Fibroblast growth factor binding protein 1 (FGFBP1) 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 a patient for the treatment of PCOS, a method for monitoring the progression of PCOS or the response to treatment, and a computer-implemented method for assessing a subject suspected of having PCOS by determining the amount or concentration of fibroblast growth factor binding protein 1 (FGFBP1) in a sample of the subject.

Description

Technical Field

[0001] The present invention relates to a method for evaluating whether a subject has polycystic ovary syndrome (PCOS) or is at risk of developing PCOS, a method for selecting a subject for treatment, and determining the amount or concentration of fibroblast growth factor binding protein 1 (FGFBP1) in a sample of a subject and comparing the determined amount or concentration with a reference value to monitor a subject suffering from or being treated for PCOS. Further, the present invention relates to a computer-implemented method for evaluating a subject suspected of having PCOS by determining the amount or concentration of FGFBP1 in a sample of the subject, optionally determining the amount or concentration of a second biomarker and / or additional diagnostic criteria, and comparing the amount or concentration of FGFBP1, optionally the second biomarker, and / or optionally the presence of diagnostic criteria to a reference.

Background Art

[0002] Polycystic ovary syndrome (PCOS) is a heterogeneous gynecological condition defined by a combination of androgen excess symptoms and ovarian dysfunction. PCOS patients can have a variety of clinical symptoms that can be reproductive and / or metabolic. Reproductive symptoms include irregular menstrual cycles, infertility, pregnancy complications, and hirsutism, while metabolic symptoms include obesity, insulin resistance, metabolic syndrome, prediabetes, type 2 diabetes, non-alcoholic fatty liver disease (NAFLD), and cardiovascular factors. These clinical symptoms are also associated with mental 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 should be excluded (Escobar-Morreale HF. Polycystic ovary syndrome: definition, aetiology, diagnosis and treatment. Nat Rev Endocrinol. 2018;14(5):270-284; Teede HJ, Misso ML, Costello MF, et al., International PCOS Network. Recommendations from the international evidence-based guideline for the assessment and management of polycystic ovary syndrome. Hum Reprod. 2018;33(9):1602-1618)). Diagnostic tests that can be used to exclude other diseases are, for example, · 17α-hydroxyprogesterone (17-OHP) to exclude NCAH (Nordenstrom and Falhammar 2018) · Prolactin to rule out hyperprolactinemia · Cortisol to exclude patients with Cushing's syndrome · Thyroid-stimulating hormone (TSH) to exclude 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 widely used for the diagnosis of PCOS 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 disorders (oligovulation, 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 of less than 21 days or more than 35 days, or less than 8 cycles per year. Clinical signs 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) calculated by measuring total testosterone and sex hormone-binding globulin (SHBG). PCOM is usually determined using a transvaginal ultrasound transducer with a frequency bandwidth including 8 MHz according to the "2018 PCOS Guidelines Based on International Evidence". The threshold for PCOM is considered to be in either ovary: more than 20 follicles per ovary and / or an ovarian volume of 10 ml or more, with the confirmation that there are no corpora lutea, cysts, or dominant follicles. When older ultrasound techniques are used, the threshold for PCOM can be an ovarian volume of 10 ml or more or a follicle count of more than 12 on either ovary.

[0007] Another way to detect PCOM is to measure anti-Müllerian hormone (AMH) in the subject. AMH is a glycoprotein hormone whose expression is important for sexual differentiation at a specific point during fetal development. Furthermore, AMH produced by the granulosa cells of growing follicles usually correlates with the number of follicles in the ovary. Therefore, serum levels 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 of 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 Guidelines 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 following three items are present: (i) oligomenorrhea (defined as an average menstrual cycle length of more than 35 days); (ii) AMH above the threshold; (iii) hyperandrogenism defined as either testosterone above the threshold and / or the presence of hirsutism (mFG score of 5 or more). 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 because only a small proportion of women with PCOS have 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 evaluation of PCOM. As a result, the diagnosis of PCOS based on the Rotterdam criteria always includes at least one subjective, device- and operator-dependent measurement that is prone to errors.

[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 form, combining its bound and free forms. There are limitations to the methods for measuring free testosterone. Direct measurement of free testosterone using radioimmunoassay is very inaccurate and does not reflect the true value. The assay has high variability within and between assays. Alternatively, especially for clinical research, higher accuracy can be obtained 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 may be improved by simultaneously measuring SHBG. To calculate the free T concentration from the levels of total testosterone and SHBG, only a quadratic equation needs to be solved (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. The 8-point modified Ferriman-Gallwey (mFG) score 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 exceeding 5 has been proposed to define hirsutism in Chinese women.There are also signs that androgen levels in the blood differ among ethnic groups. Here, the Japanese population has a low rate 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 with hyperandrogenemia, ovulatory dysfunction and / or irregular menstrual cycles, and polycystic ovarian morphology. Phenotype B is characterized by hyperandrogenemia, ovulatory dysfunction and / or irregular menstrual cycles. Phenotype C is characterized by hyperandrogenemia and polycystic ovarian morphology. Phenotype D is characterized by ovulatory dysfunction and / or irregular menstrual cycles and polycystic ovarian morphology.

[0013] Currently, there are no specific PCOS medications available. Treatments are symptom - directed and tailored to the individual's needs. Therapeutic approaches target hyperandrogenemia, irregular menstrual cycles and / or ovulatory dysfunction, and associated 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 healthcare 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 under - diagnosis and over - diagnosis. This unfavorable diagnostic experience worsens the affected women, especially during the transition from pediatric to adult care, where the tendency is pronounced and limits the timely opportunity for interventions to minimize associated comorbidities (Witchel SF, Teede HJ, Pena AS. Curtailing PCOS. Pediatr Res. 2020;87(2):353 - 361). Furthermore, timely diagnosis is extremely important 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 insufficient information. Before a diagnosis was established, more than one - third of women reported more than 2 years (33.6%) and more than 3 healthcare providers (47.1%). Only 35.2% were satisfied with the diagnosis and 15.6% were satisfied with the information received. 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] Of particular note in the diagnosis of PCOS are adolescent and young women under 25 years of age, i.e., young women in whom the characteristics of normal pubertal development overlap with adult diagnostic criteria. This makes the diagnosis controversial and difficult. Many of the symptoms used to diagnose PCOS change over time and can change during the first few years after menstruation. Normal pubertal physiological changes such as irregular menstrual cycles, acne, and PCOM overlap with adult PCOS diagnostic criteria. In adolescents and young adult women, a diagnosis of PCOS is made when both OA and HA criteria are met. In this life stage with a high incidence of polycystic ovaries, it is not recommended to perform pelvic ultrasound examinations on adolescent girls less than 8 years from menarche (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). Evaluation of irregular menstrual cycles in adolescence can be difficult. Menstrual cycles are often irregular in adolescence. Immaturity of the hypothalamic-pituitary-ovarian axis in the early years after menstruation often leads to anovulation and cycles can be somewhat longer. However, while 90% of cycles are in the range of 21 to 45 days, short cycles less than 20 days and long cycles over 45 days can occur. By 3 years from 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 evaluating what constitutes a normal menstrual cycle or bleeding pattern. Patients and their caregivers are not accustomed to what is normal, and patients may not inform their caregivers about menstrual irregularities or loss of menstruation.Furthermore, patients are often reluctant to discuss this topic with their caregivers (ACOG Committee Opinion No. 651: Menstruation in Girls and Adolescents: Using the Menstrual Cycle as a Vital Sign. Obstet Gynecol. 2015;126(6):e143-e146). Therefore, the establishment of reliable biomarkers as an aid for the diagnosis of PCOS or the identification of patients at risk of developing PCOS is of utmost importance, especially for this patient group. Delayed diagnosis in adolescent and young women is often due to a lack of willingness to diagnose adolescent women at risk due to the potential for overdiagnosis or underdiagnosis during adolescence. This can lead to obesity and insulin resistance, as well as long-term complications such as anxiety or depression. Current guidelines for the diagnosis of PCOS in adolescent and young women define oligo-ovulation, 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 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 the progression of PCOS in a subject, and / or to monitor the response to treatment in subjects with PCOS.

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

[0019] In a first aspect, the invention is a method for assessing whether a subject has PCOS or is at risk of developing PCOS, the method comprising: (a) determining the amount or concentration of FGFBP1 in a sample from the subject; and (b) comparing the determined amount or concentration to a reference. The invention relates to a method comprising these steps.

[0020] In a second aspect, the invention is a method for selecting a patient for the treatment of PCOS, the method comprising: (c) determining the amount or concentration of FGFBP1 in a sample from the subject; and (d) comparing the determined amount or concentration to a reference. The invention relates to a method comprising these steps.

[0021] In a third aspect, the invention is a method for monitoring the progression of PCOS in a subject or for monitoring the response to treatment in a subject having PCOS, the method comprising: (e) determining the level of FGFBP1 in a first sample from the subject; (f) determining the level of FGFBP1 in a second sample from the subject obtained after the first sample; (g) comparing the level of FGFBP1 in the first sample to the level of FGFBP1 in the second sample; and (h) monitoring the progression in a subject having PCOS or being treated for PCOS based on the results of step (c). The invention relates to a method comprising these steps.

[0022] In a fourth aspect, the present invention is a computer-implemented method for evaluating a patient suspected of having PCOS, comprising: (a) receiving a value for the amount or concentration of a first biomarker in a sample of interest, wherein the first biomarker is FGFBP1; (b) optionally, receiving a value for the amount or concentration of a second biomarker in the sample of interest; (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, hyperandrogenism, and polycystic ovarian morphology; (d) comparing the values for the amounts or concentrations of steps (a)-(b) with the reference for the biomarker and the value for the presence or absence of at least one additional diagnostic criterion, and / or calculating a score for evaluating a subject suspected of having PCOS based on the amount or concentration and value of the biomarker; (e) evaluating the subject based on the comparison and / or calculation performed in step (d). The present invention relates to a computer-implemented method comprising the steps of: BRIEF DESCRIPTION OF THE DRAWINGS

[0023]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

DETAILED DESCRIPTION OF THE INVENTION

[0024] The inventors of the present invention have identified fibroblast growth factor binding protein 1 (FGFBP1) as a reliable biomarker for diagnosing PCOS in a subject, determining whether a subject is at risk of developing PCOS, determining the response to treatment of a subject with PCOS, monitoring the progression of PCOS in a subject with PCOS, or monitoring the response to treatment of a subject with PCOS. FGFBP1 can be used alone or in combination with at least additional criteria such as hyperandrogenemia, oligoovulation, PCOM or irregular menstrual cycles for diagnosis, risk assessment and / or monitoring of the response to treatment in a patient. Furthermore, determination of the level of FGFBP1 compared to a control level can be used to monitor the response to treatment and / or monitor the progression of PCOS in a subject.

[0025] The inventors have for the first time shown that the FGFBP1 value in a biological fluid sample, preferably a biological fluid sample that is blood, plasma, serum, capillary blood, interstitial fluid, ascites, or menstrual fluid, more preferably a biological fluid sample that is blood, plasma, or serum, is increased in women with PCOS compared to a control. Further, the inventors have for the first time shown that the measured FGFBP1 value in a biological fluid sample, preferably a biological fluid sample that is blood, plasma, serum, capillary blood, interstitial fluid, ascites, or menstrual fluid, more preferably a biological fluid sample that is blood, plasma, or serum, is increased in women suffering from any of the phenotypes A - D of PCOS. The solution provided by the present invention is an immunoassay for detecting fibroblast growth factor binding protein 1 in a sample, preferably a biological fluid sample that is preferably blood, plasma, serum, capillary blood, interstitial fluid, ascites, or menstrual fluid, more preferably a biological fluid sample that is blood, plasma, or serum. This immunoassay can be used to diagnose women with PCOS in combination with other clinical and / or biochemical features, such as oligo-ovulation and / or irregular menstrual cycles, hyperandrogenemia or PCOM. Further, measurement of the FGFBP1 value can be used to monitor the progression of PCOS and the response to treatment in the patient. The inventors have also shown that the measured FGFBP1 value in a biological fluid sample, preferably a biological fluid sample that is blood, plasma, serum, capillary blood, interstitial fluid, ascites, or menstrual fluid, more preferably a biological fluid sample that is blood, plasma, or serum, is particularly suitable for the diagnosis of PCOS in adolescent or young women under 25 years of age, particularly under 20 years of age, particularly under 15 - 25 years of age, particularly under 15 - 20 years of age, either alone or in combination with the above additional diagnostic criteria.

[0026] The medical need for an accurate test for the reliable diagnosis of PCOS is not met. Measurement of FGFBP1 in a biological fluid sample, preferably a biological fluid sample that is blood, plasma, serum, capillary blood, interstitial fluid, ascites, or menstrual fluid, more preferably a biological fluid sample that is blood, plasma, or serum, has the advantage of being a reliable fluid-based test for identifying women suffering from currently undiagnosable PCOS. Also, measurement of FGFBP1 in a biological fluid sample, preferably a biological fluid sample that is blood, plasma, serum, capillary blood, interstitial fluid, ascites, or menstrual fluid, more preferably a biological fluid sample that is blood, plasma, or serum, can surely be used for the diagnosis of PCOS in adolescent subjects and young women under 25 years of age, particularly under 20 years of age, particularly under 15 to 25 years of age, particularly under 15 to 20 years of age. Diagnosis of PCOS in adolescent patients is difficult for the reasons stated above, and thus, the inventors provide, for the first time, an accurate test for the diagnosis of PCOS in adolescent and young women populations. Further, measurement of FGFBP1 in a biological fluid sample, preferably a biological fluid sample that is blood, plasma, serum, capillary blood, interstitial fluid, ascites, or menstrual fluid, more preferably a biological fluid sample that is blood, plasma, or serum, has the advantage of being able to identify whether a patient is responding to treatment. A further advantage of measurement of FGFBP1 in a biological fluid sample, preferably a biological fluid sample that is blood, plasma, serum, capillary blood, interstitial fluid, ascites, or menstrual fluid, more preferably a biological fluid sample that is blood, plasma, or serum, is to monitor the progression of PCOS. Further, the inventors attach a computer-implemented method for evaluating a subject suffering from PCOS by measuring the level of FGFBP1 in a biological fluid sample, preferably a biological fluid sample that is blood, plasma, serum, capillary blood, interstitial fluid, ascites, or menstrual fluid, more preferably a biological fluid sample that is blood, plasma, or serum, based on the comparison and / or calculation of the above data, together with additional criteria such as values of oligo-ovulation and / or irregular menstrual cycles, hyperandrogenism and / or polycystic ovarian morphology.

[0027] As described above, patients suffering from PCOS may exhibit two types of characteristics, reproductive or metabolic. Metabolic features of PCOS include obesity, insulin resistance, metabolic syndrome, prediabetes, type 2 diabetes, non-alcoholic fatty liver disease (NAFLD), and cardiovascular factors. The term "phenotype" may be used instead of "reproductive". The term "reproductive" (or "phenotype") refers to any characteristic of the phenotype of a woman known to have PCOS. For example, these reproductive characteristics include polycystic ovarian morphology (PCOM) and / or clinical hyperandrogenism, such as acne, seborrhea, alopecia, and / or hirsutism. Preferably, these reproductive characteristics include polycystic ovarian morphology (PCOM) and / or clinical hyperandrogenism, more preferably acne, seborrhea, alopecia, deepening of the voice, and / or hirsutism. These reproductive characteristics of clinical hyperandrogenism can be simply diagnosed by asking the woman or are evident after a short physical examination of the woman's body. Usually, the reference population shows none or only one of these phenotypic characteristics known to be indicative of PCOS.

[0028] Fibroblast growth factor-binding protein 1 (FGFBP1, also known as FGF-BP, FGFBP1, FGFBP, FGFBP-1 or HBP17) is a 26.2 kDa protein belonging to the fibroblast growth factor-binding protein family. FGFBP1 binds reversibly and non-covalently to FGF1, FGF2, FGF7, FGF10, FGF22 and HSPG2 (perlecan) (Wu DQ, Kan MK, Sato GH, et al. Characterization and molecular cloning of a putative binding protein for heparin-binding growth factors. J Biol Chem. 1991 Sep 5;266(25):16778-85; Beer HD, Bittner M, Niklaus G, et al. The fibroblast growth factor binding protein is a novel interaction partner of FGF-7, FGF-10 and FGF-22 and regulates FGF activity: implications for epithelial repair. Oncogene. 2005 Aug 11;24(34):5269-77; Abuharbeid S, Czubayko F, Aigner A. The fibroblast growth factor-binding protein FGF-BP. Int J Biochem Cell Biol. 2006;38(9):1463-8). Many members of the FGF family are immobilized in the extracellular matrix (ECM), bind to heparan sulfate proteoglycan (HSPG), and are released from this storage site by proteases and heparanase. FGFBP transports FGF from their storage sites to their receptors (Turner N, Grose R. Fibroblast growth factor signalling: from development to cancer.Nat Rev Cancer 2010, 10: 116 - 129). Biochemical studies have revealed that FGFBP1 binds to FGF2 in a dose - dependent and specific manner, and this binding is inhibited by FGF1, heparan sulfate, and heparinoids (Tassi E, Al - Attar A, Aigner A, et al. Enhancement of fibroblast growth factor (FGF) activity by an FGF - binding protein. J Biol Chem 2001, 276: 40247 - 40253). This FGFBP1 / FGF2 interaction results in a marked decrease in the affinity of FGF2 for heparin, leading to the release of FGF2 from the ECM mediated by FGFBP1 (Aigner A, Butscheid M, Kunkel P, et al. An FGF - binding protein (FGF - BP) exerts its biological function by parallel paracrine stimulation of tumor cell and endothelial cell proliferation through FGF - 2 release. Int J Cancer 2001, 92: 510 - 517). Several findings from various laboratories have shown that FGFBP1 may contribute to embryonic development, angiogenesis, wound healing, tumor growth, and malignant progression, as well as the maintenance and reinnervation of the neuromuscular junction and blood - brain barrier development (Czubayko F, Smith RV, Chung HC, Wellstein A. Tumor growth and angiogenesis induced by a secreted binding protein for fibroblast growth factors. J Biol Chem 1994, 269: 28243 - 28248; Mongiat M, Otto J Oldershaw R, et al. Fibroblast growth factor - binding protein is a novel partner for perlecan protein core.J Biol Chem 2001, 276: 10263 - 10271; Tassi E, Al - Attar A, Aigner A, et al. Enhancement of fibroblast growth factor (FGF) activity by an FGF - binding protein. J Biol Chem 2001, 276: 40247 - 40253; Gibby KA, McDonnell K, Schmidt MO, Wellstein A. A distinct role for secreted fibroblast growth factor - binding proteins in development, Proc Natl Acad Sci USA: 2009, 106: 8585 - 8590; Czubayko F, Liaudet - Coopman ED, Aigner A, et al. A secreted FGF - binding protein can serve as the angiogenic switch in human cancer. Nat Med 1997, 3: 1137 - 1140; Kurtz A, Aigner A, Cabal - Manzano RH, et al. Differential regulation of a fibroblast growth factor - binding protein during skin carcinogenesis and wound healing. Neoplasia 2004, 6: 595 - 602; Kurtz A, Wang HL, Darwiche N, et al. Expression of a binding protein for FGF is associated with epithelial development and skin carcinogenesis. Oncogene 1997, 14: 2671 - 2681; Tassi E, Henke RT, Bowden ET, et al. Expression of a fibroblast growth factor - binding protein during the development of adenocarcinoma of the pancreas and colon.Cancer Res 2006, 66: 1191 - 1198; Tassi E, Wellstein A. The angiogenic switch molecule, secreted FGF - binding protein, an indicator of early stages of pancreatic and colorectal adenocarcinoma. Semin Oncol 2006, 33: S50 - 56; Williams AH, Valdez G, Moresi V, et al. MicroRNA - 206 delays ALS progression and promotes regeneration of neuromuscular synapses in mice. Science 2009, 326: 1549 - 1554; Tassi E, McDonnell K, Gibby KA, et al. Impact of fibroblast growth factor - binding protein - 1 expression on angiogenesis and wound healing. Am J Pathol. 2011 Nov; 179(5): 2220 - 32; Cottarelli A, Corada M, Beznoussenko GV, et al. Fgfbp1 promotes blood - brain barrier development by regulating collagen IV deposition and maintaining Wnt / β - catenin signaling. Development. 2020 Aug 24; 147(16): dev185140). FGFBP1 mRNA is expressed in normal mouse skin, lung, intestine, ovary, placenta, stomach, and eye (Fon Tacer K, Bookout AL, Ding X, et al. Research resource: comprehensive expression atlas of the Fibroblast Growth Factor system in adult mouse. Mol Endocrinol 2010, 24: 2050 - 2064; Kurtz A, Wang HL, Darwiche N, et al.Expression of a binding protein for FGF is associated with epithelial development and skin carcinogenesis. (Oncogene 1997, 14: 2671-2681). Gene polymorphisms in the human FGFBP1 gene were associated with higher gene and protein expression in the human kidney and an increased risk of familial hypertension (Tomaszewski M, Charchar FJ, Nelson CP, et al. Pathway analysis shows association between FGFBP1 and hypertension. J Am Soc Nephrol. 2011 May; 22(5): 947-55). Regulation of FGF signaling by FGFBP1 has been shown to regulate vascular sensitivity to endogenous angiotensin II and, as a result, control steady-state blood pressure (Tassi E, Lai EY, Li L, et al. Blood Pressure Control by a Secreted FGFBP1 (Fibroblast Growth Factor-Binding Protein). Hypertension. 2018 Jan; 71(1): 160-167). FGF2 signaling has already been proven to be central to the maintenance of cellular plasticity in epithelial ovarian cancer, which is in the very early stages of carcinogenesis (De Cecco L, Marchionni L, Gariboldi M, et al. Gene expression profiling of advanced ovarian cancer: characterization of a molecular signature involving fibroblast growth factor 2. Oncogene. 2004 Oct 21; 23(49): 8171-83). Serum FGF2 levels have been found to be high in patients with epithelial ovarian cancer (both benign and malignant subtypes) compared to healthy controls (Barton DP, Cai A, Wendt K, et al.Angiogenic protein expression in advanced epithelial ovarian cancer.Clin Cancer Res 3:1579-1586,1997;Le Page C,Ouellet V,Madore J,ら.From gene profiling to diagnostic markers:IL-18 and FGF-2 complement CA125 as serum-based markers in epithelial ovarian cancer.Int J Cancer 118:1750-1758,2006;Madsen CV,Steffensen KD,Olsen DA,ら.Serum platelet-derived. Growth factor and fibroblast growth factor in patients with benign and malignant ovarian tumors. Anticancer Res. 2012 Sep;32(9):3817-25). In rats, treatment of ovarian cultures with FGF2 inhibited primordial follicle assembly. Furthermore, since a number of differentially expressed genes identified in the treated ovaries correlated with genes previously known to be associated with PCOS, it was suggested that FGF2-dependent abnormal follicle assembly may contribute to PCOS in later life (Nilsson E, Zhang B, Skinner MK. Gene bionetworks that regulate ovarian primordial follicle assembly. BMC Genomics. 2013 Jul 23;14:496). In cows, the expression of FGF2 mRNA is high in preovulatory follicles, decreases during the early luteal phase, and then remains at low levels during the late luteal phase and throughout pregnancy (Berisha B, Schams D, Rodler D, Pfaffl MW. Angiogenesis in The Ovary - The Most Important Regulatory Event for Follicle and Corpus Luteum Development and Function in Cow - An Overview. Anat Histol Embryol. 2016 Apr;45(2):124-30).Intraperitoneal injection of FGF2 in a mouse model of valproic acid-induced PCOS showed a protective and ameliorative effect (Moayeri A, Rostamzadeh A, Raoofi A, et al. Retinoic acid and fibroblast growth factor-2 play a key role on modulation of sex hormones and apoptosis in a mouse model of polycystic ovary syndrome induced by estradiol valerate. Taiwan J Obstet Gynecol. 2020 Nov;59(6):882-890). Conflicting results regarding serum FGF2 levels in PCOS patients have been published. Artini and co-workers reported that there was no difference in serum FGF2 levels between untreated PCOS patients and controls, but FGF2 levels increased in PCOS patients after FSH stimulation (Artini PG, Monti M, Matteucci C, et al. Vascular endothelial growth factor and basic fibroblast growth factor in polycystic ovary syndrome during controlled ovarian hyperstimulation. Gynecol Endocrinol. 2006 Aug;22(8):465-70). Patil and co-workers, instead, reported lower serum FGF2 levels in PCOS women undergoing controlled ovarian hyperstimulation for IVF compared to controls matched for age and BMI (Patil K, Hinduja I, Mukherjee S. Alteration in angiogenic potential of granulosa-lutein cells and follicular fluid contributes to luteal defects in polycystic ovary syndrome. Hum Reprod. 2021 Mar 18;36(4):1052-1064).The role of FGF2 signaling in PCOS is unclear, but its metabolic function has been studied in recent years. FGF2 can function as a positive or negative factor for in vitro adipogenesis depending on its concentration (Kim S, Ahn C, Bong N, et al. Biphasic effects of FGF2 on adipogenesis. PLoS One. 2015 Mar 19;10(3):e0120073. doi:10.1371 / journal.pone.0120073). Mathes and co-workers showed that FGF2-dependent signaling enhances the differentiation of fibrin / adipogenesis progenitors, which promote the formation of intramuscular adipose tissue (Mathes S, Fahrner A, Ghoshdastider U, et al. FGF-2-dependent signaling activated in aged human skeletal muscle promotes intramuscular adipogenesis. Proc Natl Acad Sci U S A. 2021 Sep 14;118(37):e2021013118. doi:10.1073 / pnas.2021013118). The appearance of adipose tissue between skeletal muscle fibers is a characteristic feature of aging, obesity, and type 2 diabetes, and its association with insulin resistance has been demonstrated. Furthermore, FGF2 has been shown to be a negative regulator of thermogenesis in both brown and beige adipose tissues. Disruption of FGF2 strongly enhanced the thermogenic effects of brown and beige adipose tissues, leading to increased energy consumption and improved lipid homeostasis. In addition, deletion of FGF2 protected mice from hyperlipidemia and hepatic steatosis induced by high-fat levels. (Li H, Zhang X, Huang C, et al. FGF2 disruption enhances thermogenesis in brown and beige fat to protect against adiposity and hepatic steatosis. Mol Metab. 2021 Dec;54:101358. doi:10.1016 / j.molmet.2021).

[0029] A first aspect of the present invention is a method for evaluating whether a subject has PCOS or is at risk of developing PCOS, the method comprising: (a) determining the amount or concentration of FGFBP1 in a sample from the subject; and (b) comparing the determined amount or concentration to a reference. The present invention relates to such a method.

[0030] An increase in the amount or concentration of FGFBP1 in a patient's sample indicates the presence, risk, or development of PCOS in the patient. In particular, if the amount or concentration of FGFBP1 in the patient's sample is higher than the amount or concentration of FGFBP1 in the reference or reference sample, the amount or concentration of FGFBP1 in the patient's sample indicates the presence or risk of development of PCOS in the patient. In particular, FGFBP1 is detectable in a higher amount or concentration in a biological fluid sample of a patient being evaluated for the presence or risk of development of PCOS than in the same biological fluid sample of an individual who does not have PCOS or is at risk of developing PCOS. In particular, an amount or concentration of FGFBP1 that is increased by 50% or more indicates the presence or risk of development of PCOS. In particular, an amount or concentration of FGFBP1 that is increased by 100% or more indicates the presence or risk of development of PCOS. In particular, an amount or concentration of FGFBP1 that is increased by 150% or more indicates the presence or risk of development of PCOS. In particular, an amount or concentration of FGFBP1 that is increased by 200% or more indicates the presence or risk of development of PCOS.

[0031] In a plurality of embodiments, the biological fluid sample is whole blood, serum, plasma, capillary blood, interstitial fluid, ascitic fluid, or menstrual fluid, and preferably the biological fluid sample is serum or whole blood. In a plurality of embodiments, the sample is an in vitro sample, i.e., the sample is analyzed in vitro and not returned to the body.

[0032] 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 under 25 years old. In certain embodiments, the patient is a human female patient between 15 and under 20 years old. In certain embodiments, the patient is a human female patient who has had her first menstruation less than 25 years ago and three years have passed since then. In certain embodiments, the patient is a human female patient who has had her first menstruation less than 20 years ago and three years have passed since then. In certain embodiments, the patient is a human female patient between 15 and under 25 years old who has had her first menstruation and three years have passed since then. In certain embodiments, the patient is a human female patient between 15 and under 20 years old who has had her first menstruation and three years have passed since then.

[0033] In multiple embodiments, PCOS is evaluated from the group consisting of metabolic or phenotypic PCOS. In further embodiments, PCOS is evaluated from the group consisting of PCOS of phenotype A, phenotype B, phenotype C, and phenotype D.

[0034] In multiple embodiments, the first method of the present invention is an in vitro method.

[0035] In multiple embodiments, the amount or concentration of FGFBP1 is determined using an antibody, particularly a monoclonal antibody. In multiple embodiments, step a) of determining the amount or concentration of FGFBP1 in a patient sample includes performing an immunoassay. In multiple embodiments, the immunoassay is performed in either a direct or indirect format. In multiple embodiments, 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.

[0036] In certain embodiments, step a) of determining the amount or concentration of FGFBP1 in a patient sample is i) incubating a sample from a patient with one or more antibodies that specifically bind to FGFBP1, thereby generating a complex of the antibody and FGFBP1, and ii) quantifying the complex formed in step i), thereby quantifying the amount or concentration of FGFBP1 in the patient's sample comprising.

[0037] In certain embodiments, in step i), the sample is incubated with two antibodies that specifically bind to FGFBP1. As will be apparent to those skilled in the art, the sample can be contacted with the first and second antibodies in any desired order, e.g., first with the first antibody and then with the second antibody, or first with the second antibody and then with the first antibody, or simultaneously, for a time and under conditions sufficient to form an anti-FGFBP1 antibody / FGFBP1 / second anti-FGFBP1 antibody complex. As will be readily appreciated 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-FGFBP1 antibody and an FGFBP1 antigen / analyte (= anti-FGFBP1 complex), or the formation of a secondary or sandwich complex comprising a first antibody to FGFBP1, FGFBP1 (analyte), and a second anti-FGFBP1 antibody (= first anti-FGFBP1 antibody / FGFBP1 / second anti-FGFBP1 antibody complex) is nothing other than routine experimentation.

[0038] Detection of the anti-FGFBP1 antibody / FGFBP1 complex can be carried out by any suitable means. Detection of the first anti-FGFBP1 antibody / FGFBP1 / second anti-FGFBP1 antibody complex can be carried out by any suitable means. Those skilled in the art are fully conversant with such means / methods.

[0039] In certain embodiments, a sandwich is formed comprising a first antibody to FGFBP1, FGFBP1 (analyte), and a second antibody to FGFBP1, and the second antibody is detectably labeled.

[0040] In one embodiment, a sandwich is formed that includes a first antibody to FGFBP1, FGFBP1 (the analyte), and a second antibody to FGFBP1, where the second antibody is detectably labeled and the first anti-FGFBP1 antibody is capable of binding to or binds to a solid phase.

[0041] In a plurality of 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.

[0042] In a second aspect, the invention is a method of selecting a patient for the treatment of PCOS, comprising: (c) determining the amount or concentration of FGFBP1 in a sample of the subject, and (d) comparing the determined amount or concentration to a reference. The invention relates to a method comprising these steps.

[0043] In a plurality of embodiments, if an increase in the amount of FGFBP1 in a patient's sample is detected, the patient is selected for the treatment of PCOS. In particular, if the amount of FGFBP1 is greater than the amount of FGFBP1 in a reference sample or the reference sample, the patient is selected for the treatment of PCOS. In particular, if the amount of FGFBP1 is higher in a body fluid sample of a patient being evaluated for the treatment of PCOS than in the same body fluid sample of an individual who does not have PCOS, is at risk of developing PCOS, or has not been selected for the treatment of PCOS, the patient is selected for the treatment of PCOS. In particular, if the amount of FGFBP1 has increased by 50% or more, the patient is selected for the treatment of PCOS. In particular, if the amount of FGFBP1 has increased by 100% or more, the patient is selected for the treatment of PCOS. In particular, if the amount of FGFBP1 has increased by 150% or more, the patient is selected for the treatment of PCOS. In particular, if the amount of FGFBP1 has increased by 200% or more, the patient is selected for the treatment of PCOS.

[0044] In particular, patients are selected for drug-based treatment of PCOS or improvement of lifestyle habits to control metabolic symptoms. In multiple embodiments, the drug-based therapy for PCOS is a drug for regulating the period, particularly oral contraceptives or progestin therapy, a drug for preventing or controlling diabetes, particularly type 2 diabetes, a drug for preventing or controlling high cholesterol, a hormone or drug for enhancing fertility, a drug or treatment for removing excessive hair, a drug or treatment for controlling acne, and is selected from the group consisting of.

[0045] In multiple embodiments, the biological fluid sample is whole blood, serum, plasma, capillary blood, interstitial fluid, peritoneal fluid, or menstrual fluid, and preferably the biological fluid sample is serum or whole blood. In multiple embodiments, the sample is an in vitro sample, i.e., the sample is analyzed in vitro and not returned to the body.

[0046] 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 between 15 and 25 years old. In certain embodiments, the patient is a human female patient between 15 and 20 years old. In certain embodiments, the patient is a human female patient who has had her first menstruation less than 25 years old and three years have passed. In certain embodiments, the patient is a human female patient who has had her first menstruation less than 20 years old and three years have passed. In certain embodiments, the patient is a human female patient who has had her first menstruation between 15 and 25 years old and three years have passed. In certain embodiments, the patient is a human female patient who has had her first menstruation between 15 and 20 years old and three years have passed.

[0047] In multiple embodiments, the second method of the present invention is an in vitro method.

[0048] In multiple embodiments, the amount of FGFBP1 is determined using an antibody, particularly a monoclonal antibody. In multiple embodiments, step a) of determining the amount of FGFBP1 in a patient sample comprises performing an immunoassay. In multiple embodiments, the immunoassay is performed in either a direct or indirect format. In multiple embodiments, 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.

[0049] In certain embodiments, step a) of determining the amount of FGFBP1 in a patient sample comprises i) incubating the patient sample with one or more antibodies that specifically bind to FGFBP1, thereby generating a complex of the antibody and FGFBP1, and ii) quantifying the complex formed in step i), thereby quantifying the amount of FGFBP1 in the patient sample. Including.

[0050] In certain embodiments, in step i), the sample is incubated with two antibodies that specifically bind to FGFBP1. As will be apparent to those skilled in the art, the sample can be contacted with the first and second antibodies in any desired order, i.e., first with the first antibody and then with the second antibody, or first with the second antibody and then with the first antibody, or simultaneously, for a time and under conditions sufficient to form a first anti-FGFBP1 antibody / FGFBP1 / second anti-FGFBP1 antibody complex. 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-FGFBP1 antibody and an FGFBP1 antigen / analyte (= anti-FGFBP1 complex), or the formation of a secondary or sandwich complex comprising a first antibody to FGFBP1, FGFBP1 (analyte) and a second anti-FGFBP1 antibody (= first anti-FGFBP1 antibody / FGFBP1 / second anti-FGFBP1 antibody complex) is nothing other than routine experimentation.

[0051] The detection of the anti-FGFBP1 antibody / FGFBP1 complex can be carried out by any suitable means. The detection of the first anti-FGFBP1 antibody / FGFBP1 / second anti-FGFBP1 antibody complex can be carried out by any suitable means. Those skilled in the art are fully proficient in such means / methods.

[0052] In certain embodiments, a sandwich is formed that includes a first antibody to FGFBP1, FGFBP1 (the analyte), and a second antibody to FGFBP1, and the second antibody is detectably labeled.

[0053] In one embodiment, a sandwich is formed that includes a first antibody to FGFBP1, FGFBP1 (the analyte), and a second antibody to FGFBP1, the second antibody is detectably labeled, and the first anti-FGFBP1 antibody can bind to or binds to a solid phase.

[0054] In a plurality of 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.

[0055] Furthermore, the present invention also relates to a kit containing reagents for the diagnosis of PCOS. The reagents of the kit may include an antibody or an antibody fragment. Preferably, the antibody or antibody fragment recognizes an epitope or antigen of FGFBP1. The kit may further include other reagents that recognize other biomarkers. Thus, the kit may include a combination of at least two reagents. The kit can specifically measure the amount or concentration of FGFBP1 and any other biomarker of interest. According to the present invention, the biomarker may also include hormones such as anti-Müllerian hormone, AMH. The kit can be used in any diagnostic assay.

[0056] In multiple embodiments, the amount of FGFBP1 is determined using an antibody, particularly a monoclonal antibody. In multiple embodiments, step a) of determining the amount of FGFBP1 in a patient sample comprises performing an immunoassay. In multiple embodiments, the immunoassay is performed in either a direct or indirect format. In multiple embodiments, 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.

[0057] In a third aspect, the invention is a method for monitoring PCOS progression in a subject or for monitoring the response to treatment in a subject having PCOS, comprising: (a) determining the level of FGFBP1 in a first sample of the subject; (b) determining the level of FGFBP1 in a second sample of the subject obtained after the first sample; (c) comparing the level of FGFBP1 in the first sample with the level of FGFBP1 in the second sample; and (d) monitoring the progression in a subject with or being treated for PCOS based on the result of step c). The invention relates to a method comprising the above steps.

[0058] In multiple embodiments, the progression of PCOS in a subject having PCOS is monitored to determine whether the amount or concentration of FGFBP1 changes over time in the patient sample. In particular, the progression of PCOS is monitored to determine whether the amount or concentration of FGFBP1 increases, decreases, or does not change over time. In multiple embodiments, if an increase in the amount or concentration of FGFBP1 in the subject's sample is determined, the progression of PCOS is monitored.

[0059] In multiple embodiments, a subject being treated for PCOS is monitored to determine whether the amount or concentration of FGFBP1 is changing in a sample from the subject. In particular, a subject being treated for PCOS is monitored to determine whether the amount or concentration of FGFBP1 is increasing, decreasing, or remaining unchanged. In particular, a subject being treated for PCOS is monitored to determine whether the amount or concentration of FGFBP1 is increasing, decreasing, or remaining unchanged due to the applied treatment. In some embodiments, a decrease in the amount or concentration of FGFBP1 in a subject being treated for PCOS indicates that the treatment is effective. In some embodiments, no change or an increase in the amount or concentration of FGFBP1 in a sample from a subject being treated for PCOS indicates that PCOS persists. In particular, if the amount or concentration of FGFBP1 has increased by 50% or more, treatment of PCOS is ineffective. In particular, if the amount or concentration of FGFBP1 has increased by 100% or more, treatment of PCOS is ineffective. In particular, if the amount or concentration of FGFBP1 has increased by 150% or more, treatment of PCOS is ineffective. In particular, if the amount or concentration of FGFBP1 has increased by 200% or more, treatment of PCOS is ineffective.

[0060] In certain embodiments, if an unchanged or increasing amount or concentration of FGFBP1 in a sample from a subject being treated for PCOS is determined, the treatment is adapted.

[0061] In multiple embodiments, the subject is monitored several times at various time points. In multiple embodiments, the subject is monitored several times within a time frame of several weeks, several months or several years. In certain embodiments, the subject is monitored once a month or once a year. In multiple embodiments, a subject suffering from PCOS is monitored once a month or once a year after diagnosis of PCOS. In multiple embodiments, 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.

[0062] In multiple embodiments, the treatment of PCOS is selected from the group consisting of drug-based treatment of PCOS and lifestyle changes to control metabolic symptoms. In multiple embodiments, the drug-based therapy for PCOS includes drugs for regulating the period, particularly oral contraceptives or progestin therapy, drugs for preventing or controlling diabetes, particularly type 2 diabetes, drugs for preventing or controlling high cholesterol, hormones or drugs for enhancing fertility, drugs or treatments for removing excessive hair, drugs or treatments for controlling acne.

[0063] In multiple embodiments, the biological fluid sample is whole blood, serum, plasma, capillary blood, interstitial fluid, peritoneal fluid, or menstrual fluid, preferably the biological fluid sample is serum or whole blood. In multiple embodiments, the sample is an in vitro sample, i.e., the sample is analyzed in vitro and not returned to the body.

[0064] 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 who is 3 years past menarche and under 25 years of age. In certain embodiments, the patient is a human female patient who is 3 years past menarche and under 20 years of age. In certain embodiments, the patient is a human female patient between 15 and less than 25 years of age who is 3 years past menarche. In certain embodiments, the patient is a human female patient between 15 and less than 20 years of age who is 3 years past menarche.

[0065] In multiple embodiments, the second method of the present invention is an in vitro method.

[0066] In multiple embodiments, the amount or concentration of FGFBP1 is determined using an antibody, particularly a monoclonal antibody. In multiple embodiments, step a) of determining the amount or concentration of FGFBP1 in a patient sample comprises performing an immunoassay. In multiple embodiments, the immunoassay is performed in either a direct or indirect format. In multiple embodiments, 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.

[0067] In certain embodiments, step a) of determining the amount or concentration of FGFBP1 in a patient sample comprises i) incubating the patient sample with one or more antibodies that specifically bind to FGFBP1, thereby generating a complex of the antibody and FGFBP1, and ii) Quantifying the complex formed in step i), thereby quantifying the amount or concentration of FGFBP1 in the patient's sample comprises.

[0068] In certain embodiments, in step i), the sample is incubated with two antibodies that specifically bind to FGFBP1. As will be apparent to those skilled in the art, the sample can be contacted with the first and second antibodies in any desired order, e.g., first with the first antibody and then with the second antibody, or first with the second antibody and then with the first antibody, or simultaneously, for a time and under conditions sufficient to form a first anti-FGFBP1 antibody / FGFBP1 / second anti-FGFBP1 antibody complex. As will be readily appreciated 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-FGFBP1 antibody and an FGFBP1 antigen / analyte (= anti-FGFBP1 complex), or the formation of a secondary or sandwich complex comprising a first antibody to FGFBP1, FGFBP1 (analyte), and a second anti-FGFBP1 antibody (= first anti-FGFBP1 antibody / FGFBP1 / second anti-FGFBP1 antibody complex) is routine experimentation.

[0069] Detection of the anti-FGFBP1 antibody / FGFBP1 complex can be carried out by any suitable means. Detection of the first anti-FGFBP1 antibody / FGFBP1 / second anti-FGFBP1 antibody complex can be carried out by any suitable means. Those skilled in the art are fully conversant with such means / methods.

[0070] In certain embodiments, a sandwich is formed comprising a first antibody to FGFBP1, FGFBP1 (analyte), and a second antibody to FGFBP1, and the second antibody is detectably labeled.

[0071] In one embodiment, a sandwich is formed comprising a first antibody to FGFBP1, FGFBP1 (analyte), and a second antibody to FGFBP1, the second antibody is detectably labeled, and the first anti-FGFBP1 antibody can bind to or binds to a solid phase.

[0072] In multiple 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.

[0073] In a fourth aspect, the invention is a computer-implemented method for evaluating a patient suspected of having PCOS, comprising: (a) receiving a value for the amount or concentration of a first biomarker in a sample from a subject, wherein the first biomarker is FGFBP1; (b) optionally, receiving a value for the amount or concentration of a second biomarker in the sample from 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, hyperandrogenism, and cystic ovarian morphology; (d) comparing the values for the amounts or concentrations in steps (a)-(b) with the reference for the biomarker and the values for the presence or absence of at least one additional diagnostic criterion, and / or calculating a score for evaluating a subject suspected of having PCOS based on the amount or concentration and values of the biomarker; (e) evaluating the subject based on the comparison and / or calculation performed in step (d). The invention relates to a computer-implemented method comprising the above steps.

[0074] In multiple embodiments, the computer-implemented method for evaluating a subject suspected of having PCOS comprises a method that consists essentially of the methods described above 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, additional steps may relate to further determining markers and / or collecting pre-treatment samples or evaluating the results obtained by the above methods. The method may be performed manually or assisted by automation.

[0075] 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 is assumed to receive a value of the amount of a biomarker. Such a value may be an amount, a relative amount, or any other calculated value reflecting an amount as detailed elsewhere in this specification. Thus, it should be understood that the methods described above do not require the determination of the amount of a biomarker, but rather use values of amounts that have already been determined.

[0076] The present invention also contemplates, in principle, a computer program, a computer program product, or a computer-readable storage medium into which the computer program is connectably incorporated, the computer program including 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: - A computer or computer network including at least one processor, the processor being adapted to perform a method according to one of the embodiments described herein, - A computer loadable data structure adapted to perform a method according to one of the embodiments described herein while being implemented on a computer, - A computer script, adapted such that, while the program is being executed on a computer, the computer program executes the method according to one of the embodiments described herein. - A computer program comprising program means for performing the method according to one of the embodiments described herein while the computer program is being executed on a computer or on a computer network. - A computer program comprising program means according to the preceding embodiment, wherein the program means are stored on a computer-readable storage medium. - A storage medium on which a data structure is stored, the data structure being adapted to execute the method according to one of the embodiments described herein after being loaded into the main memory device and / or the working memory device of a computer or a computer network. - A computer program product having program code means, wherein the program code means, when executed on a computer or a computer network, are stored in or on a storage medium for performing the method according to one of the embodiments described herein. - Typically an encrypted data stream signal, comprising data of parameters defined elsewhere in this specification, and - Typically an encrypted data stream signal, comprising the evaluation provided by the method of the present invention. are included.

[0077] Definition: In the context of the kit of the present invention, the term "reagent" represents a substance or compound added to a sample that enables the amount or concentration of a particular component in the sample to be indicated.

[0078] In the context of the kit of the present invention, the term "specifically measure" means detecting the exact amount or concentration of a 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, and it is not necessary to specify such conditions.

[0079] In the context of the kit of the present invention, the term "reagent" can represent 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.

[0080] 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. Any reporter moiety or label can be used with the reagents of the kit according to the second aspect of the invention as long as its signal can be directly related to 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 radioactive group, scintillation counting or autoradiography is 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). Spectrophotometry can be used to detect dyes (including, for example, the colorimetric products of enzyme reactions), luminescent groups and fluorescent groups. Biotin can be detected by binding to avidin or streptavidin and coupling 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.

[0081] The reagent may also be a substance that can 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.

[0082] 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 above-mentioned female are well known to those skilled in the art.

[0083] Preferably, the reagent can be used in electrochemiluminescence immunoassay, and more preferably, the reagent is an antibody that can be used in electrochemiluminescence immunoassay.

[0084] Furthermore, the kit may include two or more reagents, such as two reagents, three reagents, four reagents, or more different reagents, preferably two reagents that interact with one molecule specifically measured in the sample. For example, if the molecule to be measured is measured by an electrochemiluminescence immunoassay, the kit may include two antibodies that bind to the same molecule to be measured. Preferably, the two 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 may be linked to different detectable reporter moieties or labels.

[0085] The kit may further include a buffer and / or a salt for adjusting the pH as well as the reaction conditions and measurement conditions. Furthermore, the kit may include a stabilizer for maintaining the stability of the reagents and / or hormones 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 additional 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 reagents or buffers.

[0086] The kit may also include all the equipment necessary to collect 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 may include a syringe.

[0087] 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 may be sent to the physician. However, the kit may also be applied by the physician or the physician's assistant themselves. The kit may be applied during a physician's outpatient visit, stationary treatment, or home visit.

[0088] All components of the kit may be separately packaged in individual containers. However, two or more components of the kit may be packaged together in one or more containers.

[0089] The kit may further include a label containing instructions, for example, explaining how to use the kit or explaining the contents of the kit. However, this information may also be provided in any other form, such as on a storage medium like a CD-ROM or a USB stick.

[0090] It is to be understood that the words "comprise", "comprises" and "comprising" as used herein 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.

[0091] 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.

[0092] 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 listed as limiting the range, but also all or sub-ranges of the individual numerical values that are encompassed within that range as if each numerical value and sub-range were explicitly listed. By way of example, the numerical range of "150 mg to 600 mg" should be interpreted to include not only the explicitly listed 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 - 200, 150 - 250, 250 - 300, 350 - 600, etc. This same principle applies to ranges that list only a single numerical value. Further, such an interpretation should apply regardless of the width of the range or the property being described.

[0093] The term "about", when used in relation to a numerical value, means to include numerical values within a range that has 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.

[0094] As used herein, the term "indicator" refers to a sign or signal for a condition or is used to monitor a condition. Such a "condition" may refer to the biological condition of a cell, tissue or organ, or to 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.

[0095] 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 system. In the art, the term "biomarker" may also be applied as a means of detecting 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 means of detection. Thus, a biomarker can be any type 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 specific modifications of other molecules (e.g., sugar moieties or phosphoryl residues on proteins, methyl residues on genomic DNA), or substances internalized by an organism or metabolites of such substances.

[0096] The biomarker referred to herein 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 the qualitative and / or quantitative detection of a biomarker is generally known to those skilled in the art. Samples can be easily assayed for proteins using, for example, immunoassays such as Western blotting and ELISA, RIA, fluorescence and luminescence-based immunoassays, as well as commercially available proximity extension assays. Further suitable methods for detecting a biomarker include measuring physical or chemical properties specific to a peptide or polypeptide, such as its exact molecular weight or NMR spectrum, etc. The above methods include, for example, analytical devices such as biosensors, optical devices associated with immunological assays, biochips, mass spectrometers, NMR analyzers, or chromatography devices. Further, the methods include methods based on microplate ELISA, fully automated or robotic immunological assays (Elecsys (商標)Available for use in an analyzer), CBA (Cobalt Binding Assay by enzyme, e.g., Roche-Hitachi (商標) Available for use in an analyzer), and latex agglutination assay (e.g., Roche-Hitachi (商標) Available for use in an analyzer) are included.

[0097] The term "anovulation" typically refers to a state where the ovaries do not release any oocytes during a woman's menstrual cycle. A woman being evaluated for the risk of having PCOS may be determined to have anovulation if no eggs are released during at least one, preferably at least three, more preferably at least six, and most often at least nine female menstrual cycles in a year. Further, a woman being evaluated for the risk of having PCOS may 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.

[0098] The "symptoms" of a disease are obvious indications of such a disease in the tissues, organs, or organisms having the disease, and include, but are not limited to, pain, weakness, tenderness, tension, stiffness, and spasms in the tissues, organs, or individual. Typical symptoms of PCOS include, but are not limited to, oligo-ovulation, irregular menstrual cycles, hyperandrogenism, polycystic ovarian morphology, infertility, type 2 diabetes, overweight and other metabolic conditions, and psychological distress. The "signs" or "signals" of a disease include, but are not limited to, the presence, increase or elevation, decrease or decline, such as changes or alterations in specific indicators such as biomarkers or molecular markers, or the onset, presence, or worsening 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.

[0099] The terms "disease" and "disorder" are used interchangeably herein and refer to an abnormal condition, particularly an abnormal medical condition such as a disease or injury where a tissue, organ or individual can no longer perform its function efficiently. Although not necessarily so, typically a disease is associated with specific symptoms or signs that indicate 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 that can indicate "worsening" or "improvement" of the disease. "Worsening" of a disease is characterized by a decrease in the ability of a tissue, organ or organism to perform its function efficiently, whereas "improvement" of a disease typically features an increase in the ability of a tissue, organ or individual to perform its function efficiently. 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 initial or weak signs or symptoms of such a disease. In such cases, the occurrence 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.

[0100] 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. A diagnosis of PCOS is required.

[0101] The terms "sample" or "sample of interest" 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 tissue state, 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 lymph, or solid samples such as tissue extracts, cartilage, bone, synovium, and connective tissue. The analysis of the sample can be achieved on a visual or chemical basis. Visual analysis includes, but is not limited to, microscopic imaging or radiographic 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 amount, concentration or level. The sample is an in vitro sample and will be analyzed in vitro and will not be returned to the body.

[0102] As used herein, the term "amount" encompasses the absolute amount of a biomarker referred to herein, the relative amount or concentration of said 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 said peptide by direct measurement, for example, intensity values in a mass spectrum or NMR spectrum. Further included are all values or parameters obtained by indirect measurement as specified elsewhere herein, for example, the amount of response measured from a biological readout system in response to a peptide or intensity signal obtained from a specifically bound ligand. It should be understood that values correlated with the above-mentioned amounts or parameters can also be obtained by all standard mathematical operations.

[0103] 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 an absolute reference 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 can 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 for the reference amount can, for example, be compared to each other, and this comparison can be automatically performed by a computer program that executes an algorithm for the comparison. The computer program that performs the above evaluation will provide the desired evaluation in a suitable output format. In a computer-based comparison, the value of the measured amount can be compared by a computer program to a value corresponding to a suitable reference stored in a database. The computer program can further evaluate the comparison result, i.e., it can automatically provide the desired evaluation in a suitable output format. In a computer-based comparison, the value of the measured amount can be compared by a computer program to a value corresponding to a suitable reference stored in a database. The computer program can further evaluate the comparison result, i.e., it can automatically provide the desired evaluation in a suitable output format.

[0104] The expression "comparing a determined amount or concentration to a reference" is used only to further clarify what is obvious to one of ordinary skill in the art. The reference concentration is established in a control sample.

[0105] As used herein, the terms "reference sample" or "control sample" 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 reference by which the information obtained from the sample of interest can be evaluated. A control sample is derived from a healthy or normal tissue, organ, or individual, thereby providing a reference for the health status of the tissue, organ, or individual. The difference between the state of the 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 reference for the pathological state of the tissue, organ, or individual. The difference between the state of the 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 taken at an earlier time point. The difference between the state of the 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.

[0106] A control sample can be an internal or external control sample. Using an internal control sample, i.e., evaluating one or more marker levels in a test sample as well as one or more other samples taken from the same subject to determine whether there is a change in the level of the one or more markers. 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 be suffering from or at risk of a given condition, or in a solid known not to have a given condition, i.e., a "normal individual".

[0107] Those skilled in the art will understand that such external control samples may be obtained from a single individual or may be obtained from a reference population of the same age and free of confounding diseases. Typically, a "reference value" is set using a sample from 100 individuals that have been well characterized from an appropriate reference population. However, the reference population may also be selected to consist of 20, 30, 50, 200, 500 or 1000 individuals. Healthy individuals represent a preferred reference population for setting control values.

[0108] 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. Typically, the marker concentration of a sample is directly or indirectly correlated with a 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 of a 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.

[0109] As used herein, the term "assessing" refers to assessing whether a patient has PCOS or is at risk of developing PCOS. Thus, the assessment as used herein includes determining the amount or concentration of FGFBP1 in a patient sample and diagnosing PCOS, predicting the risk of developing PCOS, selecting a treatment for PCOS, or monitoring a patient who has or is being treated for PCOS by comparing the determined amount or concentration to a reference.

[0110] 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 and Sons, New York 1983. Typically 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.

[0111] The terms "decrease" or "reduction" level, amount and / or concentration of an indicator refer to the level, amount and / or concentration of such an indicator in a sample that has decreased compared to a reference or reference sample.

[0112] The terms "increase" or "elevation" 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 than in the same fluid sample of an individual not suffering from the disease has an elevated level.

[0113] The terms "measure", "measuring" or "determining" preferably include qualitative, semi-quantitative or quantitative measurements.

[0114] As used herein, the term "immunoglobulin (Ig)" refers to a glycoprotein that confers immunity of the immunoglobulin superfamily. "Surface immunoglobulin" is bound to the membrane of effector cells by their transmembrane regions and includes, but is not limited to, molecules such as B cell receptors, T cell receptors, class I and II major histocompatibility complex (MHC) proteins, beta2 microglobulin (about 2M), CD3, CD4 and CDS.

[0115] Typically, as used herein, the term "antibody" refers to a secreted immunoglobulin that lacks a transmembrane region and can be released into the bloodstream and body cavities. Human antibodies are classified into various isotypes based on the heavy chains they possess. There are five types of human Ig heavy chains, denoted by the Greek letters α, γ, δ, ε, and μ. The type of heavy chain present defines the class of the antibody, and these chains each play different roles, as seen in IgA, IgD, IgE, IgG, and IgM antibodies, which result in appropriate immune responses to different types of antigens. The different heavy chains vary in size and composition and contain approximately 450 amino acids (Janeway et al., (2001) Immunobiology, Garland Science). IgA is found in mucosal areas such as the intestine, respiratory tract, and urogenital tract, as well as in saliva, tears, and breast milk, and prevents colonization by pathogens (Underdown and Schiff (1986) Annu. Rev. Immunol. 4:389-417). IgD functions primarily as an antigen receptor on B cells that have not been exposed to antigen 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 the majority of 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). There are four different IgG subclasses in humans (IgG1, 2, 3, and 4), named in order of their abundance in serum, with IgG1 being the most abundant (approximately 66%), followed by IgG2 (approximately 23%), IgG3 (approximately 7%), and IgG (approximately 4%). The biological characteristics of the various 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 secreted pentameric form with very high binding affinity. IgM is involved in eliminating pathogens in the early stage of B cell-mediated (humoral) immunity before sufficient IgG is produced (Geisberger et al., (2006) Immunology 118:429-437). Antibodies are found not only as monomers, but also form dimers of two Ig units (e.g., IgA), tetramers of four Ig units (e.g., IgM of teleost fish), or pentamers of five Ig units (e.g., mammalian IgM). Antibodies typically consist of four polypeptide chains, two identical heavy chains and two identical light chains, linked via disulfide bonds and resembling a "Y"-shaped macromolecule. Each chain contains several immunoglobulin domains, some of which are constant domains and others are variable domains. The immunoglobulin domain consists of a two-layer sandwich of 7-9 antiparallel strands arranged within two β-sheets. 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" usually contains one constant Ig domain (CL) and one variable Ig domain (VL). For example, the human IgG heavy chain is composed of four Ig domains (also called VγCγ1-Cγ2-Cγ3) linked from the N-terminus to the C-terminus in the order of VγCH1-CH2-CH3, while the human IgG light chain is composed of two immunoglobulin domains linked from the N-terminus to the C-terminus in the order of VL-CL and is either of the kappa or lambda type (VK-CK or Vλ-Cλ). For example, the constant chain of human IgG contains 447 amino acids. Throughout this specification and the claims, the numbering of amino acid positions of immunoglobulins is according to Kabat, E.A., Wu, T.T., Perry, H.M., Gottesman, K.S., and Foeller, C., (1991) Sequences of proteins of immunological interest, 5th. 版} are numbered according to the "EU index" such as the U.S. Department of Health and Human Service, National Institutes of Health, Bethesda, MD. The "Kabat EU index" refers to the numbering of residues of human IgG1 EU antibodies. Thus, the CH domains in the context of IgG are as follows: "CH1" refers to amino acids 118 - 220 according to the EU index as in Kabat. "CH2" refers to amino acids 237 - 340 according to the EU index as in Kabat; "CH3" refers to amino acids 341 - 447 according to the EU index as in Kabat.

[0116] The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form that is not the defined antibody fragment below. Specifically, these terms refer to an antibody that includes a heavy chain containing an Fc region.

[0117] Papain digestion of an antibody produces two identical antigen-binding fragments (also referred to as "Fab portions" or "Fab regions"), called "Fab" fragments, each having a single antigen-binding site, and a 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 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; in IgM and IgE isotypes, the Fc region contains three heavy-chain constant domains (CH2-4) in each polypeptide chain. Furthermore, smaller immunoglobulin molecules exist naturally or are constructed artificially. The term "Fab' fragment" refers to a Fab fragment that further includes the hinge region of the Ig molecule, and "F(ab')2 fragment" is understood to include two Fab' fragments that are chemically linked or linked via a disulfide bond. "Single-domain antibodies (sdAb)" (Desmyter et al. (1996) Nat. Struct. 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). A bivalent single-chain variable fragment (di-scFv) can be designed by linking two scFvs (scFvA-scFvB). This can be done by generating a single peptide chain with two VH regions and two VL regions, obtaining a "tandem scFv" (VHA-VLA-VHB-VLB). Another possibility is to create an scFv with a linker that is too short for the two variable regions to fold together and force the scFv to dimerize. Usually, a linker with a length of 5 residues is used to create these dimers. This type is known as a "diabody".Shorter linkers (one or two amino acids) between the VH and VL domains result in the formation of single-specific trimers, so-called "triabodies" or "tribodies". Bispecific diabodies are formed by expressing chains having the sequences VHA-VLB and VHB-VLA or VLA-VHB and VLB-VHA, respectively. Single-chain diabodies (scDb) contain VHA-VLB and VHB-VLA fragments linked by a linker peptide (P) of 12-20 amino acids, preferably 14 amino acids (VHA-VLB-P-VHB-VLA). "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). Bispecific affinity retargeting molecules ("DART" molecules) are diabodies further stabilized by a C-terminal disulfide bridge.

[0118] Accordingly, the term "antibody fragment" refers to a part 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, scFv, di-scFv, tandem scFv, triabodies, diabodies, scDb, BiTE and DART.

[0119] 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 members 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 common 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 for longer. Various methods for measuring binding affinity are known in the art, and any of these can be used in the present invention.

[0120] "Sandwich immunoassay" is widely used for the detection of the analyte of interest. 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 analyte of interest. By appropriate means, such a sandwich complex is measured, thereby quantifying the analyte. In a typical sandwich-type method, a first antibody that is bound to a solid phase or can be bound to a solid phase, and a second antibody that is detectably labeled each bind to the analyte at different non-overlapping epitopes. The first analyte-specific binding agent (e.g., an antibody) 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 may be a tube, beads, the disk of a microplate, or other surface suitable for performing the immunoassay. The binding process is well known in the art and generally consists of crosslinkable covalent bonds or physical adsorption, and the polymer-antibody complex is washed in the preparation for the test sample. Then, a portion 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 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 conjugated to a reporter molecule used to indicate the binding of the second antibody to the complex of the first antibody and the antigen of interest.

[0121] A very widely applicable alternative sandwich assay method involves the use of a solid phase coated with a first partner of a binding pair, e.g., microparticles coated with paramagnetic streptavidin. Such microparticles are mixed and incubated with an analyte-specific binding agent bound to a second partner of the binding pair (e.g., a biotinylated antibody), a sample suspected of containing or containing an analyte to which the second partner of the binding pair is bound to the analyte-specific binding agent, and a second analyte-specific binding agent detectably labeled, e.g., with an electrochemiluminescent label as used herein. As will be apparent to those skilled in the art, these components are incubated for a time sufficient to bind the labeled antibody to the solid phase microparticles via the analyte, the second partner of the binding pair, and the analyte-specific binding agent (bound to) the first partner of the binding pair under appropriate conditions. Optionally, such an assay may include one or more washing steps.

[0122] The term "detectably labeled" encompasses labels that can be detected directly or indirectly.

[0123] A directly detectable label either provides a detectable signal or modifies a detectable signal provided by a first or second label by interacting with a second label to give, e.g., FRET (fluorescence resonance energy transfer). Labels such as fluorescent dyes and luminescent (including chemiluminescent and electrochemiluminescent) dyes (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 labeling methods. In one embodiment, detectably labeled refers to a label that results in or is derivable to result in 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.

[0124] 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.

[0125] (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).

[0126] 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 are commercially available from Invitrogen / Molecular Probes (Eugene, Oregon, USA) and Pierce Biotechnology, Inc. (Rockford, Ill.).

[0127] (b) Luminescent dyes Luminescent dyes or labels can be further subclassified into chemiluminescent dyes and electrochemiluminescent dyes.

[0128] Different classes of chemiluminescent labels include systems based on luminol, acridinium compounds, selenotellurazine and analogs, dioxetane, peroxyoxalic acid and peroxyoxalic acid derivatives. For immunoassay procedures, mainly acridinium-based labels are used (a detailed overview is given in Dodeigne C. et al., Talanta 51 (2000) 415-439).

[0129] The main relevant labels used as electrochemiluminescent labels are ruthenium- and iridium-based electrochemiluminescent complexes, respectively. Electrochemiluminescence (ECL) has proven to be very useful in analytical applications as a sensitive and selective method. ECL combines the analytical advantages of chemiluminescence 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 approximately 620 nm) regenerated with TPA (tripropylamine) at a liquid phase or liquid-solid interface, are used as ECL labels.

[0130] Electrochemiluminescence (ECL) assays provide sensitive and accurate measurement of the presence and concentration of the analyte of interest.

[0131] Such techniques use labels or other reactants that can be induced to emit light when electrochemically oxidized or reduced in a suitable chemical environment. Such electrochemiluminescence is caused by a voltage applied to a working electrode in a particular pattern at a particular 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 Nos. 5,221,605, 5,591,581, 5,597,910, PCT Application Publication Nos. WO90 / 05296, WO92 / 14139, WO90 / 05301, WO96 / 24690, US95 / 03190, US97 / 16942, US96 / 06763, WO95 / 08644, WO96 / 06946, WO96 / 33411, WO87 / 06706, WO96 / 39534, WO96 / 41175, WO96 / 40978, PCT / US97 / 03653 and U.S. Patent Application No. 08 / 437,348 (U.S. Patent No. 5,679,519). Also, reference is made to the 1994 review of the analytical applications of ECL by Knight, et al. (Analyst, 1994, 119:879-890) and the references cited therein. In one embodiment, the method according to the present specification is implemented using an electrochemiluminescent label.

[0132] In recent years, iridium-based ECL labels have also been described (International Publication No. 2012107419).

[0133] (c) Radioactive labels use radioisotopes (radionuclides), for example, 3H, 11C, 14C, 18F, 32P, 35S, 64Cu, 68Gn, 86Y, 89Zr, 99TC, 111In, 123I, 124I, 125I, 131I, 133Xe, 177Lu, 211At, or 131Bi.

[0134] (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

[0135] The present invention is merely illustrated by the following examples. In no case should the examples be construed in a manner that limits the scope of the present invention.

[0136] Example 1: Diagnostic performance of biomarker FGFBP1 in women with PCOS (phenotype A) and controls determined by proximity extension assay (PEA) technology developed by Olink As part of the measurement, 85 serum samples from human females were analyzed. The case group included 48 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 proximity extension assay (PEA) technology developed by Olink. Briefly, a matched antibody pair bound to unique partially complementary oligonucleotides corresponds to each biomarker. Quantification is then performed by quantitative real - time PCR.

[0137] For each selected panel, after diluting the samples according to the manufacturer's protocol, the Olink protocol consists of three core steps: 1. Incubation, 2. Extension and amplification, 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, 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 on the plate. The samples were incubated overnight at +4 °C. During this step, the antibody pairs bind to each respective protein in the sample. Once 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, 20 min, 95 °C, 5 min (95 °C, 30 s, 54 °C, 1 min, 60 °C, 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. According to the manufacturer's instructions, a 96.96 Dynamic Array (trademark) Integrated Fluidic Circuit (IFC) was used. 7 points of 2 μl of detection mix were added to 2.8 μl of each sample, and 5 μl of these were transferred to the primed 96.96 Dynamic Array IFC left inlet. 5 μl of primer solution was transferred to the primed 96.96 Dynamic Array IFC right inlet. The chip was loaded into a Fluidigm IFC Controller HX according to the manufacturer's instructions.The Olink protein expression 96 x 96 program was run on a Fluidigm Biomark (trademark) reader according to the manufacturer's instructions (50 °C, 120 seconds, 70 °C, 1800 seconds, 25 °C, 600 seconds, 95 °C, 300 seconds (95 °C, 15 seconds, 60 °C, 60 seconds) x 35; following settings: application gene expression, passive reference ROX; assay - single probe; probe - FAM - MGB). 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:.

[0138] Expansion control: Ct Analyte -Ct Extension Control =dCt Analyte

[0139] Inter - plate control: dCt Analyte -dCt Inter-plate Control =ddCt Analyte

[0140] Adjustment for correction factor: Correction factor - ddCt Analyte =NPX Analyte

[0141] Quality control and normalization were achieved using Olink NPX Manager software.

[0142] 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 0.985 (95% CI 0.967 - 1.0, Figure 1), indicating that FGFBP1 has high diagnostic accuracy for PCOS.

[0143] Table 1 shows the diagnostic performance of FGFBP1 for differentiating women with PCOS having a complete growth phenotype A (cases) from healthy controls using ROC analysis, with the AUC of the ROC curve analysis and the associated 95% confidence interval explained. The results were obtained using the Olink proximity extension technology.

Table 1

[0144] Using the 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 scores of the group), 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. In women with PCOS, the serum FGFBP1 concentration is increased compared to healthy controls (Figure 2).

[0145] Example 2: Diagnostic performance of biomarker FGFBP1 in women with PCOS (phenotypes A, B, C, and D) and control women determined by ELISA technology Performance verification was carried out in a sample population of 90 cases (serum samples from women with PCOS) and 44 controls (serum samples from healthy women).

[0146] The concentration of the analyte was determined by ELISA (enzyme-linked immunosorbent assay). The case group consisted of patients diagnosed with PCOS (30 with phenotype A, 20 with phenotype B, 20 with phenotype C, and 20 with phenotype D) according to the Rotterdam criteria. The control group included healthy women without PCOS.

[0147] The concentration of FGFBP1 in human serum was determined using a Human FGFBP1 ELISA Kit (Catalog No.: RAB1460) from Sigma-Aldrich. The kit is a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) designed to detect and quantify the levels of human FGFBP1 in cell culture supernatants, plasma, and serum.

[0148] A human FGFBP1 antibody pre-coated plate is provided in the kit. Samples were measured at a 75-fold dilution. After bringing all reagents to room temperature, 100 μL of each sample and standard were added to the plate. Samples and standards were measured in duplicate. While incubating at room temperature for 2.5 hours with gentle shaking, any FGFBP1 present bound to the immobilized capture antibody on the microtiter plate. During the washing step (4 × 300 μL), unbound substances were removed from the plate before adding 100 μL of the diluted detection antibody to the wells. After incubating for 1 hour with gentle shaking and another washing step (4 × 300 μL) to remove unbound detection antibody, 100 μL of the prepared streptavidin-HRP solution was added to the plate. Incubate at room temperature for 45 minutes with gentle shaking and perform a washing step (4 × 300 μL). After the final wash, 100 μL of TMB One-Step Substrate Reagent was added to the plate. The plate was incubated in the dark at room temperature for 30 minutes with gentle shaking. During incubation, the substrate changed to blue. FGFBP1 developed color in proportion to the amount bound in the first step. Color development was stopped by adding 50 μL of the stop solution, changing the solution in the wells from blue to yellow, and measuring the color intensity using a plate reader at 450 nm for detection and at 570 nm for background correction. A 7-point standard curve was obtained using 2.5-fold serial dilutions of recombinant FGFBP1 in the assay diluent. The calibration curve was fitted using 4-parameter nonlinear regression (Newton / Raphson) without weighting.

[0149] The receiver operating characteristic (ROC) curve was created (Figure 3). The model performance is 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 FGFBP1 for PCOS cases (phenotypes A - D) when all phenotypes were combined showed an AUC of 0.87 (95% CI 0.78 - 0.95), confirming the high diagnostic accuracy of FGFBP1 for PCOS (Figure 3).

[0150] Table 2, which describes the AUC of the ROC curve analysis and the associated 95% confidence intervals, shows the diagnostic performance of FGFBP1 for differentiating women with PCOS (cases, PCOS phenotypes A - D) from healthy controls using ROC analysis. The results were obtained using an ELISA immunoassay.

Table 2

[0151] 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 FGFBP1 concentration (pg / mL) increases in women with PCOS compared to healthy controls (Figure 4).

[0152] Table 3 shows the diagnostic performance of FGFBP1 for differentiating women with PCOS separated by various phenotypes A, B, C, and D from healthy controls. The results were obtained using an ELISA immunoassay. The AUC for each phenotype is reported in the table.

Table 3

[0153] ROC curve analysis of FGFBP1 for PCOS cases separated by various phenotypes against healthy controls showed AUCs of 0.88 (95% CI 0.79 - 0.96), 0.88 (95% CI 0.79 - 0.96), 0.88 (95% CI 0.80 - 0.96) and 0.84 (95% CI 0.74 - 0.95) for phenotypes A - D, respectively (Figure 5). The results confirm the high diagnostic accuracy of FGFBP1 for PCOS.

[0154] Serum FGFBP1 concentrations (pg / mL) in all various PCOS phenotypes (phenotypes A - D) showed increased levels compared to healthy controls (Figure 6, results obtained using ELISA immunoassay).

[0155] Table 4 shows the diagnostic performance of FGFBP1 in young women (under 25 years old) for distinguishing young women with PCOS from young healthy controls when all phenotypes are combined (phenotypes A - D). Results were obtained using ELISA assay.

Table 4

[0156] ROC curve analysis of FGFBP1 for young PCOS cases (under 25 years old) when all phenotypes are combined (phenotypes A - D) showed an AUC of 0.84, confirming high diagnostic accuracy for women under 25 years old when distinguishing PCOS cases from controls (95% CI 0.61 - 1, Figure 7). When only young women (under 25 years old) were included in the analysis, PCOS cases (all phenotypes A - D combined) showed an increase in serum FGFBP1 concentration (pg / mL) compared to young controls (age ≤ 25, Figure 8).

[0157] The diagnostic ability of FGFBP1 to distinguish young women (under 25 years old) with PCOS separated by various phenotypes A, B, C and D from healthy subjects (under 25 years old) was evaluated and the results are reported in Table 5 (AUC for PCOS phenotypes vs controls). Results were obtained using ELISA immunoassay.

Table 5

[0158] For the ROC curve analysis of FGFBP1 in young PCOS cases (aged 25 years or younger, phenotypes A - D), for each phenotype, the AUC was 0.93 (95% CI 0.81 - 1), 0.85 (95% CI 0.54 - 1), 0.84 (95% CI 0.57 - 1), and 0.63 (95% CI 0.19 - 1) respectively, confirming the high diagnostic accuracy of FGFBP1 for PCOS in the subgroup of women aged 25 years or younger (Figure 9). The concentration of FGFBP1 increased in all various PCOS phenotypes (phenotypes A - D, aged 25 years or younger) when compared with the FGFBP1 concentration of young healthy controls (aged 25 years or younger, Figure 10).

[0159] Example 3: Diagnostic performance of biomarker FGFBP1 in women with PCOS (phenotypes A, B, C, and D) and controls determined by ELISA technology in various age groups Performance verification was carried out in an additional sample group of 240 samples (serum samples from women with PCOS) and 48 controls (serum samples from healthy women).

[0160] The concentration of the analyte was determined by ELISA (enzyme - linked immunosorbent assay). The case group consisted of patients diagnosed with PCOS (155 cases of phenotype A, 5 cases of phenotype B, 8 cases of phenotype C, and 72 cases of phenotype D) according to the Rotterdam criteria and belonged to three different age groups: (15 years or older and less than 20 years old (n = 70), 20 years or older and less than 25 years old (n = 99), 25 years or older and less than 40 years old (n = 71)). The control group included healthy women without PCOS.

[0161] The concentration of FGFBP1 in human serum was determined as described in Example 2 using a Human FGFBP1 ELISA kit (Catalog number: RAB1460) from Sigma - Aldrich.

[0162] The receiver operating characteristic (ROC) curve was created (Figure 11). The model performance was determined by examining the area under the curve (AUC). ROC curve analysis of FGFBP1 for PCOS cases (phenotypes A-D) when all phenotypes were combined showed an AUC of 0.9 (95% CI 0.83-0.96), confirming the high diagnostic accuracy of FGFBP1 for PCOS (Figure 11).

[0163] Table 6, which describes the AUC of the ROC curve analysis and the associated 95% confidence intervals, shows the diagnostic performance of FGFBP1 for differentiating women with PCOS (cases, PCOS phenotypes A-D) from healthy controls using ROC analysis. The results were obtained using an ELISA immunoassay.

Table 6

[0164] 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 FGFBP1 concentration (pg / mL) increases in women with PCOS when compared to healthy controls (Figure 12).

[0165] Table 7 shows the diagnostic performance of FGFBP1 for differentiating women with PCOS separated by various phenotypes A, B, C, and D from healthy controls. The results were obtained using an ELISA immunoassay. The AUC for each phenotype is reported in the table.

Table 7

[0166] The ROC curve analysis of FGFBP1 for PCOS cases separated by various phenotypes against healthy controls showed AUCs of 0.90 (95% CI 0.84 - 0.97), 0.90 (95% CI 0.81 - 0.99), 0.82 (95% CI 0.69 - 0.96), and 0.90 (95% CI 0.83 - 0.97) for phenotypes A - D, respectively (Figure 13). The results confirm the high diagnostic accuracy of FGFBP1 for PCOS.

[0167] Serum FGFBP1 concentrations (pg / mL) in all various PCOS phenotypes (phenotypes A - D) showed increased levels compared to healthy controls (Figure 14, results obtained using ELISA immunoassay).

[0168] Table 8 shows the diagnostic performance of FGFBP1 in various age groups (15 ≤ age < 20, 20 ≤ age < 25, 25 ≤ age < 40) for differentiating women with PCOS from healthy controls when all phenotypes are combined (phenotypes A - D). Results were obtained using ELISA assay.

Table 8

[0169] The ROC curve analysis of FGFBP1 for PCOS cases separated by various age groups against healthy controls showed AUCs of 0.90 (95% CI 0.84 - 0.97), 0.88 (95% CI 0.81 - 0.95), and 0.91 (95% CI 0.84 - 0.98) for the age groups of 15 years or older and less than 20 years, 20 years or older and less than 25 years, and 25 years or older and less than 40 years, respectively (Figure 15). The results confirm the high diagnostic accuracy of FGFBP1 for PCOS in all different age groups.

[0170] An increase in serum FGFBP1 concentration (pg / mL) in women with PCOS compared to controls was confirmed in all different age groups (Figure 16).

[0171] Given the lack of reliable biomarkers for diagnosing PCOS, especially in young women (under 25 years old), a separate analysis was conducted for the age group of 15 to less than 25 years old.

[0172] Table 9 shows the diagnostic performance of FGFBP1 in young women (15 ≤ age < 25) for differentiating young women with PCOS from young healthy controls when all phenotypes are combined (Phenotypes A - D). Results were obtained using an ELISA assay.

Table 9

[0173] ROC curve analysis of FGFBP1 for young PCOS cases (15 ≤ age < 25) when all phenotypes (Phenotypes A - D) are combined showed an AUC of 0.88, confirming high diagnostic accuracy for women aged 15 to less than 25 years in differentiating PCOS cases from controls (95% CI 0.76 - 0.99, Figure 17). When only young women were included in the analysis (15 ≤ age < 25), PCOS cases (all phenotypes A - D combined) showed an increase in serum FGFBP1 concentration (pg / mL) compared to young controls (15 ≤ age < 25, Figure 18).

[0174] The diagnostic ability of FGFBP1 to distinguish young women with PCOS (15 ≤ age < 25) separated by various phenotypes A, B, C, and D from young controls (15 ≤ age < 25) was evaluated, and the results are reported in Table 10 (AUC for PCOS phenotypes vs controls). Results were obtained using an ELISA immunoassay.

Table 10

[0175] ROC curve analysis for FGFBP1 in young PCOS cases (15 ≤ age < 25, phenotypes A - D) showed AUC values of 0.88 (95% CI 0.77 - 0.99), 0.87 (95% CI 0.72 - 1), 0.80 (95% CI 0.63 - 0.97), and 0.87 (95% CI 0.75 - 0.99) for each phenotype, respectively, confirming the high diagnostic accuracy of FGFBP1 for PCOS in the subgroup of women aged 15 years or older and less than 25 years (Figure 19). When compared with the FGFBP1 concentration in young healthy controls, the FGFBP1 concentration increased in all different PCOS phenotypes (phenotypes A - D, 15 ≤ age < 25) (15 ≤ age < 25, Figure 20).

Claims

Claim 1 A method for assessing whether a subject has polycystic ovary syndrome (PCOS) or is at risk of developing PCOS, comprising: (a) determining the amount or concentration of FGFBP1 in a sample from the subject; and (b) comparing the determined amount or concentration to a reference. A method comprising the above steps. Claim 2 A method for selecting a patient for the treatment of PCOS, comprising: (a) determining the amount or concentration of FGFBP1 in a sample from the subject; and (b) comparing the determined amount or concentration to a reference. A method comprising the above steps. Claim 3 A method for monitoring the progression of PCOS in a subject having PCOS or for monitoring the response to treatment in a subject having PCOS, comprising: (a) determining the level of FGFBP1 in a first sample from the subject; (b) determining the level of FGFBP1 in a second sample from the subject obtained after the first sample; (c) comparing the level of FGFBP1 in the first sample to the level of FGFBP1 in the second sample; and (d) monitoring the progression of PCOS in the subject having PCOS or the response to treatment in the subject having PCOS based on the result of step (c). A method comprising the above steps. Claim 4 The method according to any one of claims 1 to 3, wherein an increase in the amount or concentration of FGFBP1 in the sample from the subject indicates the presence of PCOS in the subject. Claim 5 The method according to any one of claims 1 to 4, wherein the sample is a blood, serum, or plasma sample. Claim 6 The method according to any one of claims 1 to 5, wherein PCOS is selected from the group consisting of phenotype A PCOS, phenotype B PCOS, phenotype C PCOS, and phenotype D PCOS according to the Rotterdam scale. Claim 7 The method according to any one of claims 1 to 6, wherein phenotype A PCOS is detected. Claim 8 The method according to any one of claims 1 to 6, wherein phenotype B PCOS is detected. Claim 9 The method according to any one of claims 1 to 6, wherein phenotype C PCOS is detected. Claim 10 The method according to any one of claims 1 to 6, wherein phenotype D PCOS is detected. Claim 11 The method according to any one of claims 1 to 10, wherein PCOS is detected in adolescent or young adult females. Claim 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 FGFBP1; (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, hyperandrogenicity, and polycystic ovarian morphology; (d) comparing the values for the amounts or concentrations in steps (a) to (b) with the reference for the biomarker and the values 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 values; (e) evaluating the subject based on the comparison and / or the calculation performed in step (d). A computer-implemented method.

14. The computer-implemented method according to claim 13, wherein the amount or concentration of FGFBP1 is increased compared to a standard reference.

Images