 580, 590, 600 mg, etc., and sub-ranges such as 150 to 200, 150 to 250, 250 to 300, 350 to 600, etc. This same principle applies to ranges that enumerate only a single numerical value. Further, such an interpretation should apply regardless of the width of the range or the property being described.

[0095] The term "about", when used in connection with a numerical value, means a range of numerical values having a lower limit that is 5% less than the indicated numerical value and an upper limit that is 5% greater than the indicated numerical value.

[0096] As used herein, the term "indicator" refers to a sign or signal for a symptom or is used to monitor a condition. Such "symptoms" refer to the biological state of a cell, tissue or organ, or the health and / or disease state of an individual. An indicator can be, but is not limited to, the presence or absence of a molecule including a peptide, protein, and nucleic acid, or a change in the expression level or pattern of such a molecule in a cell, or in a tissue, organ or individual. An indicator can be a sign of the occurrence, onset or presence of a disease in an individual, or a sign of further progression of such a disease. An indicator can also be a sign of the risk of developing a disease in an individual.

[0097] In the context of the present invention, the term "biomarker" refers to a substance within a biological system that is used as an indicator of the biological state of the biological system. In the art, the term "biomarker" may also be applied to means for detecting such endogenous substances (e.g., antibodies, nucleic acid probes, etc., imaging systems). In the context of the present invention, the term "biomarker" shall be applied only to substances and not to detection means. Thus, a biomarker can be any kind of molecule present in a living body, such as nucleic acids (DNA, mRNA, miRNA, rRNA, etc.), proteins (cell surface receptors, cytosolic proteins, etc.), metabolites or hormones (blood glucose, insulin, estrogen, etc.), molecules characteristic of a specific modification of another molecule (e.g., sugar moieties or phosphoryl residues on a protein, methyl residues on genomic DNA), or substances internalized by an organism or metabolites of such substances.

[0098] As used herein, biomarkers can be detected using methods generally known in the art. Detection methods generally include methods for quantifying the level of a biomarker in a sample (quantitative methods). Which of the following methods is suitable for qualitative and / or quantitative detection of a biomarker is generally known to those skilled in the art. Samples can be readily assayed for proteins, for example, using Western methods as well as immunoassays such as ELISA, RIA, fluorescence- and luminescence-based immunoassays, and commercially available proximity extension assays. Further suitable methods for detecting biomarkers include measuring physical or chemical properties specific to a peptide or polypeptide, such as its exact molecular weight or NMR spectrum, etc. Such methods include, for example, analytical devices such as biosensors, optical devices associated with immunoassays, biochips, mass spectrometers, NMR analyzers, or chromatography devices. Further, methods include microplate ELISA-based methods, fully automated or robotic immunoassays (available on Elecsys™ analyzers), CBA (Cobalt Binding Assay by enzyme, available on Roche-Hitachi™ analyzers), and latex agglutination assays (available on Roche-Hitachi™ analyzers).

[0099] The term "anovulation" typically refers to a condition in which the ovaries do not release any oocytes during the female menstrual cycle. A female being assessed for risk of having PCOS can be determined to be suffering from anovulation if no oocytes are released over a period of at least one female menstrual cycle per year, preferably at least three female menstrual cycles per year, more preferably at least six female menstrual cycles per year, and most preferably at least nine female menstrual cycles per year. Further, a female being assessed for risk of having PCOS can be determined to be suffering from anovulation if no oocytes are released for at least six months, preferably at least nine months, more preferably at least one year.

[0100] The "symptoms" of a disease are obvious hints of such a disease by the tissues, organs or organisms having such a disease, including, but not limited to, pain, weakness, tenderness, tension, stiffness and spasm of tissues, organs or individuals. Typical symptoms of PCOS include, but are not limited to, oligo-anovulation, irregular cycles, hyperandrogenemia, polycystic ovarian morphology, infertility, type 2 diabetes, overweight and other metabolic symptoms, as well as psychological distress. The "signs" or "signals" of a disease include, but are not limited to, the presence, increase or elevation, decrease or decline of specific indicators such as biomarkers or molecular markers, changes or alterations such as changes (change) or alterations (alteration), or the onset, presence or exacerbation of symptoms. The symptoms of pain include, but are not limited to, unpleasant sensations that can be felt as persistent or various burning pains, throbbing pains, itching or stabbing pains.

[0101] The terms "disease" and "disorder" are used interchangeably herein and refer to abnormal medical conditions, such as abnormal symptoms, particularly illnesses or injuries where a tissue, organ, or individual can no longer efficiently perform its function. Although not necessarily the case, typically, a disease is associated with specific symptoms or signs indicating the presence of such a disease. Thus, the presence of such symptoms or signs can indicate the tissue, organ, or individual suffering from the disease. Changes in these symptoms or signs can indicate the progression of such a disease. The progression of a disease typically features an increase or decrease in such symptoms or signs, which can indicate "deterioration" or "improvement" of the disease. "Deterioration" of a disease is characterized by a decrease in the ability of a tissue, organ, or organism to efficiently perform its function, whereas "improvement" of a disease typically features an increase in the ability of a tissue, organ, or individual to efficiently perform its function. A tissue, organ, or individual at "risk of developing" a disease is in a healthy state but shows the potential for the disease to manifest. Typically, the risk of developing a disease is associated with early or weak symptoms or signs of such a disease. In such cases, the onset of the disease can still be prevented by treatment. Examples of diseases include, but are not limited to, inflammatory diseases, infectious diseases, skin conditions, endocrine diseases, intestinal diseases, neuropathies, joint diseases, genetic disorders, autoimmune diseases, traumatic diseases, and various types of cancer.

[0102] The terms "patient" and "subject" are used interchangeably herein and refer to an animal, preferably a mammal, more typically a human. The patient is preferably a human female. There is a need for a diagnosis of PCOS.

[0103] The terms "sample" or "target sample" are used interchangeably herein and refer to a part or piece of a tissue, organ or individual, and are usually smaller than such a tissue, organ or individual which is intended to represent the whole of the tissue, organ or individual. In the analysis, the sample provides information regarding the state of the tissue, or the health or disease state of the organ or individual. Examples of samples include, but are not limited to, fluid samples such as blood, serum, plasma, synovial fluid, urine, saliva, and lymphatic fluid, or solid samples such as tissue extracts, cartilage, bone, synovium, and connective tissue. The analysis of the sample can be achieved visually or chemically. Visual analysis includes, but is not limited to, microscopic imaging or radiation scanning of the tissue, organ or individual that enables morphological evaluation of the sample. Chemical analysis includes, but is not limited to, detection of the presence or absence of specific indicators or changes in their amounts, concentrations or levels. The sample is an in vitro sample and will be analyzed in vitro and will not be returned to the body.

[0104] As used herein, the term "amount" encompasses the absolute amount of a biomarker referred to herein, the relative amount or concentration of the biomarker, and any value or parameter that can be correlated with or derived from them. Such values or parameters include intensity signal values derived from all specific physical or chemical properties obtained from the peptide by direct measurement, for example, intensity values in a mass spectrum or an NMR spectrum. Further included are values or parameters obtained by indirect measurements as specified elsewhere herein, for example, the amount of response measured by a biological readout system in response to the peptide, or the intensity signal obtained from a specifically bound ligand. It should be understood that values correlated with the above-described amounts or parameters can also be obtained by all standard mathematical operations.

[0105] As used herein, the term "comparing" refers to comparing the amount of a biomarker in a sample from a subject to a reference amount of the biomarker as specified elsewhere herein. Comparing, as used herein, typically refers to comparing corresponding parameters or values. For example, an absolute amount is compared to a reference absolute amount, a concentration is compared to a reference concentration, or an intensity signal obtained from a biomarker in a sample is compared to the same type of intensity signal obtained from a reference sample. The comparison may be performed manually or using a computer. Thus, the comparison can be performed by a computing device. The measured or detected amount of a biomarker in a sample from a subject and the value of the reference amount can, for example, be compared to each other, and the comparison can be automatically performed by a computer program that executes an algorithm for the comparison. The computer program that performs the above evaluation provides the desired rating in a suitable output format. In a computer-aided comparison, the value of the measured amount may be compared by a computer program to a value corresponding to a suitable reference stored in a database. The computer program may further evaluate the result of the comparison, i.e., it may automatically provide the desired rating in a suitable output format. In a computer-aided comparison, the value of the measured amount may be compared by a computer program to a value corresponding to a suitable reference stored in a database. The computer program may further evaluate the comparison result, i.e., it may automatically provide the desired rating in a suitable output format.

[0106] The expression "comparing the determined amount or concentration to a reference" is used merely to further clarify what would be apparent to one of ordinary skill in the art in any event. The reference concentration is established in a control sample

[0107] The terms "reference sample" or "control sample", as used herein, refer to a sample that is analyzed in substantially the same manner as the sample of interest and whose information is compared to the information of the sample of interest. Thereby, the reference sample provides a standard by which to evaluate the information obtained from the sample of interest. A control sample is derived from a healthy or normal tissue, organ, or individual, thereby providing a standard for the health status of the tissue, organ, or individual. A difference between the state of a normal reference sample and the state of the sample of interest may indicate the risk of disease onset or the presence or further progression of such a disease or disorder. A control sample may be derived from an abnormal or diseased tissue, organ, or individual, thereby providing a standard for the pathological state of the tissue, organ, or individual. A difference between the state of an abnormal reference sample and the state of the sample of interest may indicate a reduced risk of disease onset or the absence or improvement of such a disease or disorder. A reference sample may also be derived from the same tissue, organ, or individual as the sample of interest but was taken at an earlier time point. A difference between the state of a previously taken reference sample and the state of the sample of interest may indicate the progression of the disease, i.e., the improvement or worsening of the disease over time.

[0108] A control sample can be an internal or external control sample. An internal control sample is used, i.e., in the test sample as well as in one or more other samples (s) taken from the same subject, to assess the marker level (s) to determine whether there is a change in the level (s) of the marker (s). For an external control sample, the presence or amount of a marker in a sample derived from an individual is compared to the presence or amount of the marker in an individual known to have or be at risk of a given condition; or in an individual known to be free of a given condition (i.e., a "normal individual").

[0109] Those skilled in the art will understand that such external control samples may be obtained from a single individual or from a reference population of the same age and free of confounding diseases. Typically, samples from 100 individuals well-characterized from an appropriate reference population are used to set "reference values". However, the reference population can also be selected to consist of 20, 30, 50, 200, 500 or 1000 individuals. Healthy individuals are a preferred reference population for establishing control values.

[0110] For example, the marker concentration in a patient sample can be compared to a concentration known to be associated with a particular course of a particular disease. Usually, the marker concentration in the sample is directly or indirectly correlated with the diagnosis, and the marker concentration is used, for example, to determine whether an individual is at risk of a particular disease. Alternatively, the marker concentration in the sample can be compared to a marker concentration known to be relevant, for example, in response to treatment in a particular disease, diagnosis of a particular disease, assessment of the severity of a particular disease, guidance for selecting an appropriate drug for a particular disease, determining the risk of disease progression, or in patient follow-up. Depending on the intended diagnostic use, an appropriate control sample is selected and a control value or reference value for the marker is set therein. As will be apparent to those skilled in the art, the absolute marker value set in the control sample depends on the assay used.

[0111] As used herein, the term "assessing" refers to assessing whether a patient has PCOS or is at risk of developing PCOS. Thus, the assessment used herein includes diagnosing PCOS, predicting the risk of developing PCOS, selecting a treatment for PCOS, monitoring patients with or being treated for PCOS, determining the amount or concentration of METRNL in a patient's sample, and comparing the determined amount or concentration to a reference.

[0112] As will be understood by those skilled in the art, the assessments made in accordance with the present invention, while preferred, are not typically correct for 100% of the subjects investigated. This term typically requires that a statistically significant portion of the subjects can be accurately assessed. Whether a portion is statistically significant can be readily determined by those skilled in the art using various well-known statistical assessment tools, such as determination of confidence intervals, determination of 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. Typically, the assumed confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. The p-values are typically 0.2, 0.1, 0.05.

[0113] The terms "reduced" or "decreased" level, amount and / or concentration of an indicator refer to the level, amount and / or concentration of such an indicator in a sample that is decreased compared to a reference or reference sample.

[0114] The terms "elevated" or "increased" level, amount and / or concentration of an indicator refer to the level, amount and / or concentration of such an indicator in a sample that is higher compared to a reference or reference sample. For example, in a fluid sample of an individual suffering from a given disease, a protein that can be detected in a higher amount or concentration than in the same fluid sample of an individual not suffering from the disease has an elevated level.

[0115] The terms "measure", "measuring" or "determining" preferably include qualitative, semi-quantitative or quantitative measurements.

[0116] The term "immunoglobulin (Ig)", as used herein, refers to glycoproteins of the immunoglobulin superfamily that confer immunity. "Surface immunoglobulin" is attached to the membrane of effector cells by their transmembrane regions and includes molecules such as, but not limited to, B cell receptors, T cell receptors, class I and II major histocompatibility complex (MHC) proteins, beta2 microglobulin (about 2M), CD3, CD4, and CDS.

[0117] Typically, the term "antibody" as used herein refers to a secreted immunoglobulin that lacks a transmembrane region and can thus be released into the bloodstream and body cavities. Human antibodies are classified into different isotypes based on the heavy chains they possess. There are five types of human Ig heavy chains denoted by Greek letters: α, γ, δ, ε, and μ. The type of heavy chain present defines the class of the antibody (i.e., these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively), each playing a different role and directing an appropriate immune response against different types of antigens. The different heavy chains vary in size and composition and can contain approximately 450 amino acids (Janeway et al. (2001) Immunobiology, Garland Science). IgA is found in mucosal areas such as the gastrointestinal tract, respiratory tract, and urogenital tract, as well as in saliva, tears, and breast milk, and it prevents colonization by pathogens (Underdown & Schiff (1986) Annu. Rev. Immunol. 4:389 - 417). IgD functions mainly as an antigen receptor on B cells that have not been exposed to antigens and is involved in activating basophils and mast cells to produce antimicrobial factors (Geisberger et al. (2006) Immunology 118:429 - 437; Chen et al. (2009) Nat. Immunol. 10:889 - 898). IgE is involved in allergic reactions through binding to allergens that cause the release of histamine from mast cells and basophils. IgE is also involved in protection against parasites (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). IgG provides most of the antibody - based immunity against invading pathogens and is the only antibody isotype that can pass through the placenta to confer passive immunity to the fetus (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). In humans, there are four different IgG subclasses (IgG1, 2, 3, and 4), named in the order of their abundance in serum, with IgG1 being the most abundant (about 66%), followed by IgG2 (about 23%), IgG3 (about 7%), and IgG4 (about 4%).The biological profiles of different IgG classes are determined by the structure of their respective hinge regions. IgM is expressed on the surface of B cells in monomeric form and as a secreted pentameric form with very high avidity. IgM is involved in eliminating pathogens in the early stages of B cell-mediated (humoral) immunity before sufficient IgG is produced (Geisberger et al. (2006) Immunology 118:429-437). Antibodies are not only found as monomers but are also well known to form dimers of two Ig units (e.g., IgA), tetramers of four Ig units (e.g., IgM in teleost fish), or pentamers of five Ig units (e.g., mammalian IgM). Antibodies are typically made up of four polypeptide chains, two identical heavy chains and two identical light chains, linked via disulfide bonds, and resemble a "Y"-shaped macromolecule. Each of the chains contains several immunoglobulin domains, some of which are constant domains and others are variable domains. The immunoglobulin domain consists of two layers of 7-9 antiparallel strands arranged in a β-sheet-like manner. Typically, the heavy chain of an antibody contains four Ig domains, three of which are constant (CH domains: CH1, CH2, CH3) domains and one of which is a variable domain (VH). The light chain typically contains one constant Ig domain (CL) and one variable Ig domain (VL). By way of example, the human IgG heavy chain is composed of four Ig domains linked in the order VH-CH1-CH2-CH3 (also referred to as Vγ1-Cγ2-Cγ3) from the N-terminus to the C-terminus, while the human IgG light chain is composed of two immunoglobulin domains linked in the order VL-CL from the N-terminus to the C-terminus and is either of the kappa or lambda type (VK-CK or Vλ-Cλ). By way of example, the constant chain of human IgG contains 447 amino acids.Throughout this specification and the claims, the numbering of the amino acid positions of immunoglobulins is that of the "EU index" as in Kabat, E.A., Wu, T.T., Perry, H.M., Gottesman, K.S., and Foeller, C., (1991) Sequences of proteins of immunological interest, 5th ed. U.S. Department of Health and Human Service, National Institutes of Health, Bethesda, MD. The "EU index similar to Kabat" refers to the residue numbering of human IgG1 EU antibody. Thus, in the context of IgG, the CH domains are as follows: "CH1" refers to amino acid positions 118 - 220 according to the EU index as in Kabat; "CH2" refers to amino acid positions 237 - 340 according to the EU index as in Kabat; "CH3" refers to amino acid positions 341 - 447 according to the EU index as in Kabat. th The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form, which is not the antibody fragments defined below. Specifically, these terms refer to an antibody having heavy chains that include the Fc region.

[0118] The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form, which is not the antibody fragments defined below. Specifically, these terms refer to an antibody having heavy chains that include the Fc region.

[0119] Papain digestion of an antibody produces two identical antigen-binding fragments called "Fab fragments" (also referred to as "Fab portions" or "Fab regions"), each having a single antigen-binding site, and the remaining "Fc fragment" (also referred to as "Fc portion" or "Fc region"), named to reflect its ability to readily crystallize. The crystal structure of the human IgG Fc region has been determined (Deisenhofer (1981) Biochemistry 20:2361-2370). In the IgG, IgA, and IgD isotypes, the Fc region consists of two identical protein fragments derived from the CH2 and CH3 domains of the two heavy chains of the antibody, and in the IgM and IgE isotypes, the Fc region contains three heavy-chain constant domains (CH2-4) in each polypeptide chain. Additionally, smaller immunoglobulin molecules exist naturally or are artificially constructed. The term "Fab' fragment" refers to a Fab fragment that additionally includes the hinge region of the Ig molecule, while the "F(ab')2 fragment" is understood to contain two Fab' fragments that are chemically linked or linked via disulfide bonds. "Single-domain antibodies (sdAb)" (Desmyter et al. (1996) Nat. Structure Biol. 3:803-811)) and "nanobodies" contain only a single VH domain, while "single-chain Fv (scFv)" fragments contain a heavy-chain variable domain linked to a light-chain variable domain via a short linker peptide (Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85,5879-5883). Bispecific single-chain variable fragments (di-scFv) can be engineered by linking two scFvs (scFvA-scFvB). This can be done by generating a single peptide chain with two VH regions and two VL regions, resulting in a "tandem scFv" (VHA-VLA-VHB-VLB). Another possibility is the generation of an scFv with a linker that is too short for the two variable regions to fold together, forcing the scFv to dimerize. Usually, a linker of 5 residues in length is used to generate these dimers. This type is known as a "diabody".A shorter linker (one or two amino acids) between the VH domain and the VL domain results in the formation of a single-specificity trimer, the so-called "triabody" or "tribody". The bispecific diabody is formed by expressing it in chains having the sequences VHA-VLB and VHB-VLA or VLA-VHB and VLB-VHA, respectively. The single-chain diabody (scDb) contains VHA-VLB and VHB-VLA fragments (VHA-VLB-P-VHB-VLA) linked by a linker peptide (P) of 12 to 20 amino acids, preferably 14 amino acids. The "bispecific T cell engager (BiTE)" is a fusion protein consisting of two scFvs of different antibodies, one of which binds to T cells via the CD3 receptor and the other binds to tumor cells via a tumor-specific molecule (Kufer et al. (2004) Trends Biotechnol. 22:238-244). The bispecific affinity retargeting molecule ("DART" molecule) is a diabody further stabilized by a C-terminal disulfide bridge.

[0120] Accordingly, the term "antibody fragment" refers to a portion of an intact antibody, preferably including its antigen-binding region. Antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, Fv fragments; diabodies; sdAbs, nanobodies, scFvs, di-scFvs, tandem scFvs, triabodies, diabodies, scDbs, BiTEs, and DARTs.

[0121] The term "binding affinity" generally refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between the parts of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including, but not limited to, assays based on surface plasmon resonance (e.g., BIAcore assays as described in PCT application publication WO2005 / 012359); enzyme-linked immunosorbent assay (ELISA); and competitive assays (e.g., RIA). Low-affinity antibodies generally tend to bind antigens slowly and dissociate readily, while high-affinity antibodies generally tend to bind antigens rapidly and remain bound longer. Various methods for measuring binding affinity are known in the art, and any of these can be used for the purposes of the present invention.

[0122] "Sandwich immunoassay" is widely used for the detection of a target analyte. In such an assay, the analyte is "sandwiched" between a first antibody and a second antibody. Typically, a sandwich assay requires that the capture and detection antibodies bind to different, non-overlapping epitopes on the target analyte. By appropriate means, such a sandwich complex is measured, thereby quantifying the analyte. In a typical sandwich-type assay, a first antibody that is bound to a solid phase or can bind to a solid phase, and a second antibody that is detectably labeled, each bind to the analyte at different, non-overlapping epitopes. A binding agent (e.g., an antibody) specific for the first analyte is either covalently bound or passively bound to the solid surface. The solid surface is typically glass or a polymer, and the most commonly used polymers are cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. The solid support can be a tube, a bead, a disk of a microplate, or any other surface suitable for performing an immunoassay. The binding process is well known in the art and generally consists of cross-linking covalent bonding or physical adsorption, and the polymer-antibody complex is washed in the preparation of the test sample. Next, an aliquot of the sample to be tested is added to the solid phase complex and incubated for a period sufficient to allow binding between the first antibody or capture antibody and the corresponding antigen (e.g., 2 to 40 minutes or, more conveniently, overnight) and under appropriate conditions (e.g., room temperature to 40 °C, e.g., 25 °C to 37 °C (including both ends)). Following the incubation period, the solid phase containing the first antibody or capture antibody and the antigen bound to the antibody can be washed and incubated with a secondary antibody or labeled antibody that binds to another epitope on the antigen. The second antibody is bound to a reporter molecule used to indicate the binding of the second antibody to the complex of the first antibody and the target antigen.

[0123] A very widely used alternative sandwich assay format involves the use of a solid phase coated with the first partner of a binding pair, e.g., microparticles coated with paramagnetic streptavidin. Such microparticles are incubated with a binder specific for an analyte bound to the second partner of the binding pair (e.g., a biotinylated antibody), a sample suspected of containing or containing an analyte in which the second partner of the binding pair is bound to a binder specific for the analyte, and a binder specific for a second detectable-labeled analyte. As will be apparent to those skilled in the art, these components are incubated for a period sufficient to bind the labeled antibody to the solid phase microparticles via the analyte, the binder specific for the analyte (bound to) the second partner of the binding pair, and the first partner of the binding pair under appropriate conditions. Optionally, such an assay may include one or more washing steps (plural available).

[0124] The term "detectably labeled" encompasses labels that can be detected directly or indirectly.

[0125] Either a directly detectable label provides a detectable signal or the label interacts with a second label to modify the detectable signal provided by the first or second label to give, for example, FRET (fluorescence resonance energy transfer). Labels such as fluorescent dyes and luminescent dyes (including chemiluminescence and electrochemiluminescence) (Briggs et al ‘‘Synthesis of Functionalised Fluorescent Dyes and Their Coupling to Amines and Amino Acids,’’ J.Chem.Soc., Perkin-Trans.1(1997)1051-1058) provide a detectable signal and are generally applicable to labels. In one embodiment, detectably labeled refers to a label that provides or is derivable to provide a detectable signal, i.e., a fluorescent label, a luminescent label (e.g., a chemiluminescent label or an electrochemiluminescent label), a radioactive label, or a metal chelate-based label, respectively.

[0126] A number of labels (also referred to as dyes) are available and can generally be classified into the following categories, all of which are grouped together and each of which represents an embodiment according to the present disclosure.

[0127] (a) Fluorescent dyes Fluorescent dyes are described, for example, by Briggs et al ‘‘Synthesis of Functionalized Fluorescent Dyes and Their Coupling to Amines and Amino Acids,’’ J.Chem.Soc., Perkin-Trans.1 (1997) 1051 - 1058).

[0128] Fluorescent labels or fluorophores include rare earth chelates (europium chelates), fluorescein-type labels (including FITC, 5-carboxyfluorescein, 6-carboxyfluorescein), rhodamine-type labels (including TAMRA), dansyl, lysamine, cyanine, phycoerythrin, Texas Red, and analogs thereof. Fluorescent labels can be attached to aldehyde groups contained within a target molecule using the techniques disclosed herein. Fluorescent dyes and fluorescent labeling reagents include those commercially available from Invitrogen / Molecular Probes (Eugene, Oregon, USA) and Pierce Biotechnology, Inc. (Rockford, Ill.).

[0129] (b) Luminescent dyes Luminescent dyes or labels can be further subclassified into chemiluminescent dyes and electrochemiluminescent dyes.

[0130] Different classes of chemiluminescent labels include systems based on luminol, acridinium compounds, selenotellurazine and analogs, dioxetane, peroxysuccinic acid and peroxysuccinic acid derivatives. For immunoassay procedures, mainly acridinium-based labels are used (a detailed overview is shown in Dodeigne C. et al., Talanta 51 (2000) 415 - 439).

[0131] The main relevant labels used as electrochemiluminescent labels are ruthenium- and iridium-based electrochemiluminescent complexes, respectively. Electrochemiluminescence (ECL) has proven to be very useful for analytical applications as a sensitive and selective method. ECL combines the analytical advantages of chemiluminescent analysis (absence of background light signal) with the ease of reaction control by applying an electrode potential. Generally, ruthenium complexes, especially [Ru(Bpy)3]2+ (which emits photons at about 620 nm) regenerated with TPA (tripropylamine) at a liquid phase or liquid-solid interface, are used as ECL labels.

[0132] Electrochemiluminescence (ECL) assays provide sensitive and accurate measurement of the presence and concentration of an analyte of interest. Such techniques use labels or other reactants that can be induced to luminesce when electrochemically oxidized or reduced in an appropriate chemical environment. Such electrochemiluminescence is caused by a voltage applied to a working electrode in a specific pattern at a specific time. The light generated by the label is measured and indicates the presence or amount of the analyte. For a more complete description of such ECL techniques, reference is made to U.S. Patent No. 5,221,605, U.S. Patent No. 5,591,581, U.S. Patent No. 5,597,910, PCT Application Publication No. 90 / 05296, PCT Application Publication No. 92 / 14139, PCT Application Publication No. 90 / 05301, PCT Application Publication No. 96 / 24690, PCT Application Publication US95 / 03190, PCT Application US97 / 16942, PCT Application Publication US96 / 06763, PCT Application Publication No. 95 / 08644, PCT Application Publication No. 96 / 06946, PCT Application Publication No. 96 / 33411, PCT Application Publication No. 87 / 06706, PCT Application Publication No. 96 / 39534, PCT Application Publication No. 96 / 41175, PCT Application Publication No. 96 / 40978, PCT / US97 / 03653 and U.S. Patent Application 08 / 437,348 (U.S. Patent No. 5,679,519). Also, reference is made to the 1994 review by Knight, et al. (Analyst, 1994, 119:879-890) on the analytical uses of ECL and the references cited therein. In one embodiment, the method according to the present specification is implemented using an electrochemiluminescence label.

[0133] In recent years, iridium-based ECL labels have also been described (International Publication No. 2012107419).

[0134] (c) Radioactive labels employ radioisotopes (radionuclides), such as 3H, 11C, 14C, 18F, 32P, 35S, 64Cu, 68Gn, 86Y, 89Zr, 99TC, 111In, 123I, 124I, 125I, 131I, 133Xe, 177Lu, 211At, or 131Bi.

[0135] (d) Metal chelate complexes suitable as labels for imaging and therapeutic purposes are well known in the art (U.S. Patent Application Publication No. 2010 / 0111861; U.S. Patent No. 5,342,606; U.S. Patent No. 5,428,155; U.S. Patent No. 5,316,757; U.S. Patent No. 5,480,990; U.S. Patent No. 5,462,725; U.S. Patent No. 5,428,139; U.S. Patent No. 5,385,893; U.S. Patent No. 5,739,294; U.S. Patent No. 5,750,660; U.S. Patent No. 5,834,461; Hnatowich et al, J. Immunol. Methods 65 (1983) 147-157; Meares et al, Anal. Biochem. 142 (1984) 68-78; Mirzadeh et al, Bioconjugate Chem. 1 (1990) 59-65; Meares et al, J. Cancer (1990), Suppl. 10:21-26; Izard et al, Bioconjugate Chem. 3 (1992) 346-350; Nikula et al, Nucl. Med. Biol. 22 (1995) 387-90; Camera et al, Nucl. Med. Biol. 20 (1993) 955-62; Kukis et al, J. Nucl. Med. 39 (1998) 2105-2110; Verel et al., J. Nucl. Med. 44 (2003) 1663-1670; Camera et al, J. Nucl. Med. 21 (1994) 640-646; Ruegg et al, Cancer Res. 50 (1990) 4221-4226; Verel et al, J. Nucl. Med. 44 (2003) 1663-1670; Lee et al, Cancer Res. 61 (2001) 4474-4482; Mitchell, et al, J. Nucl. Med. 44 (2003) 1105-1112; Kobayashi et al Bioconjugate Chem. 10 (1999) 103-111; Miederer et al, J. Nucl. Med.45(2004)129 - 137; DeNardo et al, Clinical Cancer Research 4(1998)2483 - 90; Blend et al, Cancer Biotherapy&Radiopharmaceuticals 18(2003)355 - 363; Nikula et al J.Nucl.Med.40(1999)166 - 76; Kobayashi et al, J.Nucl.Med.39(1998)829 - 36; Mardirossian et al, Nucl.Med.Biol.20(1993)65 - 74; Roselli et al, Cancer Biotherapy&Radiopharmaceuticals,14(1999)209 - 20).

Example

[0136] The present invention is merely illustrated by the following examples. The examples shall not be construed in any way as limiting the scope of the present invention.

[0137] Example 1: Diagnostic performance of biomarker METRNL in women with PCOS (phenotype A) and controls determined by the proximity extension assay (PEA) technology developed by Olink As part of the measurement, 88 serum samples from human women were analyzed. The case group included 51 samples from patients diagnosed with PCOS (phenotype A) according to the Rotterdam criteria. The control group included 37 samples from healthy women without PCOS. The concentration of the analyte was determined using the proximity extension assay (PEA) technology developed by Olink. Briefly, a matched antibody pair conjugated to unique partially complementary oligonucleotides addresses each biomarker. Quantification is then performed by quantitative real - time PCR.

[0138] For each of the selected panels, after diluting the samples according to the manufacturer's protocol, the Olink protocol consists of three core steps: 1. Incubation, 2. Extension and amplification, and 3. Detection. 1 μl of each sample was mixed with 3 μl of incubation mix in a 96-well plate. In addition to 92 antibody pairs labeled with DNA oligonucleotides, the incubation mix also included internal controls designed to monitor the three main steps of the Olink protocol (two incubation controls, i.e., one extension control and one detection control). As external controls, three positive controls (inter-plate controls) and three negative controls, as well as two sample controls (pooled plasma samples) were included in the plate. The samples were incubated at +4 °C overnight. During this step, the antibody pairs bind to their respective proteins in the samples. When the incubation was complete, 96 μl of extension mix was added to the samples. The plate was placed in a thermal cycler for hybridization and extension was performed by DNA polymerase (50 °C for 20 min, 95 °C for 5 min (95 °C for 30 s, 54 °C for 1 min, 60 °C for 1 min) × 17, hold at 10 °C). The DNA barcodes were amplified by PCR. Finally, the amount of each DNA barcode was quantified by microfluidic qPCR. The 96.96 Dynamic Array (trademark) Integrated Fluidic Circuit (IFC) was used according to the manufacturer's instructions. 2 μl of the detection mix at 7 points was added to 2.8 μl of each sample, and 5 μl of these were transferred to the left inlet of the primed 96.96 Dynamic Array IFC. 5 μl of the primer solution was transferred to the right inlet of the primed 96.96 Dynamic Array IFC. The chip was loaded into the Fluidigm IFC Controller HX according to the manufacturer's instructions.The Olink protein expression 96 × 96 program was run on a Fluidigm Biomark™ reader according to the manufacturer's instructions (50 °C for 120 s, 70 °C for 1800 s, 25 °C for 600 s, 95 °C for 300 s (95 °C for 15 s, 60 °C for 60 s) × 35; the following settings: application - Gene Expression; passive Reference - ROX; assay - single probe; probe - FAM - MGB). The Ct values obtained from qPCR were converted into an arbitrary unit called normalized protein eXpression (NPX, relative quantification unit on a log2 scale) using the following formula. Extension control: Ct 分析物 -Ct 伸長対照 =dCt 分析物 Inter - plate control: dCt 分析物 -dCt プレート間対照 =ddCt 分析物 Adjustment for correction factor: Correction factor - ddCt 分析物 =NPX 分析物

[0139] Quality control and normalization were achieved using the Olink NPX Manager software.

[0140] Receiver operating characteristic (ROC) curves were created (Figure 1). Model performance was determined by examining the area under the curve (AUC). The best possible AUC is 1 and the lowest possible AUC is 0.5. ROC curve analysis showed an AUC of 0.972 (95% CI 0.944 - 1.0, Figure 1), indicating that METRNL has high diagnostic accuracy for PCOS. Table 1, which describes the AUC of ROC curve analysis and the associated 95% confidence intervals, shows the diagnostic performance of METRNL for distinguishing women with PCOS with a complete proliferative phenotype A (cases) from healthy control subjects. The results were obtained using the Olink proximity extension technology.

Table 1

[0141] Using data obtained by the Olink PEA technology, box-and-whisker plots of healthy controls and PCOS cases were generated. The box includes the median (middle quartile), interquartile range (representing the middle 50% of the group's scores), upper quartile (75% of the scores are below the upper quartile), and lower quartile (25% of the scores are below the lower quartile). The whiskers represent values 1.5 times the interquartile range. Serum METRNL concentration decreases in women with PCOS when compared to healthy controls (Figure 2).

[0142] Example 2: Diagnostic performance of biomarker METRNL in women with PCOS (phenotypes A, B, C, and D) and controls determined by ELISA technology Performance verification was conducted in a sample population of 85 samples (serum samples from women with PCOS) and 46 controls (serum samples from healthy women).

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

[0144] The concentration of METRNL in human serum was determined using a Human METRNL ELISA kit from R&D Systems (Catalog number: DY7867-05). The kit is a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) designed to detect and quantify the level of human METRNL in cell culture supernatants, plasma, and serum.

[0145] The capture antibody was diluted to a working concentration in carrier protein-free PBS. A 96-well microplate was incubated with 100 μL / well of the diluted capture antibody. The plate was sealed and incubated overnight at room temperature. The plate was washed three times with 400 μL of wash buffer per well. After the final wash, the wash buffer was removed completely, and the plate was blocked by adding 300 μL of Regent Diluent to each well and incubating for at least 1 hour at room temperature. The plate was washed three times with 400 μL of wash buffer per well and made ready for use. Samples were measured at a 4-fold dilution. After bringing all reagents to room temperature, 100 μL of each sample and standard were added. Samples and standards were measured in duplicate. During a 2-hour incubation at room temperature, any METRNL present was bound to the immobilized capture antibody on the microtiter plate. Unbound material was removed from the plate before adding 100 μL of anti-METRNL Detection Antibody diluted in Reagent Diluent during the wash step (3 x 400 μL). After a 2-hour incubation and another wash step (3 x 400 μL) to remove unbound detection antibody, 100 μL of the prepared streptavidin-HRP solution was added to the plate. Subsequently, a 20-minute incubation at room temperature and a wash step (3 x 400 μL) were performed to avoid 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 incubation, the substrate changed to blue. The 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, changing the color of the solution in the well from blue to yellow, and the color intensity was measured using a plate reader at 450 nm for detection and 540 or 570 nm for background subtraction. This subtraction was to correct for optical defects within the plate. To create a calibration curve, the lyophilized recombinant METRNL delivered with the kit was reconstituted and diluted with reagent diluent. The calibration range of the assay was 15.6 pg / mL to 1000 pg / mL.A seven-point standard curve was obtained using serial two-fold dilutions of recombinant METRNL in the reagent diluent. The calibration curve was fitted using a four-parameter logistic (4-PL, Newton / Raphson) curve fit.

[0146] The receiver operating characteristic (ROC) curve was created (Figure 3). Model performance was determined by examining the area under the curve (AUC). The best possible AUC is 1, and the lowest possible AUC is 0.5. ROC curve analysis of METRNL for PCOS cases when all phenotypes (Phenotypes A - D) were combined showed an AUC of 0.94 (95% CI 0.89 - 0.99), confirming the high diagnostic accuracy of METRNL for PCOS (Figure 3). Table 2 shows the diagnostic performance of METRNL for distinguishing women with PCOS (cases, PCOS phenotypes A - D) from healthy control subjects using the AUC of the ROC curve analysis and the associated 95% confidence intervals. Results were obtained using an ELISA immunoassay.

Table 2

[0147] Using data obtained by ELISA immunoassay, box and whisker plots for controls and PCOS were created when all phenotypes (Phenotypes A - D) were combined. Serum METRNL concentration (pg / mL) decreased in women with PCOS when compared to healthy controls (Figure 4).

[0148] Table 3 shows the diagnostic performance of METRNL for distinguishing women with PCOS from healthy control subjects when separated by the different phenotypes A, B, C, and D. Results were obtained using an ELISA immunoassay. The AUC for each phenotype is reported in the table.

Table 3

[0149] ROC curve analysis of METRNL for PCOS cases and healthy controls separated by different phenotypes showed AUCs of 0.9 (95% CI 0.78 - 1), 0.93 (95% CI 0.84 - 1), 0.99 (95% CI 0.97 - 1), and 0.95 (95% CI 0.86 - 1) for phenotypes A - D, respectively (Figure 5). The results confirm the high diagnostic accuracy of METRNL for PCOS. Serum METRNL concentrations (pg / mL) in all different PCOS phenotypes (phenotypes A - D) showed decreased levels compared to healthy controls (Figure 6, results obtained using ELISA immunoassay).

[0150] Table 4 shows the diagnostic performance of METRNL in young women (age ≤ 25) for differentiating young women with PCOS from young healthy control subjects when all phenotypes (phenotypes A - D) were combined. Results were obtained using ELISA assay.

Table 4

[0151] ROC curve analysis of METRNL in young PCOS cases (age ≤ 25) when all phenotypes (phenotypes A - D) were combined showed an AUC of 0.93, confirming high diagnostic accuracy for women under 25 years old when differentiating PCOS cases from controls (95% CI 0.83 - 1.00, Figure 7). When only young women were included in the analysis (age ≤ 25), PCOS cases (all phenotypes A - D combined) showed a decrease in serum METRNL concentration (pg / mL) compared to young controls (age ≤ 25, Figure 8).

[0152] The diagnostic performance of METRNL for differentiating young women with PCOS (age ≤ 25) from young healthy control subjects (age ≤ 25) when separated by different phenotypes A, B, C, and D was evaluated, and the results are reported in Table 5 (AUC for PCOS phenotypes vs. controls). Results were obtained using ELISA immunoassay.

Table 5

[0153] ROC curve analysis of METRNL in young PCOS cases (age ≤ 25, phenotypes A - D) showed AUC values of 0.93 (95% CI 0.78 - 1), 0.67 (95% CI 0.01 - 1), 1.00 (95% CI 1 - 1), and 1.00 (95% CI 1 - 1) for each phenotype respectively. In the subgroup of women under 25 years old, high diagnostic accuracy of METRNL for PCOS was confirmed (Figure 9). METRNL concentration decreased in all different PCOS phenotypes (phenotypes A - D, age ≤ 25) when compared with the METRNL concentration of young healthy controls (age ≤ 25, Figure 10).

[0154] Example 3: Diagnostic performance of biomarker METRNL in women with PCOS (phenotypes A, B, C, and D) and controls determined by ELISA technique in different age groups Performance verification was performed on an additional sample set of 240 cases (serum samples from women with PCOS) and 48 controls (serum samples from healthy women).

[0155] The concentration of the analyte was determined by ELISA (enzyme - linked immunosorbent assay). The case group consisted of patients diagnosed with PCOS (155 phenotype A, 5 phenotype B, 8 phenotype C, and 72 phenotype D) according to the Rotterdam criteria and belonged to three different age groups: 15 - 20 (n = 70), 20 - 25 (n = 99), 25 - 40 (n = 71). The control group included healthy women without PCOS.

[0156] The concentration of METRNL in human serum was determined using a Human METRNL ELISA kit from R&D Systems (Catalog number: DY7867 - 05) as described in Example 2.

[0157] The receiver operating characteristic (ROC) curve was created (Figure 11). Model performance was determined by examining the area under the curve (AUC). ROC curve analysis for METRNL in PCOS cases when all phenotypes (Phenotypes A - D) were combined showed an AUC of 0.91 (95% CI 0.88 - 0.95), confirming the high diagnostic accuracy of METRNL for PCOS (Figure 11).

[0158] Table 6, which describes the AUC of the ROC curve analysis and the associated 95% confidence intervals, shows the diagnostic performance of METRNL for differentiating women with PCOS (cases, PCOS phenotypes A - D) from healthy control subjects using ROC analysis. The results were obtained using an ELISA immunoassay.

Table 6

[0159] Using the data obtained by ELISA immunoassay, box - and - whisker plots for controls and PCOS were created when all phenotypes (Phenotypes A - D) were combined. Serum METRNL concentration (pg / mL) decreased in women with PCOS compared to healthy controls (Figure 12).

[0160] Table 7 shows the diagnostic performance of METRNL for differentiating women with PCOS from healthy control subjects when separated by different phenotypes A, B, C, and D. The results were obtained using an ELISA immunoassay. The AUC for each phenotype is reported in the table.

Table 7

[0161] ROC curve analysis of METRNL for PCOS cases and healthy controls separated by different phenotypes showed AUCs of 0.91 (95% CI 0.87 - 0.95), 1.00 (95% CI 1.00 - 1.00), 1.00 (95% CI 1.00 - 1.00), and 0.90 (95% CI 0.84 - 0.97) for phenotypes A - D, respectively (Figure 13). The results confirm the high diagnostic accuracy of METRNL for PCOS.

[0162] Serum METRNL concentrations (pg / mL) in all different PCOS phenotypes (phenotypes A - D) showed decreased levels compared to healthy controls (Figure 14, results obtained using ELISA immunoassay).

[0163] Table 8 shows the diagnostic performance of METRNL in different age groups (15 ≤ age < 20, 20 ≤ age < 25, 25 ≤ age < 40) to distinguish women with PCOS from healthy control subjects when all phenotypes (phenotypes A - D) are combined. Results were obtained using ELISA assay.

Table 8

[0164] ROC curve analysis of METRNL for PCOS cases separated by different age groups from healthy controls showed AUCs of 0.88 (95% CI 0.80 - 0.95), 0.91 (95% CI 0.86 - 0.96), and 0.96 (95% CI 0.91 - 1.00) for the 15 - 20, 20 - 25, and 25 - 40 age groups, respectively (Figure 15). The results confirm the high diagnostic accuracy of METRNL for PCOS in all different age groups.

[0165] A decrease in serum METRNL concentration (pg / mL) in women with PCOS compared to controls was confirmed in all different age groups (Figure 16).

[0166] Taking into account the lack of a reliable biomarker for diagnosing PCOS, especially in young women (age < 25), a separate analysis was conducted for the age group of 15 years or older and less than 25 years old.

[0167] Table 9 shows the diagnostic performance of METRNL in young women (15 ≤ age < 25) for distinguishing young women with PCOS from young healthy control subjects when all phenotypes (Phenotypes A - D) are combined. The results were obtained using an ELISA assay.

Table 9

[0168] ROC curve analysis of METRNL for young PCOS cases (15 ≤ age < 25) when all phenotypes (Phenotypes A - D) are combined showed an AUC of 0.90, confirming high diagnostic accuracy for women aged 15 - 25 when distinguishing PCOS cases from controls (95% CI 0.85 - 0.94, Figure 17). When only young women were included in the analysis (15 ≤ age < 25), PCOS cases (combining all phenotypes A - D) showed a decrease in serum METRNL concentration (pg / mL) compared to young controls (15 ≤ age < 25, Figure 18).

[0169] The diagnostic performance of METRNL for distinguishing young women with PCOS (15 ≤ age < 25) from young healthy control subjects (15 ≤ age < 25) when separated by different phenotypes A, B, C, and D was evaluated, and the results are reported in Table 10 (AUC for PCOS phenotypes vs. controls). The results were obtained using an ELISA immunoassay.

Table 10

[0170] ROC curve analysis of METRNL for young PCOS cases (15 ≤ age < 25, phenotypes A - D) showed AUC values of 0.89 (95% CI 0.83 - 0.95), 1.00 (95% CI 1.00 - 1.00), 1.00 (95% CI 1.00 - 1.00), and 0.89 (95% CI 0.81 - 0.97) for each phenotype, respectively, confirming the high diagnostic accuracy of METRNL for PCOS in the subgroup of women aged 15 - 25 (Figure 19). METRNL concentration decreased in all different PCOS phenotypes (phenotypes A - D, 15 ≤ age < 25) when compared to the METRNL concentration of young healthy controls (15 ≤ age < 25, Figure 20).

Claims

1. A method for evaluating whether a subject has polycystic ovary syndrome (PCOS) or is at risk of developing PCOS, comprising: a) determining the amount or concentration of METRNL in a sample of the subject; and b) comparing the determined amount or concentration with a reference. A method comprising the above.

2. A method for selecting a patient for the treatment of PCOS, comprising: a) determining the amount or concentration of METRNL in a sample of the subject; and b) comparing the determined amount or concentration with a reference. A method comprising the above.

3. A method for monitoring the progression of PCOS in a subject with PCOS or for monitoring the response to treatment in a subject with PCOS, the method comprising: a) determining the level of METRNL in a first sample of the subject; b) determining the level of METRNL in a second sample of the subject obtained after the first sample; c) comparing the level of METRNL in the first sample with the level of METRNL in the second sample; and d) monitoring the progression in the subject suffering from or being treated for PCOS based on the result of step c). A method comprising the above.

4. The method according to claims 1 to 3, wherein a decrease in the amount or concentration of METRNL in the sample of the subject indicates the presence of PCOS in the subject.

5. The method according to claims 1 to 4, wherein the sample is a sample of blood, serum, or plasma.

6. The method according to claims 1 to 5, wherein PCOS is selected from the group consisting of PCOS of phenotype A, PCOS of phenotype B, PCOS of phenotype C, and PCOS of phenotype D according to the Rotterdam criteria.

7. The method according to claims 1 to 6, wherein PCOS of phenotype A is detected.

8. The method according to claims 1 to 6, wherein PCOS of phenotype B is detected.

9. The method according to claims 1 to 6, wherein PCOS of phenotype C is detected.

10. The method according to claims 1 to 6, wherein PCOS of phenotype D is detected.

11. The method according to claims 1 to 10, wherein PCOS is detected in adolescent or young adult females.

12. The method according to any one of claims 1 to 11, wherein the patient suffers from one or more of the following symptoms: oligo-ovulation and / or irregular cycles, hyperandrogenemia, and polycystic ovarian morphology.

13. A computer-implemented method for evaluating a subject suspected of having PCOS, comprising: a) receiving a value for the amount or concentration of a first biomarker in a sample of the subject, wherein the first biomarker is METRNL; b) optionally, receiving a value for the amount or concentration of a second biomarker in a sample of the subject; c) optionally, receiving a value for the presence or absence of at least one additional diagnostic criterion selected from the group consisting of oligo-ovulation and / or irregular cycles, hyperandrogenemia, and polycystic ovarian morphology; d) comparing the value for the amount or concentration in steps (a) to (b) with a reference for the biomarker and a value for the presence or absence of the at least one additional diagnostic criterion, and / or calculating a score for evaluating the subject suspected of having PCOS based on the amount or concentration of the biomarker and the value; e) evaluating the subject based on the comparison and / or calculation performed in step (d). A computer-implemented method as claimed in claim 13.

14. The computer-implemented method according to claim 13, wherein the amount or concentration of METRNL is decreased compared to a standard reference.

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