Leukotriene A4 hydrolase (LTA4H) as (blood) biomarker for diagnosis of polycystic ovarian syndrome

Abstract

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

Description

[0001] The present invention relates to methods for assessing whether a patient suffers from polycystic ovary syndrome (PCOS) or is at risk of developing PCOS by determining the amount or concentration of leukotriene A4 hydrolase (LTA4H) in a sample of the patient and comparing the determined amount or concentration and the determined amount or concentration with a reference, methods for selecting patients for therapy, and methods for monitoring patients suffering from PCOS or being treated for PCOS. In addition, the present invention relates to a computer-implemented method for assessing a patient suffering from suspected PCOS by determining the amount or concentration of LTA4H in a sample of the patient, optionally by determining the amount or concentration of a second biomarker and / or additional diagnostic criteria, and comparing the presence of LTA4H, the amount or concentration of the optional second biomarker and / or the optional diagnostic criteria with a reference. Background Art

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

[0003] These symptoms are not specific to PCOS, and patients are usually diagnosed only after a longer evaluation for infertility. For the final diagnosis of PCOS, other conditions or diseases (such as pregnancy, atypical adrenal hyperplasia (NCAH), congenital adrenal hyperplasia, androgen-secreting tumors, Cushing's syndrome, thyroid disease, or hyperprolactinemia) should be excluded (Escobar-Morreale HF. Polycystic ovary syndrome: definition, aetiology, diagnosis and treatment. Nat Rev Endocrinol. 2018; 14 (5): 270-284; Teede HJ, Misso ML, Costello MF et al. International PCOS Network. Recommendations from the international evidence-based guideline for the assessment and management of polycystic ovary syndrome. Hum Reprod. 2018; 33 (9): 1602-1618). Diagnostic tests that can be used to exclude other diseases, such as:

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

[0006] Prolactin to exclude hyperprolactinemia

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

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

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

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

[0011] The most widely used criteria for the diagnosis of PCOS are the so-called Rotterdam criteria. PCOS is indicated if at least 2 of the following criteria apply: (i) irregular cycles (oligomenorrhea) and / or ovulatory dysfunction (oligo-anovulation, OA), (ii) clinical and / or biochemical hyperandrogenism (HA) and (iii) polycystic ovarian morphology (PCOM) (PCOS Consensus Workshop Group, FertilSteril 2004; 81: 19-25). The first criterion is defined as a menstrual cycle with a cycle length of less than 21 days or more than 35 days or less than 8 cycles per year. Clinical and / or biochemical signs of hyperandrogenism, where clinical hyperandrogenism is defined as hirsutism (excessive and male-pattern hair growth) and / or acne, and biochemical hyperandrogenism is defined as higher levels of free androgens compared to healthy controls. Clinical hyperandrogenism is also defined as a modified Ferriman-Gallwey score>8. Biochemical hyperandrogenism can be assessed using free testosterone or the free androgen index (FAI), which can be calculated by measuring total testosterone and sex hormone binding globulin (SHBG). PCOM is usually determined according to the "PCOS 2018 International Evidence-Based Clinical Guidelines" using an intravaginal ultrasound transducer with a bandwidth of up to 8 MHz. The threshold for PCOM is considered to be in either ovary: ensuring that there is no corpus luteum, cysts, or dominant follicles, the number of follicles per ovary is >20, and / or the ovarian volume is ≥10 ml. If using older ultrasound technology, the threshold for PCOM can be an ovarian volume ≥10 ml or a follicle count >12 in either ovary.

[0012] Another method to detect PCOM is to measure anti-Mullerian hormone (AMH) in the subject. AMH is a glycoprotein hormone whose expression is essential for sex differentiation at specific times during fetal development. In addition, AMH produced by granulosa cells of growing follicles generally correlates with the number of antral follicles in the ovaries. Therefore, serum AMH levels can be an alternative biomarker to the number / count of antral follicles (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 polycysticovary syndrome 2018", serum AMH levels should not be used as an alternative method for detecting PCOM or diagnosing PCOS.

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

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

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

[0016] In order to assess biochemical hyperandrogenism, the androgen measured must have a clear normal range. Testosterone is the most abundant androgen detected, whether in total, bound or free form. The operation of measuring free testosterone has its limitations. Direct measurement of free testosterone using radioimmunoassay is very inaccurate and does not reflect the true value. The assay has high intra- and inter-batch variability. Alternatively, higher accuracy can be obtained by measuring total testosterone concentration using extraction chromatography, gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS), especially for clinical studies. The diagnostic performance of measuring serum testosterone can be enhanced by measuring SHBG at the same time, so that the calculation of free T concentration based on total testosterone and SHBG levels only requires solving a quadratic equation (Azziz R, Carmina E, Dewailly D, et al. Task Force on the Phenotype of the Polycystic Ovary Syndrome of The Androgen Excess and PCOS Society. The Androgen Excess and PCOS Society criteria for the polycysticovary syndrome: the complete task force report. Fertil Steril. 2009; 91 (2): 456-88). The definition of HA may vary by race. An mFG score of >8 to diagnose hirsutism in women with PCOS may not be suitable for all races. The prevalence of hirsutism in East Asian women is lower than that in Caucasians, and a score of >5 is recommended to define hirsutism in Chinese women. There are also indications that there are differences in blood androgen levels between different ethnic groups, with the Japanese population having a lower incidence of elevated androgens, and testosterone being recommended only as a supplementary factor for 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).

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

[0018] Currently, no specific PCOS medications are available. Treatment is symptom-directed and tailored to individual needs. Treatment targets hyperandrogenism, irregular cycles and / or ovulatory dysfunction, and associated metabolic disorders, such as diabetes. The 2018 International Evidence-Based Clinical Guidelines for the Assessment and Management of Polycystic Ovary Syndrome provide information to support clinical decision making and patient management.

[0019] Inconsistent diagnostic criteria, varying provider knowledge, and lack of consensus present unique challenges to the diagnosis and care of women with PCOS. These factors contribute to inaccurate diagnosis, including underdiagnosis and overdiagnosis. This adverse diagnostic experience worsens the condition for women and limits opportunities for timely intervention to reduce associated comorbidities, particularly during the transition from pediatric to adult care (Witchel SF, Teede HJ, AS. Curtailing PCOS. Pediatr Res. 2020; 87(2): 353-361). In addition, timely diagnosis is crucial to prevent further metabolic complications (such as type 2 diabetes) in women with the disease.

[0020] In the largest study of the experience with PCOS diagnosis, many women reported delayed diagnosis and inadequate information. One-third or more of women reported more than 2 years (33.6%) and more than 3 health care professionals (47.1%) before diagnosis. Few were satisfied with their diagnosis experience (35.2%) or the information they received (15.6%). These gaps in early diagnosis, education, and support are 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).

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

[0022] To date, there are no universal biomarkers available that can be used alone or in combination with the above symptoms or the aforementioned hormone levels to assess whether a patient has PCOS or is at risk for PCOS, and / or to determine the response of a patient with PCOS to therapy, and / or to monitor the progression of PCOS in a patient, and / or to monitor the response of a patient with PCOS to treatment.

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

[0024] In a first aspect, the present invention relates to a method for assessing whether a patient suffers from PCOS or has a risk of developing PCOS, comprising:

[0025] (a) determining the amount or concentration of LTA4H in a sample from the patient, and

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

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

[0028] (a) determining the amount or concentration of LTA4H in a sample from said patient, and

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

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

[0031] (a) determining the level of LTA4H in a first sample from the patient,

[0032] (b) determining the level of LTA4H in a second sample of the patient that has been obtained after the first sample, and

[0033] (c) comparing the level of LTA4H in the first sample to the level of LTA4H in the second sample, and

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

[0035] In a fourth aspect, the present invention relates to a computer-implemented method for assessing a patient suffering from suspected PCOS, comprising the following steps:

[0036] (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,

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

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

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

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

[0041] Figure 1 . ROC curve analysis of leukotriene A4 hydrolase. Results obtained using Olink proximity extension technology.

[0042] Figure 2 Serum leukotriene A4 hydrolase NPX in PCOS cases (phenotype A only) relative to controls. Results obtained using Olink proximity extension technology.

[0043] Figure 3 .ROC curve analysis of leukotriene A4 hydrolase in PCOS cases when all phenotypes were combined (phenotypes A to D). Results were obtained by ELISA immunoassay.

[0044] Figure 4. Serum leukotriene A4 hydrolase concentrations (ng / mL) in controls versus PCOS cases when all phenotypes were combined (phenotypes A to D). Results were obtained using an ELISA immunoassay.

[0045] Figure 5 . ROC curves for PCOS phenotypes A to D compared with the control group. Results were obtained by ELISA immunoassay.

[0046] Figure 6 Serum leukotriene A4 hydrolase concentrations (ng / mL) in healthy controls and PCOS phenotypes A to D. Results were obtained using an ELISA immunoassay.

[0047] Figure 7 ROC curve analysis of leukotriene A4 hydrolase in young PCOS cases (age ≤ 25 years) when all phenotypes A to D were combined. Results were obtained using ELISA immunoassay.

[0048] Figure 8 Serum leukotriene A4 hydrolase concentrations (ng / mL) in young controls versus young PCOS cases (age ≤ 25 years) of combined phenotypes A to D. Results were obtained using an ELISA immunoassay.

[0049] Fig. 9 . ROC curves for leukotriene A4 hydrolase in young PCOS cases (age ≤ 25 years) versus young healthy controls (age ≤ 25 years) classified by phenotypes A to D. Results were obtained using an ELISA immunoassay.

[0050] Fig.10 Serum leukotriene A4 hydrolase concentrations (ng / mL) in young healthy controls (age ≤ 25 years) and PCOS phenotypes A to D (age ≤ 25 years). Results were obtained by ELISA immunoassay.

[0051] Fig.11 .ROC curve analysis of leukotriene A4 hydrolase in PCOS cases when all phenotypes were combined (phenotypes A to D). Results were obtained by ELISA immunoassay.

[0052] Fig.12 . Serum leukotriene A4 hydrolase concentrations (ng / mL) in controls versus PCOS cases when all phenotypes were combined (phenotypes A to D). Results were obtained using an ELISA immunoassay.

[0053] Fig.13 ROC curves for leukotriene A4 hydrolase in PCOS phenotypes A to D compared with controls. Results were obtained by ELISA immunoassay.

[0054] Fig.14Serum leukotriene A4 hydrolase (ng / mL) in healthy controls and PCOS phenotypes A to D. Results were obtained using an ELISA immunoassay.

[0055] Fig.15 . ROC curves for leukotriene A4 hydrolase in different PCOS age groups (15 to 20 years, 20 to 25 years, 25 to 45 years) compared to controls. Results were obtained by ELISA immunoassay.

[0056] Fig.16 Serum leukotriene A4 hydrolase (ng / mL) in healthy controls and PCOS age groups (15 to 20 years, 20 to 25 years, 25 to 45 years). Results were obtained using ELISA immunoassay.

[0057] Fig.17 ROC curve analysis of leukotriene A4 hydrolase in young PCOS cases (15 to 25 years old) when all phenotypes A to D were combined. Results were obtained using ELISA immunoassay.

[0058] Fig.18 Serum leukotriene A4 hydrolase (ng / mL) in young controls versus young PCOS cases (15 to 25 years old) of combined phenotypes A to D. Results were obtained using an ELISA immunoassay.

[0059] Fig.19 . ROC curves for leukotriene A4 hydrolase in young PCOS cases (15 to 25 years) versus young healthy controls (15 to 25 years) classified by phenotypes A to D. Results were obtained using an ELISA immunoassay.

[0060] Fig. 20 Serum leukotriene A4 hydrolase concentrations (ng / mL) in young healthy controls (15 to 25 years) and PCOS phenotypes A to D (15 to 25 years). Results were obtained by ELISA immunoassay. DETAILED DESCRIPTION

[0061] The inventors of the present invention have identified leukotriene A4 hydrolase (LTA4H) as a reliable biomarker for diagnosing PCOS in a patient, for determining whether a patient is at risk for developing PCOS, for determining the response of a patient suffering from PCOS to therapy, or for monitoring the progression of PCOS in a patient suffering from PCOS, or for monitoring the response of a patient suffering from PCOS to therapy. LTA4H can be used alone or in combination with at least one additional criterion (such as hyperandrogenemia, oligo-anovulation, PCOM, or irregular cycles) for diagnosis, risk assessment, and / or monitoring the response of a patient to treatment. In addition, the determination of LTA4H levels compared to control levels can be used to monitor the patient's response to treatment and / or monitor the progression of PCOS.

[0062] We show for the first time that the LTA4H values ​​measured in a sample of women suffering from PCOS are increased compared to a control group, the sample preferably being a biological fluid sample, the biological fluid sample preferably being blood, plasma, serum, capillary blood, tissue fluid, peritoneal fluid or menstrual blood, and wherein the biological fluid sample is more preferably blood, plasma or serum. In addition, we show for the first time that the LTA4H values ​​measured in a sample of women suffering from any phenotype A to D of PCOS are elevated, the sample preferably being a biological fluid sample, the biological fluid sample preferably being blood, plasma, serum, capillary blood, tissue fluid, peritoneal fluid or menstrual blood, and wherein the biological fluid sample is more preferably blood, plasma or serum. The solution provided by the present invention is an immunoassay for detecting leukotriene A4 hydrolase in a sample, the sample preferably being a biological fluid sample, the biological fluid sample preferably being blood, plasma, serum, capillary blood, tissue fluid, peritoneal fluid or menstrual blood, and wherein the biological fluid sample is more preferably blood, plasma or serum. This immunoassay can also be used in combination with other clinical and / or biochemical features (such as oligo-anovulation and / or irregular cycles, hyperandrogenemia or PCOM) to diagnose women suffering from PCOS. In addition, measuring LTA4H values ​​can be used to monitor the progression of PCOS in the patient and monitor the response to therapy. We also show the measurement of LTA4H values ​​in a sample, which is preferably a biological fluid sample, which is preferably blood, plasma, serum, capillary blood, tissue fluid, peritoneal fluid or menstrual blood, and wherein the biological fluid sample is more preferably blood, plasma or serum, which value alone or in combination with additional diagnostic criteria as described above is particularly suitable for the diagnosis of PCOS in adolescents or young women under 25 years of age, particularly under 20 years of age, particularly under 15 to 25 years of age, particularly under 15 to 20 years of age.

[0063] There is an unmet medical need for an accurate test for reliable diagnosis of PCOS. The measurement of LTA4H in a sample, preferably a biofluid sample, preferably blood, plasma, serum, capillary blood, tissue fluid, peritoneal fluid or menstrual blood, and wherein the biofluid sample is more preferably blood, plasma or serum, has the advantage of a reliable biofluid-based test that can identify women with PCOS, which is currently not possible. The measurement of LTA4H in a sample, preferably a biofluid sample, preferably blood, plasma, serum, capillary blood, tissue fluid, peritoneal fluid or menstrual blood, and wherein the biofluid sample is more preferably blood, plasma or serum, can also be reliably used for the diagnosis of PCOS in adolescent patients and young women under 25 years of age, especially under 20 years of age, especially under 15 to 25 years of age, especially under 15 to 20 years of age. Due to the above reasons, the diagnosis of PCOS in adolescent patients is difficult, therefore, for the first time, we provide an accurate test for the diagnosis of PCOS in adolescent and young female populations. In addition, measuring LTA4H in a sample is advantageous for determining whether a patient is responding to treatment, and the sample is preferably a biological fluid sample, and the biological fluid sample is preferably blood, plasma, serum, capillary blood, tissue fluid, peritoneal fluid or menstrual blood, and wherein the biological fluid sample is more preferably blood, plasma or serum. Another advantage of measuring LTA4H in a patient sample is that it is used to monitor the progression of PCOS, and the sample is preferably a biological fluid sample, and it is preferably blood, plasma, serum, capillary blood, tissue fluid, peritoneal fluid or menstrual blood, and wherein the biological fluid sample is more preferably blood, plasma or serum. In addition, we attach a computer-implemented method for assessing a patient suffering from PCOS by measuring the level of LTA4H in a sample, which is preferably a biological fluid sample, which is preferably blood, plasma, serum, capillary blood, tissue fluid, peritoneal fluid or menstrual blood, and wherein the biological fluid sample is more preferably blood, plasma or serum, and optionally the level is combined with additional criteria, such as values ​​for oligo-anovulation and / or irregular cycles, hyperandrogenemia and / or polycystic ovary morphology or with additional biomarkers or hormones, for assessing the patient based on comparison and / or calculation of the above data.

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

[0065] Leukotriene A4 hydrolase (LTA4H) is part of the 5-lipoxygenase (5-LO) pathway and converts arachidonic acid (AA) into pro-inflammatory leukotrienes and anti-inflammatory lipoxins. LTA4H is widely expressed in various organs, tissues, and individual cell types ( JZ.Leukotriene A4hydrolase and the committed step in leukotrieneB4 biosynthesis. Clin Rev Allergy Immunol. 1999 Spring-Summer; 17(1-2): 111-31). According to the human protein atlas, moderate expression of LTA4H was detected in normal ovarian tissue (http: / / www.proteinatlas.org). The expression pattern of LTA4H has been studied in human ovarian tissue, and the results showed weak expression and moderate expression on granulosa cells and endofemoral cells in preovulatory follicles, respectively. After ovulation, the intensity of LTA4H on large luteal cells increases and is highest in the mid-luteal phase. High expression is 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 August; 4(8): 803-10). LTA4H is a unique bifunctional zinc metalloenzyme with epoxide hydrolase (intracellular) and aminopeptidase (extracellular) activities. As an epoxide hydrolase, LTA4H catalyzes the final critical step in the biosynthesis of leukotriene B4 (LTB4), an eicosanoid with potent chemotactic and proinflammatory 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 act as potent chemical mediators in various allergic and inflammatory responses. Emerging data suggest that leukotrienes play an important role in carcinogenesis.LTB4 levels are elevated in a variety of human cancers, including skin, lung, colon, and prostate cancers (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 reticulummolecular chaperone GRP94 and GRP78 in human lung cancer tissues and its significance. Cancer Detect Prev. 2005;29(6):544-51; Larré 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). LTB4 receptor expression 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, aleukotriene B4 receptor, in Ras transformation. Oncogene. 2004 Dec 9;23(57):9259-68). Inhibition of LTB4 synthesis by treatment with the LTA4H inhibitor bestatin reduced tumorigenesis 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). In addition, topical application of LTB4 to the skin causes not only inflammation but also massive epidermal hyperplasia (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; Ruzika 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, hypoxia marker HIF1α and macrophage marker CD68 is positively correlated in ovarian cancer tissues. In addition, hypoxic ovarian cancer cell lines show increased production of LTA4H and 5-LOX metabolites, leading to enhanced 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 March 5; 34 (10): 1241-52). LTA4H can also fight inflammation through its aminopeptidase activity, which inactivates the tripeptide Pro-Gly-Pro (PGP) by cleaving it (Stsiapanava A, Olsson U, Wan M et al. Binding of Pro-Gly-Pro at the active site of leukotriene A4hydrolase / aminopeptidase and development of anepoxide 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 can promote neutrophil accumulation (Snelgrove RJ, Jackson PL, Hardison MT et al. A critical role for LTA4H inlimiting 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 efficiency 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 variation is associated with increased survival after glucocorticoid therapy in patients with moderate tuberculous meningitis (Whitworth L, Coxon J, van Laarhoven A, et al. A Bayesian analysis of the association between Leukotriene A4 Hydrolasegenotype and survival in tuberculous meningitis. Elife. 2021 Jan 8;10:e61722).

[0066] A first aspect of the present invention relates to a method for assessing whether a patient suffers from PCOS or has a risk of developing PCOS, comprising:

[0067] (a) determining the amount or concentration of LTA4H in a sample from the patient, and

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

[0069] The amount or concentration of LTA4H in the patient's sample increases, indicating the presence of PCOS in the patient or the risk of developing PCOS. In particular, if the amount or concentration of LTA4H in the patient's sample is higher than the amount or concentration of LTA4H in the reference or reference sample, the amount or concentration of LTA4H in the patient's sample indicates the presence of PCOS in the patient or the risk of developing PCOS. In particular, a higher amount or concentration of LTA4H can be detected in the same biofluid sample of a patient assessed to have PCOS or a risk of developing PCOS than in a biofluid sample of an individual who does not suffer from PCOS or does not have a risk of developing PCOS. In particular, the amount or concentration of LTA4H increases by 50% or more, indicating the presence of PCOS or the risk of developing PCOS. In particular, the amount or concentration of LTA4H increases by 100% or more, indicating the presence of PCOS or the risk of developing PCOS. In particular, the amount or concentration of LTA4H increases by 150% or more, indicating the presence of PCOS or the risk of developing PCOS. In particular, an increase in the amount or concentration of LTA4H by 200% or more indicates the presence of PCOS or a risk of developing PCOS.

[0070] In an embodiment, the biological fluid sample is whole blood, serum, plasma, capillary blood, interstitial fluid, peritoneal fluid or menstrual blood, preferably the biological fluid sample is serum or whole blood. In an embodiment, the sample is an in vitro sample, that is, it is analyzed in vitro and will not be moved back into the body.

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

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

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

[0074] In an embodiment, the amount or concentration of LTA4H is determined using an antibody, in particular 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 a direct or indirect form. In an embodiment, such an immunoassay is selected from the group consisting of an enzyme-linked immunosorbent assay (ELISA), an enzyme immunoassay (EIA), a radioimmunoassay (RIA), or an immunoassay based on luminescence, fluorescence, chemiluminescence, or electrochemiluminescence detection.

[0075] In certain embodiments, step a) of determining the amount or concentration of LTA4H in a sample from a patient comprises the following steps:

[0076] i) incubating the patient's sample with one or more antibodies that specifically bind to LTA4H, thereby generating a complex between the antibody and LTA4H, and

[0077] ii) quantifying the complex formed in step i), thereby quantifying the amount or concentration of LTA4H in the patient sample.

[0078] In a specific embodiment, in step i), the sample is incubated with two antibodies that specifically bind to LTA4H. As will be apparent to the skilled person, the sample may be contacted with the first and second antibodies in any desired order, i.e. first with the first antibody and then with the second antibody; or first with the second antibody and then with the first antibody; or simultaneously with the first and second antibodies. The contacting is carried out for a sufficient time and under conditions to form a first anti-LTA4H antibody / LTA4H / second anti-LTA4H antibody complex. As will be readily appreciated by the skilled person, this is merely routine experimentation to set suitable or sufficient time and conditions for the formation of a complex of a specific anti-LTA4H antibody and an LTA4H antigen / analyte (=anti-LTA4H complex), or for the formation of a secondary complex or sandwich complex comprising a first antibody to LTA4H, LTA4H (analyte) and a second anti-LTA4H antibody (=first anti-LTA4H antibody / LTA4H / second anti-LTA4H antibody complex).

[0079] The anti-LTA4H antibody / LTA4H complex may be detected by any suitable means. The detection of the first anti-LTA4H antibody / LTA4H / second anti-LTA4H antibody complex may be performed by any suitable means. Those skilled in the art are fully familiar with such means / methods.

[0080] In certain embodiments, a sandwich will be formed comprising a first antibody to LTA4H, LTA4H (analyte), and a second antibody to LTA4H, wherein the second antibody is detectably labeled.

[0081] In one embodiment, a sandwich will be formed comprising a first antibody to LTA4H, LTA4H (analyte), and a second antibody to LTA4H, wherein the second antibody is detectably labeled, and wherein the first anti-LTA4H antibody is capable of binding to or to a solid phase.

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

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

[0084] (c) determining the amount or concentration of LTA4H in a sample from the patient, and

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

[0086] In an embodiment, if the amount of LTA4H in a patient sample is determined to be elevated, the patient is selected for treatment of PCOS. In particular, if the amount of LTA4H is higher than the amount of LTA4H in a reference or reference sample, the patient is selected for treatment of PCOS. In particular, if the amount of LTA4H in a biological fluid sample of a patient being assessed for treatment of PCOS is higher than the amount in the same biological fluid sample of an individual who does not suffer from PCOS or is at risk of developing PCOS or is not selected for treatment of PCOS, the patient is selected for treatment of PCOS. In particular, if the amount of LTA4H is elevated by 50% or more, the patient is selected for treatment of PCOS. In particular, if the amount of LTA4H is elevated by 100% or more, the patient is selected for treatment of PCOS. In particular, if the amount of LTA4H is elevated by 150% or more, the patient is selected for treatment of PCOS. In particular, if the amount of LTA4H is elevated by 200% or more, the patient is selected for treatment of PCOS.

[0087] In particular, the patient is selected for drug-based therapy for PCOS or lifestyle changes to control metabolic symptoms. In an embodiment, the PCOS drug therapy is selected from the group consisting of drugs for regulating the menstrual cycle (particularly oral contraceptives or progestin therapy), drugs for preventing or controlling diabetes (particularly type 2 diabetes), drugs for preventing or controlling high cholesterol, hormones or drugs for increasing fertility, drugs, hormones or surgery for removing excess hair, drugs or surgery for controlling acne.

[0088] In an embodiment, the biological fluid sample is whole blood, serum, plasma, capillary blood, interstitial fluid, peritoneal fluid or menstrual blood, preferably the biological fluid sample is serum or whole blood. In an embodiment, the sample is an in vitro sample, that is, it is analyzed in vitro and will not be moved back into the body.

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

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

[0091] In an embodiment, the amount of LTA4H is determined using an antibody, in particular 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 a direct or indirect form. In an embodiment, such an immunoassay is selected from the group consisting of an enzyme-linked immunosorbent assay (ELISA), an enzyme immunoassay (EIA), a radioimmunoassay (RIA), or an immunoassay based on luminescence, fluorescence, chemiluminescence, or electrochemiluminescence detection.

[0092] In certain embodiments, step a) of determining the amount or concentration of LTA4H in a sample from a patient comprises the following steps:

[0093] i) incubating the patient's sample with one or more antibodies that specifically bind to LTA4H, thereby generating a complex between the antibody and LTA4H, and

[0094] ii) quantifying the complex formed in step i) and thereby quantifying the amount of LTA4H in the patient sample.

[0095] In a specific embodiment, in step i), the sample is incubated with two antibodies that specifically bind to LTA4H. It is obvious to the skilled person that the sample can be contacted with the first and second antibodies in any desired order, i.e. first with the first antibody and then with the second antibody or first with the second antibody and then with the first antibody, or simultaneously, for a time and under conditions sufficient to form a first anti-LTA4H antibody / LTA4H / second anti-LTA4H antibody complex. As will be readily appreciated by the skilled person, this is merely routine experimentation to set suitable or sufficient times and conditions for the formation of a complex of a specific anti-LTA4H antibody and a LTA4H antigen / analyte (=anti-LTA4H complex), or a secondary complex or sandwich complex comprising a first antibody to LTA4H, LTA4H (analyte) and a second anti-LTA4H antibody (=first anti-LTA4H antibody / LTA4H / second anti-LTA4H antibody complex).

[0096] The anti-LTA4H antibody / LTA4H complex may be detected by any suitable means. The detection of the first anti-LTA4H antibody / LTA4H / second anti-LTA4H antibody complex may be performed by any suitable means. Those skilled in the art are fully familiar with such means / methods.

[0097] In certain embodiments, a sandwich will be formed comprising a first antibody to LTA4H, LTA4H (analyte), and a second antibody to LTA4H, wherein the second antibody is detectably labeled.

[0098] In one embodiment, a sandwich will be formed comprising a first antibody to LTA4H, LTA4H (analyte), and a second antibody to LTA4H, wherein the second antibody is detectably labeled, and wherein the first anti-LTA4H antibody is capable of binding to or to a solid phase.

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

[0100] In a third aspect, the present invention relates to a method for monitoring the progression of PCOS in a patient or for monitoring the response of a patient suffering from PCOS to treatment, the method comprising:

[0101] (a) determining the level of LTA4H in a first sample from the patient,

[0102] (b) determining the level of LTA4H in a second sample of the patient that has been obtained after the first sample, and

[0103] (c) comparing the level of LTA4H in the first sample to the level of LTA4H in the second sample, and

[0104] (d) monitoring the progress of the patient suffering from or being treated for PCOS based on the results of step c).

[0105] In an embodiment, the PCOS progression of a patient suffering from PCOS is monitored to determine whether the amount or concentration of LTA4H in a patient sample changes over time. In particular, the PCOS progression is monitored to determine whether the amount or concentration of LTA4H increases, decreases, or remains unchanged over time. In an embodiment, the PCOS progression is monitored to determine whether the amount or concentration of LTA4H in a patient sample increases.

[0106] In an embodiment, a patient receiving treatment for PCOS is monitored to determine whether the amount or concentration of LTA4H in a patient sample is changing. In particular, a patient receiving treatment for PCOS is monitored to determine whether the amount or concentration of LTA4H is increasing, decreasing, or unchanged. In particular, a patient receiving treatment for PCOS is monitored to determine whether the amount or concentration of LTA4H is increasing, decreasing, or unchanged due to the applied treatment. In an embodiment, a decrease in the amount or concentration of LTA4H in a patient receiving treatment for PCOS indicates that the treatment is effective. In an embodiment, an unchanged or increased amount or concentration of LTA4H in a sample of a patient receiving treatment for PCOS indicates that PCOS persists. In particular, if the amount or concentration of LTA4H increases to 50% or more, the treatment for PCOS is ineffective. In particular, if the amount or concentration of LTA4H increases to 100% or more, the treatment for PCOS is ineffective. In particular, if the amount or concentration of LTA4H increases to 150% or more, the treatment for PCOS is ineffective. In particular, if the amount or concentration of LTA4H increases to 200% or more, treatment of PCOS is ineffective.

[0107] In certain embodiments, if the amount or concentration of LTA4H is determined to be unchanged or increased in a sample from a patient being treated for PCOS, the treatment is adjusted.

[0108] In an embodiment, the patient is monitored multiple times at different time points. In an embodiment, the patient is monitored several times over a time frame of weeks, months, or years. In a specific embodiment, the patient is monitored once a month or once a year. In an embodiment, the patient suffering from PCOS is monitored once a month or once a year after the diagnosis of PCOS. In an embodiment, the patient treated for PCOS is monitored once after therapy. In particular, the patient being treated for PCOS is monitored once a month or once a year to determine the effectiveness of the treatment.

[0109] In an embodiment, the PCOS therapy is selected from the group consisting of drug-based therapy for PCOS and lifestyle changes to control metabolic symptoms. In an embodiment, the PCOS drug therapy is selected from the group consisting of drugs for regulating the menstrual cycle (particularly oral contraceptives or progestin therapy), drugs for preventing or controlling diabetes (particularly type 2 diabetes), drugs for preventing or controlling high cholesterol, hormones or drugs for increasing fertility, drugs, hormones or surgery for removing excess hair, drugs or surgery for controlling acne.

[0110] In an embodiment, the biological fluid sample is whole blood, serum, plasma, capillary blood, interstitial fluid, peritoneal fluid or menstrual blood, preferably the biological fluid sample is serum or whole blood. In an embodiment, the sample is an in vitro sample, that is, it is analyzed in vitro and will not be moved back into the body.

[0111] In a specific embodiment, the patient is a laboratory animal, livestock, or primate. In a specific embodiment, the patient is a human patient. In a specific embodiment, the patient is a human female patient. In a specific embodiment, the patient is a female human patient less than 25 years old. In a specific embodiment, the patient is a female human patient less than 20 years old. In a specific embodiment, the patient is a female human patient from 15 years old to less than 25 years old. In a specific embodiment, the patient is a female human patient from 15 years old to less than 20 years old. In a specific embodiment, the patient is a female human patient less than 25 years old and three years after menarche. In a specific embodiment, the patient is a female human patient less than 20 years old and three years after menarche. In a specific embodiment, the patient is a female human patient from 15 years old to less than 25 years old and three years after menarche. In a specific embodiment, the patient is a female human patient from 15 years old to less than 20 years old and three years after menarche.

[0112] In an embodiment, the second method of the invention is an in vitro method.

[0113] In an embodiment, the amount or concentration of LTA4H is determined using an antibody, in particular 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 a direct or indirect form. In an embodiment, such an immunoassay is selected from the group consisting of an enzyme-linked immunosorbent assay (ELISA), an enzyme immunoassay (EIA), a radioimmunoassay (RIA), or an immunoassay based on luminescence, fluorescence, chemiluminescence, or electrochemiluminescence detection.

[0114] In certain embodiments, step a) of determining the amount or concentration of LTA4H in a sample from a patient comprises the following steps:

[0115] i) incubating the patient's sample with one or more antibodies that specifically bind to LTA4H, thereby generating a complex between the antibody and LTA4H, and

[0116] ii) quantifying the complex formed in step i), thereby quantifying the amount or concentration of LTA4H in the patient sample.

[0117] In a specific embodiment, in step i), the sample is incubated with two antibodies that specifically bind to LTA4H. As will be apparent to the skilled person, the sample may be contacted with the first and second antibodies in any desired order, i.e. first with the first antibody and then with the second antibody; or first with the second antibody and then with the first antibody; or simultaneously with the first and second antibodies. The contacting is carried out for a sufficient time and under conditions to form a first anti-LTA4H antibody / LTA4H / second anti-LTA4H antibody complex. As will be readily appreciated by the skilled person, this is merely routine experimentation to set suitable or sufficient time and conditions for the formation of a complex of a specific anti-LTA4H antibody and an LTA4H antigen / analyte (=anti-LTA4H complex), or for the formation of a secondary complex or sandwich complex comprising a first antibody to LTA4H, LTA4H (analyte) and a second anti-LTA4H antibody (=first anti-LTA4H antibody / LTA4H / second anti-LTA4H antibody complex).

[0118] The anti-LTA4H antibody / LTA4H complex may be detected by any suitable means. The detection of the first anti-LTA4H antibody / LTA4H / second anti-LTA4H antibody complex may be performed by any suitable means. Those skilled in the art are fully familiar with such means / methods.

[0119] In certain embodiments, a sandwich will be formed comprising a first antibody to LTA4H, LTA4H (analyte), and a second antibody to LTA4H, wherein the second antibody is detectably labeled.

[0120] In one embodiment, a sandwich will be formed comprising a first antibody to LTA4H, LTA4H (analyte), and a second antibody to LTA4H, wherein the second antibody is detectably labeled, and wherein the first anti-LTA4H antibody is capable of binding to or to a solid phase.

[0121] In embodiments, the second antibody is detectably labeled directly or indirectly. In specific embodiments, the second antibody is detectably labeled with a luminescent dye, particularly a chemiluminescent dye or an electrochemiluminescent dye.

[0122] In a fourth aspect, the present invention relates to a computer-implemented method for assessing a patient suffering from suspected PCOS, comprising the following steps:

[0123] (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,

[0124] (b) optionally, receiving a value for the amount or concentration of a second biomarker in the sample from the patient,

[0125] (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, hyperandrogenemia, and polycystic ovarian morphology;

[0126] (d) comparing the value for the amount or concentration of steps (a) to (b) with a reference for the biomarker and a value for the presence or absence of at least one additional diagnostic criterion and / or calculating a score for assessing a patient with suspected PCOS based on the amount or concentration of the biomarker and the value; and

[0127] (e) assessing the patient based on the comparison and / or calculation performed in step (d).

[0128] In an embodiment, the computer-implemented method for assessing a patient suffering from suspected PCOS comprises a method consisting essentially of the above steps or a method comprising other steps. In addition, the inventive method is preferably an ex vivo method, and more preferably an in vitro method. In addition, it may also include steps other than the steps explicitly mentioned above. For example, other steps may involve determining other markers and / or sample pretreatment or evaluating the results obtained by the method. The method may be performed manually or assisted by automation.

[0129] As used herein, the term "computer-implemented" means that the method is performed in an automated manner on a data processing unit, which is typically included in a computer or similar data processing device. The data processing unit should receive the value of the amount of the biomarker. Such a value can be an amount, a relative amount, or any other calculated value reflecting the amount as described in detail elsewhere herein. Therefore, it should be understood that the above method does not require the determination of the amount of the biomarker, but uses the value of the amount that has been predetermined.

[0130] In principle, the present invention also envisages a computer program, a computer program product or a computer-readable storage medium having said computer program tangibly embedded therein, wherein the computer program comprises instructions for performing the above-described method of the present invention when the instructions are run on a data processing device or a computer.

[0131] Specifically, the present disclosure further includes:

[0132] - a computer or a computer network comprising at least one processor, wherein the processor is adapted to perform a method according to one of the embodiments described in this specification,

[0133] - a computer-loadable data structure adapted to carry out a method according to one of the embodiments described in this specification when the data structure is executed on a computer,

[0134] - a computer script, wherein the computer program is adapted to perform a method according to one of the embodiments described in the present description when the program is executed on a computer,

[0135] - a computer program comprising program means for executing a method according to one of the embodiments described in the present description when the computer program is being executed on a computer or on a computer network,

[0136] - a computer program comprising program means according to the preceding embodiment, wherein the program means is stored on a computer-readable storage medium,

[0137] a storage medium, on which a data structure is stored and on which the data structure is suitable, after being loaded into a main storage device and / or a working storage device of a computer or a computer network, for executing a method according to one of the embodiments described in the present description,

[0138] - a computer program product having program code means, wherein these program code means can be stored or are stored on a storage medium for executing the method according to one of the embodiments described in the present description when the program code means are executed on a computer or on a computer network,

[0139] - a data stream signal, usually encrypted, comprising data of the parameters defined elsewhere herein,

[0140] as well as

[0141] - A data stream signal, usually encrypted, comprising the assessment provided by the method of the invention.

[0142] In addition, the present invention also relates to a kit comprising reagents for diagnosing PCOS. The reagents of the kit may include antibodies or antibody fragments. Preferably, the antibody or antibody fragment recognizes an epitope or antigen of LTA4H. The kit may also include other reagents that recognize other biomarkers. Therefore, the kit may include a combination of at least two reagents. The kit may specifically measure the amount or concentration of LTA4H and any target biomarker. According to the present invention, biomarkers may also include hormones, such as anti-mullerian hormone, AMH. The kit can be used for any diagnostic assay.

[0143] definition:

[0144] In the context of the kit of the invention, the term "reagent" describes a substance or compound added to a sample which allows the amount or concentration of a specific component in the sample to be revealed.

[0145] In the context of the kit of the present invention, the term "specific measurement" refers to the detection of an exact amount or concentration of a well-defined molecule. For a specific measurement, a sample obtained from a female can be incubated with a reagent under conditions suitable for forming a binding agent marker-complex. Such conditions do not need to be specified, as such suitable incubation conditions are well known to those skilled in the art.

[0146] In the context of the kit of the present invention, the term "reagent" may describe a protein molecule (such as an antibody), a nucleic acid molecule (such as any form of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)), or another biochemical, organic or inorganic substance that can interact with a molecule to be specifically measured in a sample.

[0147] Further, the reagent can be connected to a detectable reporter part or label, such as an enzyme, dye, radionuclide, luminescent group, fluorescent group or biotin, such as a fluorescent marker that can be used for immunoassay analysis. Any reporter part or label can be used with the reagent of the kit according to the second aspect of the present invention, as long as its signal can be directly related to or proportional to the amount of the binding agent retained on the support after washing. The amount of the optional second binding agent that remains bound to the solid support can then be determined using a method suitable for a specific detectable reporter part or label. For radioactive groups, scintillation counting or autoradiographic artifact methods are generally suitable. Antibody-enzyme conjugates can be prepared using a variety of coupling techniques (for review, see, for example, Scouten, WH, Methods in Enzymology 135: 30-65, 1987). Spectroscopy can be used to detect dyes (including, for example, colorimetric products of enzyme reactions), luminescent groups and fluorescent groups. Biotin can be detected using avidin or streptavidin, coupled to different reporter groups (usually radioactive groups or fluorescent groups or enzymes). Enzyme reporter groups can typically be detected by adding substrate (usually for a specific time) followed by spectroscopic, spectrophotometric or other analysis of the reaction products.Standards and standard additives can be used to determine the level of antigen in a sample using techniques well known to those skilled in the art.

[0148] The reagent may also be a substance that is additionally capable of being connected to the matrix of a column for chromatography for purification and / or further analysis, such as mass spectrometry. Furthermore, the reagent may be connected to a test strip.

[0149] Preferably, the reagent is an antibody. Suitable antibodies for measuring the amount or concentration of one of the molecules to be specifically measured in a sample obtained from the above female are well known to those skilled in the art.

[0150] Preferably, the reagent is usable in electrochemiluminescence-immunoassay, and more preferably, the reagent is an antibody usable in electrochemiluminescence-immunoassay.

[0151] In addition, the kit may contain more than one reagent (such as two different reagents, three different reagents, four different reagents or more different reagents), preferably two different reagents to interact with a molecule specifically measured in the sample. For example, if the molecule specifically measured is measured in an electrochemiluminescence-immunoassay, the kit may contain two different antibodies that bind to the same molecule to be measured. Preferably, the two different antibodies that bind to the same molecule do not compete for the binding site at the molecule, but bind to the molecule at different positions. Further, both antibodies can be connected to different detectable reporting parts or labels.

[0152] The kit may further comprise a buffer and / or salt to adjust pH and reaction and measurement conditions. In addition, the kit may comprise a stabilizer, for example, to support the stability of the reagent and / or hormone during a specific measurement of (i) the amount or concentration of FT or (ii) the amount or concentration of TT and the amount or concentration of SHBG; the amount or concentration of AMH; and the amount or concentration of one or more additional hormones indicating PCOS. Suitable buffers, salts and stabilizers are well known to those skilled in the art. In addition, sodium azide can be added to all liquid solutions (such as reagents or buffers) of the kit.

[0153] The kit may also include all the equipment required to collect a blood sample from a female (such as a container for the blood sample, a needle and a device for connecting the container and the needle). Preferably, the kit may include a syringe.

[0154] Generally speaking, a doctor or a doctor's assistant can collect blood from a female. Subsequently, the blood can be sent to a laboratory where the sample is measured using a kit on a designated analyzer and the data is sent to the doctor. However, the kit can also be used by the doctor or the doctor's assistant himself. The kit can be applied during a doctor's office visit, a fixed treatment, or a home visit.

[0155] All components of the kit may be packaged separately in a single container. However, two or more components of the kit may also be packaged together in one or more containers.

[0156] The test kit may further include a label, for example, a label including instructions on how to use the test kit or describing the contents of the test kit. However, this information may also be provided in any other form, such as on a storage medium, such as a CD-ROM or a USB flash drive.

[0157] The word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of stated integers or steps or groups of integers or steps but not the exclusion of any other integers or steps or groups of integers or steps.

[0158] As used in this specification and the appended claims, the singular forms "a," "an," "the," and "said" include plural referents unless the content clearly dictates otherwise.

[0159] Concentration, amount and other numerical data can be expressed or presented in "range" format herein.It should be understood that such range format is used only for convenience and simplicity, and therefore should be flexibly interpreted as not only including the numerical value clearly listed as the range limit, but also including all individual numerical values ​​or sub-ranges covered by the range, just as each numerical value and sub-range are clearly listed.As an illustration, the numerical range "150mg to 600mg" should be interpreted as not only including the value 150mg to 600mg clearly listed, but also including individual values ​​and sub-ranges within the indicated range.Therefore, individual values, such as 150, 160, 170, 180, 190, 580, 590, 600mg and sub-ranges are included in this numerical range, such as from 150 to 200, 150 to 250, 250 to 300, 350 to 600, etc.This same principle applies to the range of only quoting one numerical value.In addition, no matter how wide the range or feature is, such explanation is applicable.

[0160] When used in connection with a numerical value, the term "about" is meant to encompass the numerical value within a range having a lower limit that is 5% less than the indicated numerical value and an upper limit that is 5% greater than the indicated numerical value.

[0161] As used herein, the term "indicator" refers to a sign or signal of a condition or is used to monitor a condition. Such a "condition" refers to a biological state of a cell, tissue, or organ or refers to the health and / or disease state of an individual. An indicator can be the presence or absence of a molecule (including but not limited to peptides, proteins, and nucleic acids), or can be a change in the expression level or pattern of such a molecule in a cell, tissue, organ, or individual. An indicator can be a sign of the onset, development, or presence of a disease in an individual or further progression of such a disease. An indicator can also be a sign of the risk of developing a disease in an individual.

[0162] In the context of the present invention, the term "biomarker" refers to a substance within a biological system that is used as an indicator of the biological state of the system. In the art, the term "biomarker" is sometimes also applied to means for detecting the endogenous substance (e.g., antibodies, nucleic acid probes, etc., imaging systems). In the context of the present invention, the term "biomarker" should only apply to substances, not to detection means. Therefore, a biomarker can be any type of molecule present in a living organism, such as nucleic acids (DNA, mRNA, miRNA, rRNA, etc.), proteins (cell surface receptors, cytoplasmic proteins, etc.), metabolites or hormones (blood sugar, insulin, estrogen, etc.), molecules with certain modification characteristics of another molecule (e.g., sugar moieties or phosphoryl residues on proteins, methyl residues on genomic DNA) or substances that have been internalized by an organism or metabolites of such substances.

[0163] The biomarkers mentioned herein can be detected using methods generally known in the art. Detection methods generally include methods for quantifying the level of biomarkers in samples (quantitative methods). Those skilled in the art generally know which of the following methods are suitable for qualitative and / or quantitative detection of biomarkers. Commercially available Western and immunoassays, such as ELISA, RIA, fluorescence and luminescence-based immunoassays and proximity extension assays can be used to conveniently measure, for example, proteins in samples. Other suitable methods for detecting biomarkers include measuring physical or chemical properties specific to peptides or polypeptides, such as their precise molecular mass or NMR spectra. The method includes, for example, biosensors, optical devices coupled to immunoassays, biochips, analytical devices (such as mass spectrometers, NMR analyzers or chromatographic devices). Further, the method includes methods based on microplate ELISA, fully automatic or robotic immunoassays (e.g., in Elecsys TM Analyzers), CBA (e.g., at Roche-Hitachi TM Enzyme cobalt binding assays available on analyzers) and latex agglutination assays (e.g., on Roche-Hitachi TM available on the analyzer).

[0164] The term "anovulation" generally describes a situation in which the ovaries do not release any oocytes at all during the female menstrual cycle. If there is no oocyte release during the duration of at least one female menstrual cycle, preferably at least three female menstrual cycles, more preferably at least six female menstrual cycles, and most preferably at least nine female menstrual cycles within a year, it can be determined that the women assessed for PCOS risk have anovulation. Further, if there is no oocyte release during the duration of at least 6 months, preferably at least 9 months, and more preferably at least 1 year, it can be determined that the females assessed for PCOS risk have anovulation.

[0165] "Symptoms" of a disease refer to noticeable disease in tissues, organs or organisms suffering from such diseases, including but not limited to pain, weakness, tenderness, tension, stiffness and cramping of tissues, organs or individuals. Typical symptoms of PCOS include but are not limited to oligo-anovulation, irregular cycles, hyperandrogenism, polycystic ovarian morphology, infertility, type 2 diabetes, overweight and other metabolic disorders and psychological distress. "Signs" or "signals" of a disease include but are not limited to changes or alterations, such as the presence, absence, increase or increase, decrease or decrease of specific indicators such as biomarkers or molecular markers, or the development, existence, or worsening of symptoms. Symptoms of pain include but are not limited to an unpleasant feeling that may manifest as persistent or varying degrees of burning, throbbing, itching or tingling.

[0166] The terms "disease" and "illness" are used interchangeably herein and refer to abnormal conditions, especially abnormal medical conditions, such as illness or injury, in which tissues, organs or individuals are no longer able to effectively perform their functions. Typically, but not necessarily, a disease is associated with specific symptoms or signs indicating the presence of such a disease. Therefore, the presence of such symptoms or signs may indicate a tissue, organ or individual suffering from a disease. Changes in these symptoms or signs may indicate the progression of the disease. The typical feature of disease progression is the increase or decrease of these symptoms or signs, which may indicate a "deterioration" or "improvement" of the disease. The "deterioration" of a disease is characterized by a decrease in the ability of a tissue, organ or organism to effectively perform its function, while the "improvement" of a disease is usually characterized by an increase in the ability of a tissue, organ or organism to effectively perform its function. Tissues, organs or individuals with a "risk of developing" a disease are in a healthy state, but show the possibility of developing the disease. Typically, the risk of developing a disease is associated with early or weak signs or symptoms of the disease. In this case, the onset of the disease can still be prevented by treatment. Examples of diseases include, but are not limited to, inflammatory diseases, infectious diseases, skin diseases, endocrine diseases, intestinal diseases, nervous system diseases, joint diseases, genetic diseases, autoimmune diseases, traumatic diseases, and various types of cancer.

[0167] As used herein, the terms "patient" and "subject" are used interchangeably and refer to an animal, preferably a mammal, and more typically a human. The patient is preferably a human female.

[0168] The term "sample" or "target sample" is used interchangeably herein and refers to a portion or slice of a tissue, organ or individual, usually smaller than such tissue, organ or individual, intended to represent the entire tissue, organ or individual. When analyzed, the sample provides information about the state of the tissue or the health or diseased state of an organ or individual. Examples of samples include, but are not limited to, liquid samples such as blood, serum, plasma, synovial fluid, urine, saliva and lymph; or solid samples such as tissue extracts, cartilage, bone, synovium and connective tissue. The analysis of the sample can be completed on a visual or chemical basis. Visual analysis includes, but is not limited to, microscopic imaging or radiographic scanning of tissues, organs or individuals to allow morphological assessment of the sample. Chemical analysis includes, but is not limited to, detecting the presence or absence of a specific indicator or changes in its amount, concentration or level. The sample is an in vitro sample that will be analyzed in vitro and will not be moved back into the body.

[0169] As used herein, the term "amount" encompasses the absolute amount of the biomarker mentioned herein, the relative amount or concentration of the biomarker, and any value or parameter associated with or derivable therefrom. Such values ​​or parameters include intensity signal values ​​from all specific physical or chemical properties obtained from the peptide by direct measurement, such as intensity values ​​in a mass spectrum or NMR spectrum. In addition, included are all values ​​or parameters obtained by indirect measurements specified elsewhere in this specification, for example, the amount of reaction measured from a biological readout system in response to an intensity signal obtained from a peptide or from a specifically bound ligand. It should be understood that values ​​associated with the above amounts or parameters can also be obtained by all standard mathematical operations.

[0170] As used herein, the term "comparison" refers to comparing the amount of a biomarker in a sample from a subject with a reference amount of a biomarker specified elsewhere in this specification. It should be understood that comparison as used herein generally refers to the comparison of corresponding parameters or values, for example, comparing an absolute amount with an absolute reference amount, and comparing a concentration with a reference concentration, or comparing an intensity signal obtained from a biomarker in a sample with an intensity signal of the same type obtained from a reference sample. The comparison can be performed manually or computer-assisted. Therefore, the comparison can be performed by a computing device. For example, the value of the measurement or detection amount of a biomarker in a sample from a subject can be compared with a reference amount, and the comparison can be automatically performed by a computer program that performs a comparison algorithm. The computer program performing the evaluation will provide the required assessment in an appropriate output format. For computer-assisted comparison, the value of the measured amount can be compared with the value corresponding to a suitable reference stored in a database by a computer program. The computer program can further evaluate the result of the comparison, i.e., automatically provide the required assessment in an appropriate output format. For computer-assisted comparison, the value of the measured amount can be compared with the value corresponding to a suitable reference stored in a database by a computer program. The computer program can further evaluate the result of the comparison, i.e., automatically provide the required assessment in an appropriate output format.

[0171] The expression "the determined amount or concentration is compared with a reference" is in any case only used to further explain what is obvious to the skilled person. A reference concentration is established in a control sample.

[0172] As used herein, the term "reference sample" or "control sample" refers to a sample that is analyzed in substantially the same manner as the target sample and its information is compared with that of the target sample. Therefore, a reference sample provides a standard for evaluating the information obtained from the target sample. The control sample may be derived from a healthy or normal tissue, organ or individual, thereby providing a standard for the health status of a tissue, organ or individual. The difference between the state of a normal reference sample and the state of a target sample may indicate the risk of disease development or the presence or further progression of such a disease or illness. The control sample may be derived from an abnormal or diseased tissue, organ or individual, thereby providing a standard for the diseased state of a tissue, organ or individual. The difference between the state of an abnormal reference sample and the state of a target sample may indicate a reduced risk of disease development or the absence or improvement of such a disease or illness. The reference sample may also be derived from the same tissue, organ or individual as the target sample, but has been obtained at an earlier time point. The difference between the state of the reference sample obtained earlier and the state of the target sample may indicate the progression of the disease, i.e., the disease improves or worsens over time.

[0173] The control sample can be an internal or external control sample. With an internal control sample, the marker level is assessed in the test sample and one or more other samples taken from the same subject to determine whether there is any change in the level of the marker. With an external control sample, the presence or amount of the marker in a sample taken from an individual is compared to its presence or amount in an individual known to have or known to be at risk for a given condition or an individual known not to have a given condition, i.e., a "normal individual".

[0174] Those skilled in the art will appreciate that such external control samples may be obtained from a single individual or from a reference population that is age-matched and free of confounding disease. Typically, samples of 100 well-characterized individuals from an appropriate reference population are used to establish a "reference value". However, a reference population consisting of 20, 30, 50, 200, 500 or 1000 individuals may also be selected. Healthy individuals represent a preferred reference population for establishing a control value.

[0175] For example, the marker concentration in the patient sample can be compared with the concentration known to be associated with the specific course of a certain disease. Usually the marker concentration of the sample is directly or indirectly related to the diagnosis, and the marker concentration is used, for example, to determine whether an individual has a risk of suffering from a certain disease. Alternatively, in judging the risk of disease progression or in the follow-up of the patient, the marker concentration of the sample can be compared, for example, with the marker concentration known to be associated with the following: a therapeutic response to a certain disease, a diagnosis of a certain disease, an assessment of the severity of a certain disease, and guidance for selecting appropriate drugs for a certain disease. According to the expected diagnostic use, an appropriate control sample is selected and a control or reference value of the marker is established therein. It is also clear to the technician that the absolute marker value established in the control sample will depend on the assay used.

[0176] The term "assessment" used herein refers to assessing whether a patient suffers from PCOS or is at risk of developing PCOS. Therefore, the assessment used herein includes diagnosing PCOS, predicting the risk of developing PCOS, selecting a therapy for PCOS, monitoring a patient suffering from PCOS or being treated for PCOS, by determining the amount or concentration of LTA4H in a patient sample, and comparing the determined amount or concentration with a reference.

[0177] As will be appreciated by those skilled in the art, although the assessment made according to the present invention is preferred, it may not be correct for 100% of the subjects studied. The term generally requires the ability to correctly assess a statistically significant portion of subjects. Those skilled in the art can use various well-known statistical assessment tools (e.g., determining confidence intervals, determining p-values, Student's t-tests, Mann-Whitney tests, etc.) to effortlessly determine whether a portion is statistically significant. For detailed information, see Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. The confidence intervals generally envisioned are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. The p-value is typically 0.2, 0.1, 0.05.

[0178] The term "decreased" or "decreased" level, amount and / or concentration of an indicator refers to a decrease in the level, amount and / or concentration of such indicator in a sample compared to a reference or reference sample.

[0179] The term "elevated" or "increased" level, amount and / or concentration of an indicator refers to a level, amount and / or concentration of such an indicator in a sample that is higher compared to a reference or reference sample. For example, a protein may have an elevated level that is detected in a higher amount or concentration in a fluid sample of an individual suffering from a given disease than in the same fluid sample of an individual not suffering from the disease.

[0180] The terms "measurement", "measuring" or "determining" preferably include qualitative, semi-quantitative or quantitative measurement.

[0181] As used herein, the term "immunoglobulin (Ig)" refers to the immunity-conferring glycoprotein of the immunoglobulin superfamily. "Surface immunoglobulins" are attached to the membrane of effector cells via their transmembrane regions and encompass molecules such as, but not limited to, B cell receptors, T cell receptors, class I and class II major histocompatibility complex (MHC) proteins, beta-2 microglobulin (about 2M), CD3, CD4, and CDS.

[0182] Generally, as used herein, the term "antibody" refers to a secretory immunoglobulin that lacks a transmembrane region and can therefore be released into the bloodstream and body cavity. Human antibodies are divided into different isotypes based on the heavy chains they possess. There are five types of human Ig heavy chains, represented by Greek letters: α, γ, δ, ε and μ. The type of heavy chain present defines the category of the antibody, i.e., these chains are present in IgA, IgD, IgE, IgG and IgM antibodies, respectively, each playing a different role, and guiding the appropriate immune response to different types of antigens. Different heavy chains differ in size and composition; and can contain approximately 450 amino acids (Janeway et al. (2001) Immunobiology, Garland Science). IgA is present in mucosal areas, such as the intestinal tract, respiratory tract and urogenital tract, as well as in saliva, tears and breast milk, to prevent pathogen colonization (Underdown & Schiff (1986) Annu. Rev. Immunol. 4: 389-417). IgD acts primarily as an antigen receptor on B cells that are not exposed to antigens, and is involved in activating basophils and mast cells to produce antimicrobial factors (Geisberger et al. (2006) Immunology 118: 429-437; Chen et al. (2009) Nat. Immunol. 10: 889-898). IgE participates in allergic reactions by binding to allergens, triggering mast cells and basophils to release histamine. IgE is also involved in preventing parasites (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). IgG provides most of the antibody-based immunity against invading pathogens, and is the only antibody isotype that can cross the placenta to provide passive immunity to the fetus (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). In humans, there are four different IgG subclasses (IgG1, 2, 3 and 4), named according to their abundance order in serum, with IgG1 being the most abundant (about 66%), followed by IgG2 (about 23%), IgG3 (about 7%) and IgG (about 4%). The biological properties of different IgG classes are determined by the structure of the corresponding hinge region. IgM is expressed on the surface of B cells in monomeric and secretory pentamer forms with very high avidity. Before sufficient IgG is produced, IgM participates in the elimination of pathogens in the early stages of B cell-mediated (humoral) immunity (Geisberger et al. (2006) Immunology 118: 429-437).Antibodies exist not only in monomeric form, but are also known to form dimers of two Ig units (e.g., IgA), tetramers of four Ig units (e.g., IgM of bony fish), or pentamers of five Ig units (e.g., IgM of mammals). Antibodies are usually composed of four polypeptide chains, including two identical heavy chains and two identical light chains, which are connected via disulfide bonds and resemble "Y"-shaped macromolecules. Each chain contains many immunoglobulin domains, some of which are constant domains, while others are variable domains. The immunoglobulin domain consists of a 2-layer sandwich structure, in which 7 to 9 antiparallel chains are arranged in two sheets. Typically, the heavy chain of an antibody contains four Ig domains, three of which are constant (CH domains: CHI, CH2, CH3) domains, and one of which is a variable domain (VH). The light chain usually contains a constant Ig domain (CL) and a variable Ig domain (VL). For example, the human IgG heavy chain consists of four connected Ig domains in the order of VwCH1-CH2-CH3 (also called VwCyl-Cy2-Cy3) from the N-terminus to the C-terminus, while the human IgG light chain consists of two connected immunoglobulin domains in the order of VL-CL from the N-terminus to the C-terminus, which is a κ type or a λ type (VK-CK or VA.-CA.). For example, the constant chain of human IgG contains 447 amino acids. In the present specification and claims, the numbering of amino acid positions in immunoglobulins is the numbering of the "EU index", such as in the following documents: Kabat, EA, Wu, TT, Perry, HM, Gottesman, KS and Foeller, C., (1991) Sequences of proteins of immunological interest, 5th edition US Department of Health and Human Service, National Institutes of Health, Bethesda, MD. For example, the "EU index in Kabat" refers to the residue numbering of human IgG1EU antibodies. Thus, the CH domains in the context of IgG are as follows: "CHI" refers to amino acid positions 118-220 according to the EU index as in Kabat; "CH2" refers to amino acid positions 237-340 according to the EU index as in Kabat; and "CH3" refers to amino acid positions 341-447 according to the EU index as in Kabat.

[0183] The terms "full length antibody", "intact antibody" and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form rather than an antibody fragment as defined below. The term specifically refers to an antibody having heavy chains that include an Fc region.

[0184] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab fragments" (also called "Fab portions" or "Fab regions"), each with a single antigen-binding site, and a residual "Fc fragment" (also called "Fc portion" or "Fc region"), a name that reflects its ability to crystallize easily. The crystal structure of the human IgG Fc region has been determined (Deisenhofer (1981) Biochemistry 20: 2361-2370). In IgG, IgA and IgD isotypes, the Fc region consists of two identical protein fragments derived from the CH2 and CH3 domains of the two heavy chains of the antibody; in IgM and IgE isotypes, the Fc region contains three heavy chain constant domains (CH2-4) in each polypeptide chain. In addition, smaller immunoglobulin molecules exist naturally or have been artificially constructed. The term "Fab' fragment" refers to a Fab fragment that additionally includes the hinge region of an Ig molecule, while a "F(ab')2 fragment" is understood to include two Fab' fragments that are chemically linked or connected via a disulfide bond. While "single domain antibodies (sdAb)" (Desmyter et al. (1996) Nat. Structure Biol. 3: 803-811) and "nanobodies" include only a single VH domain, "single chain Fv (scFv)" fragments include a heavy chain variable domain joined to a light chain variable domain via a short linker peptide (Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85, 5879-5883). A divalent single chain variable fragment (di-scFv) can be engineered by connecting two scFvs (scFvA-scFvB). This can be achieved by creating a single peptide chain with two VH and two VL regions, thereby creating a "tandem scFv" (VHA-VLA-VHB-VLB). Another possibility is to create an scFv with a linker that is too short for the two variable regions to fold together, thereby forcing the scFv to dimerize. These dimers are usually produced using a linker of 5 residues in length. This type is referred to as a "diabody". A shorter linker (one or two amino acids) between the VH and VL domains results in the formation of monospecific trimers, so-called "triabodies" or "tribadies". Bispecific diabodies are formed by expressing two chains arranged with VHA-VLB and VHB-VLA or VLA-VHB and VLB-VHA, respectively. Single-chain diabodies (scDb) include VHA-VLB and VHB-VLA fragments, which are connected (VHA-VLB-P-VHB-VLA) by a linker peptide (P) of 12-20 amino acids, preferably 14 amino acids."Bispecific T cell engagers (BiTEs)" are fusion proteins consisting of two scFvs of different antibodies, one of which binds to T cells via the CD3 receptor and the other to tumor cells via a tumor-specific molecule (Kufer et al. (2004) Trends Biotechnol. 22: 238-244). Dual affinity retargeting molecules ("DART" molecules) are diabodies that are additionally stabilized by a C-terminal disulfide bond.

[0185] Therefore, the term "antibody fragment" refers to a portion of an intact antibody, preferably comprising 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.

[0186] The term "binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including but not limited to surface plasmon resonance-based assays (e.g., BIAcore assays described in PCT Application Publication No. WO2005 / 012359); enzyme-linked immunosorbent assays (ELISA); and competitive assays (e.g., RIA). Low-affinity antibodies generally bind antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigens quickly and tend to maintain longer binding times. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention.

[0187] "Sandwich immunoassays" are widely used to detect target analytes. In this assay, the analyte is "sandwiched" between a first antibody and a second antibody. Typically, sandwich assays require that the capture and detection antibodies bind to different non-covering epitopes on the target analyte. This sandwich complex is measured by an appropriate means and the analyte is quantified thereby. In a typical sandwich assay, a first antibody bound to a solid support or capable of binding to a solid phase and a second antibody detectably labeled each bind to the analyte at different non-covering epitopes. A first analyte-specific binding agent (e.g., an antibody) is covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. The solid support may be in the form of a tubule, magnetic beads, microplate tray, or any other surface suitable for performing an immunoassay. Binding methods are well known in the art and typically consist of cross-linked covalent binding or physical adsorption, with the polymer-antibody complex being washed when the test sample is prepared. An aliquot of the sample to be tested is then added to the solid phase complex and incubated under appropriate conditions (e.g., from room temperature to 40°C, such as between 25°C and 37°C, inclusive) for a sufficient period of time (e.g., 2-40 minutes or overnight if more convenient) to allow binding between the first or capture antibody and the corresponding antigen. After the incubation period is complete, the solid phase, which includes the first or capture antibody and the antigen bound thereto, can be washed and incubated with a secondary antibody or labeled antibody that binds to another epitope on the antigen. The second antibody is linked to a reporter molecule that is used to indicate binding between the second antibody and the first antibody-target antigen complex.

[0188] Other sandwich assay formats that are very versatile include the use of solid phase supports coated with the first partner of the binding pair, such as paramagnetic streptavidin-coated microparticles. Such microparticles are mixed and incubated with: an analyte-specific binding agent (e.g., a biotinylated antibody) that binds to the second partner of the binding pair; a sample suspected of or including the analyte, wherein the second partner of the binding pair binds to the analyte-specific binding agent; and a detectably labeled second analyte-specific binding agent. As will be apparent to one skilled in the art, the components are incubated under appropriate conditions for a sufficient period of time to allow the labeled antibody (via the analyte), the analyte-specific binding agent that binds to the second partner of the binding pair, and the first partner of the binding pair to bind to the solid phase microparticles. Optionally, the assay may include one or more washing steps.

[0189] The term "detectably labeled" encompasses labels that are detectable either directly or indirectly.

[0190] Directly detectable labels provide a detectable signal or they interact with a second label to modify the detectable signal provided by the first or second label, for example, FRET (fluorescence resonance energy transfer) occurs. Labels such as fluorescent dyes and luminescent (including chemiluminescent and electrochemiluminescent) dyes (Briggs et al. "Synthesis of Functionalised Fluorescent Dyes and Their Coupling to Amines and Amino Acids," J. Chem. Soc., Perkin-Trans. 1 (1997) 1051-1058) provide a detectable signal and are generally suitable for labeling. In one embodiment, "detectably labeled" refers to a label that provides or induces a detectable signal, i.e., a fluorescent label, a luminescent label (e.g., a chemiluminescent label or an electrochemiluminescent label), a radioactive label, or a metal chelate-based label.

[0191] The large number of available labels (also called dyes) can be generally divided into the following categories, all of which collectively and each of which represent embodiments as described in the present disclosure:

[0192] (a) Fluorescent dyes

[0193] 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).

[0194] Fluorescent labels or fluorophores include rare earth chelates (europium chelates); fluorescein type labels, including FITC, 5-carboxyfluorescein, 6-carboxyfluorescein; rhodamine labels, including TAMRA; dansyl; Lissamine; cyanine; phycoerythrin; Texas Red; and analogs thereof. Using the techniques disclosed herein, fluorescent labels can be conjugated to aldehyde groups contained in target molecules. Fluorescent dyes and fluorescent labeling reagents include such fluorescent dyes and reagents available from Invitrogen / Molecular Probes (Eugene, Oregon, USA) and Pierce Biotechnology, Inc. (Rockford, Ill.).

[0195] (b) Luminescent dyes

[0196] Luminescent dyes or labels can be further divided into the following subcategories: chemiluminescent dyes and electrochemiluminescent dyes.

[0197] Different classes of chemiluminescent labels include luminol, acridine compounds, coelenterazine and analogs, dioxetanes, peroxyoxalic acid-based systems and their derivatives. For immunodiagnostic procedures, acridine-based labels are mainly used (for a detailed review, see Dodeigne C. et al., Talanta 51 (2000) 415-439).

[0198] The main related labels used as electrochemiluminescent labels are ruthenium-based and iridium-based electrochemiluminescent complexes, respectively. Electrochemiluminescence (ECL) has proven to be very useful in analytical applications as a highly sensitive and selective method. This method combines the analytical advantages of chemiluminescent analysis (no background light signal) with a more convenient control of the reaction by adopting electrode potentials. Typically, ruthenium complexes, especially [Ru(Bpy)3]2+ (emitting photons of about 620 nm) regenerated with TPA (tripropylamine) in the liquid phase or at the liquid-solid interface, are used as ECL labels.

[0199] Electrochemiluminescence (ECL) assays provide a method for sensitively and accurately detecting the presence and concentration of target analytes. The technology uses labels or other reactants that can be induced to emit light when electrochemically oxidized or reduced under appropriate chemical conditions. This electrochemiluminescence is triggered by a voltage applied to a working electrode at a specific time and in a specific manner. The light emitted by the label, after measurement, can indicate the presence or amount of the analyte. In order to more fully describe this type of ECL technology, the following documents are cited in this article: U.S. Patent No. 5,221,605, U.S. Patent No. 5,591,581, U.S. Patent No. 5,597,910, PCT Published Application WO90 / 05296, PCT Published Application WO92 / 14139, PCT Published Application WO90 / 05301, PCT Published Application WO96 / 24690, PCT Published Application US95 / 03190, PCT Application US97 / 16942, PCT Published Application U S96 / 06763, PCT published application WO95 / 08644, PCT published application WO96 / 06946, PCT published application WO96 / 33411, PCT published application WO87 / 06706, PCT published application WO96 / 39534, PCT published application WO96 / 41175, PCT published application WO96 / 40978, PCT / US97 / 03653 and U.S. Patent Application 08 / 437,348 (U.S. Patent No. 5,679,519). Also quoted is the ECL analysis application review (Analyst, 1994, 119: 879-890) published by Knight et al. in 1994 and the literature cited by the article. In one embodiment, the method described in this description is implemented using electrochemiluminescent labeling.

[0200] Recently, iridium-based ECL labels have also been described (WO2012107419).

[0201] (c) Radioactive labeling uses radioactive isotopes (radionuclides) such as 3H, 11C, 14C, 18F, 32P, 35S, 64Cu, 68Gn, 86Y, 89Zr, 99TC, 111In, 123I, 124I, 125I, 131I, 133Xe, 177Lu, 211At or 131Bi.

[0202] (d) Metal chelate complexes are suitable for use as labels for imaging and therapeutic purposes, as is known in the art (US 2010 / 0111861; US ​​5,342,606; US 5,428,155; US 5,316,757; US 5,480,990; US 5,462,725; US 5,428,139; US 5,385,893; US 5,739,294; US 5,750,660; US 5,834,461; Hnatowich et al., J. Immunol. Methods 65 (1983) 147-157; Meares et al., Anal. Biochem. 142 (1984) 68-78; Mirzadeh et al., Bioconjugate Chem. 1 (1990) 59-65; Meares et al., J. Cancer (1990), Suppl. 10:21-26; Izard et al., Bioconjugate Chem. 3 (1992) 346-350; Nikula et al., Nucl. Med. Biol. 22 (1995) 387-90; Camera et al., Nucl. Med. Biol. 20 (1993) 955-62; Kukis et al., J. Nucl. Med. 39 (1998) 2105-2110; Verel et al., J. Nucl. Med. 44 (2003) 1663-1670; Camera et al., J. Nucl. Med. 21 (1994) 640-646; Ruegg et al., Cancer Res.50 (1990) 4221-4226; Verel et al., J.Nucl.Med.44 (2003) 1663-1670; Lee et al., Cancer Res.61 (2001) 4474-4482; Mitchell et al., J.Nucl.Med.44 (2003) 1105-1112; Kobayashi et al., Bioconjugate Chem.10 (1999) 103-111; Miederer et al., J.Nucl.Med.45 (2004) 129-137; DeNardo et al., Clinical Cancer Research 4 (1998) 2483-90; Blend et al., Cancer Biotherapy & Radiopharmaceuticals 18 (2003) 355-363; Nikula et al., J. Nucl. Med. 40 (1999) 166-76; Kobayashi et al., J. Nucl. Med.39 (1998) 829-36; Mardirossian et al., Nucl. Med. Biol. 20 (1993) 65-74; Roselli et al., Cancer Biotherapy & Radiopharmaceuticals, 14 (1999) 209-20).

[0203] Examples:

[0204] The present invention is illustrated by the following examples only. The examples are not to be construed in any way as limiting the scope of the present invention.

[0205] Example 1: Diagnostic performance of the biomarker LTA4H determined by the proximity extension assay (PEA) technology developed by Olink in women with PCOS (phenotype A) and controls

[0206] As part of the measurements, 86 serum samples from human women were analyzed. The case group included 49 patient-sample measurements diagnosed with PCOS (phenotype A) according to the Rotterdam criteria. The control group included 37 healthy female-sample measurements without PCOS. The concentrations of the analytes were determined using the proximity extension assay (PEA) technology developed by Olink. Briefly, a pair of matched antibodies coupled to unique, partially complementary oligonucleotides are targeted to each biomarker. Quantification is then performed by quantitative real-time PCR.

[0207] After diluting the samples according to the manufacturer's protocol for each selected panel, the Olink protocol consists of three core steps: 1. Incubation, 2. Extension and amplification, 3. Detection. 1 μl of each sample was mixed with 3 μl of incubation mixture in a 96-well plate. In addition to the 92 antibody pairs labeled with DNA oligonucleotides, the incubation mixture also contains internal controls (2 incubation controls, 1 extension control, and 1 detection control) designed to monitor the three main steps of the Olink protocol. As external controls, 3 positive controls (inter-plate controls) and 3 negative controls and 2 sample controls (pooled plasma samples) were included in the plate. The samples were incubated overnight at +4°C. In this step, the antibody pairs bind to the corresponding proteins in the sample. After the incubation is complete, 96 μl of extension mixture is added to the sample. The plate was placed in a thermal cycler for hybridization and extended 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) x17, 10°C hold). DNA barcodes were amplified by PCR. Finally, the amount of each DNA barcode was quantified by microfluidic qPCR using the 96.96 Dynamic Array TMIntegrated fluidic circuit (IFC). 7.2 μl detection mixture was added to 2.8 μl of each sample, of which 5 μl was transferred to the left injection port of the 96.96 Dynamic Array IFC that had been perfused. 5 μl primer solution was transferred to the right injection port of the 96.96 Dynamic Array IFC that had been perfused. The chip was loaded into the Fluidigm IFCController HX according to the manufacturer's instructions. According to the manufacturer's instructions, the Olink protein expression 96×96 program (50°C 120 seconds, 70°C 1800 seconds, 25°C 600 seconds, 95°C 300 seconds (95°C 15 seconds, 60°C 60 seconds) x35 was run in the Fluidigm BiomarkTM Reader; the following settings were used: application-gene expression; inert reference dye-ROX; determination-single probe; probe-FAM-MGB). The Ct values ​​obtained from qPCR were converted to arbitrary units called Normalized Protein eXpression (NPX, a relative quantification unit on a log2 scale) using the following equation:

[0208] Extended control:

[0209] Ct 分析物 –Ct 延伸对照 =dCt 分析物

[0210] Inter-plate comparison:

[0211] dC 分析物 –dCt 板间对照 =ddCt 分析物

[0212] Adjust according to the correction factor:

[0213] Correction Factor – ddCt 分析物 =NPX 分析物

[0214] Quality control and standardization are achieved using Olink NPX Manager software.

[0215] A receiver operating characteristic (ROC) curve was generated ( Figure 1 ). Model performance was determined by looking at the area under the curve (AUC). The best possible AUC was 1, while the lowest possible was 0.5. ROC curve analysis showed an AUC of 1.00 (95% CI 1-1, Figure 1 ), indicating that LTA4H has a high diagnostic accuracy for PCOS.

[0216] Table 1 shows the diagnostic performance of leukotriene A4 hydrolase in differentiating women suffering from PCOS (mature phenotype A) (cases) from healthy control subjects using ROC analysis, depicting the AUC and associated 95% confidence intervals of the ROC curve analysis. The results were obtained using the Olink neighbor extension technique.

[0217] Table 1

[0218] Markers AUC (Area Under the Curve) 95% CI (Confidence Interval) N (sample size) Sample type LTA4H 1.000 1-1 86 serum

[0219] Data obtained with the Olink PEA technique were used to generate box-and-whisker plots of healthy controls and PCOS cases. The boxes include the median (middle quartile), interquartile range (representing the middle 50% of the group's scores), upper quartile (75% of the scores are below the upper quartile), and lower quartile (25% of the scores are below the lower quartile). The whiskers represent values ​​1.5 times the interquartile range. Serum LTA4H concentrations are elevated in women with PCOS compared with healthy controls ( Figure 2 ).

[0220] Example 2: Determination of the diagnostic performance of the biomarker LTA4H in women with PCOS (phenotypes A, B, C and D) and controls by ELISA technique

[0221] Performance validation has been performed in a sample set of 87 cases (serum samples from women with PCOS) and 42 controls (serum samples from healthy women).

[0222] The concentration of the analyte was determined by ELISA (enzyme-linked immunosorbent assay). The case group consisted of patients diagnosed with PCOS according to the Rotterdam criteria (28 phenotype A, 20 phenotype B, 20 phenotype C, and 19 phenotype D). The control group included healthy women without PCOS.

[0223] The concentration of LTA4H in human serum was determined using the Human LTA4H ELISA Kit Version 1 from Invitrogen (Catalog Number: EH308RB). 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 comes with a human LTA4H antibody pre-coated plate. Samples were measured at 2-fold dilution. After all reagents were brought to room temperature, 100 μL of each sample and standard was added. Samples and standards were measured in duplicate. During incubation at room temperature for 2.5 hours on a microplate shaker set to 650 rpm, any LTA4H present was bound to the capture antibody immobilized on the microtiter plate. In a wash step (4x300 μL), unbound material was removed from the plate, and then 100 μL of diluted anti-LTA4H biotin conjugate was added to the wells. After incubation on a shaker for 1 hour and another washing step (4x300 μL) to remove any unbound detection antibody, 100 μL of prepared streptavidin-HRP solution was added to the plate. Subsequently, incubated at room temperature for 45 minutes and washed (4x300 μL). After the last wash, 100 μL of TMB substrate was added to the plate. The plate was incubated at room temperature, in the dark for 30 minutes, while gently shaking. During the incubation, the substrate turned blue. The color development is proportional to the amount of LTA4H bound in the initial step. The color development was stopped by adding 50 μL of stop solution, the solution in the well turned from blue to yellow, and the color intensity was measured at 450 nm using a plate reader for detection, and the color intensity was measured at 570 nm for background subtraction. In order to generate a calibration curve, the lyophilized recombinant LTA4H provided with the kit was redissolved and diluted in a calibration diluent. The calibration range of this assay is 2.048 ng / mL to 500 ng / mL. Calibrator 1 (500 ng / mL) corresponds to the reconstituted stock solution, and calibrators 2 to 7 (2.048 ng / mL) were prepared by serial 2.5-fold dilution steps in calibrator diluent. Pure calibration diluent was used as blank (0 ng / mL). Calibration curves were fitted using unweighted 4-parameter nonlinear regression (Newton / Raphson).

[0224] A receiver operating characteristic (ROC) curve was generated ( Figure 3 ). Model performance was determined by looking at the area under the curve (AUC). The best possible AUC was 1, while the lowest possible was 0.5. When all phenotypes were combined (phenotypes A to D), ROC curve analysis of leukotriene A4 hydrolase for PCOS cases showed an AUC of 0.85 (95% CI 0.77-0.93), confirming the high diagnostic accuracy of LTA4H for PCOS ( Figure 3). Table 2 shows the diagnostic performance of leukotriene A4 hydrolase in differentiating women with PCOS (cases, PCOS phenotypes A to D) from healthy control subjects using ROC analysis, depicting the AUC and associated 95% confidence intervals of the ROC curve analysis. Results were obtained using an ELISA immunoassay.

[0225] Table 2

[0226] Markers AUC (Area Under the Curve) 95% CI (Confidence Interval) N (sample size) Sample type LTA4H 0.85 (0.77-0.93) 129 serum

[0227] The data obtained by ELISA immunoassay were used to generate box-whisker plots for controls and PCOS cases when all phenotypes were combined (phenotypes A to D). Serum LTA4H concentrations (ng / mL) were increased in women with PCOS compared with healthy controls ( Figure 4 ).

[0228] Table 3 shows the diagnostic performance of leukotriene A4 hydrolase in differentiating women suffering from PCOS (divided into different phenotypes A, B, C and D) from healthy control subjects. The results were obtained using an ELISA immunoassay. The AUC for each phenotype is reported in the table.

[0229] Table 3

[0230] Markers Phenotype A Phenotype B Phenotype C Phenotype D N (sample size) Sample type LTA4H 0.9 0.88 0.76 0.85 129 serum

[0231] ROC curve analysis of leukotriene A4 hydrolase in PCOS cases divided by different phenotypes relative to healthy controls showed that the AUCs for phenotypes A to D were 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), respectively ( Figure 5 The results confirmed the high diagnostic accuracy of LTA4H for PCOS. Serum leukotriene A4 hydrolase concentrations (ng / mL) showed elevated levels in all different PCOS phenotypes (phenotypes A to D) compared with healthy controls ( Figure 6 , using ELISA immunoassay to obtain results).

[0232] Table 4 shows the diagnostic performance of leukotriene A4 hydrolase in young women (age ≤ 25 years) in differentiating young women suffering from PCOS when all phenotypes were combined (phenotypes A to D) from young healthy control subjects. The results were obtained by using an ELISA assay.

[0233] Table 4

[0234] Markers AUC (Area Under the Curve) 95% CI (Confidence Interval) N (sample size) Sample type LTA4H 0.83 (0.61-1) 32 serum

[0235] When all phenotypes were combined (phenotypes A to D), ROC curve analysis of leukotriene A4 hydrolase in young PCOS cases (age ≤ 25) showed an AUC of 0.83, confirming the high diagnostic accuracy (95% CI 0.61-1, Figure 7 When only young women were included in the analysis (age ≤ 25 years), PCOS cases (all phenotypes A to D combined) showed elevated serum leukotriene A4 hydrolase concentrations compared with young controls (age ≤ 25 years) ( Figure 8 ).

[0236] The diagnostic performance of leukotriene A4 hydrolase in differentiating young women (age ≤ 25 years) with PCOS (divided into different phenotypes A, B, C and D) from young healthy control subjects (age ≤ 25 years) was evaluated and the results are reported in Table 5 below (AUC for PCOS phenotypes vs. controls). The results were obtained using an ELISA immunoassay.

[0237] Table 5

[0238] Markers Phenotype A Phenotype B Phenotype C Phenotype D N (sample size) Sample type LTA4H 0.97 0.8 0.72 0.67 32 serum

[0239] ROC curve analysis of leukotriene A4 hydrolase in young PCOS cases (age ≤ 25, phenotypes A to D) showed that the AUC of each phenotype was 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), respectively, confirming the high diagnostic accuracy of LTA4H for PCOS in the subgroup of women 25 or younger ( Fig. 9 Compared with LTA4H concentrations in young healthy controls (age ≤ 25 years), leukotriene A4 hydrolase concentrations were increased in all different PCOS phenotypes (phenotypes A to D, age ≤ 25 years) ( Fig.10 ).

[0240] Example 3: Diagnostic performance of the biomarker LTA4H and controls in women with PCOS (phenotypes A, B, C and D) in different age groups as determined by ELISA technique

[0241] Performance validation has been performed in an additional sample set of 240 cases (serum samples from women with PCOS) and 48 controls (serum samples from healthy women).

[0242] The concentration of the analyte was determined by ELISA (enzyme-linked immunosorbent assay). The case group consisted of patients diagnosed with PCOS according to the Rotterdam criteria (155 phenotype A, 5 phenotype B, 8 phenotype C, and 72 phenotype D) belonging to three different age groups: 15 to 20 (n = 70), 20 to 25 (n = 99), 25 to 40 (n = 71). The control group included healthy women (20 to 40 years old) without PCOS.

[0243] The concentration of LTA4H in human serum was determined using the Human LTA4H ELISA Kit Version 1 from Invitrogen (Cat. No.: EH308RB) as described in Example 2.

[0244] A receiver operating characteristic (ROC) curve was generated ( Fig.11 ). Model performance was determined by looking at the area under the curve (AUC). When all phenotypes were combined (phenotypes A to D), ROC curve analysis of LTA4H for PCOS cases showed an AUC of 0.74 (95% CI 0.64-0.83), confirming the high diagnostic accuracy of LTA4H for PCOS ( Fig.11 ).

[0245] Table 6 shows the diagnostic performance of LTA4H in differentiating women with PCOS (cases, PCOS phenotypes A to D) from healthy control subjects using ROC analysis, depicting the AUC and associated 95% confidence intervals of the ROC curve analysis. Results were obtained using an ELISA immunoassay.

[0246] Table 6

[0247] Markers AUC (Area Under the Curve) 95% CI (Confidence Interval) N (sample size) Sample type LTA4H 0.74 (0.64-0.83) 288 serum

[0248] The data obtained by ELISA immunoassay were used to generate box-whisker plots for controls and PCOS cases when all phenotypes were combined (phenotypes A to D). Serum LTA4H concentrations (ng / mL) were increased in women with PCOS compared with healthy controls ( Fig.12 ).

[0249] Table 7 shows the diagnostic performance of LTA4H in differentiating women suffering from PCOS (divided into different phenotypes A, B, C and D) from healthy control subjects. The results were obtained using an ELISA immunoassay. The AUC for each phenotype is reported in the table.

[0250] Table 7

[0251] Markers Phenotype A Phenotype B Phenotype C Phenotype D N (sample size) Sample type LTA4H 0.75 0.77 0.70 0.70 288 serum

[0252] ROC curve analysis of LTA4H in PCOS cases divided according to different phenotypes relative to healthy controls showed that the AUCs for phenotypes A to D were 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), respectively. Fig.13 ). The results confirmed the high diagnostic accuracy of LTA4H for PCOS.

[0253] Compared with healthy controls, serum LTA4H concentrations (ng / mL) of all the different PCOS phenotypes (phenotypes A to D) showed increased levels ( Fig.14 , using ELISA immunoassay to obtain results).

[0254] Table 8 shows the diagnostic performance of LTA4H in differentiating women suffering from PCOS (when all phenotypes were combined (phenotypes A to D)) from healthy control subjects in different age groups (15 years ≤ age < 20 years, 20 years ≤ age < 25 years, 25 years ≤ age < 40 years). The results were obtained by using ELISA assay.

[0255] Table 8

[0256] Markers 15≤Age<20 20≤Age<25 25≤Age<40 N (sample size) Sample type LTA4H 0.74 0.70 0.78 288 serum

[0257] ROC curve analysis of LTA4H in PCOS cases compared with healthy controls according to different age groups showed that the AUCs for the 15 to 20, 20 to 25, and 25 to 40 age groups were 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), respectively. Fig.15 ). The results confirmed that LTA4H had a high diagnostic accuracy for PCOS in all different age groups.

[0258] Increased serum LTA4H concentrations (ng / mL) were demonstrated in women with PCOS compared with controls in all age groups ( Fig.16 ).

[0259] Considering the lack of reliable biomarkers for the diagnosis of PCOS, especially in younger women (age <25 years), separate analyses were performed for age groups ≥15 and <25 years.

[0260] Table 9 shows the diagnostic performance of LTA4H in young women (15 years ≤ age < 25 years) in differentiating young women suffering from PCOS when all phenotypes were combined (phenotypes A to D) from young healthy control subjects. The results were obtained by using an ELISA assay.

[0261] Table 9

[0262] Markers AUC (Area Under the Curve) 95% CI (Confidence Interval) N (sample size) Sample type LTA4H 0.75 (0.61-0.88) 192 serum

[0263] When all phenotypes were combined (phenotypes A to D), ROC curve analysis of LTA4H in young PCOS cases (15 years ≤ age < 25 years) showed an AUC of 0.75, confirming the high diagnostic accuracy (95% CI 0.61-0.88, Fig.17 When only young women were included in the analysis (15≤age<25), PCOS cases (all phenotypes A to D combined) showed elevated serum LTA4H concentrations (ng / mL) compared with young controls (15≤age<25). Fig.18 ).

[0264] The diagnostic performance of LTA4H in differentiating young women (15 years ≤ age < 25 years) with PCOS (divided into different phenotypes A, B, C and D) relative to young healthy control subjects (15 years ≤ age < 25 years) was evaluated and the results are reported in Table 10 below (AUC for PCOS phenotypes relative to controls). The results were obtained using an ELISA immunoassay.

[0265] Table 10

[0266] Markers Phenotype A Phenotype B Phenotype C Phenotype D N (sample size) Sample type LTA4H 0.76 0.78 0.69 0.72 192 serum

[0267] ROC curve analysis of LTA4H in young PCOS cases (15≤age<25, phenotypes A to D) showed that the AUC of each phenotype was 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), respectively, confirming the high diagnostic accuracy of LTA4H for PCOS in the subgroup of women aged 15 to 25 years ( Fig.19 Compared with LTA4H concentrations in young healthy controls (15≤age<25), LTA4H concentrations were increased in all different PCOS phenotypes (phenotypes A to D, 15≤age<25) ( Fig. 20 ).

Claims

1. A method for assessing whether a patient has polycystic ovary syndrome (PCOS) or is at risk of developing PCOS, the method comprising (a) determining the amount or concentration of LTA4H in a sample from said patient, and (b) comparing the determined amount or concentration with a reference.

2. A method of selecting a patient for therapy for PCOS, the method comprising: (a) determining the amount or concentration of LTA4H in a sample from said patient, and (b) comparing the determined amount or concentration with a reference.

3. A method for monitoring the progression of PCOS in a patient suffering from PCOS or for monitoring the response of a patient suffering from PCOS to treatment, the method comprising (a) determining the level of LTA4H in a first sample from said patient, (b) determining the level of LTA4H in a second sample of said patient which has been obtained after said first sample, and (c) comparing said level of LTA4H in said first sample with said level of LTA4H in said second sample, and (d) monitoring the progress of said patient suffering from or being treated for PCOS based on the results of step c).

4. The method according to claims 1 to 3, wherein an increased amount or concentration of LTA4H in the sample of the patient indicates the presence of PCOS in the patient.

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

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

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

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

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

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

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

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

13. A computer-implemented method for assessing a patient with suspected PCOS, the computer-implemented method comprising the steps of: (a) receiving a value for the amount or concentration of a first biomarker in a sample from the patient, the first biomarker being 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-anovulation and / or irregular cycles, hyperandrogenemia, and polycystic ovarian morphology; (d) comparing the values ​​for the amounts or concentrations of steps (a) to (b) with a reference for the biomarker and the values ​​for the presence or absence of the at least one additional diagnostic criterion and / or calculating a score for assessing the patient as having suspected PCOS based on the amounts or concentrations and the values ​​of the biomarkers; as well as (e) assessing said patient based on said comparing and / or said calculating performed in step (d).

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

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