CN109187794B - Seminal plasma deoxycytidine and cytidine detection as idiopathic male infertility diagnostic marker and application thereof - Google Patents

Abstract

The invention belongs to the fields of analytical chemistry and clinical medicine, and discloses seminal plasma deoxycytidine and cytidine detection as idiopathic male sterility diagnostic markers and application thereof. The marker is deoxycytidine and/or cytidine, has high sensitivity and specificity in diagnosing idiopathic male infertility, and can be used for preparing diagnostic or monitoring reagents for idiopathic male infertility.

Description

Seminal plasma deoxycytidine and cytidine detection as idiopathic male infertility diagnostic marker and application thereof

Technical Field

The invention belongs to the fields of analytical chemistry and clinical medicine, and relates to detection of deoxycytidine and cytidine related to idiopathic male sterility in seminal plasma based on UPLC-Q active MS and application thereof.

Background

In 1988, the WHO first proposed the concept of reproductive health, namely, a fully harmonious state of physical, psychological and social health during the whole life of the human and its individuals in the processes of structure, function and behavior of the organism related to reproduction. Currently, about 10-15% of women of childbearing age worldwide suffer from fertility disorders. Due to the large population base, the number of people suffering from infertility among newly-married couples is far more than one million, and the infertility caused by male factors is higher than 50%. Current studies suggest genetic factors such as changes in the sequence of AZF gene region of the Y chromosome, changes in the copy number of the DAZ gene, changes in the sequence of sex hormone receptor genes, and the like. Environmental factors such as exposure to harmful factors, nutrition and lifestyle habits are all associated with male infertility. It is noteworthy that a significant portion of male infertility is manifested as unexplained etiology, which makes diagnosing idiopathic male infertility extremely difficult and mistaking treatment opportunities. The WHO diagnosis standard of male infertility is that the couple lives together for more than 1 year after marriage, and no contraceptive measures are taken, so that the male causes the female to be infertile. However, the observation time of the same residence as 1 year greatly delays the time for early treatment and intervention of male infertility; many couples cannot guarantee strict co-existence for 1 year, so that the last judgment of male sterility becomes very difficult; in order to eliminate the reason for the female, the female also needs to perform detailed examination, which brings heavy economic and medical burden. The existing male sterility related examination depends on the conventional semen routine examination, and only focuses on the routine parameters of the number, the vitality, the semen volume, the pH value, the liquefaction time and the like of the sperms. The conventional analysis result of the conventional semen parameters shows larger fluctuation because of the influence of factors such as abstinence time and the like. Thus, clinical diagnosis often requires reference to multiple semen routine analyses. More importantly, conventional semen parameter examination does not fully reflect the entire condition of the semen. Therefore, male infertility is often manifested as no obvious abnormality in conventional semen parameters, and the traditional semen parameter examination cannot effectively diagnose male infertility. Thus, there is a clinical need for new diagnostic methods for idiopathic male infertility.

Metabonomics (metabonomics) is a branch of system biology which is based on group index analysis, takes high-throughput detection and data processing as means and aims at information modeling and system integration, is a new important research field of system biology after genomics, transcriptomics and proteomics, is the science for researching the change of all metabolites generated by a biological system under external stimulation, and focuses on the change of small-molecule metabolites with the molecular weight of less than 1000 in a metabolic cycle and reflects the response change of organisms under a disease state or environmental stimulation. Metabonomics has extremely high application potential and value in the diagnosis of complex diseases, and the analyzed samples can be non-invasive and low-invasive samples of various body fluids of blood and urine, so that the compliance of patients can be greatly improved, and the metabonomics has the characteristics of high sensitivity and stability. The root cause of male infertility is the semen problem. The seminal plasma as the liquid component of the seminal fluid can well reflect the quality change of the seminal fluid, and has the advantages of no wound and easy collection. However, the use of metabolomics for the analysis of small molecules of plasma metabolism in the diagnostic monitoring of idiopathic male infertility has not received corresponding attention.

Currently, the commonly used 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 combination has good sensitivity and reproducibility, but a derivatization method is generally adopted to carry out pretreatment on a sample, so that the experimental steps become complicated. And LC-MS has the characteristics of simple sample treatment, high sensitivity and strong clinical practicability. UPLC-Q active MS is a combination of a new generation of high resolution mass spectrum and an ultra-high performance liquid phase, and has higher sensitivity, specificity and stability compared with the traditional LC-MS. Therefore, UPLC-Q active MSS is adopted to carry out metabonomics analysis of seminal plasma metabolism micromolecules, and if stable specific seminal plasma metabolism micromolecules related to idiopathic male infertility can be found to serve as biomarkers, and a UPLC-Q active MS detection method of corresponding metabolism micromolecule markers is developed, great contribution is made to male reproductive health.

Disclosure of Invention

The invention aims to provide related deoxycytidine and cytidine for idiopathic male infertility.

Another object of the present invention is to provide UPLC-Q active MS-based applications for the use of seminal plasma deoxycytidine and cytidine in the diagnosis of idiopathic male infertility.

It is still another object of the present invention to provide a detection and diagnosis kit for mass spectrometry for the above seminal plasma deoxycytidine and cytidine.

The aim of the invention is achieved by the following technical measures:

a seminal plasma marker associated with idiopathic male infertility, the marker being deoxycytidine and/or cytidine.

The seminal plasma marker is applied to the preparation of an idiopathic male infertility diagnosis or monitoring reagent.

A kit for diagnosing or monitoring idiopathic male infertility, the kit comprising reagents for detecting deoxycytidine and/or cytidine in seminal plasma.

The kit contains a reagent for detecting deoxycytidine and/or cytidine in seminal plasma by using a UPLC-Q active MS method.

The kit comprises the following reagents:

deoxycytidine and/or cytidine standards;

internal standard A: isotopic internal standards of one or more of creatinine, valine, nicotinic acid, thymine, glutaric acid, L-phenylalanine, N-acetyl-p-aminophenol, hippuric acid;

internal standard B: an isotopic internal standard of pentadecanoic acid;

internal standard C: isotopic internal standards of tetracosanoic acid.

The kit further comprises:

hypersil GOLD C18 chromatography column;

reagent A: precipitating protein with 100% methanol;

and (3) reagent B: for the mobile phase, water containing 0.1% formic acid;

and (3) reagent C: the mobile phase is acetonitrile containing 0.1 percent of formic acid;

and (3) reagent D: for reconstitution, ultra pure water.

A method for detecting the seminal plasma biomarker related to the idiopathic male infertility adopts an UPLC-Q active MS method to detect the content of deoxycytidine and/or cytidine in the seminal plasma.

The method comprises the following steps:

firstly, liquid phase conditions:

the liquid chromatographic column is Hypersil GOLD C18 chromatographic column, and the column temperature is 40 ℃;

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

and (3) sample introduction mode: volume 5. mu.l;

second, Mass Spectrometry Condition

Analysis was performed using a heating electrospray ionization (HESI) mode, positive ion mode spray voltage: 3.5 kV; negative ion mode spray voltage: 2.5 kV; capillary temperature in two modes: 250 ℃, heater temperature: 425 ℃, sheath gas flow: 50AU, auxiliary gas flow: 13AU, reverse air flow: 0 AU; lens voltage: 60V; adopting a full-scanning mode, wherein the scanning range is as follows: 70 to 1050 m/z; resolution ratio: 70000.

the invention is described in detail below:

the invention collects seminal plasma samples meeting the standard by a Standard Operation Program (SOP), and the system collects complete population basic information and clinical data and adopts a UPLC-Q active MS-based metabonomics method for analysis.

The experimental method of research mainly includes the following parts:

first, research object selection and grouping basis

First stage screening stage

Specifically diagnosed idiopathic male infertility 148 and healthy control 148 were included at random for a total of 296.

Group A: healthy control group (148 persons):

1. age between 19 and 39 years;

2. a body mass index between 17 and 31;

3. a male with healthy reproductive capacity and healthy offspring after 5-8 months;

4. there is no serious disease of the whole body.

Group B: idiopathic male infertility disease group (148 people):

1. age matched to control group;

2. the body constitution index is matched with the control group

3. A male with no success in attempting pregnancy for 12 months and a spouse without infertility disorder; .

4. There is no clear cause of male infertility;

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

6. matching nationality with a control group;

7. there is no serious disease of the whole body.

Second phase verification phase

Specifically diagnosed idiopathic male infertility 15 and healthy controls 15 were included, for a total of 30.

Group A: healthy control group (15 persons):

1. age between 24 and 36 years;

2. a body mass index between 19 and 24;

3. a male with healthy reproductive capacity and healthy offspring after 5-8 months;

4. there is no serious disease of the whole body.

Group B: idiopathic male infertility disease group (15 people):

1. age matched to control group;

2. the body mass index is matched with a control group;

3. a male with no success in attempting pregnancy for 12 months and a spouse without infertility disorder;

4. there is no clear cause of male infertility;

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

6. matching nationality with a control group;

7. there is no serious disease of the whole body.

Second, UPLC-Q active MS metabonomics analysis and deoxycytidine screening and verification for idiopathic male sterility diagnosis

1. Sample pretreatment

1.1. Taking 10 mu L of seminal plasma, adding 10 mu L of internal standard A, adding 10 mu L of internal standard B, adding 10 mu L of internal standard C, adding 40 mu L of methanol (reagent A), whirling for 30s, and precipitating protein.

1.2. The supernatant was transferred to a 1.5mL inlet EP tube after centrifugation at 16000g in a centrifuge at 4 ℃ for 15min and concentrated to dryness in a centrifugal concentration desiccator at room temperature.

1.3. Reconstituted with 10. mu.L of ultrapure water (reagent D) for analysis.

2. Instrumental detection

2.1. An analytical instrument: UPLC Ultimate 3000system (dionex) hplc; q-active high resolution mass spectrometer.

2.2. Liquid phase conditions:

2.2.1 the liquid chromatography column was a Hypersil GOLD C18 column (100 mm. times.2.1 mm, particle size 1.9 μm, Thermo Scientific, Germany) at a column temperature of 40 ℃.

2.2.2 the mobile phases used were (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 introduction mode: volume 5. mu.l.

2.3. Conditions of Mass Spectrometry

2.3.1 analysis by heated electrospray ionization (HESI).

2.3.2 using the heating electrospray ionization mode (HESI), positive ion mode spray voltage: 3.5 kV; negative ion mode spray voltage: 2.5 kV; capillary temperature in two modes: 250 ℃, heater temperature: 425 ℃, sheath gas flow: 50AU, auxiliary gas flow: 13AU, reverse air flow: 0 AU; lens voltage: 60V. Adopting a full-scanning mode, wherein the scanning range is as follows: 70 to 1050 m/z; resolution ratio: 70000.

3. characterization of the substance

The metabolic small molecule qualitative method is characterized by comparing chromatographic information (retention time) and mass spectrum information (accurate molecular weight) with standard deoxycytidine and cytidine, and comparing the chromatographic information of isotope internal standard substance series in a sample in real time to correct the retention time.

4. And (3) data analysis:

biomarker screening key metabolites were confirmed using multiple Logistic regression.

5. Difference and diagnostic significance of metabolic small molecules in seminal plasma samples of healthy control groups and idiopathic male infertility groups.

Through correcting the information of age, body mass index, smoking and drinking history, multivariate Logistic regression analysis finds that the content of seminal plasma deoxycytidine and cytidine is related to idiopathic male infertility. The metabolic small molecule combination is applied to random population to diagnose idiopathic male infertility, the sensitivity is 93.33%, the specificity is 93.33%, the area under an ROC curve is 0.9822, and the diagnostic value is high.

Third, preparation method of diagnostic kit

According to the series of experimental results, the inventor also prepares a diagnostic kit for the dynamic monitoring of idiopathic male infertility, wherein the diagnostic kit comprises a standard substance for determining deoxycytidine and cytidine which are stably existed and can be detected in seminal plasma of a subject and an internal standard substance series for auxiliary analysis. The diagnostic kit also comprises a set of reagents and devices for extracting the seminal plasma metabolism micromolecules and separating the chromatogram.

The invention has the beneficial effects that:

the inventor discovers that deoxycytidine and cytidine combination with diagnostic value and application of UPLC-Q active MS for detecting the deoxycytidine and the cytidine in seminal plasma can be used for evaluating whether idiopathic male sterility is caused by comparing metabolic small molecules in normal control and the idiopathic male sterility seminal plasma by using the UPLC-Q active MS, and develops the idiopathic male sterility diagnosis and monitoring kit convenient for clinical application.

The invention adopts seminal plasma metabolism micromolecules as markers for evaluating the idiopathic male sterility, and has the advantages that:

(1) the seminal plasma metabolism micromolecule is a novel biomarker which is strongly associated with disease fate, is stable, noninvasive, easy to detect and accurate in quantification, can greatly improve the sensitivity and specificity of idiopathic male infertility diagnosis, and the successful development of the micromolecule biomarker can create a brand new situation for the prevention and treatment of the idiopathic male infertility and provide reference for the development of other disease biomarkers.

(2) The deoxycytidine and the cytidine provided by the invention can be used as diagnostic markers of idiopathic male infertility, provide basis for further in-depth examination of clinicians, provide support for rapidly and accurately mastering the disease state and the severity of the disease of a patient and timely adopting a more personalized prevention and treatment scheme, and delay and stop the disease progression.

(3) The seminal plasma samples of idiopathic male infertility and healthy control random population are adopted for verification, and the levels of deoxycytidine and cytidine in the seminal plasma are proved to have higher sensitivity and specificity in diagnosing the idiopathic male infertility and can be used as markers.

(4) The invention adopts a strict and multistage verification and evaluation system, screens a plurality of seminal plasma metabolism micromolecules through preliminary experiments at the initial stage, and uses UPLC-Q active MS to verify independent population, thereby ensuring the reliability of the seminal plasma metabolism biomarker and the diagnosis method.

(5) The UPLC-Q active MS technology has the advantages of simple sample processing, rapid and accurate instrument analysis and higher clinical diagnosis practical value.

Drawings

In the screening stage shown in fig. 1, the information of age, body mass index, smoking and drinking history is corrected, and multivariate Logistic regression analysis finds that the content of seminal plasma deoxycytidine and cytidine is closely related to idiopathic male infertility.^aThe one-way Logistic regression results for confounding factors were not adjusted.^bAdjusting multiple Logistic regression results after age, body mass index, smoking and drinking history.

FIG. 2 Small Metabolic molecule detection level variability (mean. + -. standard deviation).

FIG. 3 shows the ROC curve between the normal control group and the idiopathic male infertility group prepared by using the content information of seminal plasma deoxycytidine at the verification stage.

FIG. 4 shows ROC curves between normal control group and idiopathic male sterility group prepared by using seminal plasma cytidine content information in the verification stage.

FIG. 5 shows the ROC curve between the normal control group and the idiopathic male infertility group prepared by using the content information of seminal plasma deoxycytidine and cytidine at the verification stage.

Detailed Description

The invention is further illustrated by the following examples.

Example 1 study selection and grouping basis

The study subjects were from the first-visit idiopathic male infertility case and healthy fertility control of the subsidiary hospital of the Nanjing medical university. The research content and the informed consent were approved by the ethical committee of the university of medical Nanjing, and were in compliance with the requirements of the relevant regulations. Cases and controls signed informed consent after understanding the content. All subjects performed a complete physical examination and completed a questionnaire that included personal basic data, lifestyle habits, occupational and environmental exposure, genetic risk factors, sexual and reproductive function, disease history, and physical activity. In the first stage, 148 cases of idiopathic male infertility and 148 healthy controls were included as required; 15 cases of idiopathic male infertility and 15 healthy controls meeting the requirements of the second stage are used as screening experimental objects of the seminal plasma metabolism small molecule biomarker of the idiopathic male infertility.

The specific sample classification criteria are as follows:

first stage screening stage

Specifically diagnosed idiopathic male infertility 148 and healthy control 148 were included at random for a total of 296.

Group A: healthy control group (148 persons):

1. age between 19 and 39 years;

2. a body mass index between 17 and 31;

3. a male with healthy reproductive capacity and healthy offspring after 5-8 months;

4. there is no serious disease of the whole body.

Group B: idiopathic male infertility disease group (148 people):

1. age matched to control group;

2. the body constitution index is matched with the control group

3. A male with no success in attempting pregnancy for 12 months and a spouse without infertility disorder; .

4. There is no clear cause of male infertility;

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

6. matching nationality with a control group;

7. there is no serious disease of the whole body.

Second phase verification phase

Specifically diagnosed idiopathic male infertility 15 and healthy controls 15 were included, for a total of 30.

Group A: healthy control group (15 persons):

1. age between 24 and 36 years;

2. a body mass index between 19 and 24;

3. a male with healthy reproductive capacity and healthy offspring after 5-8 months;

4. there is no serious disease of the whole body.

Group B: idiopathic male infertility disease group (15 people):

1. age matched to control group;

2. the body mass index is matched with a control group;

3. a male with no success in attempting pregnancy for 12 months and a spouse without infertility disorder;

4. there is no clear cause of male infertility;

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

6. matching nationality with a control group;

7. there is no serious disease of the whole body.

Example 2 UPLC-MS metabolomics idiopathic male infertility deoxycytidine and cytidine screening

1. Sample pretreatment

1.1. Taking 10 mu L of seminal plasma, adding 10 mu L of internal standard A, adding 10 mu L of internal standard B, adding 10 mu L of internal standard C, adding 40 mu L of methanol (reagent A), whirling for 30s, and precipitating protein.

1.2. The supernatant was transferred to a 1.5mL inlet EP tube after centrifugation at 16000g in a centrifuge at 4 ℃ for 15min and concentrated to dryness in a centrifugal concentration desiccator at room temperature.

1.3. Reconstituted with 10. mu.L of ultrapure water (reagent D) for analysis.

2. Instrumental detection

2.1. An analytical instrument: UPLC Ultimate 3000system (dionex) hplc; q-active high resolution mass spectrometer.

2.2. Liquid phase conditions:

2.2.1 the liquid chromatography column was a Hypersil GOLD C18 column (100 mm. times.2.1 mm, particle size 1.9 μm, Thermo Scientific, Germany) at a column temperature of 40 ℃.

2.2.2 the mobile phases used were (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 introduction mode: volume 5. mu.l.

2.3. Conditions of Mass Spectrometry

2.3.1 analysis by heated electrospray ionization (HESI).

2.3.2 using the heating electrospray ionization mode (HESI), positive ion mode spray voltage: 3.5 kV; negative ion mode spray voltage: 2.5 kV; capillary temperature in two modes: 250 ℃, heater temperature: 425 ℃, sheath gas flow: 50AU, auxiliary gas flow: 13AU, reverse air flow: 0 AU; lens voltage: 60V. Adopting a full-scanning mode, wherein the scanning range is as follows: 70 to 1050 m/z; resolution ratio: 70000.

3. characterization of the substance

The metabolic small molecule qualitative method is characterized by comparing chromatographic information (retention time) and mass spectrum information (accurate molecular weight) with standard deoxycytidine and cytidine, and comparing the chromatographic information of isotope internal standard substance series in a sample in real time to correct the retention time.

4. And (3) data analysis:

biomarker screening key metabolites were confirmed using multiple Logistic regression.

5. Difference and diagnostic significance of metabolic small molecules in seminal plasma samples of healthy control groups and idiopathic male infertility groups.

After correcting the information of age, body mass index, smoking and drinking history, multivariate Logistic regression analysis finds that the content of seminal plasma deoxycytidine and cytidine is closely related to idiopathic male infertility (figure 1).

Example 3 stability analysis of seminal plasma deoxycytidine and cytidine

The stability of the levels of seminal plasma deoxycytidine and cytidine was evaluated using the method of example 2 (2 weeks apart). The results showed that the levels of deoxycytidine and cytidine measured in seminal plasma were stable (fig. 2), possessing the properties as diagnostic/monitoring markers.

Example 4 diagnosis of idiopathic male infertility by combinations of deoxycytidine and cytidine

According to the UPLC-Q active MS metabonomics method, the inventor detects deoxycytidine and cytidine by 15 cases of random population and 15 contrasted seminal plasma samples, draws an ROC curve and evaluates the predicted sensitivity and specificity, and further evaluates the evaluation capability of detecting the 2 metabolic small molecule levels in the seminal plasma on idiopathic male infertility.

The sensitivity of deoxycytidine is 93.33%, the specificity is 73.33%, and the area under the ROC curve is 0.8756; cytidine sensitivity was 80.00%, specificity was 86.67%, and area under the ROC curve was 0.9244.

The sensitivity of the combination of deoxycytidine and cytidine was 93.33%, the specificity was 93.33%, and the area under the ROC curve was 0.9822.

Therefore, the combination of deoxycytidine and cytidine has better capability of diagnosing idiopathic male infertility.

Example 5 preparation of a kit for detecting and diagnosing idiopathic male infertility seminal plasma deoxycytidine and cytidine

Firstly, determining metabolic small molecules with higher abundance in normal control and idiopathic male infertility seminal plasma by using a UPLC-Q active MS method. Then, metabolic small molecules related to the idiopathic male sterility are screened by the UPLC-Q active MS-based metabonomics technology as a diagnostic index of whether the idiopathic male sterility is detected. And finally, controlling the number of the screened corresponding seminal plasma metabolism micromolecules to be 2, which is the simplification of optimization on the basis of a preliminary experiment. The kit comprises a batch of reagents and consumables for detecting the seminal plasma metabolism micromolecules, wherein the qualitative and quantitative analysis of the metabolism micromolecules adopts deoxycytidine and cytidine standard products, and the auxiliary analysis adopts an internal standard A: the deuterium labeled isotope internal standard of eight substances of creatinine, valine, nicotinic acid, thymine, glutaric acid, L-phenylalanine, N-acetyl-p-aminophenol and hippuric acid. Internal standard B: deuterium-labelled isotopic internal standard of pentadecanoic acid. Internal standard C: deuterium-labelled isotopic internal standard of tetracosanoic acid. Other examples are a reversed phase chromatographic column (Hypersil GOLD C18 column, 100 mm. times.2.1 mm, particle size 1.9 μm), a reagent for precipitating seminal plasma proteins (100% methanol), a reagent for mobile phase (water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid), and a reagent for extracting metabolic small molecules (100% ultrapure water) for UPLC chromatographic separation. The kit has the value that only 10 mul seminal plasma is needed to detect the content of deoxycytidine and cytidine, then the idiopathic male infertility is diagnosed by the content, and dynamic monitoring and observation of treatment effect are easy to carry out.

The specific kit comprises the following components:

deoxycytidine standard substance

Cytidine standards

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

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

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

Further, the method may further comprise:

chromatographic column (Thermo 100mm X2.1 mm, particle size 1.9 μm, Hypersil GOLD C18 chromatographic column)

Reagent A (100% methanol)

Reagent B (Water containing 0.1% formic acid)

Reagent C (acetonitrile containing 0.1% formic acid)

Reagent D (100% ultrapure water).

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1. The application of a seminal plasma marker related to idiopathic male sterility in preparing a reagent for diagnosing or monitoring the idiopathic male sterility is characterized in that the seminal plasma marker is deoxycytidine and cytidine.

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