CN109187792B - Tryptophanol and xanthosine in urine as diagnostic markers of idiopathic male infertility and application thereof - Google Patents

Abstract

The invention belongs to the fields of analytical chemistry and clinical medicine, and discloses tryptophanol and xanthosine in urine as idiopathic male sterility diagnostic markers and application thereof. The marker is tryptophanol and xanthosine, has high sensitivity and specificity in diagnosing idiopathic male sterility, and can be used for preparing diagnostic or monitoring reagent for idiopathic male sterility.

Description

Urine tryptophanol and xanthoside as diagnostic markers for idiopathic male infertility and its application

Field of Invention

The invention belongs to the fields of analytical chemistry and clinical medicine, and relates to tryptophanol and xanthoside related to idiopathic male infertility in urine as biomarkers and a detection method and application based on UPLC-Q exactive MS.

Background technique

At present, the number of infertility patients among newlyweds in my country is far more than one million. Current research suggests that genetic factors such as Y chromosome AZF gene region sequence changes, DAZ gene copy number changes, sex hormone receptor gene sequence changes, and environmental factors such as exposure to harmful factors, nutrition and living habits are all related to male infertility. It is worth noting that a considerable portion of male infertility manifests as an unexplained cause, i.e. idiopathic male infertility, which makes the diagnosis of idiopathic male infertility extremely difficult and delays the timing of treatment. The WHO's diagnostic criteria for male infertility are couples who have lived together for more than 1 year after marriage, do not take contraceptive measures, and cause the female infertility due to the male's reasons. However, due to practical reasons, many couples cannot guarantee strict cohabitation for 1 year, which makes the final judgment of male infertility very difficult; the observation time of cohabitation for up to 1 year greatly delays the early treatment and intervention of male infertility. Time; in order to rule out the reasons of the woman, the woman also needs to carry out a detailed examination, which brings a heavy economic and medical burden. Existing male infertility-related tests rely on conventional semen examinations, which only focus on routine parameters such as sperm count, motility, semen volume, pH, and liquefaction time. Because of the influence of factors such as abstinence time, the results of routine analysis of routine semen parameters show great volatility. Therefore, clinical diagnosis often needs to refer to multiple routine semen analysis, which brings a burden to doctors and patients. More importantly, the traditional semen parameter examination cannot comprehensively reflect the full status of the semen. Therefore, male infertility is often manifested as no obvious abnormality in conventional semen parameters, which also makes the traditional semen parameter examination ineffective in diagnosing male infertility. Therefore, there is an urgent need for new diagnostic methods for idiopathic male infertility.

Metabonomics is another important new research field of systems biology after genomics, transcriptomics and proteomics. The focus is on the changes of small molecule metabolites with molecular weight less than 1000 in the metabolic cycle, reflecting the changes in the body's response to disease states or environmental stimuli. Metabolomics can systematically review, analyze and explore the small molecule biomarkers produced by cells, tissues and organs and their changes, and more intuitively understand the material basis of functional phenotype changes, and its objects involve lipid metabolism, glucose metabolism, amino acid The metabolic network of organisms such as metabolism, nucleic acid metabolism, and coenzyme metabolism is an omics that is closer to "phenotype" than genomics, proteomics, and transcriptomics. Metabolomics can greatly reduce the trauma of patient sampling because the samples it analyzes can be non-invasive samples such as urine. Metabolomics has shown extremely high application potential and value in the diagnosis of complex diseases, and has the characteristics of high sensitivity and stability. Metabolomics analyzes the overall metabolic components of the body and finds specific biomarkers, which can diagnose clinical diseases in a timely, accurate, highly sensitive and specific manner. At present, metabolomics is mainly used in the diagnosis of coronary heart disease, liver disease, diabetes, hypertension, obesity and tumor. It is worth noting that urine is a clinically easily obtained biological sample, which has the advantages of non-invasiveness and large volume. Particularly suitable for disease diagnosis and screening. However, the application of metabolomic analysis of urinary metabolic small molecules in the diagnostic monitoring of idiopathic male infertility has not received corresponding attention.

UPLC-Q exactive MS is a combination of a new generation of high-resolution mass spectrometry and ultra-high performance liquid chromatography, which has stronger sensitivity, specificity and stability than traditional LC-MS. Therefore, UPLC-Q exactive MS was used for metabolomic analysis of urinary metabolic small molecules. If stable specific urinary metabolic small molecules related to the pathogenesis of idiopathic male infertility can be found as biomarkers, and the corresponding metabolic molecules can be developed. The UPLC-Q exactive MS detection method of small molecule markers is not only in the international leading position in this field, but also can create eye-catching economic benefits, and also has extensive significance for promoting male reproductive health in my country.

SUMMARY OF THE INVENTION

The object of the present invention is to provide urinary tryptophanol and xanthoside markers associated with idiopathic male infertility.

Another object of the present invention is to provide a method for detecting the above-mentioned urine tryptophanol and xanthoside markers.

Another object of the present invention is to provide a kit for detecting the above-mentioned urine tryptophanol and xanthoside markers.

The purpose of this invention is to realize through the following technical measures:

Urine markers associated with idiopathic male infertility, the markers are urinary tryptophanol and xanthoside.

The application of the urinary tryptophanol and xanthoside markers in the preparation of a diagnostic or monitoring kit for idiopathic male infertility.

A kit for diagnosing or monitoring idiopathic male infertility, the kit contains reagents for detecting urine tryptophanol and xanthoside.

The described kit contains reagents for detecting urine tryptophanol and xanthoside using the UPLC-Q exactive MS method.

Said kit, the kit contains the following reagents:

Tryptophanol and xanthoside standards;

Internal standard A: Isotopic internal standard (deuterium standard) of one or more substances in creatinine, valine, niacin, thymine, glutaric acid, L-phenylalanine, N-acetaminophen, hippuric acid , aqueous solution);

Internal standard B: Isotopic internal standard of pentadecanoic acid (deuterium standard, methanol solution);

Internal standard C: the isotopic internal standard of tetracosanoic acid (deuterium standard, methanol solution).

Further, the kit also contains:

Hypersil GOLD C18 column;

Reagent A: for protein precipitation, containing 100% methanol;

Reagent B: for mobile phase, water containing 0.1% formic acid;

Reagent C: for mobile phase, acetonitrile containing 0.1% formic acid;

Reagent D: for reconstitution, ultrapure water.

A method for the detection of urine tryptophanol and xanthoside markers associated with idiopathic male infertility as previously described using the UPLC-Q exactive MS method for the detection of urine tryptophanol and xanthoside levels .

In the above detection methods:

1. Liquid phase conditions:

The liquid chromatography column is a Hypersil GOLD C18 column, and the column temperature is 40 °C;

Mobile phase A is water containing 0.1% formic acid, mobile phase B is acetonitrile containing 0.1% formic acid, and the flow rate is 400 μL/min;

The instrument gradient was: 0-3min 1%B, 3-10min 1% to 99%B, 10-13min 99%B, 13-13.1min 99% to 1%B, 13.1-17min 1%B; (flow in each gradient The amount of phase A and the corresponding amount of mobile phase B are 100%, the same below)

Injection method: volume 5μl;

2. Mass spectrometry conditions

Heated electrospray ionization (HESI) was used for analysis. Positive ion mode spray voltage: 3.5kV; negative ion mode spray voltage: 2.5kV; Capillary temperature: 250°C, heater temperature: 425°C, sheath gas flow: 50AU, auxiliary gas flow: 13AU, backflush gas flow: 0AU; lens voltage: 60V; using full scan mode, scanning range: 70 to 1050m/z; resolution: 70000.

The present invention is described in detail as follows:

The inventors collected standard-compliant urine samples using standard operating procedures (SOPs), systematically collected complete basic population information and clinical data, and used UPLC-Q exactive MS-based metabolomics methods for analysis.

Specifically, the experimental method of the research mainly includes the following parts:

1. Research object selection and grouping basis

first stage screening stage

A total of 1037 people with 607 diagnosed idiopathic male infertility and 430 healthy controls were randomly included.

Group A: healthy control group (430 people):

1. Be between the ages of 19 and 39;

2. Body mass index between 17 and 31;

3. Males with healthy reproductive capacity and healthy offspring after 6-8 months;

4. No major systemic diseases.

Group B: Idiopathic male infertility group (607 people):

1. Age matched with the control group;

2. Body mass index matched with the control group;

3. Men who have tried to conceive for 12 months without success and whose spouse has no infertility disease;

4. No clear cause of male infertility;

5. The history of smoking and drinking is matched with the control group;

6. Ethnic and control groups are matched;

7. No major systemic diseases.

Phase 2 Verification Phase

A total of 30 patients, 15 diagnosed idiopathic male infertility and 15 healthy controls, were included.

Group A: healthy control group (15 people):

1. Aged between 24 and 36 years old;

2. Body mass index between 19 and 24;

3. Males with healthy reproductive capacity and healthy offspring after 6-8 months;

4. No major systemic diseases.

Group B: Idiopathic male infertility group (15 people):

1. Age matched with the control group;

2. Body mass index matched with the control group;

3. Men who have tried to conceive for 12 months without success and whose spouse has no infertility disease;

4. No clear cause of male infertility;

5. The history of smoking and drinking is matched with the control group;

6. Ethnic and control groups are matched;

7. No major systemic diseases.

2. UPLC-Q exactive MS metabolomic analysis and screening and validation of urinary biomarkers tryptophanol and xanthoside for the diagnosis of idiopathic male infertility

1. Sample pretreatment

1.1. Take 300 μL of urine, add 10 μL of internal standard A, add 10 μL of internal standard B, add 10 μL of internal standard C, add 900 μL of methanol (reagent A), and vortex for 30 s.

1.2. Centrifuge at 16000g for 15min at 4°C in a centrifuge, transfer the supernatant to a 1.5mL inlet EP tube, and concentrate the supernatant to dryness in a centrifugal concentration dryer at room temperature.

1.3. Reconstitute with 5 μL of ultrapure water (reagent D) for analysis.

2. Instrument testing

2.1. Analytical instruments: UPLC Ultimate 3000system (Dionex) high performance liquid chromatograph; Q-Exactive high resolution mass spectrometer.

2.2. Liquid phase conditions:

2.2.1. The liquid chromatography column was a Hypersil GOLD C18 column (100 mm×2.1 mm, particle size 1.9 μm, Thermo Scientific, Germany), and the column temperature was 40°C.

2.2.2 The mobile phases used were (A) water containing 0.1% formic acid (reagent B) and (B) acetonitrile (reagent C) containing 0.1% formic acid, and the flow rate was 400 μL/min.

2.2.3 The instrument gradient was: 0-3min 1%B, 3-10min 1% to 99%B, 10-13min 99%B, 13-13.1min 99% to 1%B, 13.1-17min 1%B.

2.2.4 Sampling method: volume 5μl.

2.3. Mass spectrometry conditions

2.3.1 Heating electrospray ionization (HESI) was used for analysis.

2.3.2 Heating electrospray ionization (HESI) was used, with positive ion mode spray voltage: 3.5kV; negative ion mode spray voltage: 2.5kV; capillary temperature: 250°C, heater temperature: 425°C, sheath gas flow in both modes : 50AU, auxiliary gas flow: 13AU, backflush gas flow: 0AU; lens voltage: 60V. Using full scan mode, scanning range: 70 to 1050m/z; resolution: 70000.

3. Material characterization

The chromatographic information (retention time) and mass spectrometry information (exact molecular weight) were compared with the standard tryptophanol and xanthoside for biomarker characterization, and the chromatographic information of the isotopic internal standard series in the sample was compared in real time to correct the retention time.

4. Data analysis:

Biomarker screening used multiple logistic regression to identify key metabolites.

5. Difference and diagnostic significance of urine tryptophanol and xanthoside in healthy control group and idiopathic male infertility group.

After adjustment for age, body mass index, smoking and drinking history, multivariate logistic regression analysis found that the levels of tryptophanol and xanthoside in urine samples were closely associated with idiopathic male infertility. Using the above-mentioned metabolic small molecule combination in random population to diagnose idiopathic male infertility, the sensitivity is 93.33%, the specificity is 93.33%, and the area under the ROC curve is 0.9822, which has a high auxiliary diagnostic value.

3. Preparation method of diagnostic kit

According to the above-mentioned series of experimental results, the inventors have also prepared a kit for diagnosing or monitoring idiopathic male infertility, the kit contains urine biomarkers for detecting idiopathic male infertility The reagents for identifying tryptophanol and xanthoside include the standard for determining the stable and detectable tryptophanol and xanthoside in the urine of the subject and the internal standard standard series for auxiliary analysis. The diagnostic kit may also include a set of reagents and equipment for extraction and chromatographic separation of urine tryptophanol and xanthoside.

Beneficial effects of the present invention:

By using UPLC-Q exactive MS to compare the metabolic small molecules in the urine of normal controls and idiopathic male infertility, the inventors found that there are small molecules in urine that can be used to evaluate whether idiopathic male infertility is present, which has diagnostic value The combination of urine tryptophanol and xanthoside, and the application of UPLC-Q exactive MS for the detection of urine tryptophanol and xanthoside, developed a diagnostic and monitoring kit for idiopathic male infertility that can facilitate clinical application.

The advantages of the present invention using urine metabolic small molecules as markers for evaluation of idiopathic male infertility are:

(1) Urine metabolic small molecule is a new type of biomarker, which is strongly associated with disease outcomes. It is not only stable, minimally invasive, easy to detect, but also quantitatively accurate. It will greatly improve the sensitivity and accuracy of the diagnosis of idiopathic male infertility. The successful development of such small molecule biomarkers will open up a new situation for the prevention and treatment of idiopathic male infertility, and provide reference for the development of biomarkers for other diseases.

(2) The urine metabolic small molecule marker provided by the present invention can be used as a diagnostic marker for idiopathic male infertility, and can assist in the diagnosis of idiopathic male infertility in the early stage, thereby providing a basis for further in-depth examination by clinicians, Provide support for quickly and accurately grasp the patient's disease state and severity, and adopt more personalized prevention and treatment plans in a timely manner, so as to delay and prevent disease progression.

(3) The present invention uses the urine samples of idiopathic male infertility and healthy control random population for verification, which proves that the levels of tryptophanol and xanthoside in urine have high sensitivity and specificity in diagnosing idiopathic male infertility degree, can be used as a marker.

(4) The present invention adopts a rigorous, multi-stage verification and evaluation system, initially screening a variety of small urine metabolic molecules through pre-experiments, and using UPLC-Q exactive MS for independent population verification, ensuring that the urinary metabolic biomarkers and Reliability of diagnostic methods.

(5) UPLC-Q exactive MS technology sample processing is simple, instrument analysis is rapid and accurate, and has high practical value for clinical diagnosis.

Description of drawings

Figure 1 During the screening phase, after adjusting for information on age, body mass index, smoking and drinking history, multivariate logistic regression analysis found that urinary tryptophanol and xanthoside were closely associated with idiopathic male infertility. ^a Univariate logistic regression results without adjustment for confounders. ^b Results of multivariate logistic regression adjusted for age, body mass index, smoking and drinking history.

Figure 2 Metabolic assay level variability (mean ± SD).

Figure 3. ROC curves between the normal control group and the idiopathic male infertility group using the information of urine tryptophanol content in the validation stage.

Figure 4. ROC curve between the normal control group and the idiopathic male infertility group prepared using the information of the urinary xanthoside content in the validation stage.

Figure 5. ROC curves between the normal control group and the idiopathic male infertility group using the information of urine tryptophanol and xanthoside content in the validation stage.

Detailed ways

The present invention will be further described below through examples.

Example 1: Research object selection and grouping basis

The subjects of this part of the study were first-diagnosed cases of idiopathic male infertility and healthy reproductive controls from the Affiliated Hospital of Nanjing Medical University. The research content and informed consent form were approved by the Ethics Committee of Nanjing Medical University and met the requirements of relevant regulations. Cases and controls signed the informed consent after learning the content. All study subjects underwent a complete physical examination and completed a questionnaire including basic personal information, living habits, occupational and environmental exposures, genetic risk factors, sexual and reproductive function, disease history, and physical activity. The first phase included 607 eligible cases of idiopathic male infertility and 430 healthy controls; the second phase included 15 eligible cases of idiopathic male infertility and 15 healthy controls as idiopathic male infertility Screening subjects for urine biomarkers. The specific sample classification standards are as follows:

first stage screening stage

A total of 1037 people with 607 diagnosed idiopathic male infertility and 430 healthy controls were randomly included.

Group A: healthy control group (430 people):

1. Be between the ages of 19 and 39;

2. Body mass index between 17 and 31;

3. Males with healthy reproductive capacity and healthy offspring after 6-8 months;

4. No major systemic diseases.

Group B: Idiopathic male infertility group (607 people):

1. Age matched with the control group;

2. Body mass index matched with the control group;

3. Men who have tried to conceive for 12 months without success and whose spouse has no infertility disease;

4. No clear cause of male infertility;

5. The history of smoking and drinking is matched with the control group;

6. Ethnic and control groups are matched;

7. No major systemic diseases.

Phase 2 Verification Phase

A total of 30 patients, 15 diagnosed idiopathic male infertility and 15 healthy controls, were included.

Group A: healthy control group (15 people):

1. Aged between 24 and 36 years old;

2. Body mass index between 19 and 24;

3. Males with healthy reproductive capacity and healthy offspring after 6-8 months;

4. No major systemic diseases.

Group B: Idiopathic male infertility group (15 people):

1. Age matched with the control group;

2. Body mass index matched with the control group;

3. Men who have tried to conceive for 12 months without success and whose spouse has no infertility disease;

4. No clear cause of male infertility;

5. The history of smoking and drinking is matched with the control group;

6. Ethnic and control groups are matched;

7. No major systemic diseases.

Example 2: UPLC-MS Metabolomics Biomarker Screening for Idiopathic Male Infertility

1. Sample pretreatment

1.1. Take 300 μL of urine, add 10 μL of internal standard A, add 10 μL of internal standard B, add 10 μL of internal standard C, add 40 μL of methanol (reagent A), and vortex for 30 s.

1.2. Centrifuge at 16000g for 15min at 4°C in a centrifuge, transfer the supernatant to a 1.5mL inlet EP tube, and concentrate the supernatant to dryness in a centrifugal concentration dryer at room temperature.

1.3. Reconstitute with 5 μL of ultrapure water (reagent D) for analysis.

2. Instrument testing

2.1. Analytical instruments: UPLC Ultimate 3000system (Dionex) high performance liquid chromatograph; Q-Exactive high resolution mass spectrometer.

2.2. Liquid phase conditions:

2.2.1 The liquid chromatographic column was a Hypersil GOLD C18 chromatographic column (100 mm×2.1 mm, particle size 1.9 μm, Thermo Scientific, Germany), and the column temperature was 40°C.

2.2.2 The mobile phases used were (A) water containing 0.1% formic acid (reagent B) and (B) acetonitrile (reagent C) containing 0.1% formic acid, and the flow rate was 400 μL/min.

2.2.3 The instrument gradient was: 0-3min 1%B, 3-10min 1% to 99%B, 10-13min 99%B, 13-13.1min 99% to 1%B, 13.1-17min 1%B.

2.2.4 Sampling method: volume 5μl.

2.3. Mass spectrometry conditions

2.3.1 Heating electrospray ionization (HESI) was used for analysis.

2.3.2 Heating electrospray ionization (HESI) was used, with positive ion mode spray voltage: 3.5kV; negative ion mode spray voltage: 2.5kV; capillary temperature: 250°C, heater temperature: 425°C, sheath gas flow in both modes : 50AU, auxiliary gas flow: 13AU, backflush gas flow: 0AU; lens voltage: 60V. Using full scan mode, scanning range: 70 to 1050m/z; resolution: 70000.

3. Material characterization

The chromatographic information (retention time) and mass spectrometry information (exact molecular weight) were compared with the standard tryptophanol and xanthoside for biomarker characterization, and the chromatographic information of the isotopic internal standard series in the sample was compared in real time to correct the retention time.

4. Data analysis:

Biomarker screening used multivariate logistic regression to identify key metabolites.

5. Differences and diagnostic significance of tryptophanol and xanthoside in urine samples of healthy control group and idiopathic male infertility group.

After adjustment for information on age, body mass index, smoking and drinking history, multivariate logistic regression analysis found that urinary tryptophanol and xanthoside were strongly associated with idiopathic male infertility (Figure 1).

Example 3 Stability analysis of tryptophanol and xanthoside in urine

The stability of tryptophanol and xanthoside levels in urine was evaluated using the method of Example 2 (interval time was 2 weeks). The results showed that the measured levels of tryptophanol and xanthoside in urine were stable (Figure 2), with properties as diagnostic/monitoring markers.

Example 4 Diagnosis of idiopathic male infertility by the combination of tryptophanol and xanthoside in urine

According to the above-mentioned UPLC-Q exactive MS metabolomics method, the inventors drew ROC curves and evaluated the sensitivity and specificity of diagnosis by detecting tryptophanol and xanthoside in urine samples of 15 cases and 15 controls in a random population, Then, the diagnostic ability of detecting the levels of these two substances in urine for idiopathic male infertility was evaluated.

The sensitivity of tryptophanol was 80.00%, the specificity was 86.67%, and the area under the ROC curve was 0.9689 (Figure 3); the sensitivity of xanthoside was 80.00%, the specificity was 80.00%, and the area under the ROC curve was 0.8978 (Figure 4).

The combined tryptophanol and xanthoside had a sensitivity of 93.33%, a specificity of 93.33%, and an area under the ROC curve of 0.9822 (Figure 5).

Therefore, the combination of tryptophanol and xanthoside has a good ability to diagnose idiopathic male infertility.

Embodiment 5 is used for idiopathic male infertility urine tryptophanol and xanthoside detection and production of diagnostic kit

First, metabolic small molecules with higher abundance in normal control and idiopathic male infertility urine were identified by UPLC-Q exactive MS method. Then, biomarkers related to idiopathic male infertility were screened by metabolomics technology based on UPLC-Q exactive MS, as a diagnostic indicator of idiopathic male infertility. It is preferable to control the number of corresponding urine biomarkers to be screened to 2, which is an optimized simplification based on the pre-experiment. The use of two biomarkers, tryptophanol and xanthoside in urine, can not only ensure better sensitivity and specificity, but also save costs, reduce the burden on patients, and reduce the detection time. It is fast, accurate and economical. The advantages are convenient for clinical promotion and use. Of course, one of the markers can be used, and the effect of using two markers is better. This kit includes a batch of reagents and consumables for the detection of urine tryptophanol and xanthoside. The qualitative and quantitative biomarkers use tryptophanol and xanthoside standards, and the auxiliary analysis uses internal standard A: creatinine, valine, cigarette smoke Deuterium standard isotope internal standard for eight substances: acid, thymine, glutaric acid, L-phenylalanine, N-acetyl-p-aminophenol and hippuric acid. Internal Standard B: Deuterium-labeled isotopic internal standard of pentadecanoic acid. Internal Standard C: Deuterium-labeled isotopic internal standard of tetracosanoic acid. There are other supporting reverse chromatographic columns for UPLC chromatographic separation (Hypersil GOLDC18 chromatographic column, 100mm×2.1mm, particle size 1.9μm), reagents for precipitating urine protein (100% methanol), reagents for mobile phase (0.1% formic acid in water and 0.1% formic acid in acetonitrile), reagents for extraction of tryptophanol and xanthoside (100% ultrapure water). The value of this kit is that it only needs 300 μl of urine to detect the content of tryptophanol and xanthoside markers in urine, and then diagnose idiopathic male infertility through the content, and it is easy to dynamically monitor and observe the treatment effect.

The specific kit composition is as follows:

Tryptophanol Standard

Xanthoside standard

Internal standard A (deuterium isotope internal standard aqueous solution of eight substances including creatinine, valine, niacin, thymine, glutaric acid, L-phenylalanine, N-acetyl-p-aminophenol, hippuric acid)

Internal standard B (deuterium standard isotope internal standard methanol solution of pentadecanoic acid)

Internal standard C (deuterium standard isotope internal standard methanol solution of tetracosanoic acid)

Further, it may also contain:

Chromatographic column (Thermo 100mm×2.1mm, particle size 1.9μm, Hypersil GOLD C18 column)

Reagent A (100% methanol)

Reagent B (water with 0.1% formic acid)

Reagent C (0.1% formic acid in acetonitrile)

Reagent D (100% ultrapure water).

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Claims (1)

1. Application of tryptophanol and xanthosine in preparing diagnostic or monitoring reagent for idiopathic male infertility is provided.

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