Fibroblast growth factor binding protein 1 (FGFBP1) as (blood) biomarker for diagnosis of polycystic ovarian syndrome

Abstract

The present invention relates to a method for assessing whether a subject has or is at risk of developing polycystic ovarian syndrome (PCOS) by determining the amount or concentration of fibroblast growth factor binding protein 1 (FGFBP1) in a sample of the subject, a method for selecting a patient for treatment of PCOS, and a method for determining whether the subject has or is at risk of developing PCOS. Methods for monitoring PCOS progression or for monitoring response to treatment, and computer-implemented methods for assessing subjects with suspected PCOS.

Description

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

[0002] Polycystic ovary syndrome (PCOS) is a heterogeneous gynecological condition, which is defined by a combination of signs of androgen excess and ovarian dysfunction. Patients with PCOS may have a series of clinical manifestations, which can be reproductive and / or metabolic. Reproductive manifestations include irregular menstrual cycles, infertility, pregnancy complications and hirsutism, while metabolic manifestations include obesity, insulin resistance, metabolic syndrome, prediabetes, type 2 diabetes, non-alcoholic fatty liver disease (NAFLD) and cardiovascular factors. These clinical manifestations are also associated with psychological disorders such as anxiety and depression (Escobar-Morreale, HF2018; International evidence-based guideline for the assessment and management of polycystic ovary syndrome 2018).

[0003] Symptoms are not specific to PCOS, and patients are usually diagnosed only after a long period of evaluation for infertility. For the final diagnosis of PCOS, other conditions or diseases (such as pregnancy, atypical adrenal hyperplasia (NCAH), congenital adrenal hyperplasia, androgen-secreting tumors, Cushing's syndrome, thyroid disease or hyperprolactinemia (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) should be excluded. Diagnostic tests that can be used to exclude other diseases, such as:

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

[0006] Prolactin to exclude hyperprolactinemia

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

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

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

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

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

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

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

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

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

[0016] In order to assess biochemical hyperandrogenism, the androgen measured must exist in a clear normal range. Testosterone is the highest measured androgen in abundance, no matter in its total amount, in bound form or in free form. The practice of measuring free testosterone has its limitations. Using radioimmunoassay to directly measure free testosterone is very inaccurate and can not reflect the true value. These assays have higher intra- and inter-assay variability. Alternatively, by using extraction and chromatography or gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS) to measure total testosterone concentration, higher accuracy will be obtained, particularly for clinical research. The diagnostic performance of measuring serum testosterone can be enhanced by measuring SHBG at the same time, so that the calculation of free T concentration from total testosterone and SHBG levels only requires solving a quadratic equation (Azziz R, Carmina E, Dewailly D, et al., Task Force on the Phenotype of the Polycystic Ovary Syndrome of The Androgen Excess and PCOS Society. The Androgen Excess and PCOS Society criteria for the polycystic ovary syndrome: the complete task force report. Fertil Steril. 2009; 91 (2): 456-88). The definition of HA may vary by race. The mFG score of >8 for diagnosing hirsutism in women with PCOS may not be suitable for diagnosis in all races. East Asian women have a lower prevalence of hirsutism than Caucasians, and a score of >5 has been proposed to define hirsutism in Chinese women. There are also indications that androgen levels in the blood differ between ethnic groups, with the Japanese population having a lower prevalence of elevated androgens, and testosterone is only recommended as a supplementary factor for diagnosing PCOS in this population (Huang Z, Yong EL. Ethnic differences: Is there an Asian phenotype for polycystic ovarian syndrome? Best Pract Res Clin Obstet Gynaecol. 2016; 37: 46-55; Kubota T. Update in polycystic ovary syndrome: new criteria of diagnosis and treatment in Japan. Reprod Med Biol. 2013; 12(3): 71-77).

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

[0018] Currently, no specific PCOS medications are available. Treatment is symptom-directed and tailored to individual needs. Treatment targets hyperandrogenism, irregular cycles, and / or ovulatory dysfunction, and associated metabolic disorders such as diabetes. The International evidence-based guideline for the assessment and management of polycystic ovary syndrome of 2018 provides information to support clinical decision making and patient management.

[0019] Inconsistent diagnostic guidelines, variable provider knowledge, and lack of consensus pose particular challenges to the diagnosis and care of women with PCOS. These factors lead to inaccurate diagnoses, resulting in both under- and overdiagnosis. This adverse diagnostic experience worsens the condition of affected women and can limit timely opportunities to intervene to minimize associated comorbidities, especially during the transition from pediatric to adult care (Witchel SF, Teede HJ, AS. Curtailing PCOS. Pediatr Res. 2020; 87(2): 353-361). Furthermore, timely diagnosis is crucial to prevent further metabolic complications (such as, for example, type 2 diabetes) in affected women.

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

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

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

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

[0024] In a first aspect, the present invention relates to a method for assessing whether a subject has PCOS or is at risk of developing PCOS, the method comprising;

[0025] (a) determining the amount or concentration of FGFBP1 in a sample from a subject, and

[0026] (b) comparing the determined amount or concentration with a reference.

[0027] In a second aspect, the invention relates to a method of selecting a patient for therapy for PCOS, the method comprising:

[0028] (c) determining the amount or concentration of FGFBP1 in a sample from the subject, and

[0029] (d) comparing the determined amount or concentration with a reference.

[0030] In a third aspect, the present invention relates to a method for monitoring the progression of PCOS in a subject or for monitoring the response of a subject with PCOS to treatment, the method comprising:

[0031] (e) determining the level of FGFBP1 in a first sample from the subject,

[0032] (f) determining the level of FGFBP1 in a second sample of the subject that has been obtained subsequent to the first sample, and

[0033] (g) comparing the level of FGFBP1 in the first sample to the level of FGFBP1 in the second sample, and

[0034] (h) monitoring the progress of the subject suffering from or being treated for PCOS based on the results of step c).

[0035] In a fourth aspect, the present invention relates to a computer-implemented method for assessing a subject with suspected PCOS, the computer-implemented method comprising the steps of:

[0036] (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,

[0037] (b) optionally, receiving a value for the amount or concentration of a second biomarker in a sample from the subject,

[0038] (c) optionally, receiving a value for the presence or absence of at least one additional diagnostic criterion selected from the group consisting of oligo-anovulation, hyperandrogenism, and polycystic ovarian morphology;

[0039] (d) comparing the value for the amount or concentration of steps (a) to (b) with a reference for the biomarker and a value for the presence or absence of at least one additional diagnostic criterion and / or calculating a score for assessing a subject with suspected PCOS based on the amount or concentration of the biomarker and the value; and

[0040] (e) assessing the subject based on the comparison and / or calculation performed in step (d). BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 .ROC curve analysis of fibroblast growth factor binding protein 1. Results obtained using Olink proximity extension technology

[0042] Figure 2 Serum fibroblast growth factor binding protein 1 in PCOS cases (phenotype A only) and controls. Results were obtained using the Olink proximity extension technique.

[0043] Figure 3 . ROC curve analysis of fibroblast growth factor binding protein 1 for PCOS cases when all phenotypes (phenotypes A to D) were combined. Results were obtained by ELISA immunoassay.

[0044] Figure 4. Serum fibroblast growth factor binding protein 1 concentrations (pg / mL) in controls and PCOS cases when all phenotypes (phenotypes A to D) were combined. Results were obtained using an ELISA immunoassay.

[0045] Figure 5 . ROC curves for fibroblast growth factor binding protein 1 in PCOS phenotypes A to D when compared to controls. Results were obtained by ELISA immunoassay.

[0046] Figure 6 Serum fibroblast growth factor binding protein 1 (pg / mL) in healthy controls and in PCOS phenotypes A to D. Results were obtained using an ELISA immunoassay.

[0047] Figure 7 ROC curve analysis of fibroblast growth factor binding protein 1 for young PCOS cases (age ≤ 25 years) when all phenotypes A to D were combined. Results were obtained using ELISA immunoassay.

[0048] Figure 8 Serum fibroblast growth factor binding protein 1 (pg / mL) in young controls and young PCOS cases (age ≤ 25 years) for combined phenotypes A to D. Results were obtained using an ELISA immunoassay.

[0049] Fig. 9 ROC curve for fibroblast growth factor binding protein 1 in young PCOS cases (age ≤ 25 years) versus young healthy controls (age ≤ 25 years) separated by phenotypes A to D. Results were obtained using an ELISA immunoassay.

[0050] Fig.10 Serum fibroblast growth factor binding protein 1 concentrations (pg / mL) in young healthy controls (age ≤ 25 years) and in PCOS phenotypes A to D (age ≤ 25 years). Results were obtained by ELISA immunoassay.

[0051] Fig.11 . ROC curve analysis of fibroblast growth factor binding protein 1 for PCOS cases when all phenotypes (phenotypes A to D) were combined. Results were obtained by ELISA immunoassay.

[0052] Fig.12 . Serum fibroblast growth factor binding protein 1 concentrations (pg / mL) in controls and PCOS cases when all phenotypes (phenotypes A to D) were combined. Results were obtained using an ELISA immunoassay.

[0053] Fig.13. ROC curves for fibroblast growth factor binding protein 1 in PCOS phenotypes A to D when compared to controls. Results were obtained by ELISA immunoassay.

[0054] Fig.14 Serum fibroblast growth factor binding protein 1 (pg / mL) in healthy controls and in PCOS phenotypes A to D. Results were obtained using an ELISA immunoassay.

[0055] Fig.15 . ROC curves for fibroblast growth factor binding protein 1 in different PCOS age groups (15 to <20, 20 to <25, 25 to <40) when compared to controls. Results were obtained by ELISA immunoassay.

[0056] Fig.16 . Serum fibroblast growth factor binding protein 1 (pg / mL) in healthy controls and in PCOS age groups (15 to <20, 20 to <25, 25 to <40). Results were obtained using an ELISA immunoassay.

[0057] Fig.17 ROC curve analysis of fibroblast growth factor binding protein 1 for young PCOS cases (age 15 to <25 years) when all phenotypes A to D were combined. Results were obtained using ELISA immunoassay.

[0058] Fig.18 Serum fibroblast growth factor binding protein 1 (pg / mL) in young controls and young PCOS cases (age 15 to <25 years) combined phenotypes A to D. Results were obtained using an ELISA immunoassay.

[0059] Fig.19 ROC curve for fibroblast growth factor binding protein 1 in young PCOS cases (age 15 to <25 years) versus young healthy controls (age 15 to <25 years) separated by phenotypes A to D. Results were obtained using an ELISA immunoassay.

[0060] Fig. 20 Serum fibroblast growth factor binding protein 1 concentrations (pg / mL) in young healthy controls (age 15 to <25 years) and in PCOS phenotypes A to D (age 15 to <25 years). Results were obtained by ELISA immunoassay. DETAILED DESCRIPTION

[0061] The inventors of the present invention have identified fibroblast growth factor binding protein 1 (FGFBP1) as a reliable biomarker to diagnose PCOS in a subject, determine whether a subject is at risk for developing PCOS, determine the response of a subject with PCOS to therapy, or monitor the progression of PCOS in a subject with PCOS, or monitor the response of a subject with PCOS to therapy. FGFBP1 can be used alone or in combination with at least one additional criterion (such as hyperandrogenemia, oligo-anovulation, PCOM, or irregular cycles) to diagnose, risk assess, and / or monitor a patient's response to therapy. Further, determining the level of FGFBP1 compared to a control level can be used to monitor the subject's response to treatment and / or monitor the progression of PCOS in a subject.

[0062] We show for the first time that the FGFBP1 value measured in a sample (preferably a biological fluid sample, which is preferably blood, plasma, serum, capillary blood, interstitial fluid, peritoneal fluid or menstrual fluid, and wherein the biological fluid sample is more preferably blood, plasma or serum) is increased in women suffering from PCOS compared to a control group. In addition, we show for the first time that the FGFBP1 value measured in a sample (preferably a biological fluid sample, which is preferably blood, plasma, serum, capillary blood, interstitial fluid, peritoneal fluid or menstrual fluid, and wherein the biological fluid sample is more preferably blood, plasma or serum) is increased in women suffering from any phenotype A to D of PCOS. The solution provided by the present invention is an immunoassay for detecting fibroblast growth factor binding protein 1 in a sample, which is preferably a biological fluid sample, which is preferably blood, plasma, serum, capillary blood, interstitial fluid, peritoneal fluid or menstrual fluid, and wherein the biological fluid sample is more preferably blood, plasma or serum. This immunoassay can also be used in combination with other clinical and / or biochemical features (such as oligo-anovulation and / or irregular cycles, hyperandrogenemia or PCOM) to diagnose women with PCOS. Further, measuring FGFBP1 values ​​can be used to monitor the progression of PCOS in the patient and monitor the response to therapy. We also show that measuring FGFBP1 values ​​in a sample (preferably a biological fluid sample, which is preferably blood, plasma, serum, capillary blood, interstitial fluid, peritoneal fluid or menstrual fluid, and wherein the biological fluid sample is more preferably blood, plasma or serum) alone or in combination with additional diagnostic criteria as described above is particularly suitable for the diagnosis of PCOS in adolescents or young women under the age of 25, particularly under the age of 20, particularly under the age of 15 to 25, particularly under the age of 15 to 20.

[0063] There is an unmet medical need for an accurate test for reliable diagnosis of PCOS. Measuring FGFBP1 in a sample (preferably a biological fluid sample, which is preferably blood, plasma, serum, capillary blood, interstitial fluid, peritoneal fluid or menstrual fluid, and wherein the biological fluid sample is more preferably blood, plasma or serum) has the following advantages: a reliable biological fluid-based test can identify women suffering from PCOS, which is currently not possible. Measuring FGFBP1 in a sample (preferably a biological fluid sample, which is preferably blood, plasma, serum, capillary blood, interstitial fluid, peritoneal fluid or menstrual fluid, and wherein the biological fluid sample is more preferably blood, plasma or serum) can also be reliably used for the diagnosis of PCOS in adolescent subjects or young women under the age of 25, particularly under the age of 20, particularly under the age of 15 to 25, particularly under the age of 15 to 20. Due to the reasons described above, the diagnosis of PCOS in adolescent patients is difficult, and therefore, we provide an accurate test for the diagnosis of PCOS in adolescent and young female populations for the first time. In addition, measuring FGFBP1 in a sample (preferably a biological fluid sample, which is preferably blood, plasma, serum, capillary blood, interstitial fluid, peritoneal fluid or menstrual fluid, and wherein the biological fluid sample is more preferably blood, plasma or serum) has the advantage of identifying whether the patient is responsive to therapy. An additional advantage of measuring FGFBP1 in a patient's sample (preferably a biological fluid sample, which is preferably blood, plasma, serum, capillary blood, interstitial fluid, peritoneal fluid or menstrual fluid, and wherein the biological fluid sample is more preferably blood, plasma or serum) is monitoring the progression of PCOS. Furthermore, we attach a computer-implemented method for assessing a subject suffering from PCOS by measuring the level of FGFBP1 in a sample, preferably a biological fluid sample, which is preferably blood, plasma, serum, capillary blood, interstitial fluid, peritoneal fluid or menstrual fluid, and wherein the biological fluid sample is more preferably blood, plasma or serum, and optionally by further criteria (such as values ​​for oligo-anovulation and / or irregular cycles, hyperandrogenemia and / or polycystic ovary morphology) or by further biomarkers or hormones (to assess the subject based on comparison and / or calculation of the data described above).

[0064] As described above, patients suffering from PCOS may show two types of characteristics-reproductive or metabolic. Metabolic PCOS includes obesity, insulin resistance, metabolic syndrome, prediabetes, type 2 diabetes, non-alcoholic fatty liver disease (NAFLD) and cardiovascular factors. The term "phenotype" can be used instead of "reproductive". The term "reproductive" (or "phenotype") describes any feature of the phenotype of females known to indicate PCOS. For example, these reproductive characteristics include polycystic ovarian morphology (PCOM) and / or clinical hyperandrogenism, such as acne, seborrhea, alopecia and / or hirsutism. Preferably, these reproductive characteristics include polycystic ovarian morphology (PCOM) and / or clinical hyperandrogenism, more preferably acne, seborrhea, alopecia, deep voice and / or hirsutism. These reproductive characteristics of clinical hyperandrogenism can be simply diagnosed by asking females, or it is obvious after a brief physical examination of the female body. Usually, the reference population does not show any or no more than one of these phenotypic characteristics known to indicate PCOS.

[0065] Fibroblast growth factor binding protein 1 (FGFBP1, also known as FGF-BP, FGFBP1, FGFBP, FGFBP-1 or HBP17) is a 26.2 kDa protein that belongs to the fibroblast growth factor binding protein family. FGFBP1 binds to FGF1, FGF2, FGF7, FGF10, FGF22 and HSPG2 (perlecan) in a reversible non-covalent manner (Wu DQ, Kan MK, Sato GH, et al., Characterization and molecular cloning of a putative binding protein for heparin-binding growth factors. J Biol Chem. 1991 Sep 5; 266(25): 16778-85; Beer HD, Bittner M, Niklaus G, et al., The fibroblast growth factor binding protein is a novel interaction partner of FGF-7, FGF-10 and FGF-22 and regulates FGF activity: implications for epithelial repair. Oncogene. 2005 Aug 11; 24(34): 5269-77; Abuharbeid S, Czubayko F, ​​Aigner A. The fibroblast growth factor-binding protein FGF-BP. Int J Biochem Cell Biol. 2006; 38(9): 1463-8). Many members of the FGF family are fixed in the extracellular matrix (ECM), bound to heparin sulfate proteoglycans (HSPGs), and released from this storage site by proteases and heparanase. FGFBP transports FGF from its storage site to its receptor (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 heparanoids (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 leads to a significant decrease in the affinity of FGF2 for heparin and, therefore, results in FGFBP1-mediated release of FGF2 from the ECM (Aigner A, Butscheid M, Kunkel P, et al., An FGF-binding protein (FGF-BP) exercises its biological function by parallel paracrine stimulation of tumor cell and endothelial cell proliferation through FGF-2 release. Int J Cancer 2001, 92: 510–517). Several findings from different laboratories indicate that FGFBP1 can contribute to embryonic development, angiogenesis, wound healing, tumor growth and malignant progression, and contribute to the maintenance and reinnervation of neuromuscular junctions and the development of the blood-brain barrier (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 switchmolecule, secreted FGFbinding protein, an indicator of early stages of pancreatic and colorectal adenocarcinoma. Semin Oncol 2006,33:S50–56; WilliamsAH, Valdez G, Moresi V, et al., MicroRNA-206delays ALS progression and promotesregeneration of neuromuscular synapses in mice. Science 2009,326:1549–1554; Tassi E, McDonnell K, Gibby KA, et al. Impact of fibroblast growth factor-bindingprotein-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 eyes (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 skincarcinogenesis. Oncogene 1997, 14: 2671–2681). Genetic polymorphisms of the human FGFBP1 gene are associated with higher gene and protein expression in human kidneys 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). It has been shown that modulation of FGF signaling by FGFBP1 can adjust vascular sensitivity to endogenous angiotensin II and thereby 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 January; 71(1): 160-167). FGF2 signaling has been shown to be important for maintaining the cellular plasticity of epithelial ovarian cancer that already exists in the initial steps 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 October 21; 23(49): 8171-83). Higher levels of serum FGF2 have been found 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, et al., From gene profiling to diagnostic markers: IL-18 and FGF-2 complement CA125 as serum-based markers in epithelial ovarian cancer. Int J Cancer 118:1750-1758,2006; Madsen CV, Steffensen KD, Olsen DA, et al., Serum platelet-derived growth factor and fibroblast growth factor in patients with benign and malignant ovarian tumors. Anticancer Res. 2012 September; 32(9):3817-25). In rats, FGF2 treatment of ovarian cultures inhibited primordial follicle assembly. In addition, many of the differentially expressed genes identified in the treated ovaries were associated with genes previously known to be associated with PCOS, suggesting that FGF2-dependent abnormal follicle assembly may be a component of PCOS in later life (Nilsson E, Zhang B, Skinner MK. Genebionetworks that regulate ovarian primordial follicle assembly. BMC Genomics. 2013 Jul 23; 14: 496). In cows, FGF2 mRNA expression is high in preovulatory follicles and in the early luteal phase, then the expression decreases in the late luteal phase and remains low during 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 April; 45(2): 124-30). Intraperitoneal injection of FGF2 has protective and ameliorative effects in a mouse model of PCOS induced by estradiol valerate (Moayeri A, Rostamzadeh A, Raoofi A, et al., Retinoic acid and fibroblast growth factor-2play a key role onmodulation of sex hormones and apoptosis in a mouse model of polycystic ovarysyndrome induced by estradiol valerate. Taiwan J Obstet Gynecol. 2020 November; 59(6): 882-890). Controversial results have been published on serum FGF2 levels in PCOS patients. Artini and colleagues reported that serum FGF2 levels did not differ between untreated PCOS patients and controls, but that FGF2 levels increased in PCOS patients after FSH stimulation (Artini PG, Monti M, Matteucci C, et al., Vascular endothelialgrowth factor and basic fibroblast growth factor in polycystic ovary syndrome during controlled ovarian hyperstimulation. Gynecol Endocrinol 2006 Aug; 22(8): 465-70). In contrast, Patil and colleagues reported that serum FGF2 levels were lower in PCOS women undergoing controlled ovarian hyperstimulation for IVF compared with age- and BMI-matched controls (Patil K, Hinduja I, Mukherjee S. Alteration in angiogenic potential of granulosa-lutein cells and follicularfluid contributes to luteal defects in polycystic ovary syndrome. Hum Reprod.2021 Mar 18;36(4):1052-1064). Although the role of FGF2 signaling in PCOS is still unclear, its metabolic functions have been studied in recent years. FGF2, depending on concentration, can act as a positive or negative factor for adipogenesis in vitro (Kim S, Ahn C, Bong N, et al., Biphasic effects of FGF2 on adipogenesis. PLoS One. 2015 Mar 19;10(3):e0120073. doi:10.1371 / journal.pone.0120073). Mathes and colleagues show that FGF2-dependent signaling enhances the differentiation of fiber / adipose-derived progenitor cells and promotes 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 USA September 14, 2021; 118 (37): e2021013118. doi: 10.1073 / pnas.2021013118). The appearance of adipose tissue between skeletal muscle fibers is a unique feature of aging, obesity and type 2 diabetes, indicating association with insulin resistance. In addition, FGF2 has been shown to be a negative regulator of thermogenesis in both brown fat and beige fat. The destruction of FGF2 strongly enhances the thermogenesis of brown fat and beige fat, resulting in increased energy expenditure and improved lipid homeostasis. Furthermore, the absence of FGF2 protected mice from obesity and hepatic steatosis caused by high fat levels. (Li H, Zhang X, Huang C, et al., FGF2 disruption enhancesthermogenesis in brown and beige fat to protect against adiposity and hepatic steatosis. Mol Metab. 2021 Dec;54:101358. doi:10.1016 / j.molmet.2021). .

[0066] A first aspect of the present invention relates to a method for assessing whether a subject has PCOS or is at risk of developing PCOS, the method comprising;

[0067] (a) determining the amount or concentration of FGFBP1 in a sample from a subject, and

[0068] (b) comparing the determined amount or concentration with a reference.

[0069] The amount or concentration of FGFBP1 in the patient's sample indicates the presence of PCOS or the risk of developing 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 of PCOS in the patient or the risk of developing PCOS. In particular, FGFBP1 can be detected in a higher amount or concentration in a biological fluid sample of a patient assessed to have PCOS or to be at risk of developing PCOS than in the same biological fluid sample of an individual who does not suffer from PCOS or does not have the risk of developing PCOS. In particular, the amount or concentration of FGFBP1 increases by 50% or more, indicating the presence of PCOS or the risk of developing PCOS. In particular, the amount or concentration of FGFBP1 increases by 100% or more, indicating the presence of PCOS or the risk of developing PCOS. In particular, the amount or concentration of FGFBP1 increases by 150% or more, indicating the presence of PCOS or the risk of developing PCOS. In particular, an increase in the amount or concentration of FGFBP1 by 200% or more indicates the presence of PCOS or a risk of developing PCOS.

[0070] In an embodiment, 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 an embodiment, the sample is an in vitro sample, i.e. the sample is analyzed in vitro without transferring it back to the body.

[0071] In a specific embodiment, the patient is a laboratory animal, a domestic animal, or a primate. In a specific embodiment, the patient is a human patient. In a specific embodiment, the patient is a human female patient. In a specific embodiment, the patient is a female human patient less than 25 years old. In a specific embodiment, the patient is a female human patient less than 20 years old. In a specific embodiment, the patient is a female human patient from 15 years old to less than 25 years old. In a specific embodiment, the patient is a female human patient from 15 years old to less than 20 years old. In a specific embodiment, the patient is a female human patient less than 25 years old and three years after menarche. In a specific embodiment, the patient is a female human patient less than 20 years old and three years after menarche. In a specific embodiment, the patient is a female human patient from 15 years old to less than 25 years old and three years after menarche. In a specific embodiment, the patient is a female human patient from 15 years old to less than 20 years old and three years after menarche.

[0072] In embodiments, PCOS is assessed from the group consisting of metabolic or phenotypic PCOS. In further embodiments, PCOS is assessed from the group consisting of phenotype A, phenotype B, phenotype C, and phenotype D PCOS.

[0073] In an embodiment, the first method of the invention is an in vitro method.

[0074] In an embodiment, the amount or concentration of FGFBP1 is determined using an antibody, in particular a monoclonal antibody. In an embodiment, step a) of determining the amount or concentration of FGFBP1 in a sample of a patient comprises performing an immunoassay. In an embodiment, the immunoassay is performed in a direct or indirect format. In an embodiment, the immunoassay is selected from the group consisting of an enzyme-linked immunosorbent assay (ELISA), an enzyme immunoassay (EIA), a radioimmunoassay (RIA), or an immunoassay based on luminescence, fluorescence, chemiluminescence, or electrochemiluminescence detection.

[0075] In certain embodiments, step a) of determining the amount or concentration of FGFBP1 in a sample from a patient comprises the following steps:

[0076] i) incubating the patient's sample with one or more antibodies that specifically bind to FGFBP1, thereby generating a complex between the antibody and FGFBP1, and

[0077] ii) quantifying the complex formed in step i), thereby quantifying the amount or concentration of FGFBP1 in the patient's sample.

[0078] In a specific embodiment, in step i), the sample is incubated with two antibodies that specifically bind to FGFBP1. As will be apparent to the skilled person, the sample may be contacted with the first and second antibodies in any desired order (i.e. first contact with the first antibody and then the second antibody; or first contact with the second antibody and then the first antibody; or simultaneously contact with the first and second antibodies) for a sufficient time and under conditions to form a first anti-FGFBP1 antibody / FGFBP1 / second anti-FGFBP1 antibody complex. As will be readily appreciated by the skilled person, this is merely a matter of routine experimentation to establish suitable or sufficient times and conditions for the formation of a complex between a specific anti-FGFBP1 antibody and an FGFBP1 antigen / analyte (=anti-FGFBP1 complex), or a secondary complex or sandwich complex comprising the first antibody to FGFBP1, FGFBP1 (analyte) and the second anti-FGFBP1 antibody (=first anti-FGFBP1 antibody / FGFBP1 / second anti-FGFBP1 antibody complex).

[0079] The anti-FGFBP1 antibody / FGFBP1 complex may be detected by any suitable means. The first anti-FGFBP1 antibody / FGFBP1 / second anti-FGFBP1 antibody complex may be detected by any suitable means. Those skilled in the art are fully familiar with such means / methods.

[0080] In certain embodiments, a sandwich will be formed comprising a first antibody to FGFBP1, FGFBP1 (analyte), and a second antibody to FGFBP1, wherein the second antibody is detectably labeled.

[0081] In one embodiment, a sandwich will be formed comprising a first antibody to FGFBP1, FGFBP1 (analyte), and a second antibody to FGFBP1, wherein the second antibody is detectably labeled and wherein the first anti-FGFBP1 antibody can be or is bound to a solid phase.

[0082] In embodiments, the second antibody is detectably labeled, directly or indirectly. In specific embodiments, the second antibody is detectably labeled with a luminescent dye, particularly a chemiluminescent dye or an electrochemiluminescent dye.

[0083] In a second aspect, the invention relates to a method of selecting a patient for therapy for PCOS, the method comprising:

[0084] (c) determining the amount or concentration of FGFBP1 in a sample from the subject, and

[0085] (d) comparing the determined amount or concentration with a reference.

[0086] In an embodiment, if the amount of FGFBP1 in the patient's sample is determined to be elevated, the patient is selected for a PCOS therapy. In particular, if the amount of FGFBP1 is higher than the amount of FGFBP1 in a reference or reference sample, the patient is selected for a PCOS therapy. In particular, if the amount of FGFBP1 in a biological fluid sample of a patient assessed for a PCOS therapy is higher than the amount in the same biological fluid sample of an individual who does not suffer from PCOS or is at risk of developing PCOS or is not selected for a PCOS therapy, the patient is selected for a PCOS therapy. In particular, if the amount of FGFBP1 is elevated by 50% or more, the patient is selected for a PCOS therapy. In particular, if the amount of FGFBP1 is elevated by 100% or more, the patient is selected for a PCOS therapy. In particular, if the amount of FGFBP1 is elevated by 150% or more, the patient is selected for a PCOS therapy. In particular, if the amount of FGFBP1 is elevated by 200% or more, the patient is selected for a PCOS therapy.

[0087] In particular, the patient is selected to undergo a drug-based therapy for PCOS or to undergo lifestyle changes to control metabolic symptoms. In an embodiment, the drug-based therapy for PCOS is selected from the group consisting of: drugs for regulating menstrual periods, in particular oral contraceptives or progestin therapy; drugs for preventing or controlling diabetes, in particular type 2 diabetes; drugs for preventing or controlling high cholesterol; hormones or drugs to increase fertility; drugs, hormones or procedures to remove excess hair; drugs or procedures to control acne.

[0088] In an embodiment, 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 an embodiment, the sample is an in vitro sample, i.e. the sample is analyzed in vitro without transferring it back to the body.

[0089] In a specific embodiment, the patient is a laboratory animal, a domestic animal, or a primate. In a specific embodiment, the patient is a human patient. In a specific embodiment, the patient is a human female patient. In a specific embodiment, the patient is a female human patient less than 25 years old. In a specific embodiment, the patient is a female human patient less than 20 years old. In a specific embodiment, the patient is a female human patient from 15 years old to less than 25 years old. In a specific embodiment, the patient is a female human patient from 15 years old to less than 20 years old. In a specific embodiment, the patient is a female human patient less than 25 years old and three years after menarche. In a specific embodiment, the patient is a female human patient less than 20 years old and three years after menarche. In a specific embodiment, the patient is a female human patient from 15 years old to less than 25 years old and three years after menarche. In a specific embodiment, the patient is a female human patient from 15 years old to less than 20 years old and three years after menarche.

[0090] In an embodiment, the second method of the invention is an in vitro method.

[0091] In an embodiment, the amount of FGFBP1 is determined using an antibody, in particular a monoclonal antibody. In an embodiment, step a) of determining the amount of FGFBP1 in a patient sample comprises performing an immunoassay. In an embodiment, the immunoassay is performed in a direct or indirect format. In an embodiment, the immunoassay is selected from the group consisting of an enzyme-linked immunosorbent assay (ELISA), an enzyme immunoassay (EIA), a radioimmunoassay (RIA), or an immunoassay based on luminescence, fluorescence, chemiluminescence or electrochemiluminescence detection.

[0092] In a specific embodiment, step a) of determining the amount of FGFBP1 in a sample from a patient comprises the following steps:

[0093] i) incubating the patient's sample with one or more antibodies that specifically bind to FGFBP1, thereby generating a complex between the antibody and FGFBP1, and

[0094] ii) quantifying the complex formed in step i), thereby quantifying the amount of FGFBP1 in the patient's sample.

[0095] In a specific embodiment, in step i), the sample is incubated with two antibodies that specifically bind to FGFBP1. As will be apparent to a person skilled in the art, the sample may be contacted with the first and second antibodies in any desired order (i.e. first contact with the first antibody and then the second antibody; or first contact with the second antibody and then the first antibody; or simultaneously contact with the first and second antibodies) for a sufficient time and under conditions to form a first anti-FGFBP1 antibody / FGFBP1 / second anti-FGFBP1 antibody complex. As will be readily appreciated by a person skilled in the art, this is merely a matter of routine experimentation to establish suitable or sufficient times and conditions for the formation of a complex between a specific anti-FGFBP1 antibody and an FGFBP1 antigen / analyte (=anti-FGFBP1 complex), or a secondary complex 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).

[0096] The anti-FGFBP1 antibody / FGFBP1 complex may be detected by any suitable means. The first anti-FGFBP1 antibody / FGFBP1 / second anti-FGFBP1 antibody complex may be detected by any suitable means. Those skilled in the art are fully familiar with such means / methods.

[0097] In certain embodiments, a sandwich will be formed comprising a first antibody to FGFBP1, FGFBP1 (analyte), and a second antibody to FGFBP1, wherein the second antibody is detectably labeled.

[0098] In one embodiment, a sandwich will be formed comprising a first antibody to FGFBP1, FGFBP1 (analyte), and a second antibody to FGFBP1, wherein the second antibody is detectably labeled and wherein the first anti-FGFBP1 antibody can be or is bound to a solid phase.

[0099] In embodiments, the second antibody is detectably labeled, directly or indirectly. In specific embodiments, the second antibody is detectably labeled with a luminescent dye, particularly a chemiluminescent dye or an electrochemiluminescent dye.

[0100] Further, the present invention also relates to a kit comprising reagents for diagnosing PCOS. The reagents of the kit may include antibodies or antibody fragments. Preferably, the antibody or antibody fragment recognizes an epitope or antigen of FGFBP1. The kit may further contain other reagents that recognize other biomarkers. Therefore, the kit may include a combination of at least two reagents. The kit may specifically measure the amount or concentration of FGFBP1 and any other biomarker of interest. According to the present invention, biomarkers may also include hormones, such as anti-Mullerian hormone AMH. The kit can be used for any diagnostic assay.

[0101] In an embodiment, the amount of FGFBP1 is determined using an antibody, in particular a monoclonal antibody. In an embodiment, step a) of determining the amount of FGFBP1 in a patient sample comprises performing an immunoassay. In an embodiment, the immunoassay is performed in a direct or indirect format. In an embodiment, such immunoassay is selected from the group consisting of: an enzyme-linked immunosorbent assay (ELISA), an enzyme immunoassay (EIA), a radioimmunoassay (RIA), or an immunoassay based on luminescence, fluorescence, chemiluminescence or electrochemiluminescence detection.

[0102] In a third aspect, the present invention relates to a method for monitoring the progression of PCOS in a subject or for monitoring the response of a subject with PCOS to treatment, the method comprising:

[0103] (a) determining the level of FGFBP1 in a first sample from a subject,

[0104] (b) determining the level of FGFBP1 in a second sample of the subject that has been obtained subsequent to the first sample, and

[0105] (c) comparing the level of FGFBP1 in the first sample to the level of FGFBP1 in the second sample, and

[0106] (d) monitoring the progress of the subject suffering from or being treated for PCOS based on the results of step c).

[0107] In embodiments, a subject with PCOS is monitored for PCOS progression to determine whether the amount or concentration of FGFBP1 in a sample of the patient changes over time. In particular, PCOS progression is monitored to determine whether the amount or concentration of FGFBP1 increases, decreases, or does not change over time. In embodiments, PCOS progression is monitored if an elevated amount or concentration of FGFBP1 in a sample of the subject is determined.

[0108] In embodiments, a subject being treated for PCOS is monitored to determine whether the amount or concentration of FGFBP1 in a sample of the subject changes. In particular, a subject being treated for PCOS is monitored to determine whether the amount or concentration of FGFBP1 increases, decreases, or does not change. In particular, a subject being treated for PCOS is monitored to determine whether the amount or concentration of FGFBP1 increases, decreases, or does not change as a result of the administered therapy. In embodiments, a decreased amount or concentration of FGFBP1 in a subject being treated for PCOS indicates that the therapy is effective. In embodiments, an unchanged or increased amount or concentration of FGFBP1 in a sample of a subject being treated for PCOS indicates persistent PCOS. In particular, if the amount or concentration of FGFBP1 increases to 50% or more, the treatment for PCOS is ineffective. In particular, if the amount or concentration of FGFBP1 increases to 100% or more, the treatment for PCOS is ineffective. Particularly, if the amount or concentration of FGFBP1 increases to 150% or more, the treatment for PCOS is ineffective. Particularly, if the amount or concentration of FGFBP1 increases to 200% or more, the treatment for PCOS is ineffective.

[0109] In certain embodiments, if an unchanged or increased amount or concentration of FGFBP1 is determined in a sample of a subject being treated for PCOS, therapy is adjusted.

[0110] In an embodiment, the subject is monitored several times at different time points. In an embodiment, the subject is monitored several times over a time frame of weeks, months, or years. In a specific embodiment, the subject is monitored monthly or annually. In an embodiment, the subject suffering from PCOS is monitored monthly or annually after the diagnosis of PCOS. In an embodiment, the subject being treated for PCOS is monitored once after therapy. In particular, the subject being treated for PCOS is monitored monthly or annually to determine the effect of treatment.

[0111] In an embodiment, the treatment of PCOS is selected from the group consisting of: drug-based treatment of PCOS and making lifestyle changes to control metabolic symptoms. In an embodiment, the drug-based treatment of PCOS is selected from the group consisting of: drugs for regulating menstrual periods, 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 to increase fertility; drugs, hormones or procedures to remove excess hair; drugs or procedures to control acne.

[0112] In an embodiment, 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 an embodiment, the sample is an in vitro sample, i.e. the sample is analyzed in vitro without transferring it back to the body.

[0113] In a specific embodiment, the patient is a laboratory animal, a domestic animal, or a primate. In a specific embodiment, the patient is a human patient. In a specific embodiment, the patient is a human female patient. In a specific embodiment, the patient is a female human patient less than 25 years old. In a specific embodiment, the patient is a female human patient less than 20 years old. In a specific embodiment, the patient is a female human patient from 15 years old to less than 25 years old. In a specific embodiment, the patient is a female human patient from 15 years old to less than 20 years old. In a specific embodiment, the patient is a female human patient less than 25 years old and three years after menarche. In a specific embodiment, the patient is a female human patient less than 20 years old and three years after menarche. In a specific embodiment, the patient is a female human patient from 15 years old to less than 25 years old and three years after menarche. In a specific embodiment, the patient is a female human patient from 15 years old to less than 20 years old and three years after menarche.

[0114] In an embodiment, the second method of the invention is an in vitro method.

[0115] In an embodiment, the amount or concentration of FGFBP1 is determined using an antibody, in particular a monoclonal antibody. In an embodiment, step a) of determining the amount or concentration of FGFBP1 in a sample of a patient comprises performing an immunoassay. In an embodiment, the immunoassay is performed in a direct or indirect format. In an embodiment, the immunoassay is selected from the group consisting of an enzyme-linked immunosorbent assay (ELISA), an enzyme immunoassay (EIA), a radioimmunoassay (RIA), or an immunoassay based on luminescence, fluorescence, chemiluminescence, or electrochemiluminescence detection.

[0116] In certain embodiments, step a) of determining the amount or concentration of FGFBP1 in a sample from a patient comprises the following steps:

[0117] i) incubating the patient's sample with one or more antibodies that specifically bind to FGFBP1, thereby generating a complex between the antibody and FGFBP1, and

[0118] ii) quantifying the complex formed in step i), thereby quantifying the amount or concentration of FGFBP1 in the patient's sample.

[0119] In a specific embodiment, in step i), the sample is incubated with two antibodies that specifically bind to FGFBP1. As will be apparent to the skilled person, the sample may be contacted with the first and second antibodies in any desired order (i.e. first contact with the first antibody and then the second antibody; or first contact with the second antibody and then the first antibody; or simultaneously contact with the first and second antibodies) for a sufficient time and under conditions to form a first anti-FGFBP1 antibody / FGFBP1 / second anti-FGFBP1 antibody complex. As will be readily appreciated by the skilled person, this is merely a matter of routine experimentation to establish suitable or sufficient times and conditions for the formation of a complex between a specific anti-FGFBP1 antibody and an FGFBP1 antigen / analyte (=anti-FGFBP1 complex), or a secondary complex or sandwich complex comprising the first antibody to FGFBP1, FGFBP1 (analyte) and the second anti-FGFBP1 antibody (=first anti-FGFBP1 antibody / FGFBP1 / second anti-FGFBP1 antibody complex).

[0120] The anti-FGFBP1 antibody / FGFBP1 complex may be detected by any suitable means. The first anti-FGFBP1 antibody / FGFBP1 / second anti-FGFBP1 antibody complex may be detected by any suitable means. Those skilled in the art are fully familiar with such means / methods.

[0121] In certain embodiments, a sandwich will be formed comprising a first antibody to FGFBP1, FGFBP1 (analyte), and a second antibody to FGFBP1, wherein the second antibody is detectably labeled.

[0122] In one embodiment, a sandwich will be formed comprising a first antibody to FGFBP1, FGFBP1 (analyte), and a second antibody to FGFBP1, wherein the second antibody is detectably labeled and wherein the first anti-FGFBP1 antibody can be or is bound to a solid phase.

[0123] In embodiments, the second antibody is detectably labeled, directly or indirectly. In specific embodiments, the second antibody is detectably labeled with a luminescent dye, particularly a chemiluminescent dye or an electrochemiluminescent dye.

[0124] In a fourth aspect, the present invention relates to a computer-implemented method for assessing a subject with suspected PCOS, the computer-implemented method comprising the steps of:

[0125] (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,

[0126] (b) optionally, receiving a value for the amount or concentration of a second biomarker in a sample from the subject,

[0127] (c) optionally, receiving a value for the presence or absence of at least one additional diagnostic criterion selected from the group consisting of oligo-anovulation, hyperandrogenism, and polycystic ovarian morphology;

[0128] (d) comparing the value for the amount or concentration of steps (a) to (b) with a reference for the biomarker and a value for the presence or absence of at least one additional diagnostic criterion and / or calculating a score for assessing a subject with suspected PCOS based on the amount or concentration of the biomarker and the value; and

[0129] (e) assessing the subject based on the comparison and / or calculation performed in step (d).

[0130] In an embodiment, the computer-implemented method for assessing a subject with suspected PCOS comprises a method consisting essentially of the aforementioned steps or a method comprising additional steps. In addition, the inventive method is preferably an ex vivo method, and more preferably an in vitro method. In addition, it may also include steps other than the steps explicitly mentioned above. For example, other steps may involve determining other markers and / or sample pretreatment or evaluating the results obtained by the method. The method may be performed manually or assisted by automation.

[0131] As used herein, the term "computer-implemented" means that the method is performed in an automated manner on a data processing unit, which is typically included in a computer or similar data processing device. The data processing unit should receive a value for the amount of a biomarker. Such a value can be an amount, a relative amount, or any other calculated value reflecting an amount as described in detail elsewhere herein. Therefore, it should be understood that the above method does not require determining the amount of a biomarker, but rather uses a value for an already predetermined amount.

[0132] In principle, the present invention also envisages a computer program, a computer program product or a computer-readable storage medium having said computer program tangibly embedded therein, wherein the computer program comprises instructions for performing the above-described method of the present invention when the instructions are run on a data processing device or a computer.

[0133] Specifically, the present disclosure also includes:

[0134] - a computer or a computer network comprising at least one processor, wherein the processor is adapted to perform a method according to one of the embodiments described in this specification,

[0135] - a computer-loadable data structure adapted to carry out a method according to one of the embodiments described in this specification when the data structure is executed on a computer,

[0136] - a computer script, wherein the computer program is adapted to perform a method according to one of the embodiments described in this specification when the program is executed on a computer,

[0137] - a computer program comprising program means for carrying out a method according to one of the embodiments described in the present description when the computer program is executed on a computer or on a computer network,

[0138] - a computer program comprising program means according to the preceding embodiment, wherein the program means is stored on a computer-readable storage medium,

[0139] a storage medium, on which a data structure is stored and on which the data structure is adapted to carry out a method according to one of the embodiments described in the present description after having been loaded into a main storage device and / or a working storage device of a computer or a computer network,

[0140] - a computer program product having program code means, wherein the program code means can be stored or stored on a storage medium for carrying out a method according to one of the embodiments described in the present description when the program code means is executed on a computer or on a computer network,

[0141] - a data stream signal, usually encrypted, comprising data of the parameters defined elsewhere herein,

[0142] as well as

[0143] - A data stream signal, usually encrypted, comprising the assessment provided by the method of the invention.

[0144] definition:

[0145] In the context of the kit of the invention, the term "reagent" describes a substance or compound added to a sample which allows the amount or concentration of a specific component in the sample to be revealed.

[0146] In the context of the kit of the present invention, the term "specific measurement" refers to the detection of an exact amount or concentration of a well-defined molecule. For a specific measurement, a sample obtained from a female can be incubated with a reagent under conditions suitable for forming a binding agent marker-complex. Such conditions do not need to be specified, as such suitable incubation conditions are well known to those skilled in the art.

[0147] In the context of the kit of the present invention, the term "reagent" may describe 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 a molecule to be specifically measured in a sample.

[0148] Further, the reagent can be connected to a detectable reporter part or label, such as an enzyme, dye, radionuclide, luminescent group, fluorescent group or biotin, such as a fluorescent marker that can be used for immunoassay analysis. Any reporter part or label can be used with the reagent of the kit according to the second aspect of the present invention, as long as its signal can be directly related to or proportional to the amount of the binding agent retained on the support after washing. The amount of the optional second binding agent that remains bound to the solid support can then be determined using a method suitable for a specific detectable reporter part or label. For radioactive groups, scintillation counting or autoradiographic artifact methods are generally suitable. Antibody-enzyme conjugates can be prepared using a variety of coupling techniques (for review, see, for example, Scouten, WH, Methods in Enzymology 135: 30-65, 1987). Spectroscopy can be used to detect dyes (including, for example, colorimetric products of enzyme reactions), luminescent groups and fluorescent groups. Biotin can be detected using avidin or streptavidin, coupled to different reporter groups (usually radioactive groups or fluorescent groups or enzymes). Enzyme reporter groups can typically be detected by adding substrate (usually for a specific time) followed by spectroscopic, spectrophotometric or other analysis of the reaction products.Standards and standard additives can be used to determine the level of antigen in a sample using techniques well known to those skilled in the art.

[0149] The reagent may also be a substance that is additionally capable of being connected to the matrix of a column for chromatography for purification and / or further analysis, such as mass spectrometry. Furthermore, the reagent may be connected to a test strip.

[0150] Preferably, the reagent is an antibody. Suitable antibodies for measuring the amount or concentration of one of the molecules to be specifically measured in a sample obtained from the above female are well known to those skilled in the art.

[0151] Preferably, the reagent is usable in electrochemiluminescence-immunoassay, and more preferably, the reagent is an antibody usable in electrochemiluminescence-immunoassay.

[0152] In addition, the kit may contain more than one reagent (such as two different reagents, three different reagents, four different reagents or more different reagents), preferably two different reagents to interact with a molecule specifically measured in the sample. For example, if the molecule specifically measured is measured in an electrochemiluminescence-immunoassay, the kit may contain two different antibodies that bind to the same molecule to be measured. Preferably, the two different antibodies that bind to the same molecule do not compete for the binding site at the molecule, but bind to the molecule at different positions. Further, both antibodies can be connected to different detectable reporting parts or labels.

[0153] The kit may further comprise a buffer and / or salt to adjust pH and reaction and measurement conditions. In addition, the kit may comprise a stabilizer, for example, to support the stability of the reagent and / or hormone during a specific measurement of (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. In addition, sodium azide can be added to all liquid solutions (such as reagents or buffers) of the kit.

[0154] The kit may also include all the equipment required 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.

[0155] Generally speaking, a doctor or a doctor's assistant can collect blood from a female. Subsequently, the blood can be sent to a laboratory where the sample is measured using a kit on a designated analyzer and the data is sent to the doctor. However, the kit can also be used by the doctor or the doctor's assistant himself. The kit can be applied during a doctor's office visit, a fixed treatment, or a home visit.

[0156] All components of the kit may be packaged separately in a single container. However, two or more components of the kit may also be packaged together in one or more containers.

[0157] The test kit may further include a label, for example, a label including instructions on how to use the test kit or describing the contents of the test kit. However, this information may also be provided in any other form, such as on a storage medium, such as a CD-ROM or a USB flash drive.

[0158] The word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of stated integers or steps or groups of integers or steps but not the exclusion of any other integers or steps or groups of integers or steps.

[0159] As used in this specification and the appended claims, the singular forms "a," "an," "the," and "said" include plural referents unless the content clearly dictates otherwise.

[0160] Concentration, amount and other numerical data can be expressed or presented in "range" format in this article.It should be understood that such range format is used only for convenience and simplicity, and therefore should be flexibly interpreted as not only including the numerical value clearly listed as range limit, but also including all individual numerical values ​​or sub-ranges covered by the range, just as each numerical value and sub-range are clearly listed.As an illustration, the numerical range "150mg to 600mg" should be interpreted as not only including the value 150mg to 600mg clearly listed, but also including individual values ​​and sub-ranges within the indicated range.Therefore, individual values ​​are included in this numerical range, such as 150, 160, 170, 180, 190, ... 580, 590, 600mg and sub-ranges, such as 150 to 200, 150 to 250, 250 to 300, 350 to 600, etc.This same principle applies to the scope of only quoting one numerical value.In addition, no matter how wide the range or feature is, such explanation is applicable.

[0161] When used in connection with a numerical value, the term "about" is meant to encompass the numerical value within a range having a lower limit that is 5% less than the indicated numerical value and an upper limit that is 5% greater than the indicated numerical value.

[0162] As used herein, the term "indicator" refers to a sign or signal of a condition or is used to monitor a condition. Such a "condition" refers to the biological state of a cell, tissue, or organ or to the health and / or disease state of an individual. An indicator can be the presence or absence of a molecule, including but not limited to peptides, proteins, and nucleic acids, or can be a change in the expression level or pattern of such a molecule in a cell, tissue, organ, or individual. An indicator can be a sign of the occurrence, development, or presence of a disease in an individual or further progression of such a disease. An indicator can also be a sign that an individual is at risk of developing a disease.

[0163] 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" is sometimes also applied to means for detecting the endogenous substance (e.g., antibodies, nucleic acid probes, etc., imaging systems). In the context of the present invention, the term "biomarker" should only apply to substances, not to detection means. Therefore, a biomarker can be any type of molecule present in a living organism, such as nucleic acids (DNA, mRNA, miRNA, rRNA, etc.), proteins (cell surface receptors, plasmin, etc.), metabolites or hormones (blood sugar, insulin, estrogen, etc.), a certain modified molecular feature of another molecule (e.g., sugar moieties or phosphoryl residues on proteins, methyl residues on genomic DNA), or a substance that has been internalized by an organism or a metabolite of such a substance.

[0164] The biomarkers mentioned herein can be detected using methods generally known in the art. Detection methods generally include methods for quantifying the level of biomarkers in samples (quantitative methods). Those skilled in the art generally know which of the following methods are suitable for qualitative and / or quantitative detection of biomarkers. Commercially available Western and immunoassays, such as ELISA, RIA, fluorescence and luminescence-based immunoassays and proximity extension assays can be used to conveniently measure, for example, proteins in samples. Other suitable methods for detecting biomarkers include measuring physical or chemical properties specific to peptides or polypeptides, such as their precise molecular mass or NMR spectra. The method includes, for example, biosensors, optical devices coupled to immunoassays, biochips, analytical devices (such as mass spectrometers, NMR analyzers or chromatographic devices). Further, the method includes methods based on microplate ELISA, fully automatic or robotic immunoassays (e.g., in Elecsys TM Analyzers), CBA (e.g., at Roche-Hitachi TM Enzyme cobalt binding assays available on analyzers) and latex agglutination assays (e.g., on Roche-Hitachi TM available on the analyzer).

[0165] The term "anovulation" generally describes a situation in which the ovaries do not release any oocytes at all during the female menstrual cycle. If there is no oocyte release during the duration of at least one female menstrual cycle, preferably at least three female menstrual cycles, more preferably at least six female menstrual cycles, and most preferably at least nine female menstrual cycles within a year, it can be determined that the women assessed for PCOS risk have anovulation. Further, if there is no oocyte release during the duration of at least 6 months, preferably at least 9 months, and more preferably at least 1 year, it can be determined that the females assessed for PCOS risk have anovulation.

[0166] The "symptoms" of a disease refer to the disease that can be noticed by the tissue, organ or organism suffering from the disease, and include but are not limited to pain, weakness, tenderness, tension, stiffness and cramps in tissues, organs or individuals. The typical symptoms of PCOS include but are not limited to rare-anovulation, irregular cycles, hyperandrogenism, polycystic ovarian morphology, infertility, type 2 diabetes, overweight and other metabolic conditions and psychological stress. The "signs" or "signals" of a disease include but are not limited to changes or variations, the presence, absence, increase or increase, decrease or decrease of specific indicators such as biomarkers or molecular markers, or the development, presence or deterioration of symptoms. Symptoms of pain include but are not limited to an unpleasant feeling that may manifest as persistent or varying degrees of burning, throbbing, itching or tingling.

[0167] The terms "disease" and "illness" are used interchangeably herein to refer to abnormal conditions, especially abnormal medical conditions, such as diseases or injuries, in which tissues, organs or individuals are no longer able to effectively perform their functions. Typically, but not necessarily, a disease is associated with specific symptoms or signs indicating the presence of the disease. Therefore, the presence of such symptoms or signs may indicate a tissue, organ or individual suffering from the disease. The changes in these symptoms or signs may indicate the progression of the disease. The typical feature of the progression of a disease is the increase or decrease of such symptoms or signs, which may indicate a "deterioration" or "improvement" of the disease. The "deterioration" of a disease is characterized by a decrease in the ability of a tissue, organ or organism to effectively perform its function, while the typical feature of a "improvement" of a disease is an increase in the ability of a tissue, organ or organism to effectively perform its function. Tissues, organs or individuals with a "risk of developing" a disease are in a healthy state, but show the possibility of developing the disease. Typically, the risk of developing a disease is associated with early or weak signs or symptoms of the disease. In this case, the onset of the disease can still be prevented by treatment. Examples of diseases include, but are not limited to, inflammatory diseases, infectious diseases, skin conditions, endocrine diseases, intestinal diseases, neurological disorders, joint diseases, genetic disorders, autoimmune diseases, traumatic diseases, and various types of cancer.

[0168] The terms "patient" and "subject" are used interchangeably herein and refer to an animal, preferably a mammal, and more typically a human. The patient is preferably a human female. There is a need to diagnose PCOS.

[0169] The terms "sample" or "sample of interest" are used interchangeably herein and refer to a portion or slice of a tissue, organ, or individual, usually smaller than such tissue, organ, or individual, intended to represent the entire tissue, organ, or individual. When analyzed, the sample provides information about the state of the tissue or the health or diseased state of the organ or individual. Examples of samples include, but are not limited to, liquid 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. Analysis of the sample can be completed on a visual or chemical basis. Visual analysis includes, but is not limited to, microscopic imaging or radiographic scanning of tissues, organs, or individuals to allow morphological assessment of the sample. Chemical analysis includes, but is not limited to, detecting the presence or absence of a specific indicator or the amount, concentration, or level of a specific indicator. The sample is an in vitro sample that will be analyzed in vitro without being transferred back to the body.

[0170] As used herein, the term "amount" encompasses the absolute amount of the biomarker mentioned herein, the relative amount or concentration of the biomarker, and any value or parameter associated with or derivable therefrom. Such values ​​or parameters include intensity signal values ​​from all specific physical or chemical properties obtained from the peptide by direct measurement, such as intensity values ​​in a mass spectrum or NMR spectrum. In addition, all values ​​or parameters obtained by indirect measurements specified elsewhere in this specification are contemplated, for example, response amounts measured from a biological readout system in response to an intensity signal obtained from a peptide or from a specifically bound ligand. It should be understood that values ​​associated with the above amounts or parameters can also be obtained by all standard mathematical operations.

[0171] As used herein, the term "comparison" refers to comparing the amount of a biomarker in a sample from a subject with a reference amount of a biomarker specified elsewhere in this specification. It should be understood that comparison as used herein generally refers to the comparison of corresponding parameters or values, for example, comparing an absolute amount with an absolute reference amount, and comparing a concentration with a reference concentration, or comparing an intensity signal obtained from a biomarker in a sample with an intensity signal of the same type obtained from a reference sample. The comparison can be performed manually or computer-assisted. Therefore, the comparison can be performed by a computing device. For example, the value of the measured or detected amount of a biomarker in a sample from a subject can be compared with a reference amount, and the comparison can be automatically performed by a computer program that performs an algorithm for comparison. The computer program performing the evaluation will provide the required assessment in an appropriate output format. For computer-assisted comparison, the value of the measured amount can be compared with a value corresponding to a suitable reference stored in a database by a computer program. The computer program can further evaluate the result of the comparison, i.e., automatically provide the desired assessment in an appropriate output format. For computer-assisted comparison, the value of the measured amount can be compared with a value corresponding to a suitable reference stored in a database by a computer program. The computer program may further evaluate the results of the comparison, ie automatically provide the desired rating in an appropriate output format.

[0172] The expression "the determined amount or concentration is compared with a reference" is in any case only used to further explain what is obvious to the skilled person. A reference concentration is established in a control sample.

[0173] As used herein, the term "reference sample" or "control sample" refers to a sample that is analyzed in a substantially identical manner to the target sample and its information is compared with that of the target sample. Therefore, a reference sample provides a standard for evaluating the information obtained from the target sample. The control sample can be derived from healthy or normal tissue, organ or individual, thereby providing a standard for the health status of a tissue, organ or individual. The difference between the state of a normal reference sample and the state of the target sample can indicate the risk of disease development or the presence or further progression of such a disease or illness. The control sample can be derived from abnormal or diseased tissue, organ or individual, thereby providing a standard for the disease status of a tissue, organ or individual. The difference between the state of an abnormal reference sample and the state of the target sample can indicate the reduced risk of disease development or the absence or improvement of such a disease or illness. The reference sample can also be derived from the same tissue, organ or individual as the target sample, but has been collected at an earlier time point. The difference between the state of the reference sample collected earlier and the state of the target sample can indicate the progression of the disease, i.e., the improvement or deterioration of the disease over time.

[0174] The control sample can be an internal or external control sample. Using an internal control sample, the marker level is assessed in the test sample and in one or more other samples taken from the same subject to determine whether there is any change in the level of the marker. For an external control sample, the presence or amount of the marker in a sample derived from an individual is compared with its presence or amount in an individual known to suffer from a given condition or known to be at risk of a given condition or an individual known to not have a given condition (i.e., a "normal individual").

[0175] Those skilled in the art will appreciate that such external control samples may be obtained from a single individual or from a reference population that is age-matched and free of confounding disease. Typically, samples of 100 well-characterized individuals from an appropriate reference population are used to establish a "reference value". However, a reference population consisting of 20, 30, 50, 200, 500 or 1000 individuals may also be selected. Healthy individuals represent a preferred reference population for establishing a control value.

[0176] For example, the marker concentration in the patient sample can be compared with the concentration known to be associated with the specific course of a certain disease. Usually the marker concentration of the sample is directly or indirectly related to the diagnosis, and for example the marker concentration is used to determine whether the individual is at risk of a certain disease. Alternatively, for example the marker concentration of the sample can be compared with the marker concentration, which is known to be associated with the response of a certain disease to therapy, the diagnosis of a certain disease, the assessment of the severity of a certain disease, the guidance of selecting appropriate drugs for a certain disease, the risk of judging disease progression or the follow-up of the patient. Depending on the diagnostic use expected, select appropriate control samples and establish a control or reference value for the opposing marker therein. It is also clear to the technician that the absolute marker value set up in the control sample will depend on the mensuration used.

[0177] As used herein, the term "assessment" refers to assessing whether a patient suffers from PCOS or is at risk of developing PCOS. Therefore, as used herein, assessment includes diagnosing PCOS by determining the amount or concentration of FGFBP1 in a sample of a patient and comparing the determined amount or concentration with a reference, predicting the risk of developing PCOS, selecting a therapy for PCOS, and monitoring a patient suffering from PCOS or being treated for PCOS.

[0178] As will be appreciated by those skilled in the art, although the assessment made according to the present invention is preferred, it may not be correct for 100% of the subjects studied. The term generally requires the ability to correctly assess a statistically significant portion of subjects. Those skilled in the art can use various well-known statistical evaluation tools (e.g., determining confidence intervals, determining p values, Student's t-tests, Mann-Whitney tests, etc.) to effortlessly determine whether a portion is statistically significant. Detailed information can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. The confidence intervals generally envisioned are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. The p-value is typically 0.2, 0.1, 0.05.

[0179] The term "decreased" or "reduced" level, amount and / or concentration of an indicator means that the level, amount and / or concentration of such indicator in a sample is reduced compared to a reference or reference sample.

[0180] The term "elevated" or "increased" level, amount and / or concentration of an indicator refers to a level, amount and / or concentration of such an indicator in a sample that is higher compared to a reference or reference sample. For example, a protein detectable in a higher amount or concentration in a fluid sample of an individual suffering from a given disease has an elevated level compared to the same fluid sample of an individual not suffering from the disease.

[0181] The terms "measurement", "measuring" or "determining" preferably include qualitative, semi-quantitative or quantitative measurement.

[0182] As used herein, the term "immunoglobulin (Ig)" refers to the immunity-conferring glycoprotein of the immunoglobulin superfamily. "Surface immunoglobulins" are attached to the membrane of effector cells via their transmembrane regions and encompass molecules such as, but not limited to, B cell receptors, T cell receptors, class I and class II major histocompatibility complex (MHC) proteins, beta-2 microglobulin (about 2M), CD3, CD4, and CDS.

[0183] Generally, as used herein, the term "antibody" refers to a secreted immunoglobulin that lacks a transmembrane region and can therefore be released into the bloodstream and body cavity. Human antibodies are grouped into different isotypes based on the heavy chains they possess. There are five types of human Ig heavy chains, represented by Greek letters: α, γ, δ, ε and μ. The heavy chain types present define the categories of antibodies, i.e., these chains are present in IgA, IgD, IgE, IgG and IgM antibodies, respectively, each playing a different role, and guiding appropriate immune responses for different types of antigens. Different heavy chains differ in size and composition; and can contain approximately 450 amino acids (Janeway et al. (2001) Immunobiology, Garland Science). IgA is present in mucosal areas, such as the intestinal tract, respiratory tract and urogenital tract, as well as in saliva, tears and breast milk, to prevent pathogen colonization (Underdown & Schiff (1986) Annu. Rev. Immunol. 4: 389-417). IgD acts primarily as an antigen receptor on B cells that are not exposed to antigens, and is involved in activating basophils and mast cells to produce antimicrobial factors (Geisberger et al. (2006) Immunology 118: 429-437; Chen et al. (2009) Nat. Immunol. 10: 889-898). IgE participates in allergic reactions by binding to allergens, triggering mast cells and basophils to release histamine. IgE is also involved in preventing parasites (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). IgG provides most of the antibody-based immunity against invading pathogens, and is the only isotype of antibodies that can cross the placenta to provide passive immunity to the fetus (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). In humans, there are four different IgG subclasses (IgG1, 2, 3, and 4), named in order of their abundance in serum, with IgG1 being the most abundant (~66%), followed by IgG2 (~23%), IgG3 (~7%), and IgG (~4%). The biological characteristics of the different IgG classes are determined by the structure of the corresponding hinge region. IgM is expressed on the surface of B cells in monomeric and secretory pentamer forms with very high affinity. Before sufficient IgG is produced, IgM participates in the elimination of pathogens in the early stages of B cell-mediated (humoral) immunity (Geisberger et al. (2006) Immunology 118:429-437).Antibodies exist not only in monomeric form, but are also known to form dimers of two Ig units (e.g., IgA), tetramers of four Ig units (e.g., IgM of bony fish), or pentamers of five Ig units (e.g., IgM of mammals). Antibodies are usually composed of four polypeptide chains, including two identical heavy chains and two identical light chains, which are connected via disulfide bonds and resemble "Y"-shaped macromolecules. Each chain contains many immunoglobulin domains, some of which are constant domains and others are variable domains. The immunoglobulin domain consists of a 2-layer sandwich structure, in which 7 to 9 antiparallel chains are arranged in two sheets. Typically, the heavy chain of an antibody contains four Ig domains, three of which are constant (CH domains: CHI, CH2, CH3) domains, and one of which is a variable domain (VH). The light chain usually contains a constant Ig domain (CL) and a variable Ig domain (VL). For example, the human IgG heavy chain consists of four connected Ig domains in the order of VwCH1-CH2-CH3 (also called VwCyl-Cy2-Cy3) from the N-terminus to the C-terminus, while the human IgG light chain consists of two connected immunoglobulin domains in the order of VL-CL from the N-terminus to the C-terminus, which is a κ type or a λ type (VK-CK or VA.-CA.). For example, the constant chain of human IgG contains 447 amino acids. In the present specification and claims, the numbering of amino acid positions in immunoglobulins is the numbering of the "EU index", as in the following documents: Kabat, EA, Wu, TT, Perry, HM, Gottesman, KS, and Foeller, C., (1991) Sequences of proteins of immunological interest, 5th edition. US Department of Health and Human Service, National Institutes of Health, Bethesda, MD. "EU index as in Kabat" refers to the residue numbering of human IgG lEU antibodies. Thus, the CH domains in the context of IgG are as follows: "CHI" refers to amino acid positions 118-220 according to the EU index as in Kabat; "CH2" refers to amino acid positions 237-340 according to the EU index as in Kabat; and "CH3" refers to amino acid positions 341-447 according to the EU index as in Kabat.

[0184] The terms "full length antibody", "intact antibody" and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form rather than an antibody fragment as defined below. The term specifically refers to an antibody having heavy chains that include an Fc region.

[0185] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab fragments" (also called "Fab portions" or "Fab regions"), each with a single antigen-binding site, and a residual "Fc fragment" (also called "Fc portion" or "Fc region"), a name that reflects 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. In addition, smaller immunoglobulin molecules occur naturally or have been artificially constructed. The term "Fab' fragment" refers to a Fab fragment that additionally includes the hinge region of an Ig molecule, while a "F(ab')2 fragment" is understood to include two Fab' fragments that are chemically linked or connected via a disulfide bond. Although "single domain antibodies (sdAb)" (Desmyter et al. (1996) Nat. Structure Biol. 3: 803-811) and "nano antibodies" include only a single VH domain, "single chain Fv (scFv)" fragments include a heavy chain variable domain that is joined to a light chain variable domain via a short linker peptide (Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85, 5879-5883). A divalent single chain variable fragment (di-scFv) can be engineered by connecting two scFvs (scFvA-scFvB). This can be achieved by generating a single peptide chain with two VH and two VL regions, thereby generating a "tandem scFv" (VHA-VLA-VHB-VLB). Another possibility is to create a scFv with a joint, which is too short for two variable regions to fold together, thereby forcing scFv dimerization. A joint with a length of 5 residues is generally used to produce these dimers. This type is referred to as a "diabody". A shorter joint (one or two amino acids) between VH and VL domains leads to the formation of a monospecific trimer, so-called "triabodies" or "tribadies". Bispecific diabodies are formed by expressing a chain having VHA-VLB and VHB-VLA or VLA-VHB and VLB-VHA arrangements, respectively. Single-chain diabodies (scDb) include VHA-VLB and VHB-VLA fragments, which are connected (VHA-VLB-P-VHB-VLA) by 12-20 amino acids, preferably 14 amino acid linker peptides (P)."Bispecific T cell engagers (BiTEs)" are fusion proteins 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). Dual affinity retargeting molecules ("DART" molecules) are diabodies that are additionally stabilized by a C-terminal disulfide bond.

[0186] Therefore, the term "antibody fragment" refers to a portion of an intact antibody, preferably including its antigen binding region. Antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments; diabodies; sdAb, nanobodies, scFv, di-scFv, tandem scFv, triabodies, diabodies, scDb, BiTE and DART.

[0187] The term "binding affinity" generally refers to the strength of the sum 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 intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including but not limited to surface plasmon resonance-based assays (e.g., BIAcore assays described in PCT Application Publication No. WO2005 / 012359); enzyme-linked immunosorbent assays (ELISAs); and competition assays (e.g., RIAs). Low-affinity antibodies generally bind antigen slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigen quickly and tend to remain bound for longer periods of time. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention.

[0188] "Sandwich immunoassays" are widely used to detect target analytes. In this assay, the analyte is "sandwiched" between a first antibody and a second antibody. Typically, sandwich assays require that the capture and detection antibodies bind to different non-covering epitopes on the target analyte. This sandwich complex is measured by an appropriate means and the analyte is quantified thereby. In a typical sandwich assay, a first antibody bound to a solid support or capable of binding to a solid phase and a second antibody detectably labeled each bind to the analyte at different non-covering epitopes. A first analyte-specific binding agent (e.g., an antibody) is covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. The solid support may be in the form of a tubule, magnetic beads, microplate tray, or any other surface suitable for performing an immunoassay. Binding methods are well known in the art and typically consist of cross-linked covalent binding or physical adsorption, with the polymer-antibody complex being washed when the test sample is prepared. An aliquot of the sample to be tested is then added to the solid phase complex and incubated under appropriate conditions (e.g., from room temperature to 40°C, such as between 25°C and 37°C, inclusive) for a sufficient period of time (e.g., 2-40 minutes or overnight if more convenient) to allow binding between the first or capture antibody and the corresponding antigen. After the incubation period is complete, the solid phase, which includes the first or capture antibody and the antigen bound thereto, can be washed and incubated with a secondary antibody or labeled antibody that binds to another epitope on the antigen. The second antibody is linked to a reporter molecule that is used to indicate binding between the second antibody and the first antibody-target antigen complex.

[0189] An extremely versatile alternative sandwich assay format involves the use of a solid phase coated with a first partner of a binding pair, such as paramagnetic streptavidin-coated microparticles. Such microparticles are mixed and incubated with: an analyte-specific binding agent (e.g., a biotinylated antibody) that binds to a second partner of a binding pair; a sample suspected of or including an analyte, wherein the second partner of the binding pair binds to the analyte-specific binding agent; and a detectably labeled second analyte-specific binding agent. As will be apparent to one skilled in the art, the components are incubated under appropriate conditions for a sufficient period of time to allow the labeled antibody (via the analyte), the analyte-specific binding agent that binds to the second partner of the binding pair, and the first partner of the binding pair to bind to the solid phase microparticles. Optionally, the assay may include one or more washing steps.

[0190] The term "detectably labeled" encompasses labels that are detectable either directly or indirectly.

[0191] Directly detectable labels provide a detectable signal or they interact with a second label to modify the detectable signal provided by the first or second label, for example, FRET (fluorescence resonance energy transfer) occurs. 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 suitable for labeling. In one embodiment, "detectably labeled" refers to a label that provides or induces 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.

[0192] The large number of available labels (also called dyes) can be generally divided into the following categories, all of which collectively and each of which represent embodiments as described in the present disclosure:

[0193] (a) Fluorescent dyes

[0194] Fluorescent dyes are described, for example, in 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).

[0195] Fluorescent labels or fluorophores include rare earth chelates (europium chelates); fluorescein type labels, including FITC, 5-carboxyfluorescein, 6-carboxyfluorescein; rhodamine labels, including TAMRA; dansyl; Lissamine; cyanine; phycoerythrin; Texas Red; and analogs thereof. Using the techniques disclosed herein, fluorescent labels can be conjugated to aldehyde groups contained in target molecules. Fluorescent dyes and fluorescent labeling reagents include such fluorescent dyes and reagents available from Invitrogen / Molecular Probes (Eugene, Oregon, USA) and Pierce Biotechnology, Inc. (Rockford, Ill.).

[0196] (b) Luminescent dyes

[0197] Luminescent dyes or labels can be further divided into the following subcategories: chemiluminescent dyes and electrochemiluminescent dyes.

[0198] Different classes of chemiluminescent labels include luminol, acridine compounds, coelenterazine and analogs, dioxetanes, peroxyoxalic acid-based systems and their derivatives. For immunodiagnostic procedures, mainly acridine-based labels are used (a detailed review is given in Dodeigne C. et al., Talanta 51 (2000) 415-439).

[0199] The main correlation labels used as electrochemiluminescent labels are ruthenium-based and iridium-based electrochemiluminescent complexes, respectively. It has been proven that electrochemiluminescence (ECL) is very useful in analytical applications as a highly sensitive and selective method. This method combines the analytical advantages of chemiluminescent analysis (no background light signal) with a more convenient control of the reaction by adopting electrode potential. Typically, ruthenium complexes, especially [Ru(Bpy)3]2+ (which releases photons at about 620nm) regenerated in liquid phase or liquid-solid interface with TPA (tripropylamine) are used as ECL labels.

[0200] Electrochemiluminescence (ECL) assays provide sensitive and accurate measurements of the presence and concentration of an analyte of interest. The technique employs a label or other reactant that is induced to emit light when electrochemically oxidized or reduced under appropriate chemical conditions. This electrochemiluminescence is triggered by a voltage applied to a working electrode at a specific time and in a specific manner. The light emitted by the label, when measured, indicates the presence or amount of the analyte. For a more complete description of such ECL techniques, the following references are cited: U.S. Patent No. 5,221,605, U.S. Patent No. 5,591,581, U.S. Patent No. 5,597,910, PCT Published Application WO90 / 05296, PCT Published Application WO92 / 14139, PCT Published Application WO90 / 05301, PCT Published Application WO96 / 24690, PCT Published Application US95 / 03190, PCT Application US97 / 16942, PCT Published Application US9 6 / 06763, PCT published application WO95 / 08644, PCT published application WO96 / 06946, PCT published application WO96 / 33411, PCT published application WO87 / 06706, PCT published application WO96 / 39534, PCT published application WO96 / 41175, PCT published application WO96 / 40978, PCT / US97 / 03653 and U.S. Patent Application 08 / 437,348 (U.S. Patent No. 5,679,519). Also quoted is the ECL analysis application review (Analyst, 1994, 119: 879-890) published by Knight et al. in 1994 and the literature cited by the article. In one embodiment, the method described in this description is implemented using electrochemiluminescent labeling.

[0201] Recently, iridium-based ECL labels have also been described (WO2012107419).

[0202] (c) Radioactive labeling uses radioactive isotopes (radionuclides) such as 3H, 11C, 14C, 18F, 32P, 35S, 64Cu, 68Gn, 86Y, 89Zr, 99TC, 111In, 123I, 124I, 125I, 131I, 133Xe, 177Lu, 211At or 131Bi.

[0203] (d) Labeled metal chelate complexes suitable for use as imaging and therapeutic targets are known in the art (US 2010 / 0111861; US ​​5,342,606; US 5,428,155; US 5,316,757; US 5,480,990; US 5,462,725; US 5,428,139; US 5,385,893; US 5,739,294; US 5,750,660; US 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).

[0204] Examples

[0205] The present invention is illustrated by the following examples only. The examples are not to be construed in any way as limiting the scope of the present invention.

[0206] Example 1: Diagnostic performance of the biomarker FGFBP1 in women with PCOS (phenotype A) and controls determined by the proximity extension assay (PEA) technology developed at Olink

[0207] As part of the measurement, 85 serum samples from human women were analyzed. The case group included 48 samples from patients diagnosed with PCOS (A phenotype) according to the Rotterdam criteria. The control group included 37 samples from healthy women without PCOS. The concentration of the analyte was determined using the proximity extension assay (PEA) technology developed at Olink. In short, a matching pair of antibodies coupled to a unique, partially complementary oligonucleotide will respond to each biomarker. Then quantification is performed by quantitative real-time PCR.

[0208] After sample dilution according to the manufacturer's protocol for each of the selected panels, the Olink protocol consists of three core steps: 1. Incubation; 2. Extension and amplification; 3. Detection. One μl of each sample is mixed with 3 μl of incubation mixture in a 96-well plate. In addition to the 92 antibody pairs labeled with DNA oligonucleotides, the incubation mixture also contains internal controls designed to monitor the three main steps of the Olink protocol (2 incubation controls, 1 extension control, and 1 detection control). As external controls, 3 positive controls (inter-plate controls) and 3 negative controls and 2 sample controls (pooled plasma samples) are included in the plate. The samples were incubated overnight at +4°C. During this step, the antibody pairs bind to the corresponding proteins in the sample. When the incubation is complete, 96 μl of extension mixture is added to the sample. The plate is placed in a thermal cycler for hybridization and extended by DNA polymerase (50°C 20min, 95°C 5min (95°C 30s, 54°C 1min, 60°C 1min) x17, 10°C freeze). DNA barcodes were amplified by PCR. Finally, the amount of each DNA barcode was quantified by microfluidic qPCR. The 96.96 Dynamic Array was used according to the manufacturer's instructions. TMIntegrated fluid circuit (IFC). Seven point two μl of detection mixture is added to each sample of 2.8 μl, of which 5 μl is transferred to the left inlet of the 96.96 dynamic array IFC that is perfused. Five μl of primer solution is transferred to the right inlet of the 96.96 Dynamic Array IFC that is perfused. The chip is loaded in the Fluidigm IFC controller HX according to the manufacturer's instructions. According to the manufacturer's instructions, the Olink protein expression 96×96 program is run in the Fluidigm BiomarkTM Reader (50°C 120s, 70°C 1800s, 25°C 600s, 95°C 300s (95°C 15s, 60°C 60s) x35; using the following settings: application-gene expression; passive reference-ROX; determination-single probe; probe-FAM-MGB). The Ct value obtained from qPCR is transformed into an arbitrary unit called normalized protein expression (NPX, relative quantification unit on log2 scale) using the following equation:

[0209] Extended control:

[0210] Ct 分析物 –Ct 延伸对照 =dCt 分析物

[0211] Inter-plate comparison:

[0212] dC 分析物 –dCt 板间对照 =ddCt 分析物

[0213] Adjust according to the correction factor:

[0214] Correction Factor – ddCt 分析物 =NPX 分析物

[0215] Quality control and normalization were performed using Olink NPX Manager software.

[0216] Generate a receiver operating characteristic (ROC) curve ( Figure 1 ). Model performance was determined by looking at the area under the curve (AUC). The best possible AUC was 1, while the lowest possible was 0.5. ROC curve analysis showed an AUC of 0.985 (95% CI 0.967 to 1.0, Figure 1 ), indicating that FGFBP1 has a high diagnostic accuracy for PCOS.

[0217] The diagnostic performance of FGFBP1 using ROC analysis to distinguish women with PCOS with mature phenotype A (cases) from healthy control subjects is shown in Table 1, which depicts the AUC and associated 95% confidence intervals of the ROC curve analysis. The results were obtained using the Olink neighbor extension technique.

[0218] Table 1

[0219] Markers AUC (Area Under the Curve) 95% CI (Confidence Interval) N (sample size) Sample type FGFBP1 0.985 0.967-1 85 serum

[0220] Box and whisker plots of healthy controls and PCOS cases were generated using data obtained using Olink PEA technology. The boxes include the median (middle quartile), the interquartile range (which represents the middle 50% of the scores for the group), the upper quartile (75% of the scores are below the upper quartile), and the lower quartile (25% of the scores are below the lower quartile). The whiskers represent values ​​1.5 times the interquartile range. Serum FGFBP1 concentrations were increased in women with PCOS when compared to healthy controls ( Figure 2 ).

[0221] Example 2: Diagnostic performance of the biomarker FGFBP1 in women with PCOS (phenotypes A, B, C and D) and controls determined by ELISA technique

[0222] Performance validation has been performed on a sample set of 90 cases (serum samples from women with PCOS) and 44 controls (serum samples from healthy women).

[0223] The concentration of the analyte was determined by ELISA (enzyme-linked immunosorbent assay). The case group consisted of patients diagnosed with PCOS according to the Rotterdam criteria (30 phenotype A, 20 phenotype B, 20 phenotype C, and 20 phenotype D). The control group included healthy women without PCOS.

[0224] The concentration of FGFBP1 in human serum was determined using the Human FGFBP1 ELISA Kit from Sigma-Aldrich (Cat. No.: RAB1460). 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.

[0225] The kit is provided with human FGFBP1 antibody pre-coated plates. Samples are measured with 75-fold dilution. After all reagents are placed at room temperature, 100 μL of each sample and standard are added to the plate. Samples and standards are measured in duplicate. During the 2.5-hour incubation at room temperature with gentle shaking, any FGFBP1 present is combined with the fixed capture antibody on the microtiter plate. During the cleaning step (4x300 μL), unbound substances are removed from the plate, and then 100 μL of diluted detection antibodies are added to the wells. After 1h incubation with gentle shaking and another washing step (4x300 μL) to remove any unbound detection antibodies, 100 μL of prepared streptavidin HRP solution is added to the plate. Afterwards, 45 minutes incubation and washing steps (4x300 μL) are carried out at room temperature with gentle shaking. After the last wash, 100 μL of TMB one-step substrate reagent is added to the plate. The plate was incubated in the dark at room temperature for 30 minutes with gentle shaking. During the incubation period, the substrate turned blue. The color development was proportional to the amount of FGFBP1 combined in the initial step. The color development was stopped by adding 50 μL stop solution, the solution in the well changed from blue to yellow, and the color intensity was measured at 450nm using a plate reader for detection, and the color intensity was measured at 570nm for background subtraction. A seven-point standard curve was obtained using 2.5-fold serial dilutions of the recombinant FGFBP1 in the assay diluent. The calibration curve was fitted using unweighted 4-parameter nonlinear regression (Newton / Raphson).

[0226] Generate a receiver operating characteristic (ROC) curve ( Figure 3 ). Model performance was determined by looking at the area under the curve (AUC). The best possible AUC was 1, while the lowest possible was 0.5. ROC curve analysis of FGFBP1 for PCOS cases when all phenotypes (phenotypes A to D) were combined showed an AUC of 0.87 (95% CI 0.78 to 0.95), confirming the high diagnostic accuracy of FGFBP1 for PCOS ( Figure 3 ).

[0227] The diagnostic performance of FGFBP1 using ROC analysis to distinguish women with PCOS (cases, PCOS phenotypes A to D) from healthy control subjects is shown in Table 2, which describes the AUC and associated 95% confidence intervals of the ROC curve analysis. The results were obtained using an ELISA immunoassay.

[0228] Table 2

[0229] Markers AUC (Area Under the Curve) 95% CI (Confidence Interval) N (sample size) Sample type FGFBP1 0.87 (0.78-0.95) 134 serum

[0230] The data obtained by ELISA immunoassay were used to generate box-whisker plots of controls and PCOS cases when all phenotypes (phenotypes A to D) were combined. Serum FGFBP1 concentrations (pg / mL) were increased in women with PCOS when compared to healthy controls ( Figure 4 ).

[0231] Table 3 shows the diagnostic performance of FGFBP1 in differentiating women with PCOS from healthy control subjects when separated by different phenotypes A, B, C and D. The results were obtained using an ELISA immunoassay. The AUC for each phenotype is reported in the table.

[0232] Table 3

[0233] Markers Phenotype A Phenotype B Phenotype C Phenotype D N (sample size) Sample type FGFBP1 0.88 0.88 0.88 0.84 134 serum

[0234] ROC curve analysis of FGFBP1 in PCOS cases and healthy controls separated by different phenotypes showed that the AUCs for phenotypes A to D were 0.88 (95% CI 0.79 to 0.96), 0.88 (95% CI 0.79 to 0.96), 0.88 (95% CI 0.80 to 0.96), and 0.84 (95% CI 0.74 to 0.95), respectively ( Figure 5 ). The results confirmed the high diagnostic accuracy of FGFBP1 for PCOS.

[0235] Compared with healthy controls, serum FGFBP1 concentrations (pg / mL) in all different PCOS phenotypes (phenotypes A to D) showed increased levels ( Figure 6 , results were obtained using ELISA immunoassay).

[0236] Table 4 shows the diagnostic performance of FGFBP1 in discriminating young women with PCOS from young healthy control subjects when all phenotypes (Phenotypes A to D) were combined. Results were obtained using an ELISA assay.

[0237] Table 4

[0238] Markers AUC (Area Under the Curve) 95% CI (Confidence Interval) N (sample size) Sample type FGFBP1 0.84 (0.61-1) 32 serum

[0239] ROC curve analysis of FGFBP1 for young PCOS cases (age ≤ 25 years) when all phenotypes (phenotypes A to D) were combined showed an AUC of 0.84, confirming the high diagnostic accuracy (95% CI 0.61 to 1, Figure 7 When only young women (age ≤25 years) were included in the analysis, PCOS cases (all phenotypes A to D combined) were significantly more likely to develop leukemia than young controls (age ≤25 years, Figure 8) showed increased serum FGFBP1 concentrations (pg / mL).

[0240] The diagnostic performance of FGFBP1 in differentiating young women (age≤25 years) with PCOS from young healthy control subjects (age≤25 years) when separated by different phenotypes A, B, C and D has been evaluated and the results are reported in Table 5 (AUC for PCOS phenotypes vs. controls). The results were obtained using an ELISA immunoassay.

[0241] Table 5

[0242] Markers Phenotype A Phenotype B Phenotype C Phenotype D N (sample size) Sample type FGFBP1 0.93 0.85 0.84 0.63 32 serum

[0243] ROC curve analysis of FGFBP1 for young PCOS cases (age ≤ 25 years, phenotypes A to D) showed that for each phenotype, the AUC was 0.93 (95% CI 0.81 to 1), 0.85 (95% CI 0.54 to 1), 0.84 (95% CI 0.57 to 1), and 0.63 (95% CI 0.19 to 1), respectively, confirming the high diagnostic accuracy of FGFBP1 for PCOS in the subgroup of women aged 25 years or younger ( Fig. 9 ). When compared to young healthy controls (age ≤ 25 years, Fig.10 ), the FGFBP1 concentrations were increased in all the different PCOS phenotypes (phenotypes A to D, age ≤25 years).

[0244] Example 3: Diagnostic performance of the biomarker FGFBP1 in all age groups in women with PCOS (phenotypes A, B, C and D) and controls determined by ELISA technique

[0245] Performance validation has been performed in an additional sample set of 240 cases (serum samples from women with PCOS) and 48 controls (serum samples from healthy women).

[0246] The concentration of the analyte was determined by ELISA (enzyme-linked immunosorbent assay). The case group consisted of patients diagnosed with PCOS according to the Rotterdam criteria (155 phenotype A, 5 phenotype B, 8 phenotype C and 72 phenotype D), belonging to three different age groups: 15 to <20 (n=70), 20 to <25 (n=99), 25 to <40 (n=71). The control group included healthy women without PCOS.

[0247] The concentration of FGFBP1 in human serum was determined as described in Example 2 using the Human FGFBP1 ELISA kit from Sigma-Aldrich (Cat. No.: RAB1460).

[0248] Generate a receiver operating characteristic (ROC) curve ( Fig.11 ). Model performance was determined by looking at the area under the curve (AUC). ROC curve analysis of FGFBP1 for PCOS cases when all phenotypes (phenotypes A to D) were combined showed an AUC of 0.9 (95% CI 0.83 to 0.96), confirming the high diagnostic accuracy of FGFBP1 for PCOS ( Fig.11 ).

[0249] The diagnostic performance of FGFBP1 using ROC analysis to distinguish women with PCOS (cases, PCOS phenotypes A to D) from healthy control subjects is shown in Table 6, which depicts the AUC and associated 95% confidence intervals of the ROC curve analysis. The results were obtained using an ELISA immunoassay.

[0250] Table 6

[0251] Markers AUC (Area Under the Curve) 95% CI (Confidence Interval) N (sample size) Sample type FGFBP1 0.90 (0.83-0.96) 288 serum

[0252] The data obtained by ELISA immunoassay were used to generate box-whisker plots of controls and PCOS cases when all phenotypes (phenotypes A to D) were combined. Serum FGFBP1 concentrations (pg / mL) were increased in women with PCOS when compared to healthy controls ( Fig.12 ).

[0253] Table 7 shows the diagnostic performance of FGFBP1 in differentiating women with PCOS from healthy control subjects when separated by different phenotypes A, B, C and D. The results were obtained using an ELISA immunoassay. The AUC for each phenotype is reported in the table.

[0254] Table 7

[0255] Markers Phenotype A Phenotype B Phenotype C Phenotype D N (sample size) Sample type FGFBP1 0.90 0.90 0.82 0.90 288 serum

[0256] ROC curve analysis of FGFBP1 in PCOS cases and healthy controls separated by different phenotypes showed that the AUCs for phenotypes A to D were 0.90 (95% CI 0.84 to 0.97), 0.90 (95% CI 0.81 to 0.99), 0.82 (95% CI 0.69 to 0.96), and 0.90 (95% CI 0.83 to 0.97), respectively ( Fig.13 ). The results confirmed the high diagnostic accuracy of FGFBP1 for PCOS.

[0257] Compared with healthy controls, serum FGFBP1 concentrations (pg / mL) in all different PCOS phenotypes (phenotypes A to D) showed increased levels ( Fig.14 , results were obtained using ELISA immunoassay).

[0258] Table 8 shows the diagnostic performance of FGFBP1 in differentiating women with PCOS from healthy control subjects in different age groups (15≤age<20, 20≤age<25, 25≤age<40) when all phenotypes (Phenotypes A to D) were combined. The results were obtained using an ELISA assay.

[0259] Table 8

[0260] Markers 15≤Age<20 20≤Age<25 25≤Age<40 N (sample size) Sample type FGFBP1 0.90 0.88 0.91 288 serum

[0261] ROC curve analysis of FGFBP1 in PCOS cases and healthy controls separated by different age groups showed that the AUCs were 0.90 (95% CI 0.84 to 0.97), 0.88 (95% CI 0.81 to 0.95), and 0.91 (95% CI 0.84 to 0.98) for the age groups 15 to <20, 20 to <25, and 25 to <40, respectively ( Fig.15 ). The results confirmed the high diagnostic accuracy of FGFBP1 for PCOS in all different age groups.

[0262] Increased serum FGFBP1 concentrations (pg / mL) in women with PCOS compared to controls were confirmed in all different age groups ( Fig.16 ).

[0263] Considering the lack of reliable biomarkers for the diagnosis of PCOS, especially in younger women (age <25 years), separate analyses were performed for age groups ≥15 years and <25 years.

[0264] Table 9 shows the diagnostic performance of FGFBP1 in discriminating young women with PCOS from young healthy control subjects in young women (15≤age<25 years) when all phenotypes (Phenotypes A to D) were combined. Results were obtained using an ELISA assay.

[0265] Table 9

[0266] Markers AUC (Area Under the Curve) 95% CI (Confidence Interval) N (sample size) Sample type FGFBP1 0.88 (0.76-0.99) 192 serum

[0267] ROC curve analysis of FGFBP1 for young PCOS cases (15≤age<25 years) when all phenotypes (phenotypes A to D) were combined showed an AUC of 0.88, confirming the high diagnostic accuracy (95% CI 0.76 to 0.99, Fig.17 When only young women (15 ≤ age < 25 years) were included in the analysis, PCOS cases (all phenotypes A to D combined) were significantly more likely to have a positive PCOS risk than young controls (15 ≤ age < 25 years, Fig.18) showed increased serum FGFBP1 concentrations (pg / mL).

[0268] The diagnostic performance of FGFBP1 in differentiating young women with PCOS (15≤age<25 years) from young healthy control subjects (15≤age<25 years) when separated by different phenotypes A, B, C and D has been evaluated and the results are reported in Table 10 (AUC for PCOS phenotypes vs. controls). The results were obtained using an ELISA immunoassay.

[0269] Table 10

[0270] Markers Phenotype A Phenotype B Phenotype C Phenotype D N (sample size) Sample type FGFBP1 0.88 0.87 0.80 0.87 192 serum

[0271] ROC curve analysis of FGFBP1 for young PCOS cases (15 ≤ age < 25 years, phenotypes A to D) showed that for each phenotype, the AUC was 0.88 (95% CI 0.77 to 0.99), 0.87 (95% CI 0.72 to 1), 0.80 (95% CI 0.63 to 0.97), 0.87 (95% CI 0.75 to 0.99), respectively, confirming the high diagnostic accuracy of FGFBP1 for PCOS in the subgroups of women aged ≥ 15 and < 25 years ( Fig.19 ). When compared to young healthy controls (15≤age<25 years, Fig. 20 ), the FGFBP1 concentrations were increased in all the different PCOS phenotypes (phenotypes A to D, 15 ≤ age < 25 years).

Claims

1. A method for assessing whether a subject has polycystic ovary syndrome (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 with a reference.

2. A method of selecting a patient for therapy for PCOS, the method comprising: (a) determining the amount or concentration of FGFBP1 in a sample from a subject, and (b) comparing the determined amount or concentration with a reference.

3. A method for monitoring the progression of PCOS in a subject with PCOS or for monitoring the response of a subject with PCOS to treatment, the method comprising (a) determining the level of FGFBP1 in a first sample from the subject, (b) determining the level of FGFBP1 in a second sample of the subject that has been obtained after the first sample, and (c) comparing said level of FGFBP1 in said first sample with said level of FGFBP1 in said second sample, and (d) monitoring the progress of the subject suffering from or being treated for PCOS based on the results of step c).

4. The method of claims 1 to 3, wherein an increased amount or concentration of FGFBP1 in the sample of the subject indicates the presence of PCOS in the subject.

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

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

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

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

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

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

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

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

13. A computer-implemented method for assessing a subject for suspected PCOS, the computer-implemented method comprising the steps of: (a) receiving a value for the amount or concentration of a first biomarker in a sample of the subject, the first biomarker being FGFBP1, (b) optionally, receiving a value for the amount or concentration of a second biomarker in a 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-anovulation and / or irregular cycles, hyperandrogenemia, and polycystic ovarian morphology; (d) comparing the values ​​for the amounts or concentrations of steps (a) to (b) with a 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 assessing the subject as having suspected PCOS based on the amounts or concentrations and the values ​​of the biomarkers; as well as (e) assessing the subject based on the comparing and / or the calculating performed in step (d).

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

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