Leukotriene A4 hydrolase (LTA4H) as a (blood) biomarker for the diagnosis of polycystic ovary syndrome

Abstract

The present invention relates to a method for assessing whether a subject has polycystic ovary syndrome (PCOS) or is at risk of developing PCOS, a method for selecting patients for the treatment of PCOS, a method for monitoring PCOS progression or monitoring response to treatment, and a computer-implemented method for assessing a subject suspected of having PCOS by determining the amount or concentration of leukotriene A4 hydrolase (LTA4H) in a sample of the subject.

Description

Technical Field

[0001] The present invention relates to a method for assessing whether a patient has polycystic ovarian syndrome (PCOS) or is at risk of developing PCOS, a method for selecting a patient for treatment, and determining the amount or concentration of leukotriene A4 hydrolase (LTA4H) in a patient's sample and monitoring a patient suffering from or being treated for PCOS by comparing the determined amount or concentration with a reference. Further, the present invention relates to a computer-implemented method for assessing a patient suspected of having PCOS by determining the amount or concentration of LTA4H in a patient's sample, optionally determining the amount or concentration of a second biomarker and / or additional diagnostic criteria, and by referring to the amount or concentration of LTA4H, optionally the second biomarker and / or the presence of diagnostic criteria and comparing with a reference.

Background Art

[0002] Polycystic ovarian syndrome (PCOS) is a heterogeneous gynecological disorder defined by a combination of androgen excess symptoms and ovarian dysfunction. PCOS patients can have various clinical symptoms that can be reproductive and / or metabolic in nature. Reproductive findings include irregular menstrual cycles, infertility, pregnancy complications, and hirsutism, and metabolic findings include obesity, insulin resistance, metabolic syndrome, prediabetes, type 2 diabetes, and cardiovascular factors. These clinical symptoms are also associated with psychological disorders such as anxiety and depression (Escobar-Morreale, H.F. 2018; International evidence-based guideline for the assessment and management of polycystic ovary syndrome 2018).

[0003] The symptoms are not specific to PCOS, and often, patients are diagnosed only after a longer evaluation for infertility. For a definitive diagnosis of PCOS, other conditions or diseases such as pregnancy, non-classical congenital adrenal hyperplasia (NCAH), congenital adrenal hyperplasia, androgen-secreting tumors, Cushing's syndrome, thyroid disorders, or hyperprolactinemia must be excluded (Escobar-Morreale HF. Polycystic ovary syndrome: definition, aetiology, diagnosis and treatment. Nat Rev Endocrinol. 2018;14(5):270-284; Teede HJ, Misso ML, Costello MF, et al. International PCOS Network. Recommendations from the international evidence-based guideline for the assessment and management of polycystic ovary syndrome. Hum Reprod. 2018;33(9):1602-1618). Diagnostic tests that can be used to exclude other diseases are, for example, · 17α-hydroxyprogesterone (17-OHP) to exclude NCAH (Nordenstrom and Falhammar 2018) · Prolactin to rule out hyperprolactinemia · Cortisol to exclude patients with Cushing's syndrome · Thyroid-stimulating hormone (TSH) to exclude thyroid disorders

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

[0005] PCOS is one of the most common endocrine disorders in women, and despite the fact that 10% of women of reproductive age are affected, up to 70% of affected women remain undiagnosed (March WA, Moore VM, Willson KJ, et al. The prevalence of polycystic ovary syndrome in a community sample assessed under contrasting diagnostic criteria. Hum Reprod. 2010;25(2):544-51).

[0006] The criteria mainly and widely used for PCOS diagnosis are the so-called Rotterdam criteria. PCOS is indicated when at least two of the following criteria are applied: (i) irregular cycles (oligomenorrhea) and / or ovulatory dysfunction (oligo-anovulation, OA), (ii) clinical and / or biochemical hyperandrogenism (HA), and (iii) polycystic ovarian morphology (PCOM) (PCOS Consensus Workshop Group, Fertil Steril 2004;81:19-25). The first criterion is defined as a menstrual cycle having a cycle length of less than 21 days or more than 35 days or less than 8 cycles per year. In the case of clinical and / or biochemical signs of hyperandrogenism, clinical hyperandrogenism is defined as hirsutism (excessive male-pattern hair growth) and / or acne, and biochemical hyperandrogenism is defined as higher levels of free androgens compared to healthy controls. Clinical hyperandrogenism is also defined as a modified Ferriman-Gallwey score of more than 8. Biochemical hyperandrogenism can be evaluated using free testosterone or free androgen index (FAI) which can be calculated by measuring total testosterone and sex hormone binding globulin (SHBG). PCOM is usually determined according to the "International Evidence-based Guideline for PCOS 2018" using a transvaginal ultrasound transducer with a frequency bandwidth including 8 MHz. The threshold for PCOM is considered, for the ovaries, to be either >20 follicles per ovary and / or ovarian volume ≥10 ml, thereby ensuring the absence of corpora lutea, cysts or dominant follicles. When older ultrasound techniques are used, the threshold for PCOM can be an ovarian volume of ≥10 ml or a follicle count of >12 for either ovary.

[0007] Another way to detect PCOM is to measure the subject's anti-Müllerian hormone (AMH). AMH is a glycoprotein hormone whose expression is important for sexual differentiation at a specific time during fetal development. Furthermore, AMH produced by the granulosa cells of growing follicles usually correlates with the number of follicles in the ovary. Therefore, the serum level of AMH can be a surrogate biomarker for the antral follicle count / number (AFC) determined by transvaginal ultrasound. 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 "International evidence-based guideline for the assessment and management of polycystic ovary syndrome 2018", serum AMH levels should not be used as an alternative for the detection of PCOM or the diagnosis of PCOS.

[0008] A further method for detecting PCOS is the 3-item PCOS criteria system (Indran et al. 2018). In this system, it has been proposed to diagnose PCOS if two of the three items are present: (i) oligomenorrhea (defined as an average menstrual cycle length > 35 days); (ii) AMH above the threshold; (iii) hyperandrogenemia defined as either testosterone above the threshold and / or the presence of hirsutism (mFG score ≥ 5). Alternatively, AMH has been suggested in combination with hyperandrogenemia and oligomenorrhea (Sahmay et al., 2014), or in combination with SHBG (Calzada et al., 2019).

[0009] Another way to detect PCOS is to measure other hormones, such as luteinizing hormone (LH) and follicle-stimulating hormone (FSH). However, the diagnostic utility of the LH:FSH ratio for the diagnosis of PCOS seems low because only a small fraction of women with PCOS had a significantly elevated LH:FSH ratio (Cho et al. 2005). In fact, the range of LH:FSH ratios seen in women diagnosed with PCOS is wide (Malini and George 2018).

[0010] The need to consider the results of multiple diagnostic tests and clinical examinations requires specific expertise, which makes it very difficult for less specialized physicians (such as general practitioners) to diagnose PCOS in clinical routine. For example, the determination of PCOM by transvaginal ultrasound requires an appropriate ultrasound device and a subjective analysis of the ultrasound images by a physician. Furthermore, the results can also depend on the specific ultrasound device used for the assessment of PCOM. As a result, the diagnosis of PCOS based on the Rotterdam criteria always includes at least one subjective, device- and operator-dependent, error-prone measurement.

[0011] To assess biochemical hyperandrogenemia, there must be a well-established normal range for the androgens measured. Testosterone is the most abundantly measured androgen in its total, bound, and free forms. There are limitations to the methods for measuring free testosterone. Direct measurement of free testosterone using radioimmunoassay is very inaccurate and does not reflect the true value. The assay has high variability within and between assays. Alternatively, higher accuracy can be obtained by measuring total testosterone concentration using extraction and chromatography, or gas (GC-MS) or liquid (LC-MS) chromatography–mass spectrometry, especially for clinical studies. The diagnostic performance of measuring serum testosterone can be enhanced by the simultaneous measurement of SHBG such that the calculation of free T concentration from total testosterone and SHBG levels requires only the solution of a quadratic equation (Azziz R, Carmina E, Dewailly D, et al. Task Force on the Phenotype of the Polycystic Ovary Syndrome of The Androgen Excess and PCOS Society. The Androgen Excess and PCOS Society criteria for the polycystic ovary syndrome: the complete task force report. Fertil Steril. 2009;91(2):456-88). The definition of HA may vary according to ethnicity. An mFG score of more than 8 for diagnosing hirsutism in women with PCOS is not appropriate for diagnosis in all ethnic groups. Women of East Asian origin have a lower prevalence of hirsutism compared with white people, and a score of more than 5 has been proposed to define hirsutism in Chinese women.There are also signs that androgen levels in the blood vary among ethnic groups. Here, the Japanese population has a low prevalence of increased androgen production, and testosterone is only recommended as a complementary factor in the diagnosis of PCOS in this population (Huang Z, Yong EL. Ethnic differences: Is there an Asian phenotype for polycystic ovarian syndrome? Best Pract Res Clin Obstet Gynaecol. 2016;37:46 - 55; Kubota T. Update in polycystic ovary syndrome: new criteria of diagnosis and treatment in Japan. Reprod Med Biol. 2013;12(3):71 - 77).

[0012] Patients with PCOS can be classified into four different phenotypes named A, B, C, or D (Neven ACH, Laven J, Teede HJ, Boyle JA. A Summary on Polycystic Ovary Syndrome: Diagnostic Criteria, Prevalence, Clinical Manifestations, and Management According to the Latest International Guidelines. Semin Reprod Med. 2018 Jan;36(1):5 - 12). Phenotype A is characteristic of patients with hyperandrogenemia, ovulatory dysfunction and / or irregular cycles, and polycystic ovarian morphology. Phenotype B is characterized by hyperandrogenemia, ovulatory dysfunction, and / or irregular cycles. Phenotype C is characterized by hyperandrogenemia and polycystic ovarian morphology. Phenotype D is characterized by ovulatory dysfunction and / or irregular cycles and polycystic ovarian morphology.

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

[0014] Inconsistent diagnostic criteria, variable provider knowledge, and lack of consensus pose specific challenges to the diagnosis and care of women with PCOS. These factors contribute to inaccurate diagnoses, both underdiagnosis and overdiagnosis. This unfavorable diagnostic experience worsens affected women and limits timely opportunities for intervention to minimize associated co-morbidities, especially during the transition from pediatric to adult care (Witchel SF, Teede HJ, Pena AS. Curtailing PCOS. Pediatr Res. 2020;87(2):353-361). Additionally, timely diagnosis is crucial to prevent further metabolic complications in affected women, such as type 2 diabetes.

[0015] In the largest study of PCOS diagnostic experiences, many women reported delays in diagnosis and inadequate information. Before a diagnosis was established, over one-third of women reported more than 2 years (33.6%) and more than 3 healthcare providers (47.1%). Few were satisfied with their diagnostic experience (35.2%) or the information they received (15.6%). These gaps in early diagnosis, education, and support represent clear opportunities to improve the patient experience (Gibson-Helm M, Teede H, Dunaif A, Dokras A. Delayed Diagnosis and a Lack of Information Associated With Dissatisfaction in Women With Polycystic Ovary Syndrome. J Clin Endocrinol Metab. 2017;102(2):604-612).

[0016] Areas of particular interest in the diagnosis of PCOS are young women, i.e., adolescents and young women under 25 years of age, when the features of normal pubertal development overlap with adult diagnostic criteria. This makes the diagnosis controversial and difficult. Many of the manifestations used to diagnose PCOS progress over time and can change during the first few years after menarche. Normal pubertal physiological changes such as irregular menstrual cycles, acne, and PCOM overlap with adult PCOS diagnostic criteria. In adolescents and young adult women, PCOS is diagnosed when both OA and HA criteria are met. Pelvic ultrasound examination is not recommended in adolescents less than 8 years from menarche because of the high incidence of polycystic ovaries at this life stage (Pena AS, Witchel SF, Hoeger KM, Oberfield SE, Vogiatzi MG, Misso M, Garad R, Dabadghao P, Teede H. Adolescent polycystic ovary syndrome according to the international evidence-based guideline. BMC Med. 2020;18(1):72). Assessment of irregular menstrual cycles in adolescents can be difficult. Menstrual cycles are often irregular during adolescence. Immaturity of the hypothalamic-pituitary-ovarian axis during the first few years after menarche often results in anovulation and a possible lengthening of the cycle. However, 90% of cycles are in the range of 21 to 45 days, but short cycles less than 20 days and long cycles over 45 days can occur. By 3 years after menarche, 60 to 80% of menstrual cycles are 21 to 34 days in length, as typical in adults. Young women and their caregivers (e.g., parents or guardians) often have difficulty assessing what constitutes a normal menstrual cycle or bleeding pattern. Patients and their guardians may not know well what is normal, and patients may not inform their guardians of menstrual irregularities or loss of menstruation.Also, patients are often reluctant to discuss this topic with their guardians (ACOG Committee Opinion No. 651: Menstruation in Girls and Adolescents: Using the Menstrual Cycle as a Vital Sign. Obstet Gynecol. 2015;126(6):e143 - e146). Delayed diagnosis in adolescents and young women is often due to reluctance to diagnose at - risk adolescents because of puberty and fear of over - or under - diagnosis. This can lead to obesity and insulin resistance, as well as long - term complications such as anxiety or depression. Current guidelines for the diagnosis of PCOS in adolescents and young women define oligo - anovulation, irregular menstrual cycles, and hyperandrogenemia as criteria to improve diagnostic accuracy in this patient group (Pena AS, Witchel SF, Hoeger KM, et al. Adolescent polycystic ovary syndrome according to the international evidence - based guideline. BMC Med. 2020;18(1):72). The guidelines also recommend re - evaluation of the diagnosis at 3 - year intervals and lifestyle changes to minimize symptoms and comorbidities associated with PCOS such as anxiety and depression. Therefore, the establishment of reliable biomarkers as an aid for the diagnosis of PCOS or the identification of patients at risk of developing PCOS is of utmost importance, especially for this patient group.

[0017] To date, there are no available universal biomarkers that are used alone or in combination with the above symptoms or the aforementioned hormone levels to assess whether a patient has or is at risk of developing PCOS, and / or to determine the response to treatment in patients with PCOS, and / or to monitor PCOS progression in patients, and / or to monitor the response to treatment in patients with PCOS.

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

[0019] In one aspect, the invention is a method of assessing whether a patient has PCOS or is at risk of developing PCOS, the method comprising: (a) determining the amount or concentration of LTA4H in a sample from the patient; and (b) comparing the determined amount or concentration to a reference.

[0020] In a second aspect, the invention is a method of selecting a patient for treatment of PCOS, the method comprising: (a) determining the amount or concentration of LTA4H in a sample from the patient; and (b) comparing the determined amount or concentration to a reference.

[0021] In a third aspect, the invention is a method of monitoring the progression of PCOS in a patient or the response of a patient having PCOS to treatment, the method comprising: (a) determining the level of LTA4H in a first sample from the patient; (b) determining the level of LTA4H in a second sample from the patient obtained after the first sample; (c) comparing the level of LTA4H in the first sample to the level of LTA4H in the second sample; and (d) monitoring the progression in a patient suffering from or being treated for PCOS based on the result of step (c).

[0022] In a fourth aspect, the invention is a computer-implemented method of assessing a patient suspected of having PCOS, the method comprising: (a) Receiving a value for the amount or concentration of a first biomarker in a patient's sample, wherein the first biomarker is LTA4H, the step of receiving a value for the amount or concentration of the first biomarker; (b) Optionally, receiving a value for the amount or concentration of a second biomarker in a patient's sample; (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 hyperandrogenemia and polycystic ovarian morphology; (d) Comparing the values for the amounts or concentrations in steps (a) - (b) with the criteria for the biomarker and the values for the presence or absence of at least one additional diagnostic criterion, and / or calculating a score for assessing a patient suspected of having PCOS based on the amount or concentration and values of the biomarker; (e) Assessing the patient based on the comparison and / or calculation performed in step (d), relating to a computer - implemented method.

Brief Description of the Drawings

[0023]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

DETAILED DESCRIPTION OF THE INVENTION

[0024] The inventors of the present invention have identified leukotriene A4 hydrolase (LTA4H) as a reliable biomarker for diagnosing polycystic ovary syndrome (PCOS) in patients, determining whether a patient is at risk of developing PCOS, determining the response to treatment in PCOS patients, or monitoring the progression of PCOS in patients with PCOS, or monitoring the response to treatment in patients with PCOS. LTA4H can be used alone or in combination with at least additional criteria such as hyperandrogenemia, oligo-anovulation, PCOM or irregular cycles for diagnosis, risk assessment and / or monitoring of the response to treatment in patients. Furthermore, determination of the level of LTA4H compared to a control level can be used to monitor the response to treatment and / or monitor the progression of PCOS in the patient.

[0025] The inventors have for the first time shown that the LTA4H value measured in a sample, preferably a biological fluid sample (preferably blood, plasma, serum, capillary blood, interstitial fluid, ascites, or menstrual fluid, and more preferably the biological fluid sample is blood, plasma or serum), is increased in women suffering from PCOS compared to controls. Further, the inventors have for the first time shown that the LTA4H value measured in a sample, preferably a biological fluid sample (preferably blood, plasma, serum, capillary blood, interstitial fluid, ascites, or menstrual fluid, and more preferably the biological fluid sample is blood, plasma or serum), is increased in females suffering from any of the phenotypes A - D of PCOS. The solution provided by the present invention is an immunoassay for detecting leukotriene A4 hydrolase in a sample, preferably a biological fluid sample (preferably blood, plasma, serum, capillary blood, interstitial fluid, ascites, or menstrual fluid, and more preferably the biological fluid sample is blood, plasma or serum). This immunoassay can be used in combination with other clinical and / or biochemical features, such as oligo - anovulation and / or irregular cycles, hyperandrogenemia or PCOM, to diagnose females having PCOS. Further, the measurement of the LTA4H value can be used to monitor the progression of PCOS in the patient and the response to treatment. The inventors have also shown that the measurement of the LTA4H value in a sample, preferably a biological fluid sample (preferably blood, plasma, serum, capillary blood, interstitial fluid, ascites, or menstrual fluid, and more preferably the biological fluid sample is blood, plasma or serum), is particularly suitable for the diagnosis of PCOS in adolescents or young women under 25 years old, especially under 20 years old, particularly under 15 - 25 years old, especially under 15 - 20 years old, either alone or in combination with the above - mentioned additional diagnostic criteria.

[0026] There is an unmet medical need for accurate tests for the reliable diagnosis of PCOS. Measurement of LTA4H in a sample, preferably a biological fluid sample (preferably blood, plasma, serum, capillary blood, interstitial fluid, ascites, or menstrual fluid, and more preferably the biological fluid sample is blood, plasma or serum), has the advantage of a highly reliable body fluid-based test for identifying females suffering from currently undiagnosable PCOS. Measurement of LTA4H in a sample, preferably a biological fluid sample (preferably blood, plasma, serum, capillary blood, interstitial fluid, ascites or menstrual fluid, and more preferably the biological fluid sample is blood, plasma or serum), can surely be used for the diagnosis of PCOS even in young patients and young females under 25 years old, particularly under 20 years old, particularly under 15 to 25 years old, particularly under 15 to 20 years old. Diagnosis of PCOS in young patients is difficult for the above reasons, and thus, the inventors provide for the first time an accurate test for the diagnosis of PCOS in young and young female populations. Further, measurement of LTA4H in a sample, preferably a biological fluid sample (preferably blood, plasma, serum, capillary blood, interstitial fluid, ascites, or menstrual fluid, and more preferably the biological fluid sample is blood, plasma or serum), has the advantage of identifying whether a patient responds to treatment. A further advantage of measurement of LTA4H in a patient's sample, preferably a biological fluid sample (preferably blood, plasma, serum, capillary blood, interstitial fluid, ascites, or menstrual fluid, and more preferably the biological fluid sample is blood, plasma or serum), is to monitor the progression of PCOS.Furthermore, the inventors provide a computer-implemented method for assessing a patient suffering from PCOS, the method including measuring the level of LTA4H in a sample, preferably a biological fluid sample (preferably blood, plasma, serum, capillary blood, interstitial fluid, ascites, or menstrual fluid, more preferably blood, plasma or serum), optionally together with further criteria such as values for oligo-anovulation and / or irregular cycles, hyperandrogenemia and / or polycystic ovarian morphology, or together with further biomarkers or hormones, for assessing the patient based on the comparison and / or calculation of said data, thereby including a computer-implemented method for assessing a patient suffering from PCOS.

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

[0028] Leukotriene A4 hydrolase (LTA4H) is part of the 5-lipoxygenase (5-LO) pathway that converts arachidonic acid (AA) into pro-inflammatory leukotrienes and anti-inflammatory lipoxins. LTA4H is widely expressed in various organs, tissues, and individual cell types (Haeggstrom JZ. Leukotriene A4 hydrolase and the committed step in leukotriene B4 biosynthesis. Clin Rev Allergy Immunol. 1999 Spring-Summer;17(1-2):111-31). According to the Human Protein Atlas, moderate expression of LTA4H is detected in normal ovarian tissue (http: / / www.proteinatlas.org). The expression pattern of LTA4H has been investigated in human ovarian tissue, revealing weak and moderate expression in preovulatory follicles on granulosa and theca interna cells, respectively. After ovulation, the intensity of LTA4H on large luteal cells increased and was highest in the mid-luteal phase. High expression was also observed in the corpus luteum in early pregnancy (Hattori N, Fujiwara H, Maeda M, et al. Human large luteal cells in the menstrual cycle and early pregnancy express leukotriene A4 hydrolase. Mol Hum Reprod. 1998 Aug;4(8):803-10). LTA4H is a unique bifunctional zinc metalloenzyme with both epoxide hydrolase (intracellular) and aminopeptidase (extracellular) activities.As an epoxide hydrolase, LTA4H catalyzes the final step involved in the biosynthesis of leukotriene B4 (LTB4), an eicosanoid with potent chemotactic and pro-inflammatory properties (Serhan CN. Resolution phase of inflammation: novel endogenous anti-inflammatory and proresolving lipid mediators and pathways. Annual review of immunology. 2007;25:101-137). Leukotrienes are a family of eicosanoids that function as potent chemical mediators in various allergic and inflammatory responses. New data suggest that leukotrienes may have an important role in carcinogenesis.The LTB4 level is increased in several human cancers, including skin cancer, lung cancer, colon cancer, and prostate cancer (Dreyling KW, Hoppe U, Peskar BA, et al Leukotriene synthesis by human gastrointestinal tissues. Biochim Biophys Acta. 1986 Sep 12;878(2):184 - 93; Chen X, Wang S, Wu N, Yang CS. Leukotriene A4 hydrolase as a target for cancer prevention and therapy. Curr Cancer Drug Targets. 2004 May;4(3):267 - 83; Wang Q, He Z, Zhang J, et al. Overexpression of endoplasmic reticulum molecular chaperone GRP94 and GRP78 in human lung cancer tissues and its significance. Cancer Detect Prev. 2005;29(6):544 - 51; Larre S, Tran N, Fan C, et al. PGE2 and LTB4 tissue levels in benign and cancerous prostates. Prostaglandins Other Lipid Mediat. 2008 Dec;87(1 - 4):14 - 9; Oi N, Yamamoto H, Langfald A, et al. LTA4H regulates cell cycle and skin carcinogenesis. Carcinogenesis. 2017 Jul 1;38(7):728 - 737).The expression of the LTB4 receptor is increased in human pancreatic cancer (Hennig R, Ding XZ, Tong WG, et al. 5-Lipoxygenase and leukotriene B(4) receptor are expressed in human pancreatic cancers but not in pancreatic ducts in normal tissue. Am J Pathol. 2002 Aug;161(2):421-8). LTB4 expression is also increased in HRAS-v12 transformed cells, and the receptor BLT2 is required for RAS-induced transformation in vivo (Yoo MH, Song H, Woo CH, et al. Role of the BLT2, a leukotriene B4 receptor, in Ras transformation. Oncogene. 2004 Dec 9;23(57):9259-68). Inhibition of LTB4 synthesis by treatment with bestatin, an LTA4H inhibitor, decreased tumor formation in in vivo models of esophageal adenocarcinoma and colorectal cancer (Chen X, Li N, Wang S, et al. Leukotriene A4 hydrolase in rat and human esophageal adenocarcinomas and inhibitory effects of bestatin. J Natl Cancer Inst. 2003 Jul 16;95(14):1053-61; Zhao S, Yao K, Li D, et al. Inhibition of LTA4H by bestatin in human and mouse colorectal cancer. EBioMedicine. 2019 Jun;44:361-374).Furthermore, topical application of LTB4 to the skin resulted in not only inflammation but also substantial hyperplasia of the epidermis (Bauer RW, van der Kerhof PC, de Grood RM. Epidermal hyperproliferation following the induction of microabscesses by leukotriene B4. Br J Dermatol 1986;114:409-12; Ruzicka T, Burg G. Effects of chronic intracutaneous administration of arachidonic acid and its metabolites. Induction of leukocytoclastic vasculitis by leukotriene B4 and 12-hydroxyeicosatetraenoic acid and its prevention by prostaglandin E2. J Invest Dermatol 1987;88:120-3). The expression of 5-lipoxygenase of the hypoxia marker HIF1α and the macrophage marker CD68 was positively correlated in ovarian cancer tissues. Furthermore, hypoxic ovarian cancer cell lines showed increased production of LTA4H and 5-LOX metabolites involved in enhancing the infiltration of tumor-associated macrophages (Wen Z, Liu H, Li M, et al. Increased metabolites of 5-lipoxygenase from hypoxic ovarian cancer cells promote tumor-associated macrophage infiltration. Oncogene. 2015 Mar 5;34(10):1241-52).LTA4H can also counteract inflammation through its aminopeptidase activity, which is inactivated by cleaving the tripeptide Pro-Gly-Pro (PGP) (Stsiapanava A, Olsson U, Wan M, et al. Binding of Pro-Gly-Pro at the active site of leukotriene A4 hydrolase / aminopeptidase and development of an epoxide hydrolase selective inhibitor. Proc Natl Acad Sci USA. 2014 Mar 18;111(11):4227-32). PGP is a biomarker for chronic obstructive pulmonary disease (COPD) and promotes neutrophil accumulation (Snelgrove RJ, Jackson PL, Hardison MT, et al. A critical role for LTA4H in limiting chronic pulmonary neutrophilic inflammation. Science. 2010 Oct 1;330(6000):90-4). Serum LTA4H has recently been suggested as a potential biomarker for predicting the efficacy of allergen immunotherapy for the treatment of allergic rhinitis (Ma TT, Cao MD, Yu RL, et al. Leukotriene A4 Hydrolase Is a Candidate Predictive Biomarker for Successful Allergen Immunotherapy. Front Immunol. 2020 Nov 24;11:559746).Higher expression of LTA4H due to LTA4H gene variants was associated with increased survival during glucocorticoid treatment in patients with tuberculous meningitis having moderate disease (Whitworth L, Coxon J, van Laarhoven A, et al. A Bayesian analysis of the association between Leukotriene A4 Hydrolase genotype and survival in tuberculous meningitis. Elife. 2021 Jan 8;10:e61722).

[0029] In a first aspect of the present invention, a method for assessing whether a patient has PCOS or is at risk of developing PCOS, comprising: (a) determining the amount or concentration of LTA4H in a sample from the patient; and (b) comparing the determined amount or concentration with a reference. The present invention relates to such a method.

[0030] An increase in the amount or concentration of LTA4H in a sample from the patient indicates the presence, risk or development of PCOS in the patient. In particular, the amount or concentration of LTA4H in a sample from the patient indicates the presence or risk of development of PCOS in the patient if the amount or concentration of LTA4H in the sample from the patient is higher than the amount or concentration of LTA4H in the reference or reference sample. In particular, LTA4H is detectable in a biological fluid sample from a patient assessed for the presence or risk of development of PCOS in a greater amount or concentration than in the same biological fluid sample from an individual not suffering from PCOS or not at risk of developing PCOS. In particular, an amount or concentration of LTA4H increased by 50% or more indicates the presence or risk of development of PCOS. In particular, an amount or concentration of LTA4H increased by 100% or more indicates the presence or risk of development of PCOS. In particular, an amount or concentration of LTA4H increased by 150% or more indicates the presence or risk of development of PCOS. In particular, an amount or concentration of LTA4H increased by 200% or more indicates the presence or risk of development of PCOS.

[0031] In an embodiment, the biological fluid sample is whole blood, serum, plasma, capillary blood, interstitial fluid, ascites, or menstrual fluid, and preferably, the biological fluid sample is serum or whole blood. In an embodiment, the sample is an in vitro sample, i.e., it is analyzed in vitro and not returned to the body.

[0032] In certain embodiments, the patient is an experimental animal, a domestic animal, or a primate. In certain embodiments, the patient is a human patient. In certain embodiments, the patient is a human female patient. In certain embodiments, the patient is a human female patient under 25 years old. In certain embodiments, the patient is a human female patient under 20 years old. In certain embodiments, the patient is a human female patient between 15 and under 25 years old. In certain embodiments, the patient is a human female patient between 15 and under 20 years old. In certain embodiments, the patient is a human female patient under 25 years old and within 3 years after menarche. In certain embodiments, the patient is a human female patient under 20 years old and within 3 years after menarche. In certain embodiments, the patient is a human female patient between 15 and under 25 years old and within 3 years after menarche. In certain embodiments, the patient is a human female patient between 15 and under 20 years old and within 3 years after menarche.

[0033] In an embodiment, PCOS is evaluated from the group consisting of metabolic or phenotypic PCOS. In a further aspect, PCOS is evaluated from the group consisting of phenotypic A, phenotypic B, phenotypic C, and phenotypic D PCOS.

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

[0035] In embodiments, the amount or concentration of LTA4H is determined using an antibody, in particular a monoclonal antibody. In embodiments, step a) of determining the amount or concentration of LTA4H in a patient sample comprises performing an immunoassay. In embodiments, the immunoassay is performed in either a direct or indirect format. In embodiments, such immunoassays are selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), or immunoassays based on luminescence, fluorescence, chemiluminescence or electrochemiluminescence detection.

[0036] In certain embodiments, step a) of determining the amount or concentration of LTA4H in a patient sample comprises i) incubating the patient sample with one or more antibodies that specifically bind to LTA4H, thereby generating a complex of the antibody and LTA4H, and ii) quantifying the complex formed in step i), thereby quantifying the amount or concentration of LTA4H in the patient sample.

[0037] In certain embodiments, in step i), the sample is incubated with two antibodies that specifically bind to LTA4H. As will be apparent to those skilled in the art, the sample can be contacted with the first anti-LTA4H antibody, then the second antibody, or the second antibody first and then the first antibody, or the first antibody and the second antibody simultaneously, in any desired order, for a time and under conditions sufficient to form a first anti-LTA4H antibody / LTA4H / second anti-LTA4H antibody complex. As will be readily appreciated by those skilled in the art, establishing the time and conditions appropriate or sufficient for either the formation of a complex between a specific anti-LTA4H antibody and an LTA4H antigen / analyte (= anti-LTA4H complex), or the formation of a secondary or sandwich complex comprising a first anti-LTA4H antibody, LTA4H (analyte) and a second anti-LTA4H antibody (= anti-LTA4H antibody / LTA4H / second anti-LTA4H antibody complex) is routine experimentation.

[0038] The detection of the anti-LTA4H antibody / LTA4H complex can be carried out by any suitable means. The detection of the first anti-LTA4H antibody / LTA4H / second anti-LTA4H antibody complex can be performed by any suitable means. Those skilled in the art are fully proficient in such means / methods.

[0039] In certain embodiments, a sandwich is formed that includes a first antibody to LTA4H, LTA4H (the analyte), and a second antibody to LTA4H, and the second antibody is detectably labeled.

[0040] In one embodiment, a sandwich is formed that includes a first antibody to LTA4H, LTA4H (the analyte), and a second antibody to LTA4H, and the second antibody is detectably labeled, and the first anti-LTA4H antibody can bind to or is bound to a solid phase.

[0041] In embodiments, the second antibody is detectably labeled either directly or indirectly. In certain embodiments, the second antibody is detectably labeled with a luminescent dye, particularly a chemiluminescent dye or an electrochemiluminescent dye.

[0042] In a second aspect, the invention is a method of selecting a patient for the treatment of PCOS, comprising: (a) determining the amount or concentration of LTA4H in a sample from the patient; and (b) comparing the determined amount or concentration to a reference.

[0043] In an embodiment, when an increase in the amount of LTA4H in a patient's sample is determined, the patient is selected for the treatment of PCOS. In particular, when the amount of LTA4H is more than that in a reference sample or the amount of LTA4H in the reference sample, the patient is selected for the treatment of PCOS. In particular, when the amount of LTA4H is higher in a biological fluid sample of a patient evaluated for the treatment of PCOS than in the same biological fluid sample of an individual who does not have PCOS, is not at risk of developing PCOS, or has not been selected for the treatment of PCOS, the patient is selected for the treatment of PCOS. In particular, when the amount of LTA4H has increased by 50% or more, the patient is selected for the treatment of PCOS. In particular, when the amount of LTA4H has increased by 100% or more, the patient is selected for the treatment of PCOS. In particular, when the amount of LTA4H has increased by 150% or more, the patient is selected for the treatment of PCOS. In particular, when the amount of LTA4H has increased by 200% or more, the patient is selected for the treatment of PCOS.

[0044] In particular, the patient is selected for drug-based treatment of PCOS or lifestyle changes to control metabolic syndrome. In an embodiment, the drug-based therapy for PCOS includes drugs for regulating the period, 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, drugs for enhancing hormones or fertility, drugs or treatments for removing excessive hair, and drugs or treatments for controlling acne, selected from the group consisting of.

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

[0046] In certain embodiments, the patient is an experimental animal, a domestic animal or a primate. In certain embodiments, the patient is a human patient. In certain embodiments, the patient is a human female patient. In certain embodiments, the patient is a human female patient under 25 years of age. In certain embodiments, the patient is a human female patient under 20 years of age. In certain embodiments, the patient is a human female patient between 15 and less than 25 years of age. In certain embodiments, the patient is a human female patient between 15 and less than 20 years of age. In certain embodiments, the patient is a human female patient under 25 years of age and within 3 years after menarche. In certain embodiments, the patient is a human female patient under 20 years of age and within 3 years after menarche. In certain embodiments, the patient is a human female patient between 15 and less than 25 years of age and within 3 years after menarche. In certain embodiments, the patient is a human female patient between 15 and less than 20 years of age and within 3 years after menarche.

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

[0048] In an embodiment, the amount of LTA4H is determined using an antibody, particularly a monoclonal antibody. In an embodiment, step a) of determining the amount or concentration of LTA4H in a patient sample comprises performing an immunoassay. In an embodiment, the immunoassay is performed in either a direct or indirect format. In an embodiment, such immunoassays are selected from the group consisting of an enzyme-linked immunosorbent assay (ELISA), an enzyme immunoassay (EIA), a radioimmunoassay (RIA), or an immunoassay based on the detection of luminescence, fluorescence, chemiluminescence or electrochemiluminescence.

[0049] In certain embodiments, step a) of determining the amount or concentration of LTA4H in a patient sample comprises i) incubating the patient sample with one or more antibodies that specifically bind to LTA4H, thereby generating a complex of the antibody and LTA4H, and ii) quantifying the complex formed in step i), thereby quantifying the amount of LTA4H in the patient sample.

[0050] In certain embodiments, in step i), the sample is incubated with two antibodies that specifically bind to LTA4H. As will be apparent to those skilled in the art, the sample can be contacted with the first anti-LTA4H antibody / LTA4H / second anti-LTA4H antibody complex in any desired order, for example, first with the first antibody and then the second antibody, or first with the second antibody and then the first antibody, or simultaneously with the first and second antibodies, for a time and under conditions sufficient to form the complex. As will be readily understood by those skilled in the art, establishing the time and conditions appropriate or sufficient for either the formation of the complex between the specific anti-LTA4H antibody and the LTA4H antigen / analyte (= anti-LTA4H complex), or the formation of the secondary or sandwich complex comprising the first anti-LTA4H antibody, LTA4H (analyte) and the second anti-LTA4H antibody (= anti-LTA4H antibody / LTA4H / second anti-LTA4H antibody complex) is routine experimentation.

[0051] The detection of the anti-LTA4H antibody / LTA4H complex can be performed by any suitable means. The detection of the first anti-LTA4H antibody / LTA4H / second anti-LTA4H antibody complex can be carried out by any suitable means. Those skilled in the art are fully conversant with such means / methods.

[0052] In certain embodiments, a sandwich is formed comprising a first antibody to LTA4H, LTA4H (analyte) and a second antibody to LTA4H, and the second antibody is detectably labeled.

[0053] In one embodiment, a sandwich is formed comprising a first antibody to LTA4H, LTA4H (analyte) and a second antibody to LTA4H, the second antibody is detectably labeled, and the first anti-LTA4H antibody can bind to or is bound to a solid phase.

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

[0055] In a third aspect, the present invention is a method of monitoring the progression of PCOS in a patient or monitoring the response to treatment of a patient having PCOS, comprising: (a) determining the level of LTA4H in a first sample of the patient; (b) determining the level of LTA4H in a second sample of the patient obtained after the first sample; (c) comparing the level of LTA4H in the first sample with the level of LTA4H in the second sample; (d) monitoring the progression in a patient suffering from or being treated for PCOS based on the result of step (c).

[0056] In an embodiment, the progression of PCOS in a patient having PCOS is monitored to determine whether the amount or concentration of LTA4H in the patient's sample changes over time. In particular, the progression of PCOS is monitored to determine whether the amount or concentration of LTA4H increases, decreases, or does not change over time. In an embodiment, when an increase in the amount or concentration of LTA4H in the patient's sample is determined, the progression of PCOS is monitored.

[0057] In embodiments, patients undergoing treatment for PCOS are monitored to determine whether the amount or concentration of LTA4H in the patient's sample has changed. In particular, patients undergoing treatment for PCOS are monitored to determine whether the amount or concentration of LTA4H has increased, decreased, or remained unchanged. In particular, patients undergoing treatment for PCOS are monitored to determine whether the amount or concentration of LTA4H has increased, decreased, or remained unchanged due to the applied treatment. In embodiments, a decrease in the amount or concentration of LTA4H in a patient undergoing treatment for PCOS indicates that the treatment is effective. In embodiments, no change or an increase in the amount or concentration of LTA4H in a sample from a patient being treated for PCOS indicates that PCOS persists. In particular, if the amount or concentration of LTA4H has increased by 50% or more, the treatment of PCOS is ineffective. In particular, if the amount or concentration of LTA4H has increased by 100% or more, the treatment of PCOS is ineffective. In particular, if the amount or concentration of LTA4H has increased by 150% or more, the treatment of PCOS is ineffective. In particular, if the amount or concentration of LTA4H has increased by 200% or more, the treatment of PCOS is ineffective.

[0058] In certain embodiments, if an unchanged or increased amount or concentration of LTA4H in a sample from a patient being treated for PCOS is determined, the treatment is adapted.

[0059] In embodiments, the patient is monitored several times at different time points. In embodiments, the patient is monitored several times within a time frame of weeks, months, or years. In certain embodiments, the patient is monitored once a month or once a year. In embodiments, a patient suffering from PCOS is monitored once a month or once a year after diagnosis of PCOS. In embodiments, a patient being treated for PCOS is monitored once after treatment. In particular, a patient being treated for PCOS is monitored once a month or once a year to determine the effectiveness of the treatment.

[0060] In embodiments, the treatment of PCOS is selected from the group consisting of drug-based treatment of PCOS and lifestyle changes to control metabolic syndromes. In embodiments, the drug-based therapy for PCOS includes drugs for regulating the period, particularly oral contraceptives or progestin therapy, drugs for preventing or controlling diabetes, particularly type 2 diabetes, drugs for preventing or controlling high cholesterol, drugs for enhancing hormones or fertility, drugs or treatments for removing excessive hair, and drugs or treatments for controlling acne, and is selected from the group consisting of these.

[0061] In embodiments, the biological fluid sample is whole blood, serum, plasma, capillary blood, interstitial fluid, ascites, or menstrual fluid, and preferably, the biological fluid sample is serum or whole blood. In embodiments, the sample is an in vitro sample, that is, it is analyzed in vitro and not returned to the body.

[0062] In certain embodiments, the patient is an experimental animal, a livestock, or a primate. In certain embodiments, the patient is a human patient. In certain embodiments, the patient is a human female patient. In certain embodiments, the patient is a human female patient under 25 years old. In certain embodiments, the patient is a human female patient under 20 years old. In certain embodiments, the patient is a human female patient aged from 15 to less than 25 years old. In certain embodiments, the patient is a human female patient aged from 15 to less than 20 years old. In certain embodiments, the patient is a human female patient under 25 years old and within 3 years after menarche. In certain embodiments, the patient is a human female patient under 20 years old and within 3 years after menarche. In certain embodiments, the patient is a human female patient aged from 15 to less than 25 years old and within 3 years after menarche. In certain embodiments, the patient is a human female patient aged from 15 to less than 20 years old and within 3 years after menarche.

[0063] In embodiments, the second method of the present invention is an in vitro method.

[0064] In embodiments, the amount or concentration of LTA4H is determined using an antibody, in particular a monoclonal antibody. In embodiments, step a) of determining the amount or concentration of LTA4H in a patient sample comprises performing an immunoassay. In embodiments, the immunoassay is performed in either a direct or indirect format. In embodiments, such immunoassays are selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), or immunoassays based on the detection of luminescence, fluorescence, chemiluminescence or electrochemiluminescence.

[0065] In certain embodiments, step a) of determining the amount or concentration of LTA4H in a patient sample comprises i) incubating the patient sample with one or more antibodies that specifically bind to LTA4H, thereby generating a complex of the antibody and LTA4H, and ii) quantifying the complex formed in step i), thereby quantifying the amount or concentration of LTA4H in the patient sample.

[0066] In certain embodiments, in step i), the sample is incubated with two antibodies that specifically bind to LTA4H. As will be apparent to those skilled in the art, the sample can be contacted with the first anti-LTA4H antibody, then the second antibody, or the second antibody first and then the first antibody, or the first antibody and the second antibody simultaneously, in any desired order, for a time and under conditions sufficient to form a first anti-LTA4H antibody / LTA4H / second anti-LTA4H antibody complex. As will be readily appreciated by those skilled in the art, establishing the time and conditions appropriate or sufficient for either the formation of a complex between a specific anti-LTA4H antibody and an LTA4H antigen / analyte (= anti-LTA4H complex), or the formation of a secondary or sandwich complex comprising a first anti-LTA4H antibody, LTA4H (analyte) and a second anti-LTA4H antibody (= anti-LTA4H antibody / LTA4H / second anti-LTA4H antibody complex) is routine experimentation.

[0067] The detection of the anti-LTA4H antibody / LTA4H complex can be performed by any suitable means. The detection of the first anti-LTA4H antibody / LTA4H / second anti-LTA4H antibody complex can be carried out by any suitable means. Those skilled in the art are fully proficient in such means / methods.

[0068] In certain embodiments, a sandwich is formed that includes a first antibody to LTA4H, LTA4H (the analyte), and a second antibody to LTA4H, and the second antibody is detectably labeled.

[0069] In one embodiment, a sandwich is formed that includes a first antibody to LTA4H, LTA4H (the analyte), and a second antibody to LTA4H, the second antibody is detectably labeled, and the first anti-LTA4H antibody can bind to or is bound to a solid phase.

[0070] In embodiments, the second antibody is detectably labeled either directly or indirectly. In certain embodiments, the second antibody is detectably labeled with a luminescent dye, particularly a chemiluminescent dye or an electrochemiluminescent dye.

[0071] In a fourth aspect, the present invention is a computer-implemented method for assessing a patient suspected of having PCOS, comprising: (a) receiving a value for the amount or concentration of a first biomarker in a sample from the patient, wherein the first biomarker is LTA4H; (b) optionally, receiving a value for the amount or concentration of a second biomarker in a sample from the patient; (c) optionally, receiving a value for the presence or absence of at least one additional diagnostic criterion selected from the group consisting of oligo-ovulation and hyperandrogenemia and polycystic ovarian morphology; (d) comparing a value for the quantity or concentration of steps (a) to (b) with a value for the presence or absence of a reference for the biomarker and at least one additional diagnostic criterion, and / or calculating a score for assessing a patient suspected of having PCOS based on the quantity or concentration and value of the biomarker; (e) assessing the patient based on the comparison and / or calculation performed in step (d), relating to a computer-implemented method.

[0072] In an embodiment, a computer-implemented method for assessing a patient suspected of having PCOS includes a method that consists essentially of a method including the aforementioned steps or further steps. Further, the method of the present invention is preferably an ex vivo method, more preferably an in vitro method. Further, the method of the present invention may include steps in addition to those explicitly described above. For example, further steps may relate to further determining a marker and / or collecting a pre-treatment sample or evaluating the results obtained by the above method. The method may be performed manually or assisted by automation.

[0073] As used herein, the term "computer-implemented" means that the method is typically executed in an automated manner on a data processing unit included in a computer or similar data processing device. The data processing unit receives a value for the quantity of the biomarker. Such a value can be a quantity, relative quantity or any other calculated value that reflects the quantities described in detail elsewhere in this specification. Thus, it should be understood that the method described above does not require the determination of the quantity of the biomarker, but rather uses values for quantities that have already been predetermined.

[0074] The present invention also generally contemplates a computer program, a computer program product, or a computer-readable storage medium having the computer program tangibly incorporated therein, where the computer program, when executed on a data processing device or computer, includes instructions for performing the method of the present invention as specified above. Specifically, the present disclosure further encompasses the following: - A computer or computer network comprising at least one processor, the processor being configured to execute a method according to one of the embodiments described herein. - A computer-loadable data structure configured to execute a method according to one of the embodiments described herein when executed on a computer. - A computer script, where the computer program is adapted to execute one of the methods of the embodiments described herein while the program is being executed on a computer. - A computer program comprising program means for executing a method according to one of the embodiments described herein when the computer program is executed on a computer or a computer network. - A computer program having the program means described in the preceding embodiments stored on a computer-readable storage medium. - A storage medium having a data structure stored thereon, the data structure being adapted to execute a method according to one of the embodiments described herein after being loaded into the main memory device and / or the working memory device of a computer or a computer network. - A computer program product having program code means that can be stored or stored on a storage medium for executing a method according to one of the embodiments described herein when the program code means are executed on a computer or a computer network. - Data including data on parameters defined elsewhere in this specification, typically encrypted, data stream signals, and - Data stream signals typically encrypted, including the assessments provided by the method of the present invention.

[0075] Furthermore, the present invention also relates to a kit comprising reagents for the diagnosis of PCOS. The reagents of the kit may include antibodies or antibody fragments. Preferably, the antibody or antibody fragment recognizes an epitope or antigen of LTA4H. The kit may further include other reagents that recognize other biomarkers. Thus, the kit may include a combination of at least two reagents. The kit may specifically measure the amount or concentration of LTA4H and any other biomarker of interest. According to the present invention, the biomarker can also include hormones, such as Anti-Mullerian Hormone (AMH). The kit can be used in any diagnostic assay.

[0076] Definition: In relation to the kit of the present invention, the term "reagent" represents a substance or compound added to a sample that enables the indication of the amount or concentration of a specific component in the sample.

[0077] In relation to the kit of the present invention, the term "specifically measure" means detecting the exact amount or concentration of a clearly defined molecule. For a specific measurement, a sample obtained from a female can be incubated with a reagent under conditions appropriate for the formation of a binding agent - marker complex. Such appropriate incubation conditions are well known to those skilled in the art, so it is not necessary to specify such conditions.

[0078] In relation to the kit of the present invention, the term "reagent" can represent a protein molecule (such as an antibody, etc.), a nucleic acid molecule (any form of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), etc.), or another biochemical, organic or inorganic substance that can interact with the molecule specifically measured in the sample.

[0079] Furthermore, the reagent can be linked to a detectable reporter moiety or label such as an enzyme, a dye, a radionuclide, a luminescent group, a fluorescent group or biotin, for example a fluorescent marker that can be used in immunoassay analysis. The reporter moiety or label can be used with the reagent of the kit according to the second aspect of the present invention as long as its signal can be directly related or proportional to the amount of the binder remaining on the support after washing. Then, the amount of any second binder remaining bound to the solid support can be determined using a method suitable for the specific detectable reporter moiety or label. In the case of a radionuclide, scintillation counting or autoradiography is generally appropriate. Antibody-enzyme conjugates can be prepared using various coupling techniques (for a review, see, for example, Scouten, W.H., Methods in Enzymology 135:30-65, 1987). Dyes (including, for example, the colorimetric products of enzyme reactions), luminescent groups and fluorescent groups can be detected using spectroscopy. Biotin can be detected by binding it to avidin or streptavidin and then to a different reporter group (usually a radioactive or fluorescent group or an enzyme). Enzyme reporter groups can generally be detected by addition of a substrate (usually for a specific period), followed by spectroscopic analysis, spectrophotometry or other analysis of the reaction product. Standards and standard additions can be used to determine the level of antigen in a sample using techniques well known to those skilled in the art.

[0080] The reagent may also be a substance that can potentially bind further to the matrix of a column used in chromatography for purification and / or further analysis (such as mass spectrometry). Furthermore, the reagent may be linked to a test strip.

[0081] Preferably, the reagent is an antibody. Suitable antibodies for measuring the amount or concentration of one of the specifically measured molecules in a sample obtained from the female as described above are well known to those skilled in the art.

[0082] Preferably, the reagent can be used in an electrochemiluminescence immunoassay, and more preferably, the reagent is an antibody that can be used in an electrochemiluminescence immunoassay.

[0083] Furthermore, the kit can include more than one reagent, such as two different reagents, three different reagents, four different reagents or more different reagents, preferably two different reagents that interact with one molecule specifically measured in the sample. For example, when the molecule specifically measured is measured by an electrochemiluminescence immunoassay, the kit can include two different antibodies that bind to the same molecule being measured. Preferably, the two different antibodies that bind to the same molecule do not compete for the binding site on the molecule and bind to this molecule at different positions. Furthermore, both antibodies can be linked to different detectable reporter moieties or labels.

[0084] The kit can further include a buffer and / or a salt for adjusting the pH as well as the reaction conditions and measurement conditions. Furthermore, the kit includes a stabilizer for supporting the stability of the reagent and / or the hormone during the specific measurement of, for example, (i) the amount or concentration of FT, or (ii) the amount or concentration of TT and the amount or concentration of SHBG, the amount or concentration of AMH, and the amount or concentration of one or more additional hormones indicating PCOS. Suitable buffers, salts and stabilizers are well known to those skilled in the art. Furthermore, sodium azide can be added to all liquid solutions of the kit such as the reagent or the buffer solution.

[0085] The kit may also include all the equipment necessary to collect a blood sample from a female, such as a container for the blood sample, a needle, and a device for connecting the container and the needle. Preferably, the kit can include a syringe.

[0086] Generally, a physician or a physician's assistant may collect blood from a female. Thereafter, the blood may be sent to a laboratory where the sample is measured using the kit with a designated analyzer and the data is sent to the physician. However, the kit may also be applied by the physician or the physician's assistant themselves. The kit can be applied during a physician's outpatient, stationary treatment or home visit.

[0087] All components of the kit may be separately packaged in individual containers. However, it is also possible that two or more components of the kit may be packaged together in one or more containers.

[0088] The kit may further comprise, for example, instructions regarding the use of the kit or a label containing instructions for the contents of the kit. However, this information may also be provided in any other form, such as on a storage medium like a CD-ROM or a USB stick.

[0089] It is to be understood that the word "comprise", and variations such as "comprises" and "comprising", mean the inclusion of the stated integer or step or group of integers or steps, but do not mean the exclusion of any other integer or step or group of integers or steps.

[0090] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0091] Concentrations, amounts, and other numerical data may be expressed or presented herein in the form of a "range". It is understood that such a range format is merely used for convenience and brevity, and thus, should be interpreted flexibly to include not only the numerical values explicitly recited as the boundaries of the range, but also all of the individual numerical values or sub-ranges subsumed within that range as if each were explicitly recited. By way of illustration, a numerical range of "150 mg to 600 mg" should be interpreted to include not only the explicitly recited values of 150 mg to 600 mg, but also the individual values and sub-ranges within the indicated range. Thus, this numerical range includes the individual values such as 150, 160, 170, 180, 190, 580, 590, 600 mg, etc., and sub-ranges such as 150 to 200, 150 to 250, 250 to 300, 350 to 600, etc. This same principle applies to ranges that enumerate only a single numerical value. Further, such interpretation should apply regardless of the width of the range or the property being described.

[0092] When used in relation to a numerical value, the term "about" means a value that encompasses a range having a lower limit that is 5% less than the indicated value and an upper limit that is 5% greater than the indicated value.

[0093] As used herein, the term "indicator" refers to a sign or signal of a symptom or is used to monitor a condition. Such "symptoms" refer to the biological condition of a cell, tissue or organ, or the health and / or disease state of an individual. An indicator can be, but is not limited to, the presence or absence of a molecule including a peptide, protein, and nucleic acid, or a change in the expression level or pattern of such a molecule in a cell, or tissue, organ or individual. An indicator can be a sign of the occurrence, onset or presence of a disease in an individual, or a sign of further progression of such a disease. An indicator can also be a sign of the risk of developing a disease in an individual.

[0094] In connection with the present invention, the term "biomarker" refers to a substance within a biological system that is used as an indicator of the biological state of the biological system. In the art, the term "biomarker" may also be applied to means for detecting such endogenous substances (e.g., antibodies, nucleic acid probes, etc., imaging systems). In connection with the present invention, the term "biomarker" shall be applied only to substances and not to detection means. Thus, a biomarker can be any kind of molecule present in a living body, such as nucleic acids (DNA, mRNA, miRNA, rRNA, etc.), proteins (cell surface receptors, cytosolic proteins, etc.), metabolites or hormones (blood glucose, insulin, estrogen, etc.), molecules characteristic of a specific modification of another molecule (e.g., sugar moieties or phosphoryl residues on a protein, methyl residues on genomic DNA), or substances internalized by an organism or metabolites of such substances.

[0095] As used herein, biomarkers can be detected using methods generally known in the art. Detection methods generally include methods for quantifying the level of a biomarker in a sample (quantitative methods). Which of the following methods is suitable for qualitative and / or quantitative detection of a biomarker is generally known to those skilled in the art. Samples can be readily assayed for proteins using, for example, Western methods as well as immunoassays such as ELISA, RIA, fluorescence- and luminescence-based immunoassays, and commercially available proximity extension assays. Further suitable methods for detecting biomarkers include measuring physical or chemical properties specific to a peptide or polypeptide, such as its exact molecular weight or NMR spectrum, etc. Such methods include, for example, analytical devices such as biosensors, optical devices associated with immunoassays, biochips, mass spectrometers, NMR analyzers, or chromatography devices. Further, the methods include methods based on microplate ELISA, fully automated or robotic immunoassays (available on Elecsys™ analyzers), CBA (Cobalt Binding Assay by enzyme, available on Roche-Hitachi™ analyzers), and latex agglutination assays (available on Roche-Hitachi™ analyzers).

[0096] The term "anovulation" typically refers to the condition where the ovaries do not release any oocytes during the female menstrual cycle. A female at risk of having PCOS can be determined to be suffering from anovulation if no oocytes are released over a period of at least one female menstrual cycle per year, preferably at least three female menstrual cycles per year, more preferably at least six female menstrual cycles per year, and most preferably at least nine female menstrual cycles per year. Further, a female at risk of having PCOS can be determined to be suffering from anovulation if no oocytes are released for at least six months, preferably at least nine months, more preferably at least one year.

[0097] The "symptoms" of a disease are significant hints of such a disease by the tissues, organs or organisms having such a disease, including, but not limited to, pain, weakness, tenderness, tension, stiffness and spasm of tissues, organs or individuals. Typical symptoms of PCOS include, but are not limited to, oligo-anovulation, irregular cycles, hyperandrogenemia, polycystic ovarian morphology, infertility, type 2 diabetes, overweight and other metabolic symptoms, as well as psychological distress. The "signs" or "signals" of a disease include, but are not limited to, the presence, increase or elevation, decrease or reduction, change or alteration such as changes in specific indicators such as biomarkers or molecular markers, or the onset, presence or exacerbation of symptoms. The symptoms of pain include, but are not limited to, unpleasant sensations that can be felt as persistent or various burning pains, throbbing pains, itching or stabbing pains.

[0098] The terms "disease" and "disorder" are used interchangeably herein and refer to abnormal medical conditions such as abnormal symptoms, particularly illnesses or injuries where a tissue, organ, or individual can no longer perform its function efficiently. Although not always the case, typically, a disease is associated with specific symptoms or signs that indicate the presence of such a disease. Thus, the presence of such symptoms or signs can indicate the tissue, organ, or individual suffering from the disease. Changes in these symptoms or signs can indicate the progression of such a disease. The progression of a disease typically features an increase or decrease in such symptoms or signs that can indicate a "worsening" or "improvement" of the disease. The "worsening" of a disease is characterized by a decrease in the ability of a tissue, organ, or organism to perform its function efficiently, whereas the "improvement" of a disease typically features an increase in the ability of a tissue, organ, or individual to perform its function efficiently. A tissue, organ, or individual at "risk of developing" a disease is in a healthy state but shows the potential for the disease to manifest. Typically, the risk of developing a disease is associated with early or weak symptoms or signs of such a disease. In such cases, the onset of the disease can still be prevented by treatment. Examples of diseases include, but are not limited to, inflammatory diseases, infectious diseases, skin conditions, endocrine diseases, intestinal diseases, neurological disorders, joint diseases, genetic disorders, autoimmune diseases, traumatic diseases, and various types of cancer.

[0099] The terms "patient" and "subject" are used interchangeably herein and refer to an animal, preferably a mammal, more typically a human. A patient is preferably a human female.

[0100] The terms "sample" or "target sample" are used interchangeably herein and refer to a part or piece of a tissue, organ or individual, and are usually smaller than such a tissue, organ or individual which is intended to represent the whole of the tissue, organ or individual. In the analysis, the sample provides information regarding the state of the tissue, or the health or disease state of the organ or individual. Examples of samples include, but are not limited to, fluid samples such as blood, serum, plasma, synovial fluid, urine, saliva, and lymphatic fluid, or solid samples such as tissue extracts, cartilage, bone, synovium, and connective tissue. The analysis of the sample can be achieved visually or chemically. Visual analysis includes, but is not limited to, microscopic imaging or radiation scanning of the tissue, organ or individual enabling morphological evaluation of the sample. Chemical analysis includes, but is not limited to, detection of the presence or absence of specific indicators or changes in their amount, concentration or level. The sample is an in vitro sample and will be analyzed in vitro and will not be returned to the body.

[0101] As used herein, the term "amount" includes the absolute amount of a biomarker referred to herein, the relative amount or concentration of the biomarker, and any value or parameter that can be correlated with or derived from them. Such values or parameters include intensity signal values derived from all specific physical or chemical properties obtained from the peptide by direct measurement, for example, intensity values in a mass spectrum or an NMR spectrum. Further included are values or parameters obtained by indirect measurements as specified elsewhere herein, for example, the amount of response measured by a biological readout system in response to the peptide, or the intensity signal obtained from a specifically bound ligand. It should be understood that values correlated with the above-described amount or parameter can also be obtained by all standard mathematical operations.

[0102] As used herein, the term "comparing" refers to comparing the amount of a biomarker in a sample from a subject to a reference amount of the biomarker as specified elsewhere herein. Comparing, as used herein, generally refers to comparing corresponding parameters or values. For example, an absolute amount is compared to a reference absolute amount, a concentration is compared to a reference concentration, or an intensity signal obtained from a biomarker in a sample is compared to the same type of intensity signal obtained from a reference sample. The comparison may be performed manually or using a computer. Thus, the comparison can be performed by a computing device. The measured or detected amount of a biomarker in a sample from a subject and the value of the reference amount can, for example, be compared to each other, and the comparison can be automatically performed by a computer program that executes an algorithm for the comparison. The computer program that performs the above evaluation provides the desired rating in a suitable output format. In a computer-assisted comparison, the value of the measured amount may be compared by a computer program to a value corresponding to a suitable reference stored in a database. The computer program may further evaluate the result of the comparison, i.e., it may automatically provide the desired rating in a suitable output format. In a computer-assisted comparison, the value of the measured amount may be compared by a computer program to a value corresponding to a suitable reference stored in a database. The computer program may further evaluate the comparison result, i.e., it may automatically provide the desired rating in a suitable output format.

[0103] The expression "comparing the determined amount or concentration to a reference" is used merely to further illustrate what will be apparent to one of ordinary skill in the art. The reference concentration is established in a control sample

[0104] The terms "reference sample" or "control sample", as used herein, refer to a sample that is analyzed in substantially the same manner as the sample of interest and whose information is compared to the information of the sample of interest. Thereby, the reference sample provides a reference by which the information obtained from the sample of interest can be evaluated. A control sample is derived from a healthy or normal tissue, organ, or individual, thereby providing a reference for the health status of the tissue, organ, or individual. The difference between the state of a normal reference sample and the state of the sample of interest can indicate the risk of disease onset or the presence or further progression of such a disease or disorder. A control sample can be derived from an abnormal or diseased tissue, organ, or individual, thereby providing a reference for the pathological state of the tissue, organ, or individual. The difference between the state of an abnormal reference sample and the state of the sample of interest can indicate a reduced risk of disease onset or the absence or improvement of such a disease or disorder. A reference sample can also be derived from the same tissue, organ, or individual as the sample of interest but was taken at an earlier time point. The difference between the state of a previously taken reference sample and the state of the sample of interest can indicate the progression of the disease, i.e., the improvement or worsening of the disease over time.

[0105] A control sample can be an internal or external control sample. An internal control sample is used, i.e., in a test sample as well as one or more other samples (s) taken from the same subject, to assess the marker level (s) to determine whether there is a change in the level (s) of the marker (s). For an external control sample, the presence or amount of a marker in a sample derived from an individual is compared to the presence or amount of the marker in an individual known to have or be at risk of having a given condition; or an individual known to not have a given condition (i.e., a "normal individual").

[0106] Those skilled in the art will understand that such external control samples may be obtained from a single individual or from a reference population of the same age and free of confounding diseases. Typically, a "reference value" is set using samples from 100 individuals sufficiently characterized from an appropriate reference population. However, the reference population can also be selected to consist of 20, 30, 50, 200, 500 or 1000 individuals. Healthy individuals are a preferred reference population for establishing control values.

[0107] For example, the marker concentration in a patient sample can be compared to a concentration known to be associated with a particular course of a particular disease. Usually, the marker concentration in the sample is directly or indirectly correlated with the diagnosis, and the marker concentration is used, for example, to determine whether an individual is at risk of a particular disease. Alternatively, the marker concentration of a sample can be compared to a marker concentration known to be relevant, for example, in response to treatment in a particular disease, diagnosis of a particular disease, assessment of the severity of a particular disease, guidance for selecting an appropriate drug for a particular disease, determination of the risk of disease progression, or patient follow-up. Depending on the intended diagnostic use, an appropriate control sample is selected and a control value or reference value for the marker is set therein. As will be apparent to those skilled in the art, the absolute marker value set with the control sample depends on the assay used.

[0108] As used herein, the term "assessing" refers to assessing whether a patient has PCOS or is at risk of developing PCOS. Thus, the assessments used herein include diagnosing PCOS, predicting the risk of developing PCOS, selecting a treatment for PCOS, monitoring patients with or being treated for PCOS, determining the amount or concentration of LTA4H in a patient's sample, and comparing the determined amount or concentration to a reference.

[0109] As will be understood by those skilled in the art, the assessments made in accordance with the present invention, while preferred, will not typically be correct for 100% of the subjects investigated. This term typically requires that a statistically significant portion of the subjects can be accurately assessed. Whether a portion is statistically significant can be readily determined by those skilled in the art using various well-known statistical assessment tools, such as determination of confidence intervals, determination of p-values, Student's t-test, Mann-Whitney test, etc. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Typically, the assumed confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. The p-values are typically 0.2, 0.1, 0.05.

[0110] The terms "reduced" or "decreased" level, amount and / or concentration of an indicator refer to the level, amount and / or concentration of such an indicator in a sample that is decreased compared to a reference or reference sample.

[0111] The terms "elevated" or "increased" level, amount and / or concentration of an indicator refer to the level, amount and / or concentration of such an indicator in a sample that is higher compared to a reference or reference sample. For example, in a fluid sample of an individual suffering from a given disease, a protein that can be detected in a higher amount or concentration than in the same fluid sample of an individual not suffering from the disease has an elevated level.

[0112] The terms "measure", "measuring" or "determining" preferably include qualitative, semi-quantitative or quantitative measurements.

[0113] The term "immunoglobulin (Ig)", as used herein, refers to glycoproteins of the immunoglobulin superfamily that confer immunity. "Surface immunoglobulin" is attached to the membrane of effector cells by their transmembrane regions and includes, but is not limited to, molecules such as B cell receptors, T cell receptors, class I and II major histocompatibility complex (MHC) proteins, beta2 microglobulin (about 2M), CD3, CD4, and CDS.

[0114] Typically, the term "antibody" as used herein refers to a secreted immunoglobulin that lacks a transmembrane region and can thus be released into the bloodstream and body cavities. Human antibodies are classified into different isotypes based on the heavy chains they possess. There are five types of human Ig heavy chains denoted by Greek letters: α, γ, δ, ε, and μ. The type of heavy chain present defines the class of the antibody (i.e., these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively), each playing a different role and directing an appropriate immune response against different types of antigens. The different heavy chains vary in size and composition and can contain approximately 450 amino acids (Janeway et al. (2001) Immunobiology, Garland Science). IgA is found in mucosal areas such as the gastrointestinal tract, respiratory tract, and urogenital tract, as well as in saliva, tears, and breast milk, and prevents colonization by pathogens (Underdown & Schiff (1986) Annu. Rev. Immunol. 4:389 - 417). IgD functions mainly as an antigen receptor on B cells that have not been exposed to antigens and is involved in activating basophils and mast cells to produce antimicrobial factors (Geisberger et al. (2006) Immunology 118:429 - 437; Chen et al. (2009) Nat. Immunol. 10:889 - 898). IgE is involved in allergic reactions through binding to allergens that cause the release of histamine from mast cells and basophils. IgE is also involved in protection against parasites (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). IgG provides most of the antibody - based immunity against invading pathogens and is the only antibody isotype that can pass through the placenta to confer passive immunity to the fetus (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). There are four different IgG subclasses (IgG1, 2, 3, and 4) in humans, named in order of their abundance in serum, with IgG1 being the most abundant (about 66%), followed by IgG2 (about 23%), IgG3 (about 7%), and IgG4 (about 4%).The biological profiles of different IgG classes are determined by the structure of their respective hinge regions. IgM is expressed on the surface of B cells in monomeric form and as a secreted pentameric form with very high avidity. IgM is involved in eliminating pathogens at the initial stage of B cell-mediated (humoral) immunity before sufficient IgG is produced (Geisberger et al. (2006) Immunology 118:429-437). Antibodies are not only found as monomers but also well-known for forming dimers of two Ig units (e.g., IgA), tetramers of four Ig units (e.g., IgM in teleost fish), or pentamers of five Ig units (e.g., mammalian IgM). Antibodies are typically made up of four polypeptide chains, two identical heavy chains and two identical light chains, linked via disulfide bonds, and resemble a "Y"-shaped macromolecule. Each of the chains contains several immunoglobulin domains, some of which are constant domains and others variable domains. The immunoglobulin domain consists of two layers of 7-9 antiparallel β-strands arranged in a β-sheet. Typically, the heavy chain of an antibody contains four Ig domains, three of which are constant (CH domains: CH1, CH2, CH3) domains and one of which is a variable domain (VH). The light chain typically contains one constant Ig domain (CL) and one variable Ig domain (VL). By way of example, the human IgG heavy chain is composed of four Ig domains linked in the order V H -CH1-CH2-CH3 (also referred to as V H -Cγ1-Cγ2-Cγ3) from the N-terminus to the C-terminus, while the human IgG light chain is composed of two immunoglobulin domains linked in the order VL-CL from the N-terminus to the C-terminus and is either of the kappa or lambda type (VK-CK or Vλ-Cλ). By way of example, the constant chain of human IgG contains 447 amino acids.Throughout this specification and the claims, the numbering of amino acid positions of immunoglobulins is that of the "EU index" as in Kabat, E.A., Wu, T.T., Perry, H.M., Gottesman, K.S., and Foeller, C., (1991) Sequences of proteins of immunological interest, 5th ed. U.S. Department of Health and Human Service, National Institutes of Health, Bethesda, MD. The "EU index like Kabat" refers to the residue numbering of human IgG1 EU antibodies. Thus, in the context of IgG, the CH domains are as follows: "CH1" refers to amino acid positions 118 to 220 according to the EU index as in Kabat; "CH2" refers to amino acid positions 237 to 340 according to the EU index as in Kabat; "CH3" refers to amino acid positions 341 to 447 according to the EU index as in Kabat. th The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form that is not the antibody fragment defined below. Specifically, these terms refer to an antibody having a heavy chain that includes the Fc region.

[0115] The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form that is not the antibody fragment defined below. Specifically, these terms refer to an antibody having a heavy chain that includes the Fc region.

[0116] Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab fragments" (also referred to as "Fab portions" or "Fab regions"), each having a single antigen-binding site, and the remaining "Fc fragment" (also referred to as "Fc portion" or "Fc region"), named to reflect its ability to crystallize readily. 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, while in IgM and IgE isotypes, the Fc region contains three heavy-chain constant domains (CH2-4) in each polypeptide chain. Additionally, smaller immunoglobulin molecules exist either naturally or are artificially constructed. The term "Fab' fragment" refers to a Fab fragment that additionally includes the hinge region of the Ig molecule, while the "F(ab')2 fragment" is understood to contain two Fab' fragments that are chemically linked or linked via disulfide bonds. "Single-domain antibodies (sdAb)" (Desmyter et al. (1996) Nat. Structure Biol. 3:803-811) and "nanobodies" contain only a single VH domain, while "single-chain Fv (scFv)" fragments contain a heavy-chain variable domain linked to a light-chain variable domain via a short linker peptide (Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85,5879-5883). Bispecific single-chain variable fragments (di-scFv) can be engineered by linking two scFvs (scFvA-scFvB). This can be done by generating a single peptide chain with two VH regions and two VL regions, yielding a "tandem scFv" (VHA-VLA-VHB-VLB). Another possibility is the generation of an scFv with a linker that is too short for the two variable regions to fold together, forcing the scFv to dimerize. Usually, a linker of 5 residues in length is used to generate these dimers. This type is known as a "diabody".Shorter linkers (one or two amino acids) between the VH and VL domains result in the formation of single - specificity trimers, so - called "triabodies" or "tribodies". Bispecific diabodies are formed by expressing two chains in the VHA - VLB and VHB - VLA or VLA - VHB and VLB - VHA arrangements, respectively. Single - chain diabodies (scDb) contain VHA - VLB and VHB - VLA fragments (VHA - VLB - P - VHB - VLA) linked by a linker peptide (P) of 12 - 20 amino acids, preferably 14 amino acids. "Bispecific T - cell engagers (BiTE)" are fusion proteins consisting of two scFvs of different antibodies, where one scFv binds to T cells via the CD3 receptor and the other binds to tumor cells via a tumor - specific molecule (Kufer et al. (2004) Trends Biotechnol. 22:238 - 244). Bispecific affinity retargeting molecules ("DART" molecules) are diabodies further stabilized by a C - terminal disulfide bridge.

[0117] Accordingly, the term "antibody fragment" refers to a portion of an intact antibody, preferably including its antigen - binding region. Antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, Fv fragments; diabodies; sdAb, nanobodies, scFv, di - scFv, tandem scFv, triabodies, diabodies, scDb, BiTE, and DART.

[0118] The term "binding affinity" generally refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between the moieties of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including, but not limited to, assays based on surface plasmon resonance (e.g., BIAcore assays as described in PCT application publication WO2005 / 012359); enzyme-linked immunosorbent assay (ELISA); and competitive assays (e.g., RIA). Low-affinity antibodies generally bind antigens slowly and tend to dissociate readily, while high-affinity antibodies generally bind antigens rapidly and tend to remain bound for longer. Various methods for measuring binding affinity are known in the art, and any of these can be used for the purposes of the present invention.

[0119] "Sandwich immunoassay" is widely used for the detection of the analyte of interest. In such an assay, the analyte is "sandwiched" between a first antibody and a second antibody. Typically, a sandwich assay requires that the capture and detection antibodies bind to different, non-overlapping epitopes on the analyte of interest. By appropriate means, such a sandwich complex is measured, whereby the analyte is quantified. In a typical sandwich-type assay, a first antibody that is bound to a solid phase or can be bound to a solid phase, and a second antibody that is detectably labeled, each bind to the analyte at different, non-overlapping epitopes. The binding agent (e.g., antibody) specific for the first analyte is either covalently bound or passively bound to the solid surface. The solid surface is typically glass or a polymer, and the most commonly used polymers are cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. The solid support may be a tube, bead, disk of a microplate, or any other surface suitable for the performance of an immunoassay. The binding process is well known in the art and generally consists of cross-linking covalent bonding or physical adsorption, and the polymer-antibody complex is washed in the preparation of the test sample. Next, an aliquot of the sample to be tested is added to the solid phase complex and incubated for a period sufficient to allow binding between the first antibody or capture antibody and the corresponding antigen (e.g., 2 to 40 minutes or, more conveniently, overnight) and under appropriate conditions (e.g., room temperature to 40 °C, e.g., 25 °C to 37 °C (including both ends)). Following the incubation period, the solid phase containing the first antibody or capture antibody and the antigen bound to the antibody can be washed and incubated with a secondary antibody or labeled antibody that binds to another epitope on the antigen. The second antibody is bound to a reporter molecule used to indicate the binding of the second antibody to the complex of the first antibody and the antigen of interest.

[0120] A very widely used alternative sandwich assay format involves the use of a solid phase coated with the first partner of a binding pair, e.g., microparticles coated with paramagnetic streptavidin. Such microparticles are incubated with a binder specific for an analyte bound to the second partner of the binding pair (e.g., a biotinylated antibody), a sample suspected of containing or containing an analyte in which the second partner of the binding pair is bound to a binder specific for the analyte, and a binder specific for a second detectable-labeled analyte. As will be apparent to those skilled in the art, these components are incubated for a period sufficient to bind the labeled antibody to the solid phase microparticles via the analyte, the binder specific for the analyte (bound to) the second partner of the binding pair, and the first partner of the binding pair under appropriate conditions. Optionally, such an assay may include one or more washing steps (plural available).

[0121] The term “detectably labeled” encompasses labels that can be detected directly or indirectly.

[0122] A directly detectable label either provides a detectable signal or the label interacts with a second label to modify a detectable signal provided by the first or second label to give, for example, FRET (fluorescence resonance energy transfer). Labels such as fluorescent dyes and luminescent dyes (including chemiluminescent and electrochemiluminescent) (Briggs et al “Synthesis of Functionalised Fluorescent Dyes and Their Coupling to Amines and Amino Acids,” J.Chem.Soc.,Perkin-Trans.1(1997)1051-1058) provide a detectable signal and are generally applicable to labels. In one embodiment, detectably labeled refers to a label that either provides or is derivable to provide a detectable signal, i.e., a fluorescent label, a luminescent label (e.g., a chemiluminescent label or an electrochemiluminescent label), a radioactive label or a metal chelate-based label.

[0123] A number of labels (also referred to as dyes) are available and can generally be classified into the following categories, all of which are grouped together and each of which represents an embodiment according to the present disclosure.

[0124] (a) Fluorescent dyes Fluorescent dyes are described, for example, by Briggs et al “Synthesis of Functionalized Fluorescent Dyes and Their Coupling to Amines and Amino Acids,” J.Chem.Soc., Perkin-Trans.1 (1997) 1051 - 1058).

[0125] Fluorescent labels or fluorophores include rare earth chelates (europium chelates), fluorescein-type labels (including FITC, 5-carboxyfluorescein, 6-carboxyfluorescein), rhodamine-type labels (including TAMRA), dansyl, lysamine, cyanine, phycoerythrin, Texas red, and their analogs. Fluorescent labels can be attached to aldehyde groups contained within a target molecule using the techniques disclosed herein. Fluorescent dyes and fluorescent labeling reagents include those commercially available from Invitrogen / Molecular Probes (Eugene, Oregon, USA) and Pierce Biotechnology, Inc. (Rockford, Ill.).

[0126] (b) Luminescent dyes Luminescent dyes or labels can be further subclassified into chemiluminescent dyes and electrochemiluminescent dyes.

[0127] Different classes of chemiluminescent labels include systems based on luminol, acridinium compounds, selenotellurazine and analogs, dioxetane, peroxysuccinic acid and peroxysuccinic acid derivatives. For immunoassay procedures, mainly acridinium-based labels are used (a detailed overview is shown in Dodeigne C. et al., Talanta 51 (2000) 415 - 439).

[0128] The main relevant labels used as electrochemiluminescent labels are ruthenium- and iridium-based electrochemiluminescent complexes, respectively. Electrochemiluminescence (ECL) has proven to be very useful for analytical applications as a highly sensitive and selective method. ECL combines the analytical advantages of chemiluminescent analysis (absence of background light signal) with the ease of reaction control by applying an electrode potential. Generally, ruthenium complexes, especially [Ru(Bpy)3]2+ (which emits photons at about 620 nm) regenerated with TPA (tripropylamine) at the liquid phase or liquid-solid interface, are used as ECL labels.

[0129] Electrochemiluminescence (ECL) assays provide sensitive and accurate measurement of the presence and concentration of a target analyte. Such techniques use labels or other reactants that can be induced to emit light when electrochemically oxidized or reduced in an appropriate chemical environment. Such electrochemiluminescence is caused by a voltage applied to a working electrode in a particular pattern at a particular time. The light generated by the label is measured and indicates the presence or amount of the analyte. For a more complete description of such ECL techniques, reference is made to U.S. Patent No. 5,221,605, U.S. Patent No. 5,591,581, U.S. Patent No. 5,597,910, PCT Application Publication WO90 / 05296, PCT Application Publication WO92 / 14139, PCT Application Publication WO90 / 05301, PCT Application Publication WO96 / 24690, PCT Application Publication US95 / 03190, PCT Application US97 / 16942, PCT Application Publication US96 / 06763, PCT Application Publication WO95 / 08644, PCT Application Publication WO96 / 06946, PCT Application Publication WO96 / 33411, PCT Application Publication WO87 / 06706, PCT Application Publication WO96 / 39534, PCT Application Publication WO96 / 41175, PCT Application Publication WO96 / 40978, PCT / US97 / 03653 and U.S. Patent Application 08 / 437,348 (U.S. Patent No. 5,679,519). Also, reference is made to the 1994 review on the analytical use of ECL by Knight, et al. (Analyst, 1994, 119:879-890) and the references cited therein. In one embodiment, the method according to the present specification is implemented using an electrochemiluminescence label.

[0130] In recent years, iridium-based ECL labels have also been described (International Publication No. 2012107419).

[0131] (c) Radioactive labels employ radioisotopes (radionuclides), such as 3H, 11C, 14C, 18F, 32P, 35S, 64Cu, 68Gn, 86Y, 89Zr, 99TC, 111In, 123I, 124I, 125I, 131I, 133Xe, 177Lu, 211At, or 131Bi.

[0132] (d) Metal chelate complexes suitable as labels for imaging and therapeutic purposes are well known in the art (U.S. Patent Application Publication No. 2010 / 0111861; U.S. Patent No. 5,342,606; U.S. Patent No. 5,428,155; U.S. Patent No. 5,316,757; U.S. Patent No. 5,480,990; U.S. Patent No. 5,462,725; U.S. Patent No. 5,428,139; U.S. Patent No. 5,385,893; U.S. Patent No. 5,739,294; U.S. Patent No. 5,750,660; U.S. Patent No. 5,834,461; Hnatowich et al, J. Immunol. Methods 65 (1983) 147-157; Meares et al, Anal. Biochem. 142 (1984) 68-78; Mirzadeh et al, Bioconjugate Chem. 1 (1990) 59-65; Meares et al, J. Cancer (1990), Suppl. 10:21-26; Izard et al, Bioconjugate Chem. 3 (1992) 346-350; Nikula et al, Nucl. Med. Biol. 22 (1995) 387-90; Camera et al, Nucl. Med. Biol. 20 (1993) 955-62; Kukis et al, J. Nucl. Med. 39 (1998) 2105-2110; Verel et al., J. Nucl. Med. 44 (2003) 1663-1670; Camera et al, J. Nucl. Med. 21 (1994) 640-646; Ruegg et al, Cancer Res. 50 (1990) 4221-4226; Verel et al, J. Nucl. Med. 44 (2003) 1663-1670; Lee et al, Cancer Res. 61 (2001) 4474-4482; Mitchell, et al, J. Nucl. Med. 44 (2003) 1105-1112; Kobayashi et al Bioconjugate Chem. 10 (1999) 103-111; Miederer et al, J. Nucl. Med.45(2004)129 - 137; DeNardo et al, Clinical Cancer Research 4(1998)2483 - 90; Blend et al, Cancer Biotherapy&Radiopharmaceuticals 18(2003)355 - 363; Nikula et al J.Nucl.Med.40(1999)166 - 76; Kobayashi et al, J.Nucl.Med.39(1998)829 - 36; Mardirossian et al, Nucl.Med.Biol.20(1993)65 - 74; Roselli et al, Cancer Biotherapy&Radiopharmaceuticals,14(1999)209 - 20).

Example

[0133] The present invention is merely illustrated by the following examples. In no case should the examples be construed in a manner that limits the scope of the present invention.

[0134] Example 1: Diagnostic performance of biomarker LTA4H in women with PCOS (phenotype A) and controls determined by the proximity extension assay (PEA) technology developed by Olink As part of the measurement, 86 serum samples from human females were analyzed. The case group included 49 samples from patients diagnosed with PCOS (phenotype A) according to the Rotterdam criteria. The control group included 37 samples from healthy women without PCOS. The concentration of the analyte was determined using the proximity extension assay (PEA) technology developed by Olink. Briefly, a matched antibody pair conjugated to unique partially complementary oligonucleotides addresses each biomarker. Quantification is then performed by quantitative real - time PCR.

[0135] For each selected panel, after diluting the samples according to the manufacturer's protocol, the Olink protocol consists of three core steps: 1. Incubation, 2. Extension and amplification, and 3. Detection. 1 μl of each sample was mixed with 3 μl of incubation mix in a 96-well plate. In addition to 92 antibody pairs labeled with DNA oligonucleotides, the incubation mix also included internal controls designed to monitor the three main steps of the Olink protocol (two incubation controls, namely one extension control and one detection control). As external controls, three positive controls (inter-plate controls), three negative controls, and two sample controls (pooled plasma samples) were included in the plate. The samples were incubated overnight at +4°C. During this step, the antibody pairs bind to their respective proteins in the samples. When the incubation was complete, 96 μl of extension mix was added to the samples. The plate was placed in a thermal cycler for hybridization, and extension was performed by DNA polymerase (50°C for 20 minutes, 95°C for 5 minutes (95°C for 30 seconds, 54°C for 1 minute, 60°C for 1 minute) × 17, hold at 10°C). The DNA barcodes were amplified by PCR. Finally, the amount of each DNA barcode was quantified by microfluidic qPCR. The 96.96 Dynamic Array (trademark) Integrated Fluidic Circuit (IFC) was used according to the manufacturer's instructions. 2 μl of the detection mix at seven points was added to 2.8 μl of each sample, and 5 μl of these was transferred to the left inlet of the primed 96.96 Dynamic Array IFC. 5 μl of the primer solution was transferred to the right inlet of the primed 96.96 Dynamic Array IFC. The chip was loaded into the Fluidigm IFC Controller HX according to the manufacturer's instructions.The Olink protein expression 96 × 96 program was run on a Fluidigm Biomark™ reader according to the manufacturer's instructions (50 °C for 120 s, 70 °C for 1800 s, 25 °C for 600 s, 95 °C for 300 s (95 °C for 15 s, 60 °C for 60 s) × 35; the following settings: application - Gene Expression; passive Reference - ROX; assay - single probe; probe - FAM - MGB). The Ct values obtained from qPCR were converted to an arbitrary unit called normalized protein eXpression (NPX, a relative quantification unit on a log2 scale) using the following formula:. Extension control: Ct 分析物 -Ct 伸長対照 =dCt 分析物 Inter - plate control: dCt 分析物 -dCt プレート間対照 =ddCt 分析物 Adjustment for correction factor: Correction factor - ddCt 分析物 =NPX 分析物

[0136] Quality control and normalization were achieved using the Olink NPX Manager software.

[0137] Receiver operating characteristic (ROC) curves were created (Figure 1). Model performance was determined by examining the area under the curve (AUC). The best possible AUC is 1 and the lowest possible AUC is 0.5. ROC curve analysis showed an AUC of 1.00 (95% CI 1 - 1, Figure 1), indicating that LTA4H has high diagnostic accuracy for PCOS.

[0138] The diagnostic performance of leukotriene A4 hydrolase for distinguishing women with PCOS with a complete proliferative phenotype A (cases) from healthy control subjects using ROC analysis is shown in Table 1, which describes the AUC of the ROC curve analysis and the associated 95% confidence interval. The results were obtained using the Olink proximity extension technology.

Table 1

[0139] Using data obtained by the Olink PEA technology, box-and-whisker plots of healthy controls and PCOS cases were generated. The box includes the median (middle quartile), interquartile range (representing the central 50% of the group's scores), upper quartile (75% of the scores are below the upper quartile), and lower quartile (25% of the scores are below the lower quartile). The whiskers represent values 1.5 times the interquartile range. Serum LTA4H concentration increases in women with PCOS when compared to healthy controls (Figure 2).

[0140] Example 2: Diagnostic performance of biomarker LTA4H in women with PCOS (phenotypes A, B, C, and D) and controls as determined by ELISA technology Performance verification was conducted in a sample population of 87 cases (serum samples from women with PCOS) and 42 controls (serum samples from healthy women).

[0141] The concentration of the analyte was determined by ELISA (enzyme-linked immunosorbent assay). The case group consisted of patients diagnosed with PCOS (28 phenotype A, 20 phenotype B, 20 phenotype C, and 19 phenotype D) according to the Rotterdam criteria. The control group included healthy women without PCOS.

[0142] The concentration of LTA4H in human serum was determined using the Invitrogen (Catalog No.: EH308RB) Human LTA4H ELISA Kit Ver.1. The kit is a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) designed to detect and quantify the levels of human LTA4H in cell culture supernatants, plasma, and serum. The kit provides a human LTA4H antibody pre-coated plate. Samples were measured at a 2-fold dilution. After bringing all reagents to room temperature, 100 μL of each sample and standard were added. Samples and standards were measured in duplicate. Any LTA4H present was bound to the immobilized capture antibody on the microtiter plate during a 2.5-hour incubation at room temperature on a microplate shaker set at 650 rpm. During the washing step (4 × 300 μL), unbound substances were removed from the plate, and then 100 μL of diluted anti-LTA4H biotin conjugate was added to the wells. After a 1-hour incubation on the shaker and another washing step (4 × 300 μL) to remove unbound detection antibody, 100 μL of the prepared streptavidin-HRP solution was added to the plate. It was incubated at room temperature for 45 minutes and a washing step (4 × 300 μL) was performed. After the final wash, 100 μL of TMB substrate was added to the plate. The plate was incubated in the dark at room temperature for 30 minutes while gently shaking. During incubation, the substrate changed to blue. The color development was proportional to the amount of LTA4H bound in the first step. The color development was stopped by adding 50 μL of stop solution, changing the solution in the wells from blue to yellow, and the color intensity was measured using a plate reader at 450 nm for detection and at 570 nm for background subtraction. To generate a calibration curve, the lyophilized recombinant LTA4H provided with the kit was reconstituted and diluted with the standard diluent. The calibration range of the assay was 2.048 ng / mL to 500 ng / mL. Calibrator 1 (500 ng / mL) corresponded to the reconstituted stock solution, and Calibrators 2 to 7 (2.048 ng / mL) were prepared by successive 2.5-fold dilution steps in the calibrator diluent. Pure calibrator diluent functioned as the blank (0 ng / mL).The calibration curve was fitted using a four-parameter non-linear regression (Newton / Raphson) without weighting.

[0143] The receiver operating characteristic (ROC) curve was created (Figure 3). The model performance was determined by examining the area under the curve (AUC). The best possible AUC is 1, and the lowest possible AUC is 0.5. The ROC curve analysis for leukotriene A4 hydrolase in PCOS cases when all phenotypes (Phenotypes A - D) were combined showed an AUC of 0.85 (95% CI 0.77 - 0.93), confirming the high diagnostic accuracy of LTA4H for PCOS (Figure 3). The diagnostic performance of leukotriene A4 hydrolase for distinguishing women with PCOS (cases, PCOS phenotypes A - D) from healthy control subjects using ROC analysis is shown in Table 2, which describes the AUC of the ROC curve analysis and the associated 95% confidence intervals. The results were obtained using an ELISA immunoassay.

Table 2

[0144] Using the data obtained by ELISA immunoassay, box-and-whisker plots for controls and PCOS were created when all phenotypes (Phenotypes A - D) were combined. Serum LTA4H concentration (ng / mL) increases in women with PCOS when compared to healthy controls (Figure 4).

[0145] Table 3 shows the diagnostic performance of leukotriene A4 hydrolase for distinguishing women with PCOS from healthy control subjects when separated by different phenotypes A, B, C, and D. The results were obtained using an ELISA immunoassay. The AUC for each phenotype is reported in the table.

Table 3

[0146] ROC curve analysis of leukotriene A4 hydrolase for PCOS cases separated by different phenotypes from healthy controls showed AUCs of 0.9 (95% CI 0.82 - 0.98), 0.88 (95% CI 0.78 - 0.97), 0.76 (95% CI 0.64 - 0.88), and 0.85 (95% CI 0.75 - 0.95) for phenotypes A - D, respectively (Figure 5). The results confirm the high diagnostic accuracy of LTA4H for PCOS. Serum leukotriene A4 hydrolase concentrations (ng / mL) in all different PCOS phenotypes (phenotypes A - D) showed increased levels compared to healthy controls (Figure 6, results obtained using ELISA immunoassay).

[0147] Table 4 shows the diagnostic performance of leukotriene A4 hydrolase in young women (age ≤ 25) to distinguish young women with PCOS from young healthy control subjects when all phenotypes (phenotypes A - D) are combined. Results were obtained using ELISA assay.

Table 4

[0148] ROC curve analysis of leukotriene A4 hydrolase for young PCOS cases (age ≤ 25) when all phenotypes (phenotypes A - D) are combined showed an AUC of 0.83, confirming high diagnostic accuracy for women under 25 when distinguishing PCOS cases from controls (95% CI 0.61 - 1, Figure 7). When only young women were included in the analysis (age ≤ 25), PCOS cases (all phenotypes A - D combined) showed increased serum leukotriene A4 hydrolase concentrations compared to young controls (age ≤ 25, Figure 8).

[0149] The diagnostic performance of leukotriene A4 hydrolase to distinguish young women with PCOS (age ≤ 25) from young healthy control subjects (age ≤ 25) when separated by different phenotypes A, B, C, and D was evaluated, and the results are reported in Table 5 (AUC for PCOS phenotypes vs. controls). Results were obtained using ELISA immunoassay.

Table 5

[0150] For the ROC curve analysis of leukotriene A4 hydrolase in young PCOS cases (age ≤ 25, phenotypes A - D), the AUC values were 0.97 (95% CI 0.89 - 1), 0.80 (95% CI 0.34 - 1), 0.72 (95% CI 0.34 - 1), and 0.67 (95% CI 0.25 - 1) for each phenotype respectively. In the subgroup of women under 25 years old, a high diagnostic accuracy of LTA4H for PCOS was confirmed (Figure 9). The leukotriene A4 hydrolase concentration increased in all different PCOS phenotypes (phenotypes A - D, age ≤ 25) when compared with the LTA4H concentration in young healthy controls (age ≤ 25, Figure 10).

[0151] Example 3: Diagnostic performance of biomarker LTA4H in women with PCOS (phenotypes A, B, C, and D) and controls determined by ELISA technology in different age groups Performance verification was performed on an additional sample set of 240 cases (serum samples from women with PCOS) and 48 controls (serum samples from healthy women).

[0152] The concentration of the analyte was determined by ELISA (enzyme - linked immunosorbent assay). The case group consisted of patients diagnosed with PCOS (155 phenotype A, 5 phenotype B, 8 phenotype C, and 72 phenotype D) according to the Rotterdam criteria and belonging to three different age groups: 15 - 20 (n = 70), 20 - 25 (n = 99), 25 - 40 (n = 71). The control group included healthy women without PCOS (aged 20 - 40 years).

[0153] The concentration of LTA4H in human serum was determined using the Human LTA4H ELISA Kit Ver.1 from Invitrogen (Catalog No.: EH308RB) as described in Example 2.

[0154] The receiver operating characteristic (ROC) curve was created (Figure 11). The model performance was determined by examining the area under the curve (AUC). The ROC curve analysis of LTA4H for PCOS cases when all phenotypes (Phenotypes A - D) were combined showed an AUC of 0.74 (95% CI 0.64 - 0.83), confirming the high diagnostic accuracy of LTA4H for PCOS (Figure 11).

[0155] Table 6, which describes the AUC of the ROC curve analysis and the associated 95% confidence intervals, shows the diagnostic performance of LTA4H for differentiating women with PCOS (cases, PCOS phenotypes A - D) from healthy control subjects using ROC analysis. The results were obtained using an ELISA immunoassay.

Table 6

[0156] Using the data obtained by ELISA immunoassay, box - and - whisker plots for controls and PCOS were created when all phenotypes (Phenotypes A - D) were combined. Serum LTA4H concentration (ng / mL) increases in women with PCOS compared to healthy controls (Figure 12).

[0157] Table 7 shows the diagnostic performance of LTA4H for differentiating women with PCOS from healthy control subjects when separated by different phenotypes A, B, C, and D. The results were obtained using an ELISA immunoassay. The AUC for each phenotype is reported in the table.

Table 7

[0158] ROC curve analysis of LTA4H for PCOS cases and healthy controls separated by different phenotypes showed AUCs of 0.75 (95% CI 0.66 - 0.84), 0.77 (95% CI 0.65 - 0.89), 0.70 (95% CI 0.50 - 0.90), and 0.70 (95% CI 0.60 - 0.81) for phenotypes A - D, respectively (Figure 13). The results confirm the high diagnostic accuracy of LTA4H for PCOS.

[0159] Serum LTA4H concentrations (ng / mL) in all different PCOS phenotypes (phenotypes A - D) showed increased levels compared to healthy controls (Figure 14, results obtained using ELISA immunoassay).

[0160] Table 8 shows the diagnostic performance of LTA4H in different age groups (15 ≤ age < 20, 20 ≤ age < 25, 25 ≤ age < 40) to distinguish women with PCOS from healthy control subjects when all phenotypes (phenotypes A - D) are combined. Results were obtained using ELISA assay.

Table 8

[0161] ROC curve analysis of LTA4H for PCOS cases separated by different age groups from healthy controls showed AUCs of 0.74 (95% CI 0.64 - 0.84), 0.70 (95% CI 0.61 - 0.80), and 0.78 (95% CI 0.68 - 0.87) for the 15 - 20, 20 - 25, and 25 - 40 age groups, respectively (Figure 15). The results confirm the high diagnostic accuracy of LTA4H for PCOS in all different age groups.

[0162] An increase in serum LTA4H concentration (ng / mL) in women with PCOS compared to controls was confirmed in all different age groups (Figure 16).

[0163] Given that there is no reliable biomarker for diagnosing PCOS, especially in young women (age < 25), a separate analysis was conducted for the age group of 15 years and above but less than 25 years.

[0164] Table 9 shows the diagnostic performance of LTA4H in young women (15 ≤ age < 25) for differentiating young women with PCOS from young healthy control subjects when all phenotypes (Phenotypes A - D) are combined. The results were obtained using an ELISA assay. [Table 9]

[0165] ROC curve analysis of LTA4H for young PCOS cases (15 ≤ age < 25) when all phenotypes (Phenotypes A - D) are combined showed an AUC of 0.75, confirming high diagnostic accuracy for women aged 15 - 25 in differentiating PCOS cases from controls (95% CI 0.61 - 0.88, Figure 17). When only young women (15 ≤ age < 25) were included in the analysis, PCOS cases (all phenotypes A - D combined) showed an increase in serum LTA4H concentration (ng / mL) compared to young controls (15 ≤ age < 25, Figure 18).

[0166] The diagnostic performance of LTA4H for differentiating young women with PCOS (15 ≤ age < 25) from young healthy control subjects (15 ≤ age < 25) when separated by different phenotypes A, B, C, and D was evaluated, and the results are reported in Table 10 (AUC for PCOS phenotypes vs. controls). The results were obtained using an ELISA immunoassay. [Table 10]

[0167] ROC curve analysis of LTA4H for young PCOS cases (15 ≤ age < 25, phenotypes A - D) showed AUC values of 0.76 (95% CI 0.63 - 0.90), 0.78 (95% CI 0.61 - 0.95), 0.69 (95% CI 0.47 - 0.92), and 0.72 (95% CI 0.58 - 0.86) for each phenotype, respectively, confirming the high diagnostic accuracy of LTA4H for PCOS in the subgroup of women aged 15 - 25 (Figure 19). LTA4H concentration increased in all different PCOS phenotypes (phenotypes A - D, 15 ≤ age < 25) when compared to the LTA4H concentration of young healthy controls (15 ≤ age < 25, Figure 20).

Claims

**Claim 1** A method for assessing whether a patient has polycystic ovary syndrome (PCOS) or is at risk of developing PCOS, comprising: (a) determining the amount or concentration of LTA4H in a sample from the patient; and (b) comparing the determined amount or concentration to a reference. A method as described above. **Claim 2** A method for selecting a patient for the treatment of PCOS, comprising: (a) determining the amount or concentration of LTA4H in a sample from the patient; and (b) comparing the determined amount or concentration to a reference. A method as described above. **Claim 3** A method for monitoring the progression of PCOS in a patient with PCOS or for monitoring the response to treatment in a patient with PCOS, the method comprising: (a) determining the level of LTA4H in a first sample from the patient; (b) determining the level of LTA4H in a second sample from the patient obtained after the first sample; (c) comparing the level of LTA4H in the first sample to the level of LTA4H in the second sample; and (d) monitoring the progression in the patient suffering from or being treated for PCOS based on the result of step (c). A method as described above. **Claim 4** The method according to any one of claims 1 to 3, wherein an increase in the amount or concentration of LTA4H in the sample from the patient indicates the presence of PCOS in the patient. **Claim 5** The method according to any one of claims 1 to 4, wherein the sample is a sample of blood, serum or plasma. **Claim 6** The method according to any one of claims 1 to 5, wherein PCOS is selected from the group consisting of phenotype A PCOS, phenotype B PCOS, phenotype C PCOS and phenotype D PCOS according to the Rotterdam criteria. **Claim 7** The method according to any one of claims 1 to 6, wherein phenotype A PCOS is detected. **Claim 8** The method according to any one of claims 1 to 6, wherein phenotype B PCOS is detected. **Claim 9** The method according to any one of claims 1 to 6, wherein phenotype C PCOS is detected. **Claim 10** The method according to any one of claims 1 to 6, wherein phenotype D PCOS is detected. **Claim 11** The method according to any one of claims 1 to 10, wherein PCOS is detected in an adolescent or young adult female. **Claim 12** The method according to any one of claims 1 to 11, wherein the patient suffers from one or more of the following symptoms: oligo-anovulation and / or irregular cycles, hyperandrogenemia, and polycystic ovarian morphology.

13. A computer-implemented method for assessing a patient suspected of having PCOS, comprising: (a) receiving a value for the amount or concentration of a first biomarker in a sample of the patient, wherein the first biomarker is LTA4H; (b) optionally, receiving a value for the amount or concentration of a second biomarker in a sample of the patient; (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 value for the amount or concentration in steps (a) to (b) with the reference for the biomarker and the value for the presence or absence of the at least one additional diagnostic criterion, and / or calculating a score for assessing the patient suspected of having PCOS based on the amount or concentration of the biomarker and the value; (e) assessing the patient based on the comparison and / or the calculation performed in step (d). A computer-implemented method comprising the steps above.

14. The computer-implemented method according to claim 13, wherein the amount or concentration of LTA4H is increased compared to a standard reference.

Images