CN114414695B - Molecular marker related to azoospermia, and detection method and application thereof - Google Patents
Molecular marker related to azoospermia, and detection method and application thereof Download PDFInfo
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Abstract
The invention relates to a molecular marker 3- (3-hydroxyphenyl) propionic acid related to azoospermia, and a detection method and application thereof, belonging to the technical fields of analytical chemistry and clinical medicine. The invention adopts UPLC-Qexact MS to compare the metabolism micromolecules of semen in normal control and azoospermia, discovers that 3- (3-hydroxyphenyl) propionic acid with diagnostic value which can be used for evaluating whether azoospermia exists in the semen, and detects the application of UPLC-Q exact MS of the substance, and develops a azoospermia diagnosis and monitoring kit which can be convenient for clinical application. The metabolic small molecule is a novel biomarker which has strong association with disease outcome, is stable, noninvasive, easy to detect and accurate in quantification. The 3- (3-hydroxyphenyl) propionic acid provided by the invention can be used as a diagnosis marker of azoospermia, provides a basis for further deep examination of clinicians, provides support for rapidly and accurately grasping the disease state and the disease severity of patients and timely taking a control scheme, and delays and prevents disease progression.
Description
Technical Field
The invention relates to a molecular marker 3- (3-hydroxyphenyl) propionic acid related to azoospermia, and a detection method and application thereof, belonging to the technical fields of analytical chemistry and clinical medicine.
Background
It is counted by the World Health Organization (WHO) that about 15% of women of child bearing age all over the world suffer from infertility. The population cardinality of China is large, the number of sterile patients in new couples is far more than one million, and the sterility caused by male factors is higher than 50%. The incidence of azoospermia is 10% -20% of male infertility. Finding biomarkers associated with azoospermia to aid or replace semen parameter analysis is very urgent. The technical progress of male science can effectively make up for the deficiency of conventional semen analysis, provides a new method for diagnosing azoospermia and can provide a new visual angle for the generation mechanism of azoospermia. It is an urgent need to provide a method for objectively assessing fertility. Histology techniques include genomics, transcriptomics, proteomics and metabolomics. Metabonomics is an emerging technology that can be used for the discovery of biomarkers for disease diagnosis and can reveal the mechanism by which disease occurs. Metabonomics reveals molecular events that occur below the genetic, transcriptional, and protein levels, and is considered a recent histology approach to disease phenotypes. At present, seminal plasma metabolome change of azoospermia is still unknown, seminal plasma is the main component of seminal fluid, seminal plasma regulates the chemical components of seminal fluid, and the seminal plasma is a non-invasive sample and is particularly suitable for finding biomarkers for azoospermia diagnosis and mechanism research.
Currently, common techniques for metabonomics research include liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), and Nuclear Magnetic Resonance (NMR). The nuclear magnetic resonance technology is characterized in that the components to be detected are not damaged, the sample pretreatment is simple, but the sensitivity is lower; the gas chromatography-mass spectrometry has better sensitivity and reproducibility, but a derivatization method is generally adopted to pretreat a sample, so that the experimental steps become complicated. The UPLC-Q exact MS is a combination of a new generation of high-resolution mass spectrum and an ultra-high performance liquid phase, and has stronger sensitivity, specificity and stability compared with the traditional LC-MS. Therefore, the UPLC-Q actual MSS is adopted to carry out metabonomics analysis of the seminal plasma metabolism small molecules, if stable specific seminal plasma metabolism small molecules related to the incidence of idiopathic male infertility can be found to be used as biomarkers, and a UPLC-Q actual MS detection method of corresponding metabolism small molecule markers is developed, great contribution is made to male reproductive health.
Disclosure of Invention
It is an object of the present invention to provide a molecular marker 3- (3-hydroxyphenyl) propionic acid associated with azoospermia provision.
It is another object of the present invention to provide UPLC-Q actual MS-based applications for the use of the molecular marker 3- (3-hydroxyphenyl) propionic acid for azoospermia diagnosis.
It is still another object of the present invention to provide a detection and diagnostic kit for chromatographic mass spectrometry for the molecular marker 3- (3-hydroxyphenyl) propionic acid described above.
In order to achieve the above purpose, the present invention provides the following technical solutions: a molecular marker 3- (3-hydroxyphenyl) propionic acid associated with azoospermia, said molecular marker being 3- (3-hydroxyphenyl) propionic acid.
The invention also provides application of the molecular marker 3- (3-hydroxyphenyl) propionic acid in preparing a azoospermia diagnosis or monitoring reagent taking semen as a detection sample.
The invention further provides a kit for diagnosing or monitoring azoospermia prepared by using the molecular marker 3- (3-hydroxyphenyl) propionic acid, which comprises the following components:
3- (3-hydroxyphenyl) propionic acid standard;
internal standard A: an atmosphere standard isotope internal standard of eight substances, namely creatinine, valine, nicotinic acid, thymine, glutaric acid, L-phenylalanine, N-acetyl-p-aminophenol and hippuric acid;
internal standard B: an argon-labeled internal standard of pentadecanoic acid;
internal standard C: an atmosphere standard isotope internal standard of tetracosanoic acid and a Hypersil GOLDC18 chromatographic column are matched: 100 mm. Times.2.1 mm, particle size 1.9. Mu. m Thermo Scientific, germany;
reagent A: 100% methanol;
reagent B: water containing 0.1% formic acid;
reagent C: acetonitrile containing 0.1% formic acid;
reagent D: ultrapure water.
The invention also provides a method for diagnosing or monitoring azoospermia by UPLC-Q actual MS, which detects the molecular marker 3- (3-hydroxyphenyl) propionic acid in semen.
The aim of the invention is achieved by the following technical measures:
3- (3-hydroxyphenyl) propionic acid related to azoospermia in humans is detected by UPLC-Q real MS method to perform early diagnosis of azoospermia.
The detection and diagnosis kit contains a 3- (3-hydroxyphenyl) propionic acid standard. Internal standard A: argon standard isotope internal standard of creatinine, valine, nicotinic acid, thymine, glutaric acid, L-phenylalanine, N-acetyl-para-aminophenol and hippuric acid. Internal standard B: pentadecanoic acid. Internal standard C: and (3) an atmosphere label isotope internal standard of the tetracosanoic acid. The column was fitted with a Hypersil GOLD C18 column (100 mm. Times.2.1 mm, particle size 1.9. Mu. m Thermo Scientific, germany), reagent A (for precipitation of proteins, containing 100% methanol), reagent B (for mobile phase, containing 0.1% formic acid in water), reagent C (for mobile phase, containing 0.1% formic acid in acetonitrile), reagent D (for reconstitution, ultrapure water).
The invention is described in detail as follows:
the system collects complete crowd basic information and clinical data by collecting semen samples meeting the standard by using a Standard Operation Program (SOP), and adopts a metabonomics method based on UPLC-Q actual MS for analysis.
In particular, the experimental method studied mainly comprises the following parts:
1. study selection and grouping basis
80 individuals with azoospermia including both obstructive and non-obstructive types and 116 individuals with healthy controls, 196 total, were included for definitive diagnosis.
Group A: healthy control group (80 persons):
1. ages between 24 and 36 years;
2. a body mass index between 19 and 24;
3. men with healthy reproductive capacity, and healthy offspring after 5-8 months;
4. no serious disease of the whole body.
Group B: azoospermia disease group (116):
1. age matched the control group;
2. the body mass index is matched with the control group;
3. men who do not succeed in attempting pregnancy for 12 months and who have no infertility in spouse;
4. explicitly suffering from azoospermia;
5. matching smoking history with control group;
6. the ethnicity is matched with the control group;
7. no serious disease of the whole body.
2. UPLC-Q actual MS metabonomics analysis and screening and verification of 3- (3-hydroxyphenyl) propionic acid for azoospermia diagnosis
1. Sample pretreatment
1.1. 100. Mu.L of semen was taken, 10. Mu.L of internal standard A was added, 10. Mu.L of internal standard B was added, 10. Mu.L of internal standard C was added, 310. Mu.L of methanol (reagent A) was added, vortexed for 30s, and protein was precipitated.
1.2. Centrifugation was performed for 15min at 20000g at 4℃in a centrifuge, the supernatant was transferred to a 1.5mL inlet EP tube and the supernatant was concentrated to dryness at room temperature in a centrifugal concentration dryer.
1.3. The sample was reconstituted with 20. Mu.L of ultrapure water (reagent D) and analyzed.
2. Instrument detection
2.1. Analytical instrument: a UPLC Ultimate 3000system (Dionex) high performance liquid chromatograph; q-exact high resolution mass spectrometer.
2.2. Liquid phase conditions:
2.2.1 liquid chromatography column was a Hypersil GOLD C18 column (100 mm. Times.2.1 mm, particle size 1.9 μm, thermo Scientific, germany) with column temperature of 40 ℃.
2.2.2 the mobile phase employed was (A) water containing 0.1% formic acid (reagent B) and (B) acetonitrile containing 0.1% formic acid (reagent C), at a flow rate of 400. Mu.L/min.
2.2.3 instrument gradient: 0-3min 1% mobile phase (B), 3-10min 1% to 99% mobile phase (B), 10-13min 99% mobile phase (B), 13-13.1min 99% to 1% mobile phase (B), 13.1-17min 1% mobile phase (B).
2.2.4 sample injection mode: the volume was 10. Mu.L.
2.3. Mass spectrometry conditions
2.3.1 analysis was performed by heating electrospray ionization (HESI).
2.3.2 using a heated electrospray ionization (HESI), positive ion mode spray voltage: 3.5kV; negative ion mode spray voltage: 2.5kV; capillary temperature in two modes: 250 ℃, heater temperature: 425 ℃, sheath gas flow: 50AU, auxiliary gas flow: 13AU, blowback gas flow: 0AU; lens voltage: 60V. Full scan mode, scan range: 70 to 1050m/z; resolution ratio: 70000.
3. qualitative determination of substances
The metabolism micromolecules are qualitatively compared with the chromatographic information (retention time) and the mass spectrum information (accurate molecular weight) of the standard 3- (3-hydroxy phenyl) propionic acid, and the chromatographic information of the isotope internal standard series in the sample is compared in real time to correct the retention time.
4. Data analysis:
biomarker screening key metabolites were confirmed using a heteroscedastic T-test. The difference P value of 3- (3-hydroxyphenyl) propionic acid content in healthy control and azoospermia semen was 7.93X 10-13.
5. Differences and diagnostic significance of small metabolic molecules in semen samples of healthy control group and azoospermia group.
Analysis revealed that the content of semen 3- (3-hydroxyphenyl) acrylic acid was associated with azoospermia. The metabolism micromolecule is used for diagnosing azoospermia by adopting random crowds, the sensitivity of 3- (3-hydroxyphenyl) propionic acid is 88.1%, the specificity is 96.8%, the area under ROC curve is 0.944, and the diagnosis value is higher.
3. Diagnostic kit preparation method
According to the series of experimental results, a diagnostic kit for dynamic monitoring of azoospermia is also prepared, wherein the diagnostic kit comprises a standard substance for measuring 3- (3-hydroxyphenyl) propionic acid which exists stably and can be detected in the semen of a subject and an internal standard substance series for assisting analysis. The diagnosis kit also comprises a set of reagents and equipment for extracting and separating the metabolism micromolecule.
The invention has the beneficial effects that: through adopting UPLC-Q exact MS to compare the metabolism small molecules in normal control and azoospermia semen, the semen is found to have 3- (3-hydroxyphenyl) propionic acid with diagnostic value which can be used for evaluating whether azoospermia exists or not, and the application of UPLC-Qexact MS detected by the 3- (3-hydroxyphenyl) propionic acid, and a azoospermia diagnosis and monitoring kit which can be convenient for clinical application is developed.
The invention adopts the metabolism small molecule 3- (3-hydroxy phenyl) propionic acid as the marker for azoospermia evaluation, and has the advantages that:
(1) The metabolism small molecule is a novel biomarker, has strong relevance with disease outcome, is stable, noninvasive, easy to detect and accurate in quantification, greatly improves the sensitivity and specificity of azoospermia diagnosis, and provides reference for the development of other disease biomarkers in the brand new situation of preventing and treating azoospermia.
(2) The 3- (3-hydroxyphenyl) propionic acid provided by the invention can be used as a diagnosis marker of azoospermia, provides a basis for further deep examination of clinicians, and provides support for rapidly and accurately grasping the disease state and the disease severity of patients and timely taking more personalized prevention and treatment schemes to delay and prevent disease progression.
(3) The semen sample of the random population with azoospermia and healthy control is adopted for verification, and the level of 3- (3-hydroxyphenyl) propionic acid in the semen is proved to have higher sensitivity and specificity in the diagnosis of azoospermia, and can be used as a marker.
(4) The invention adopts a tight and multistage verification and evaluation system, screens a plurality of metabolic small molecules through preliminary experiments in the early stage, and uses UPLC-Q actual MS to carry out independent crowd verification, thereby ensuring the reliability of the metabolic biomarker and the diagnosis method.
(5) The UPLC-Q actual MS technology sample is simple to process, the instrument analysis is rapid and accurate, and the method has high clinical diagnosis practical value.
The foregoing description is only an overview of the present invention, and is intended to provide a better understanding of the present invention, as it is embodied in the following description, with reference to the preferred embodiments of the present invention and the accompanying drawings.
Drawings
FIG. 1 shows the ROC curve between the normal control group and azoospermia, which was made using the 3- (3-hydroxyphenyl) propionic acid content information.
Detailed Description
The following describes in further detail the embodiments of the present invention with reference to the drawings and examples. The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
The invention is further illustrated by the following examples.
Example 1 study selection and grouping basis
The study subjects in this section were from the first-diagnosis azoospermia cases and normal comparison of semen conventional parameters in affiliated hospitals of Nanjing medical university and affiliated medical centers of Chinese university and affiliated sixth hospital. The research content and the informed consent are approved by the ethical committee of the university of Nanjing medical science, and meet the requirements of related regulations. All study subjects were examined for complete physical examination and completed a questionnaire comprising personal basis, lifestyle, occupational and environmental exposure, genetic risk factors, sexual and reproductive functions, disease history and physical activity. Specific sample classification criteria are as follows:
the inclusion of definitive diagnosis included 62 azoospermia of the obstructive and non-obstructive type and 84 healthy controls, 146 total.
Group A: healthy control group (84 people):
1. ages between 24 and 36 years;
2. a body mass index between 19 and 24;
3. men with healthy reproductive capacity, and healthy offspring after 5-8 months;
4. no serious disease of the whole body.
Group B: azoospermia disease group (62 people):
1. age matched the control group;
2. the body mass index is matched with the control group;
3. men who do not succeed in attempting pregnancy for 12 months and who have no infertility in spouse;
4. explicitly suffering from azoospermia;
5. matching smoking history with control group;
6. the ethnicity is matched with the control group;
7. no serious disease of the whole body.
EXAMPLE 2UPLC-MS metabonomics azoospermia 3- (3-hydroxyphenyl) propionic acid screening
1. Sample pretreatment
1.1. 10. Mu.L of semen was taken, 10. Mu.L of internal standard A was added, 10. Mu.L of internal standard B was added, 10. Mu.L of internal standard C was added, 40. Mu.L of methanol (reagent A) was added, vortexed for 30s, and protein was precipitated.
1.2. The supernatant was transferred to a 1.5mL inlet EP tube by centrifugation at 16000g in a centrifuge at 4deg.C for 15min and concentrated to dryness in a centrifugal concentration dryer at room temperature.
1.3. The sample was reconstituted with 10. Mu.L of ultrapure water (reagent D) and analyzed.
2. Instrument detection
2.1. Analytical instrument: a UPLC Ultimate 3000system (Dionex) high performance liquid chromatograph; q-exact high resolution mass spectrometer.
2.2. Liquid phase conditions:
2.2.1 liquid chromatography column was a Hypersil GOLD C18 column (100 mm. Times.2.1 mm, particle size 1.9 μm, thermo Scientific, germany) with column temperature of 40 ℃.
2.2.2 the mobile phase employed was (A) water containing 0.1% formic acid (reagent B) and (B) acetonitrile containing 0.1% formic acid (reagent C), at a flow rate of 400. Mu.L/min.
2.2.3 instrument gradient: 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 sample injection mode: the volume was 5. Mu.L.
2.3. Mass spectrometry conditions
2.3.1 analysis was performed by heating electrospray ionization (HESI).
2.3.2 using a heated electrospray ionization (HESI), positive ion mode spray voltage: 3.5kV; negative ion mode spray voltage: 2.5kV; capillary temperature in two modes: 250 ℃, heater temperature: 425 ℃, sheath gas flow: 50AU, auxiliary gas flow: 13AU, blowback gas flow: 0AU; lens voltage: 60V. Full scan mode, scan range: 70 to 1050m/z; resolution ratio: 70000.
3. qualitative determination of substances
The metabolism micromolecules are qualitatively compared with the chromatographic information (retention time) and the mass spectrum information (accurate molecular weight) of the standard 3- (3-hydroxy phenyl) propionic acid, and the chromatographic information of the isotope internal standard series in the sample is compared in real time to correct the retention time.
4. Data analysis:
biomarker screening key metabolites were confirmed using a heteroscedastic T-test.
Example 3 3 (3-hydroxyphenyl) propionic acid diagnosis of azoospermia
According to the UPLC-Q actual MS metabonomics method described above, the present inventors evaluated the ability of detecting the level of small molecules of the metabolism in semen to assess azoospermia by detecting 3- (3-hydroxyphenyl) propionic acid in 62 cases and 84 control semen samples, drawing ROC curves therefrom and evaluating the predicted sensitivity and specificity.
Referring to FIG. 1,3- (3-hydroxyphenyl) propionic acid has a sensitivity of 88.7%, a specificity of 94.0% and an area under the ROC curve of 0.944, so that 3- (3-hydroxyphenyl) propionic acid has a better ability to diagnose azoospermia.
Example 4 preparation of 3- (3-hydroxyphenyl) propionic acid detection and diagnostic kit for azoospermia semen the metabolic small molecules with higher abundance in normal control and azoospermia semen were first determined by the method of UPLC-Q actual MS. Then, metabolic small molecules related to azoospermia are screened therein by a metabonomics technology based on UPLC-Q actual MS as a diagnostic index of whether azoospermia is or not. Finally, the number of the screened corresponding small metabolic molecules is controlled to be 1, which is the optimized simplification made on the basis of pre-experiments. The kit comprises a batch of reagents and consumables for detecting small metabolic molecules, wherein the qualitative and quantitative analysis of the small metabolic molecules adopts a standard substance of 3- (3-hydroxyphenyl) propionic acid, and the auxiliary analysis adopts an internal standard A: creatinine, valine, nicotinic acid, thymic power, glutaric acid, L-phenylalanine, N-acetyl-para-aminophenol, and hippuric acid. Internal standard B: pentadecanoic acid. Internal standard C: and (3) an atmosphere label isotope internal standard of the tetracosanoic acid. Other are the matched reverse chromatographic columns for UPLC chromatographic separations (HypersilGOLD C18 column, 100 mm. Times.2.1 mm, particle size 1.9 μm), reagents for precipitating proteins (100% methanol), reagents for mobile phases (water with 0.1% formic acid and acetonitrile with 0.1% formic acid), reagents for extracting metabolic small molecules (100% ultrapure water). The kit has the value that the content of 3- (3-hydroxyphenyl) acrylic acid can be detected only by 10 mu L of semen, azoospermia can be diagnosed by the content, and dynamic monitoring and observation of treatment effects are easy.
The specific kit comprises the following components:
3- (3-hydroxyphenyl) propionic acid standard;
internal standard A (atmosphere standard isotope internal standard aqueous solution of eight substances of creatinine, valine, nicotinic acid, thymine piperidine, glutaric acid, L-phenylalanine, N-acetyl-p-aminophenol and hippuric acid);
internal standard B (pentadecanoic acid in methanol solution of internal standard isotope);
internal standard C (an atmosphere-labeled isotope of tetracosanoic acid internal standard methanol solution);
further, it may further comprise:
chromatographic column (Thermo 100 mm. Times.2.1 mm, particle size 1.9 μm, hypersil GOLD C18 column);
reagent a (100% methanol);
reagent B (water with 0.1% formic acid);
reagent C (acetonitrile containing 0.1% formic acid);
reagent D (100% ultrapure water).
In another embodiment, standard Operation Procedure (SOP) is used to collect the standard-compliant seminal plasma sample, and the conclusion is also true after the test is performed, i.e. the seminal plasma can be used as the test sample for detection.
In conclusion, by comparing the metabolic small molecules in normal control semen and azoospermia semen by adopting UPLC-Q exact MS, the fact that 3- (3-hydroxyphenyl) propionic acid with diagnostic value exists in semen for evaluating whether azoospermia exists or not, and the application of UPLC-Q exact MS for detecting 3- (3-hydroxyphenyl) propionic acid are developed, and the azoospermia diagnosis and monitoring kit which is convenient for clinical application is convenient.
The invention adopts the metabolism small molecule 3- (3-hydroxy phenyl) propionic acid as the marker for azoospermia evaluation, and has the advantages that:
(1) The metabolism small molecule is a novel biomarker, has strong relevance with disease outcome, is stable, noninvasive, easy to detect and accurate in quantification, greatly improves the sensitivity and specificity of azoospermia diagnosis, and provides reference for the development of other disease biomarkers in the brand new situation of preventing and treating azoospermia.
(2) The 3- (3-hydroxyphenyl) propionic acid provided by the invention can be used as a diagnosis marker of azoospermia, provides a basis for further deep examination of clinicians, and provides support for rapidly and accurately grasping the disease state and the disease severity of patients and timely taking more personalized prevention and treatment schemes to delay and prevent disease progression.
(3) The semen sample of the random population with azoospermia and healthy control is adopted for verification, and the level of 3- (3-hydroxyphenyl) propionic acid in the semen is proved to have higher sensitivity and specificity in the diagnosis of azoospermia, and can be used as a marker.
(4) The invention adopts a tight and multistage verification and evaluation system, screens a plurality of metabolic small molecules through preliminary experiments in the early stage, and uses UPLC-Q actual MS to carry out independent crowd verification, thereby ensuring the reliability of the metabolic biomarker and the diagnosis method.
(5) The UPLC-Q actual MS technology sample is simple to process, the instrument analysis is rapid and accurate, and the method has high clinical diagnosis practical value.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
The technical features and the detection items of the above-described embodiments may be arbitrarily combined, and for brevity of description, all possible combinations of the technical features in the above-described embodiments are not described, however, as long as there is no contradiction between the combinations of the technical features, they should be regarded as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (1)
1. Application of 3- (3-hydroxyphenyl) propionic acid as a molecular marker related to azoospermia in preparing a azoospermia diagnosis or monitoring reagent by taking semen as a detection sample.
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| KATSUNORI YAMAKAWA 等.Comparative Analysis of Interindividual Variations in the Seminal Plasma Proteome of Fertile Men With Identification of Potential Markers for Azoospermia in Infertile Patients.《Journal of Andrology》.2007,第28卷(第6期),第858-865页. * |
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