Semen analysis is an important method for assessing male fertility and is also an experimental basis for the diagnosis and observation of the efficacy of male diseases. However, the parameters of semen analysis are not specific, and it is not able to determine the fertilization ability of the few spermatozoa that reach the fertilization position, so a comprehensive assessment combined with clinical and other sperm function test indicators is needed to properly evaluate male fertility.
The results of semen analysis are susceptible to bias due to many factors such as frequency of ejaculation, temperature, laboratory conditions, technical proficiency of the examiner, and subjective judgment, so semen collection and analysis must be performed in strict accordance with appropriate standardized procedures in order to provide the necessary information about the clinical status of the subject.
At the initial consultation of an infertile couple, the male partner should undergo at least two semen analyses according to standard procedures, and a third analysis should be performed if there are significant differences between the two analyses.
Semen specimens may contain pathogenic bacteria and viruses (e.g., HIV, hepatitis virus, herpes simplex virus, etc.) and should therefore be considered biohazardous. Laboratory technicians should take precautions and use disposable gloves and various utensils.
Used utensils should be disinfected. Semen culture, for bioassay, intrauterine fertilization or in vitro fertilization, must be handled strictly with sterile materials and aseptic operations.
I. Collection and delivery of semen specimens
The standardization of semen collection is a prerequisite for good semen analysis, so it is important to inform the subject in detail about the methods and precautions for semen collection and delivery before semen collection.
1. Abstinence should be at least 48 hours, but not more than 7 days, before specimen collection. To minimize fluctuations in semen analysis results, the number of days of abstinence should be as constant as possible. Each semen analysis report should state: the patient’s name, the duration of abstinence, the date and time of specimen collection, whether the specimen collection is complete, and the time interval from collection to analysis of the specimen.
2. The initial examiner should have two semen analyses, and the interval between two semen collections should be greater than 7 days, but not more than 3 weeks. If there is a significant difference in the results between the two times, the specimen should be taken again for a third analysis.
3. The collection of specimens should preferably be done separately in a sperm collection room near the laboratory. Otherwise, it should be sent to the laboratory within 1 hour after collection.
4. It is best to take semen by masturbation and collect semen in a wide-mouthed glass or plastic container that has no toxic effect on sperm. The temperature should be kept at 20~40℃ to avoid reducing sperm viability. If microbiological examination is to be done, the patient should first urinate and wash his hands and penis and collect in a sterile container.
5. If masturbation is difficult, special condoms can be used for semen collection. Because latex condoms used daily can affect the survival of sperm, they cannot be used for semen collection. The interruption of intercourse method should also not be used for semen collection because the initial portion of the ejaculate may be lost and this is usually the portion with the highest sperm density. Moreover, the specimen will be contaminated by bacteria and microorganisms in the female reproductive tract; at the same time, acidic vaginal secretions will also have a negative impact on sperm viability.
6, Semen collection must be complete, and incomplete semen should not be analyzed.
7. The temperature of the specimen should be kept above 20°C, but not more than 40°C, during transportation to the laboratory.
8. The container for semen collection must be marked with the name (and/or ID number) of the subject and the date and time of specimen collection.
II. Visual observation of semen
1. Liquefaction of semen
At room temperature, semen will coagulate immediately after ejaculation and then enter the liquefaction process, which is mostly completed within 15 minutes. If semen does not liquefy in more than 60 minutes, it should be considered abnormal. Therefore, it is recommended to observe liquefaction at 20, 30 and 60 minutes after semen extraction and record the time required for liquefaction and whether it is complete. Normal semen can contain jelly-like particles that do not liquefy; this phenomenon is not clinically significant.
Prolonged liquefaction time, incomplete liquefaction or non-liquefaction of semen is usually associated with hyposecretion of the prostate gland and deficiency of proteolytic enzymes.
Semen that does not liquefy requires separate treatment, either mechanical mixing or pineapple protease digestion (pineapple protease 1 g/L).
The addition of equal amounts of culture fluid and repeated blowing with a sparger can also liquefy some specimens. It should be noted that all these treatments may affect the results of biochemical, sperm viability and sperm morphology measurements of seminal plasma.
2. Appearance of semen
After liquefaction, the appearance of semen specimens should first be observed visually at room temperature. Normal semen has a uniform, off-white texture, and semen may be slightly yellow after a long period of abstinence. If the sperm density is very low or absent, the semen may appear thin. If there are red blood cells, semen may appear reddish-brown. If there is jaundice or taking certain vitamins, semen may appear yellow.
3. Semen volume
The amount of semen can be measured with a graduated measuring cylinder with a conical bottom. The amount of semen in a normal man is not less than 2 ml per ejaculation, and the small amount of semen is often related to the partial loss of semen when taking semen. If the semen is not lost, the amount is small and does not coagulate, there is no sperm, and the vas deferens is absent on physical examination, congenital underdevelopment of the seminal gland should be considered.
4. Semen viscosity
To assess the viscosity, a 5 ml pipette with a wide hole can be used to gently inhale semen. Normal semen forms discontinuous droplets at the mouth of the tube due to gravity, and droplets will form >2 cm of stretches when the viscosity is abnormal.
Another way to detect viscosity is to insert a glass rod into the semen, lift the rod, and observe the length of the strains, which are considered abnormal if they exceed 2 cm. Highly viscous semen can hinder the movement of sperm.
5.PH value
PH value should be measured within 1 hour after ejaculation. Put a drop of semen on the PH test paper (PH test paper range is 6.1 to 10.0 or 6.4 to 8.0) and after 30 seconds, the color of the wetted area should be uniform and the PH value should be read out in comparison with the standard tape. Regardless of which PH test paper is used, its accuracy should be verified with known standards.
Third, microscopic examination of semen
Microscopic examination of semen includes the determination of sperm density, sperm viability, sperm survival, sperm agglutination, non-sperm cell components and morphological analysis of spermatozoa.
It is recommended to use a phase contrast microscope for semen microscopy. An ordinary light microscope can also be used, but the concentrator should be adjusted.
1. Preparation of specimens for microscopic examination
The volume of semen sampling and the size of the coverslip should be standardized so that semen is analyzed at a thickness of approximately 20 μm. Sperm density should be roughly determined before precise detection of sperm density. This is done by.
Using a spiker, place a 10 μl drop of semen on a clean slide and cover with a 22L x 22L coverslip. The coverslip is weighted so that the specimen is unfolded for optimal observation, taking care to avoid creating air bubbles between the coverslip and the slide.
Let it stand for 1 minute to stabilize. The test can be performed at room temperature between 20 and 24°C. Since temperature affects sperm viability grading, laboratory temperature must be standardized. Sperm viability and motility are highly temperature dependent and it is recommended that the assessment of viability be performed preferably using an insulated mirror table with a constant table temperature of 37°C.
The prepared specimen should be observed under a 100x microscope for the formation of mucus filaments, sperm agglutination and uniformity of specimen unfolding on the slide, and then examined under a 400x microscope.
2.Primary examination of sperm density
Before the precise counting of sperm density, a preliminary examination of sperm density should be performed, and the results of the preliminary examination will be used as the basis for the dilution times of semen. The diameter of the field of view at 400 times magnification is different for different microscopes, and the general diameter ranges from 250 to 400 μm, equivalent to 1 to 2.5 nl, with a thickness of 20 μm of the sample.
The diameter of the field of view can be determined by a microscope scale or a grid in the counting cell. The number of sperm in each field of view is counted and multiplied by 106/ml to give a rough estimate of the sperm density of the semen specimen. This estimate is used to determine the dilution of the sperm density measured with the hematocrit plate: 200 sperm at a 1:50 dilution.
A large variation in the number of sperm per field of view suggests that the specimen is heterogeneous. In this case, the semen specimen should be mixed again, re-sampled and tested. Abnormal semen viscosity, incomplete liquefaction, sperm aggregation in mucus filaments or sperm agglutination can affect semen mixing.
If the sperm count is low (less than 1 to 2 per field of view at 400x), the specimen can be concentrated by centrifugation before measuring sperm density. Take 1 ml of semen (or as much semen as possible) and centrifuge at 600g for 15 minutes. A known volume of seminal plasma is discarded and counted after thorough mixing and sedimentation.
The final density is corrected for the volume of seminal plasma discarded. The specimen can also be evaluated for sperm viability and morphology after centrifugation. If the sperm count is still low after centrifugation and concentration (less than 1 or 2 per field of view), the reported sperm density is centrifuged at 3000g for 15 minutes. Only when all sediment is resuspended and no sperm are found after thorough and systematic examination, can a determination of azoospermia be made at this time.
3. Assessment of sperm motility
Sperm vitality refers to the ability of sperm to move forward. For ease of operation sperm vitality is divided into four levels: a, b, c and d.
Level “a”: fast forward motion (i.e. speed ≥ 25μm/s at 37℃, or speed ≥ 20μm/s at 20℃).
Class “b”: slow or sluggish forward motion.
Class “c”: non-forward motion.