Immunological testing for infertility

Immunological examinations of infertility include male anti-sperm autoimmune infertility, female anti-sperm allogeneic infertility, and female anti-Zona pellucida infertility. Control diet: 1, most of the test items require blood sampling on an empty stomach in the morning, should be fasted coffee, strong tea, high sugar and cola drinks; 2, liver function, blood lipids, blood coagulation and other items require fasting for 12-16 hours, and Dinner one day in advance should avoid drinking alcohol, prohibit high-fat, high-protein diet; 3, diet has a significant impact on blood lipid test, at least within 3 days before the blood draw to pay attention to maintain a normal diet. Basic Information Specialist classification: eugenics and superiority examination classification: body fluid examination Applicable gender: whether men and women apply fasting: fasting Analysis results: Below normal: Normal value: no Above normal: negative: The result of the negative test should be normal. Positive: A positive test indicates a disease that may be infertile. Tips: Stop sexual intercourse for 4 to 7 days. Testosterone propionate, testosterone phenylacetate, and nantrolone phenylpropionate could not be used 1 week before the test. Normal value The results of various immunological tests were normal and negative. Clinical significance Abnormal results Multiple immunological tests indicate infertility. The male infertility and female infertility patients need to be examined. Positive results may be diseases: infertility, male infertility, female pseudohermaphroditism, male genital malformation precautions Inappropriate people: not yet known. Taboo before inspection: First, when collecting sperm: 1. Before leaving semen, the patient should stop sexual intercourse for 4 to 7 days. Testosterone propionate, testosterone phenylacetate, and nantrolone phenylpropionate could not be used 1 week before the test. Drinking should be stopped within 1 month before the test. This must be done. 2, when taking semen, soft soap or paraffin oil can be used for penile massage, the specimens are collected in sterile test tubes; condoms can be used (washed clean, no spermicide drugs) or semen can be collected by sexual intercourse, but this way The amount collected is often small. 3. If the semen is not obtained by the above method, the seminal vesicle and the end of the vas deferens can be massaged through the rectum, and the urine can be collected to check whether there is sperm in the sediment. 4. Do not expose semen to overheated and cold environments. Hand it to the doctor for reference, no more than 30 minutes. Keep warm in cold weather, and keep it in your underwear pocket when you send it. 5. If bacterial culture is to be carried out, the urethral opening should be rinsed and disinfected, and the semen should be collected in a sterile test tube. Second, before the serum collection requirements: (1) Controlling diet 1. Most inspection items require blood sampling on an empty stomach in the morning. Coffee, tea, high sugar and cola beverages should be fasted. 2, liver function, blood lipids, blood coagulation and other items require fasting for 12-16 hours, and one day in advance of dinner should avoid drinking, prohibiting high-fat, high-protein diet. 3, diet has a significant impact on blood lipid testing, at least within 3 days before the blood draw to pay attention to maintain a normal diet. (B) to avoid drug effects The effects of drugs on biochemical tests and bacterial culture are complex. Therefore, if possible, the subject should stop taking all medications 1-2 days before the test. If medication is necessary for treatment, the effects of the drug should be considered when analyzing the results of the test. (3) Avoiding the impact of exercise 1, strenuous exercise can make many blood components change, even for more than 24 hours, so after vigorous exercise should not immediately take blood and walk quickly, at least 10-15 minutes after rest, then take blood. 2, should be in a calm state to draw blood, to avoid emotional excitement. Requirements for inspection: 1. In order to ensure that you can accurately understand your physical examination results after the medical examination, please fill in the medical examination form and each test sheet carefully before the medical examination. The writing should be clear, especially the name. 2, after taking serum, you should sit still for 15-20 minutes. Inspection process -, anti-sperm immune infertility check Antisperm immunity includes anti-sperm humoral immunity (anti-sperm antibodies) and anti-sperm cell immunity. (1) Detection of anti-sperm antibodies 1. Post-intercourse test: Take cervical mucus within 6~24h after sexual intercourse, and observe the number of sperm counted under high power microscope. In each high power field, there are ≥ 21 extremely lively sperm-specific factors. If the serum titer is ≥1:32, it can be considered to contain sperm antibodies. Others have capillary agglutination and plate agglutination, which are less used. 2. Complement-dependent cytotoxicity test and sperm-braking test: Sperm antibody binds to sperm antigen, activates the complement system, and causes damage to cell membrane permeability and integrity. If it is a brake antibody, it loses its activity and brakes. A value of ≥ 2 is positive; if it is a cytotoxic antibody, it is positively stained with certain dyes, requiring a sperm motility >70%. The micro method only needs a few microliters of sample. For samples with low sperm activity rate, the live sperm can be extracted by the upstream method, and the survival rate can reach over 90%. The practice can only detect antibodies that act on the sperm tail. Strong specificity, poor sensitivity, and high false negative rate. 3. Passive hemagglutination method: The treated red blood cells are sensitized with the sperm extract and the seminal plasma, and the sensitized red blood cells are reacted with the diluted serum to be tested on the micro-reaction plate, and the red blood cell agglutination is positive. This method is not affected by sperm viability, counting, microbial or tissue fragments in spermatozoa. The researchers believe that this method is specific and sensitive, but there are few reports, and its reliability and practicability have yet to be confirmed. 4. Enzyme-linked immunosorbent assay (ELISA) and biotin-avidin enzyme-linked immunosorbent assay (BA-ELISA): application of intact sperm or sperm membrane antigen-coated solid phase carrier, sperm antibody in the sample to be tested and cultured It can then bind to the surface of the antigen. Subsequently, the second antibody of the enzyme label can also bind to the surface, and then the enzyme substrate is added, and the result is judged according to the color development of the substrate. Can be observed with the naked eye, can also be measured with a microplate reader, the latter objective and accurate, generally more than a group of negative serum optical density plus 2 times the standard deviation is positive. Since the intact sperm is easily non-specifically adsorbed, if it is coated with soluble sperm membrane antigen, the method is sensitive, specific, objective, and quantitative, and can determine the type of antibody and is easy to operate. BA-ELISA is a new generation of quantitative assay developed on the basis of conventional ELISA, which has more sensitivity and specificity than conventional methods. 5. Immunobath test (1mmunbeadTest, IBT): Polyacrylamide microspheres coated with anti-human immunoglobulin antibody can bind to the sperm surface bound with sperm antibody, and immunobeads can be seen with sperm under phase contrast microscope or electron microscope. Run, if the proportion of sperm combined with one or more immunobeads is ≥lo% positive. This method determines the site, type, and proportion of bound sperm, and can detect antibodies bound to the surface of live sperm. Its sensitivity, specificity and reproducibility are good, the operation is slightly cumbersome, and the samples required for testing are few. It can be used for the detection of trace samples such as cervical mucus and fallopian tube fluid. 6. Radiolabeled immunoglobulin or protein A method: Isotope-labeled anti-human immunoglobulin or grape seedling A protein is bound to sperm antibodies bound to the surface of the sperm, and the radioactivity of the surface is measured by an x ​​counter. If live sperm is used as an antigen, its specificity and sensitivity are high, and the type of antibody can be determined. However, due to the different sources of live sperm in each experiment, the results are less reproducible; and the equipment is expensive. The operator is susceptible to radioactive damage, so this method is less reported. 7. Indirect immunofluorescence test: After the surface is bound to the antigen, it can bind to the second antibody labeled with fluorescein, and the proportion of positive sperm showing fluorescence is observed under a fluorescence microscope. Because the test requires methanol fixation to cause damage to the membrane, the internal antigen is exposed, and the antibody against the internal antigen is ubiquitous in the redundant people, so the false positive is too high and has no clinical practical value. In recent years, liquid phase culture has not been fixed during the experiment, and the use value of this method has been redefined. 8. Fine egg interaction direct inspection (1) Sperm-transparent band binding or penetrating test: The human egg obtained from laparoscopy is better than the spermatozoa after incubation under certain conditions, and whether the sperm can pass through the zona pellucida into the space around the egg cell, if the sperm cannot be worn Pass the zona pellucida and consider the presence of a brake antibody. This test is also interfered by other factors. (2) sperm-de-transparent hamster egg penetration test (SPA): the optimized extracted live sperm is mixed with the hamster-free eggs and observed under a phase contrast microscope. If the egg contains a swollen sperm head (with tail) penetrate. The SPA can be used to determine: 1 the fertilization ability of sperm with seminal plasma antibodies; 2 the importance of various antibodies in preventing pregnancy. This test can also be affected by a variety of factors. (two) detection of anti-sperm cell immunity There are few studies on cell-mediated anti-sperm immunity, and each conclusion is different. Animal experiments and human tests have shown that anti-sense cellular immunity may be one of the causes of infertility. The assays for detecting cell-mediated anti-sense immune responses include lymphocyte transformation assay, leukocyte adhesion inhibition assay, leukocyte or macrophage migration inhibition assay, skin test, and peripheral blood leukocyte procoagulant activity assay. The antigens used are different, such as freshly washed sperm, frozen-thawed sperm, semi-purified sperm, and the like. At present, because the antigens used are not uniform, the detection methods are different, the number of research cases is small, the specificity of each method is difficult to evaluate, and the results are difficult to compare. Second, the detection of anti-transparent immune infertility The source of human zona pellucida is limited and difficult to obtain, but the human zona pellucida and the porcine zona pellucida have cross-antigenicity. Therefore, in the actual practice, the porcine zona pellucida is used instead of the human zona pellucida to detect the zona pellucida antibody in human serum. Since the presence of heterologous lectin (mainly IgM) in some normal human serum interferes with the experimental results, fresh porcine red blood cells are required to absorb the test serum before the test. 1. Scotch band precipitation reaction: After the surface of the zona pellucida binds to the antibody, the refractive index changes under a light microscope or a dark field microscope. This method is mostly used to identify serum, has low sensitivity and is difficult to use in clinical practice. 2. Radioimmunoassay: After radiolabeled porcine zona pellucida antigen and serum to be tested, the antigen-antibody complex is isolated and its radioactivity is measured. This method can be quantitative, specific and sensitive, but due to radioactive damage, and reports are not many, this method is still difficult to affirm. 3. Indirect immunofluorescence assay: After the human anti-Zona pellucida antibody binds to the surface of the porcine egg, the anti-human immunoglobulin antibody labeled with fluorescein binds to the surface of the zona pellucida and exhibits obvious peri-fluorescence under a fluorescence microscope. The reliability of this law depends on other objective methods. 4. ELISA and BALISA method: the solid phase carrier is coated with the porcine zona pellucida antigen, and the serum, the second antibody and the substrate to be tested are added, and the washing is carried out step by step, and finally the result is judged according to the color change of the substrate. This method requires less sample size, requires no special equipment, is easy to operate, can quantitatively determine antibodies and determine antibody type, and has good specificity and sensitivity. The BA-ELISA has the advantages of a conventional ELISA, and its sensitivity is greatly improved. 5. Passive hemagglutination method The purified porcine zona pellucida antigen is used to coat the red blood cells of other species as the antigen target, and in the presence of the zona pellucida antibody, the sensitized red blood cells agglutinate. There are few reports on this method, and it is difficult to evaluate its sensitivity and specificity. 6. Sperm-transparent band binding or penetration test: This method can be positive if there is a zona pellucida antibody or an anti-sperm antibody. Because the test is greatly influenced by the culture environment factors, the research results of different laboratories are difficult to compare. This method can be applied in conjunction with other methods to complement each other to ensure the reliability of the results. Not suitable for the crowd The examination is less invasive and generally has no contraindications. Adverse reactions and risks This test is less invasive and generally does not cause serious complications or other hazards.

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