CN112129877B - Seminal plasma mannose-6-phosphate and neopterin detection as idiopathic male sterility diagnostic marker and application thereof - Google Patents
Seminal plasma mannose-6-phosphate and neopterin detection as idiopathic male sterility diagnostic marker and application thereof Download PDFInfo
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Abstract
The invention belongs to the fields of analytical chemistry and clinical medicine, and provides UPLC-Q active MS for detecting mannose-6-phosphate and neopterin related to idiopathic male sterility in seminal plasma and application thereof. According to the invention, UPLC-Q active MS is adopted to compare metabolic small molecules in normal control and idiopathic male sterility seminal plasma, and the combination of mannose-6-phosphate and neopterin with diagnostic value, which can be used for evaluating whether idiopathic male sterility is suffered, and the application of UPLC-Q active MS for detecting mannose-6-phosphate and neopterin are found in seminal plasma, so that the kit for diagnosing and monitoring idiopathic male sterility, which can be conveniently applied clinically, is developed.
Description
Technical Field
The invention belongs to the fields of analytical chemistry and clinical medicine, and relates to detection of mannose-6-phosphate and neopterin related to idiopathic male sterility in seminal plasma based on UPLC-Q active MS and application thereof.
Background
In 1988, the World Health Organization (WHO) first proposed the concept of reproductive health, namely a physically, psychologically and socially sound state of well-balanced health of the human and its individuals throughout the life and in the structural, functional and behavioral processes of the reproductive-related body. 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 research suggests that genetic and environmental factors are the major causes of male infertility. Genetic factors: such as changes in the sequence of the AZF gene region of the Y chromosome, changes in the copy number of the DAZ gene, changes in the sequence of the sex hormone receptor gene, and the like. Environmental factors: if harmful factors are exposed, nutrition and living habits are all related to male sterility. 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 standard for male sterility diagnosis is that the couple lives for more than 1 year after marriage, and no contraceptive measures are taken, and 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 another new important research field of subsequent genomics, transcriptomics, proteomics and post-system biology, 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 an organism under a disease state or environmental stimulation. Metabonomics shows 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 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 MS detection method for metabolic small molecule can make great contribution to male reproductive health if stable specific seminal plasma metabolic small molecule related to idiopathic male sterility can be found and used as biomarker of idiopathic male sterility and a UPLC-Q active MS detection method for corresponding metabolic small molecule marker is developed.
Disclosure of Invention
The invention aims to provide relevant mannose-6-phosphate and neopterin for idiopathic male infertility.
Another objective of the invention is to provide UPLC-Q active MS-based application of seminal plasma mannose-6-phosphate and neopterin for idiopathic male infertility diagnosis.
It is still another object of the present invention to provide a detection and diagnosis kit for chromatography-mass spectrometry for the above seminal plasma mannose-6-phosphate and neopterin.
The aim of the invention is achieved by the following technical measures:
mannose-6-phosphate and neopterin related to human idiopathic male sterility are combinations of the mannose-6-phosphate and the neopterin, and the metabolic small molecules are detected by an UPLC-Q active MS method to carry out early diagnosis of the idiopathic male sterility.
The detection and diagnosis kit contains mannose-6-phosphate and neopterin standard substance. 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. The kit was packed with Hypersil GOLD C18 column (100 mm. times.2.1 mm, particle size 1.9 μm Thermo Scientific, Germany), reagent A (for protein precipitation, 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, ultra pure water).
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.
II, screening and verification of mannose-6-phosphate and neopterin for UPLC-Q active MS metabonomics analysis and 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 adopts the comparison of chromatographic information (retention time) and mass spectrum information (accurate molecular weight) with standard mannose-6-phosphate and neopterin, and compares the chromatographic information of an isotope internal standard 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 contents of seminal plasma mannose-6-phosphate and neopterin are 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 0.9778, 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 which can be used for the dynamic monitoring of idiopathic male infertility and comprises standards for determining stable and detectable mannose-6-phosphate and neopterin in seminal plasma of a subject and an internal standard 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 mannose-6-phosphate and neopterin combination with diagnostic value and application of UPLC-Q active MS for detecting mannose-6-phosphate and neopterin exist in seminal plasma by comparing metabolic small molecules in normal control and idiopathic male sterility seminal plasma by using 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 mannose-6-phosphate and the neopterin 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 disease severity of patients and timely adopting more personalized prevention and treatment schemes, and delay and prevent disease progression.
(3) The seminal plasma samples of idiopathic male infertility and healthy control random population are adopted for verification, and the levels of mannose-6-phosphate and neopterin in 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 figure 1, the contents of seminal plasma mannose-6-phosphate and neopterin are closely related to idiopathic male infertility through information of corrected age, body mass index, smoking and drinking histories and multivariate Logistic regression analysis.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 seminal plasma mannose-6-phosphate content information at the verification stage.
FIG. 4 shows ROC curve between normal control group and idiopathic male infertility group prepared by using the information of neopterin content in seminal plasma at verification stage.
FIG. 5 shows the ROC curve between the normal control group and the idiopathic male infertility group prepared by using the information of the contents of seminal plasma mannose-6-phosphate and neopterin in 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):
5. age between 19 and 39 years;
6. a body mass index between 17 and 31;
7. a male with healthy reproductive capacity and healthy offspring after 5-8 months;
8. there is no serious disease of the whole body.
Group B: idiopathic male infertility disease group (148 people):
8. age matched to control group;
9. the body constitution index is matched with the control group
10. A male with no success in attempting pregnancy for 12 months and a spouse without infertility disorder; .
11. There is no clear cause of male infertility;
12. the smoking and drinking history is matched with the control group;
13. matching nationality with a control group;
14. 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):
5. age between 24 and 36 years;
6. a body mass index between 19 and 24;
7. a male with healthy reproductive capacity and healthy offspring after 5-8 months;
8. there is no serious disease of the whole body.
Group B: idiopathic male infertility disease group (15 people):
8. age matched to control group;
9. the body mass index is matched with a control group;
10. a male with no success in attempting pregnancy for 12 months and a spouse without infertility disorder;
11. there is no clear cause of male infertility;
12. the smoking and drinking history is matched with the control group;
13. matching nationality with a control group;
14. there is no serious disease of the whole body.
Example 2UPLC-MS Metabonomics idiopathic Male sterile mannose-6-phosphate and neopterin 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 adopts the comparison of chromatographic information (retention time) and mass spectrum information (accurate molecular weight) with standard mannose-6-phosphate and neopterin, and compares the chromatographic information of an isotope internal standard 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 contents of seminal plasma mannose-6-phosphate and neopterin are closely related to idiopathic male infertility (figure 1).
Example 3 stability analysis of seminal plasma mannose-6-phosphate and neopterin
The stability of seminal plasma mannose-6-phosphate and neopterin levels was evaluated using the method of example 2 (2 weeks apart). The results showed that the levels of mannose-6-phosphate and neopterin measured in seminal plasma were stable (FIG. 2), and had properties as diagnostic/monitoring markers.
Example 4 diagnosis of idiopathic Male infertility by combination of mannose-6-phosphate and neopterin
According to the UPLC-Q active MS metabonomics method, the inventor detects mannose-6-phosphate and neopterin by 15 random population seminal plasma samples and 15 control seminal plasma samples, so as to draw an ROC curve and evaluate the predicted sensitivity and specificity, and further evaluate the evaluation capability of detecting the 2 metabolic small molecule levels in the seminal plasma on idiopathic male infertility.
The sensitivity of mannose-6-phosphate was 73.33%, the specificity was 93.33%, and the area under the ROC curve was 0.8756; the sensitivity of neopterin was 80.00%, the specificity was 93.33%, and the area under the ROC curve was 0.9467.
The sensitivity of the combination of mannose-6-phosphate and neopterin was 93.33%, the specificity was 93.33%, and the area under the ROC curve was 0.9778.
Therefore, the combination of mannose-6-phosphate and neopterin has better capability of diagnosing idiopathic male infertility.
EXAMPLE 5 preparation of seminal plasma mannose-6-phosphate and neopterin detection and diagnostic kit for idiopathic male infertility
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 mannose-6-phosphate and neopterin standard substances, 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 mannose-6-phosphate and neopterin, and then the idiopathic male infertility is diagnosed according to the content, and dynamic monitoring and observation of treatment effects are easy to carry out.
The specific kit comprises the following components:
mannose-6-phosphate standard substance
Neopterin standard product
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).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (1)
1. The application of the seminal plasma metabolism small molecular marker in preparing a reagent for diagnosing or monitoring idiopathic male infertility comprises mannose-6-phosphate and neopterin.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103071147A (en) * | 2011-10-26 | 2013-05-01 | 麦德托尼克公司 | Continuous subcutaneous administration of interferon-alpha to hepatitis b infected patients |
| CN106442770A (en) * | 2016-09-05 | 2017-02-22 | 南京医科大学 | Idiopathic male infertility-related seminal plasma metabolic small molecular marker as well as detection method and application thereof |
| CN106568852A (en) * | 2016-10-10 | 2017-04-19 | 南京医科大学 | Idiopathic male infertility related steroid hormone marker in serum, detection method and application thereof |
| CN107846903A (en) * | 2015-05-28 | 2018-03-27 | 细胞结构公司 | The stem cell in placenta source and its for recovering regeneration engine, correcting protein group defect and extending the purposes in aging individuals life-span |
| CN110114043A (en) * | 2016-12-27 | 2019-08-09 | 舒沃德哈娜·萨林·洛普 | For reducing non-the trembling property low temperature heat production method of adipose tissue |
| CN111432654A (en) * | 2017-12-04 | 2020-07-17 | 雾凇科技有限公司 | Method for producing protein phospholipid complex from crustacean capture |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2014193145A (en) * | 2013-03-01 | 2014-10-09 | Naoki Yamashita | Evaluation method of human blastocyst by norepinephrine content in blastocyst culture solution |
| US20170007644A1 (en) * | 2013-12-06 | 2017-01-12 | Fuwan Pty Ltd. | A method of treating neoplasia |
| CN106556655A (en) * | 2016-10-10 | 2017-04-05 | 南京医科大学 | Medium-chain fatty acid mark related to idiopathic male infertility in serum and its detection method and application |
| EP3681504B1 (en) * | 2017-09-14 | 2023-08-23 | Icagen, Inc. | Methods of detection using x-ray fluorescence |
| CN112129876B (en) * | 2020-09-24 | 2021-07-20 | 南京医科大学 | Seminal plasma organic acid marker related to idiopathic male sterility and detection method and application thereof |
-
2020
- 2020-09-24 CN CN202011019767.0A patent/CN112129877B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103071147A (en) * | 2011-10-26 | 2013-05-01 | 麦德托尼克公司 | Continuous subcutaneous administration of interferon-alpha to hepatitis b infected patients |
| CN107846903A (en) * | 2015-05-28 | 2018-03-27 | 细胞结构公司 | The stem cell in placenta source and its for recovering regeneration engine, correcting protein group defect and extending the purposes in aging individuals life-span |
| CN106442770A (en) * | 2016-09-05 | 2017-02-22 | 南京医科大学 | Idiopathic male infertility-related seminal plasma metabolic small molecular marker as well as detection method and application thereof |
| CN106568852A (en) * | 2016-10-10 | 2017-04-19 | 南京医科大学 | Idiopathic male infertility related steroid hormone marker in serum, detection method and application thereof |
| CN110114043A (en) * | 2016-12-27 | 2019-08-09 | 舒沃德哈娜·萨林·洛普 | For reducing non-the trembling property low temperature heat production method of adipose tissue |
| CN111432654A (en) * | 2017-12-04 | 2020-07-17 | 雾凇科技有限公司 | Method for producing protein phospholipid complex from crustacean capture |
Non-Patent Citations (3)
| Title |
|---|
| Serum Levels of Neopterin and Mannose Binding Lectin in Early and Late Onset Neonatal Sepsis;Hassanin Noor Hadi AL-Mussawi et al;《International Journal of Medical Science and Current Research》;20200831;第382-388页 * |
| 基于代谢组学技术的特发性男性不育及精液参数的尿液代谢特征研宄;孙娴 等;《2017年生殖毒理药理学理论与技术及科技产品研发学术交流会论文集》;20171231;第83页 * |
| 快速发展的亚细胞蛋白质组学;郭立海 等;《中国生物化学与分子生物学报》;20050430;第143-150页 * |
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