Instructions for conducting spermogram tests
Instructions for conducting spermogram tests
Purpose
The purpose of this guideline is to describe spermogram testing.
2 - Scope of application
This guideline is used in the microbiology unit to perform spermogram tests on Semen samples.
3 - Responsibility for implementation
Responsible for monitoring the proper implementation of this instruction is the responsibility of quality control and supervisor.
The person in charge of the microbiology unit is responsible for the proper implementation of this instruction.
4 - Definitions
Semen analysis is one of the most important aspects of fertility follow-up because about 40% of infertility is caused by men, and the above test is a simple test. But a simple sperm analysis, especially if it indicates infertility, is not a definite result because the sperm count changes from one day to the next.
آزمایش Title of the experiment
Spermogram is performed.
. Principles of testing
Let's start with a brief overview of the testicular structure.
1- The organs are oval and wide, the number of which is two and their length is about 5 cm.
2. The outer layer of the testicle is made of a dense, white fibrous tissue called the tunica albuginea.
3. This layer enters the back of the testicle and creates an upright and unfinished wall that divides itself into several walls and divides the testicular tissue into 250 to 400 lobules.
4. Each of these lobules contains one to three narrow spiral tubes called seminiferous tubules.
5. Inside these tubes, other cells called sertoli or sertoli cells are found in the testicles that are responsible for protecting and nourishing sperm.
Ley 6- Leidig or Leydig interstitial cells are scattered between the tubes and are responsible for the secretion of male hormones, especially testosterone.
. 7- Testosterone secretion in Lidig cells is under LH control.
8- Testosterone together with FSH controls the production of spermatogenesis in seminiferous tubules.
9- After joining each other, the sperm-forming tubes form larger larger tubes called straight tubules, which in turn, after interference, form a network called Rete Testis, which the sperm form after being made in the tubes. They are poured into it.
. 10- About 20 small and spiral tubes called Efferent Ductule, which join the epididymis after passing through the white membrane, pass through the testicular network.
11- The epididymis of each testicle is a spiral tube that forms the first part of the testicular organ. The vas deferens is an epididymis that secretes ejaculatory ducts after attachment to the ejaculatory duct, through which the spermatozoa and secretions of the esophageal sac travel to the urethra.
The process of spermatogenesis is as follows:
1. First, the primary cells in this pathway, called spermatogonia, form the primary spermatocytes during a series of mitotic divisions.
2. Each primary spermatocyte performs the first stage of meiotic division and forms secondary spermatocytes into two cells (meiosis 1).
3. Each secondary spermatocyte is converted into two spermatid cells during the second stage of meiotic division. (Meuse 2)
Up to this point, spermatogenesis has been performed in the testes.
4- In the transfer pathway and in the epididymis, the final stage of spermatid maturation has taken place and it contains tail, head and neck.
In general, four sperm are produced from each primary spermatocyte.
Finally, sperm (male gametes or spermatozoa) combine with oocytes (female gametes or oocyte) (during the process of fertilization or conception) to form eggs or zygotes.
Semen is formed from the secretion of the following parts:
1- Prostate secretions, which make up 25-30% of the volume of semen.
It is a cone-shaped gland the size of a fig tree that is located under the bladder. Its secretion is controlled by dihydrotestosterone and produces a white, clear, thin, and alkaline fluid that contains proteolytic enzymes (fibrinolysin), citric acid, phosphatic acid, PSA, and lipids.
2- Testicular secretions, which make up 2-5% of the volume of semen.
3- Seminal secretions of vesicles (or seminal vesicles) which make up 65-75% of the volume of semen.
There are two small glands located on the back of the bladder and on either side of the prostate. Which inhibits the female immune system against foreign antigens in semen, etc.) to feed sperm. Its secretions flow through the excretory duct to the end of the vas deferens and form ejaculatory ducts.
4 - secretions of the bulbovarter glands and coupefer glands, which make up less than 1% of semen volume.
The Kupfer glands are two small pea-sized glands that lie below the prostate and on either side of the forehead. Their secretions are poured into the urethra and act as a lubricant. The mucus secreted from this area increases sperm motility in the vaginal and cervix environments and prevents sperm from being expelled from the vagina.
In spermogram, two characteristics of semen are considered:
1- Macroscopic properties
2- Microscopic properties
The macroscopic properties of semen include color, transparency, concentration of hydrogen or pH ions, volume, duration of proximity, fluidity, and viscosity.
The microscopic characteristics of semen include a study of motility, sperm count, survival rate, sperm morphology, white and red blood cell counts, and a qualitative study of semen fructose levels.
5- Sample conditions:
Minimum sample size:
All samples should be poured into a special container after masturbation.
30 micro liters (10 landa for chamber sperm, 10 landa for pH determination and 10 landa for morphological lam)
Sample type:
Freshly collected semen.
Sampling conditions:
It should be 3-5 days after the last proximity, do not use condoms, gels and soaps during sampling. The sample should be delivered to the relevant authority immediately after sampling.
Storage conditions:
The sample should be stored in a 37 سان C incubator for 20 minutes before testing.
Conditions for rejecting or accepting a sample:
If the sample is taken at home and placed in the air below 37 بیشتر C for more than 30-45 minutes, sampling should be repeated 3 days later.
6- Materials, supplies and equipment
1- Spermogram incubator
2- Microscope
3- Colors for counting, being alive and fructose
4- Sampler 10 microliters
5- pH paper
6- Lam
7- Test method:
Most of the referrals for this fluid are for sterile examination, after vasectomy, microbial culture, and confirmation or denial of semen in forensic medicine.
1- Provide written guidance and inform the patient that the most appropriate way to prepare a sample is masturbation, preferably in the laboratory.
2- Pre-test refusal period in some references is 2 to 5 days and in some, the period is equal to the usual interval between two intimacy.
3 - If the patient prefers to sample at home, it is important to deliver it quickly (up to 20 minutes) and at a temperature of 37 samples.
4 - The use of condoms or viscous substances, etc. and proximity may interfere with the results.
5- Collect all the samples of the masturbation once in the sterile Urine bottle and deliver it to the laboratory.
Duration of abstinence:
This means that each person has avoided intimacy for 2-3 days during sampling.
If the amount is less than 2 days, the color is yellowish but has no effect on motility, but if it is more than 5 days, the number of sperm increases but most of them are dead. After sampling with the above conditions, before any work, we put the sample in the incubator to get out of the clot state. This stage is called liquefaction. During this time, we check the sample every 10 minutes and rotate it. If it moves like water in a container, we can test it. let's do.
During this time, the incubation of fibrinolytic enzymes and PSA itself as a fibrinolytic secretion from the prostate can cause the clot to disappear around the sperm, so a disturbance in liquefaction means a disturbance in the enzyme prostate.
Note: Why are the Semnan fluids clotted when they leave the body?
The pH of the sperm is alkaline (6-6.5) and the pH of the vaginal acid (4.8-3.7). This clot causes the alkaline sperm to be protected against the acidic pH of the vagina and gradually reaches a neutral pH. Otherwise, the sperm die at the beginning of entering the vaginal environment. . The coagulation factor is responsible for the coagulation factors present in the vesicle gland gland.
After incubation, two parameters are checked:
1- Microscopic parameters 2 -Macroscopic
Macroscopic parameters:
- Volume: should be between 1.5 to 5 ml.
Decreased volume: The retention may be less than two days or the glandular discharge may be less. Taking a number of medications, including anticancer drugs (methotrexate, vincristine, hydroxyurea, etc.), cimetidine, estrogens, reduces the volume of semen.
Increased volume: The duration of withdrawal is more than 5 days or testosterone levels are high, as a result of elevated LH, FSH and prolactin, glandular activity increases and volume increases.
pH: should be between 7.2-8.8, above 9 indicates that the prostate secretion is high and the acidic pH is less than 7, ie the urethra and genitals have merged, causing the urine to mix with the semen, causing the sperm to die.
Consistency: Checked with pipette 5.
-Normal: Drop by drop.
Mild viscide: stretches between 2-5 cm.
Modrate viscide: Between 10-5 cm.
Severly viscide: stretch more than 10 cm.
The higher the consistency of semen, the lower the sperm motility in the vagina and cervix.
Color: It should be milky and semi-transparent.
When the blood is in the semen, it turns pink or red and is called hematospermia. If it remains stable, it can be a sign of an important medical problem, and the patient must be evaluated by a urologist. This can be caused by inflammation or infection, infection, obstruction or injury to the testicles, urethra, epididymis or prostate.
Agglutination: Formed when there is an anti-sperm antibody.
Elegotination is usually caused by a blow to the testicle. These people do IUI for pregnancy.
In the next step, 3 slides should be prepared.
1- Wet fast mount:
10 Landa puts sperm on the slide and puts 22-22 lumens on it. In this case, the space between the slide and the slide is 20-22 microns, so the sperm have enough space to move.
Before seeing the slide, set it to 37 degrees for 1 minute (to perform sample stability), look at the Prowarming stage and with the lens 40. In this slide, the percentage of sperm motility and WBC and Immature Germ Cell and agglutination are checked. It is recommended to provide two slides and the difference in parameters in the two slides should not exceed 5%.
Motile: The approximate percentage of live sperm that includes 3 categories:
Rapid progressive: Moves 5 sperm heads forward in 1 second.
Slow progressive: In 1 second, it progresses to 4-1 sperm heads.
Non progressive: They move in place of their head or eventually move one head of sperm forward.
Immotile: Approximate percentage of dead sperm.
Immature Germ Cell: The same as spermatids.
People with Azoospermia should be careful with Immature Germ Cells.
If the Immature Germ Cell is not present, we will report that the testicular tissue has a problem.
If Immature Germ Cell is present, vasodilatory secretions are problematic and can be treated with ICSI.
2- Vitality: 10 Landa sperm + 10 Landa Eosin 5% g mixed, we take 10 Landa and with 100 lenses we count about 100 sperm and determine the percentage of living things.
Sperm that are alive have a healthy membrane, so the color does not penetrate them and they remain colorless, but sperm that have died penetrate the color of their damaged membrane and turn pink.
Vitality is usually 1 to 2 percent more valuable than Motile.
3- Morphological lam: We fix 10 landasperms with fixator (ethanol) and after hot staining with lenses, we examine 100 morphologies of sperm in 100 sperm.
Recognizable spermatozoa with a single tail are considered in the morphological differential count.
Classification of morphological defects:
1- Head Defects:
Includes large, small head, Tapered, Pyriform, round, shapeless heads, vacuoled heads, double heads and heads with small acrosomal area
2- Mid-piece Defects:
Including a crooked neck, asymmetrical insertion, a thick or irregular midsection, an unusual narrow midline (such as a lack of mitochondrial membrane)
3-- Tail Defects:
Includes short, multi-tailed, hairpin, broken, crooked tails, irregularly broad tails, coiled tails, or any combination thereof.
4- Cytoplasmic droplets: Cytoplasmic droplets are usually concentrated in the middle part.
Total motile functional sperm: Sperm that have both motility and good morphology.
Total number × normal morphology% × (slow + Rapid) motility% x Volume = TMFS
TMFS must be greater than 40.
Measurement of fructose: Fructose secreted in the vesicle seminal as a main substance for the survival of sperm as well as a suitable environment for the movement and swimming of sperm in the vaginal environment.
Fructose is essential for sperm motility. If sperm motility is more than 20%, it must have fructose, but if it is below 20% or Azoospermia, it must be checked.
1.8 Fructose Solution + 200 Landa Pour the semen into a long tube and put it on the flame to boil. If it turns orange, it is Sufficient. If it does not change color, it is Deficient.
Zinc: Examines the ability of the prostate to secrete zinc. Its normal level is 135 ± 40 µg / ml. Zinc stabilizes DNA in sperm chromatin. Reducing zinc reduces infertility by increasing fragility. Decreased zinc has the opposite effect on spermatogenesis.
Centrifuge the cement liquid and measure the surface liquid.
Sperm count: done with neobar lam. After diluting the sperm, we count them in the houses related to WBC. Due to the dilution, it is multiplied by a factor such as:
1.9 Counting solution +100 Landa semen = coefficient of dilution 1/20
To obtain the required dilution, we can prepare a wet mount sample and estimate the number of sperm in each microscopic X 400 (hpf) field.
15> Spermatozoa dilute 1 to 5 ((1 semen + 4 diluent
40-15 Spermatozoa dilute 1 to 10 ((1 semen + 9 diluent
200- 40 spermatozoa dilute 1 to 20 ((1 semen + 19 diluent
200
Fructose solution:
Resorcinol - 50 mg
HCL - Concentrated 33 ml
Up to 100ml D.W.
Eosin G5%
Eosin G 0.5 gr
NaCl 0.9 gr
Up to 100ml D.W.
Sperm counting solution:
NaHCO3 5gr
Formalin35% 1ml
Gentian Violet 0.1 gr in 5 ml of distilled water and centrifuge
4- Take 3 ml of it and bring its volume to 100 ml with distilled water.
Up to 100 ml D.W.
calibration
does not have
1- If the number of spermatozoa in each field of view is significantly different when counting spermatozoa, it is a sign that the sample is not uniform. In such cases, the semen sample is thoroughly mixed and the count is repeated.
2- In examining the motility of sperms, two slides should be prepared and the difference in mobility between the two slides is acceptable up to 5%.
9- How to calculate the results in quantitative methods:
TMFS = Size ×% Motility (Slow + Rapid) × Normal Morphology% × Total number
10- Reference Interval:
1- Volume of 1.5 ml or more
2- pH 2/7 or more
3- 106 x 15 spermatozoa sperm per ml or more
4- 32% mobility with grade a and b
15% mobile with grade a
5- Morphology more than 4%
6- Being 75% or more alive
7. WBC 1-0 in each largest dry microscopic field (hpf)
8- Immature Germ Cells 5- 0
11- Reportable Range:
From 0 to 100%
12- Warning range or critical values:
Move less than 20% - sperm count less than 2 million - pH less than 6.5
Safety considerations:
Use gloves. Use a pipette and pointer to remove the sample.
13- Analytical features (test characteristics and efficiency):
People who have been observed to have agglutination in their semen are identified by this test and the report is reported to a specialist.
14- Limitations of testing (potential sources of variability):
1. If the sample is out of the incubator 30 minutes after sampling, it will change the normal condition of the sample.
2. Pregnancy should be avoided for 5-2 days. If it is less than 2 days, the volume decreases and the semen color turns yellow, and if it is more than 5 days, the percentage of dead sperm increases.
15. The next steps in dealing with abnormal results:
Inform the supervisor and the technical manager of the laboratory
16- Interfering factors:
Use gels, condoms and soaps
Drug Interactions:
Drugs that may lower semen levels include anti-neoplasm agents (such as Mustard nitrogen, procarbazine, vincristine, methotrexate), cimetidine, estrogens, and methyl testosterone.
17- How to interpret the results:
1. All specimens in which no spermatozoa are seen under a microscope should be centrifuged at 3000 g for 15 minutes, and if no spermatozoa are observed after a full sediment search, azoospermia should be reported.
2. A large number of leukocytes (leukocyte spermia) may be associated with infection and poor sperm quality.
3. An increase in small round cells is a sign of a defect in the development of acrosomes, called globozoospermia.
4- Lack of semen is called Aspermia
Normozoospermia: Natural ejaculatory fluid as defined by reference values.
6- Oligozoospermia: sperm count is less than the reference value
7- Astenozoospermia: mobility is less than the reference value
8- Teratozoospermia: Morphology is less than the reference value.
Increases:
If the prevention time is more than 5 days, it will increase the number of dead sperm.
Reduction cases:
Decreased pH occurs due to azoospermia or sperm death.
18- Other cases:
does not have
19- Related documents
does not have
20 - References and appendices
1. Faramarz Ghanbari
2. sperm tests based on WHO
3. Pagana 2011
