Semen:- Part 1 – Semen analysis, and Semen Counting Procedure
Sample for Semen analysis
- This is preferred if the patient is in the lab to collect the sample.
- Masturbation is preferred, and the entire collected semen should be submitted.
- The accepted volume is 2 to 5 mL.
- Collect the sample when the doctor or the technician should be available to evaluate the motility immediately.
- 2 to 3 days of sexual abstinence is preferred.
- Prolonged abstinence should be discouraged because of the quality of sperm cells’ quality, and especially their motility will decrease.
- The proper container is also important.
- To evaluate the vasectomy, the patient must ejaculate once or twice before the semen specimen collection day. This process will clear the distal portion of the vas deference.
- Samples are unsatisfactory If the sample is obtained at the house by coitus interruptus or masturbation.
- In the house sample, instruct the patient to deliver it within one hour of collection.
- Instruct the patient to avoid cold and heat during transportation. The best is to keep the sample in closed hands (in the first).
Precautions for Semen analysis
- Don’t use condoms, particularly with spermicide.
- The specimen should be maintained at 37 °C during transport if brought from home and examined within 3 hours of collection.
- A sterile container is needed, and the sample should be collected at a room temperature of 37 °C.
- Plastic containers are not recommended.
- Avoid extreme temperatures.
- The analysis should be done immediately when the semen is liquefied.
- Should be examined within 4 hours,
- The sample should be kept at 37 °C.
- Wait till liquefaction is complete for the examination.
- Drugs that may decrease the count are:
- Antineoplastic agents (nitrogen mustard, procarbazine, vincristine, and methotrexate).
- Direct the patient to avoid alcohol intake for several days before the semen collection.
- Instruct the patient to avoid sexual activity for 2 to 3 days before collecting the sample.
- Avoid prolonged abstinence because the quality of the sperm and their motility may be decreased.
Purpose of the test (Indications) for semen analysis
- This is the workup of the infertile couple.
- This is done to evaluate the quality of sperm.
- To confirm the efficacy of vasectomy (postvasectomy).
- Done for the forensic studies.
- To get semen for artificial insemination.
Definition of semen:
- Seminal fluid is the secretion of prostate glands, two seminal vesicles, two Cowper glands, and testes that serve as the vehicle for sperm transport and create the alkaline medium. It also provides nutrition. This serves as the medium which promotes the sperm’s motility and survival.
- Semen (seminal fluid) consists of products of various male secretion products from reproductive organs like the testes, epididymis, seminal vesicle, prostate, bulbourethral, and urethral glands.
- The ejaculated semen is a viscous, yellow-grey fluid that forms a firm gel-like clot immediately after ejaculation.
- This clotted semen liquefies at room temperature (37 °C) within 5 to 60 minutes. If it requires more than one hour, that semen is considered abnormal.
Semen analysis includes:
- Sperm count.
- Evaluation of the normal sperm.
- Evaluation of sperm motility.
- The pH of the semen.
- It occurs in the seminiferous tubules of the testes. These are matured in the epididymis.
- Semen production is dependent upon the function of the testes.
- These are stimulated by testosterone.
- The testes’ testosterone concentration is 100 times more than in the peripheral blood.
- This high concentration of testosterone is needed for spermatogenesis.
- LH stimulates Leydig’s cells to produce testosterone.
- The Semniferous tubules are lined with 46 chromosome germ cells called spermatogonia.
- These cells undergo mitotic division and ultimately give rise to primary spermatocytes.
- Secondary spermatocytes give rise to spermatids by meiotic division, which will differentiate into sperm or spermatozoa containing 23 chromosomes.
Factors for spermatogenesis:
- Semen production depends upon the function of the testis.
- Hypothalamus produces a gonadotropin-releasing hormone (GRH) in response to low testosterone levels.
- GRH stimulates the pituitary gland to produce the follicular stimulating hormone.
- Now the FSH stimulates the Sertoli cell, which is the location of sperm production.
- FSH stimulates the Sertoli cells to secrete activin, which stimulates spermatogenesis.
- Besides, the Sertoli cells secrete inhibin, which inhibits FSH secretion from the pituitary gland.
- LH stimulates the Leydig cells, which produce testosterone. Testosterone then stimulates the seminiferous tubules to produce sperm.
- Spermatogenesis (in the seminiferous tubules) takes approximately 3 months.
- The maturation of the spermatogonia to mature spermatocytes takes approximately 75 days through the epididymis.
- In the epididymis, motility is acquired; it takes another 14 days.
- Spermatocytes develop from the Sertoli cells in the seminiferous tubules. They get mature in the epididymis and are ready for fertilization.
Hormonal role in spermatogenesis:
- In primary gonadal failure:
- FSH is raised.
- LH is raised.
- In secondary gonadal failure:
- FSH is decreased.
- LH is decreased.
Classification of spermatocytes:
- It depends upon the sperm count:
- Aspermia (azoospermia) When there is no sperm.
- Oligospermia when the count is < 20 million/mL.
- Asthenospermia is low sperm motility.
- Necrospermia is the Normal count of the sperm but is non-motile.
- Hemospermia is when there are abundant RBCs in the ejaculate.
Semen analysis includes:
- Volume. Check when the sample is fresh.
- Sperm count. This can be done when the semen is completely liquified.
- Motility. This should be checked when the sample is fresh and repeated when it is liquefied.
- Morphology. This can be better appreciated if the smear is stained.
- The volume is 2 to 6 ml.
- The color is gray to white or opalescent.
- Liquefaction time is normally 10 to 30 minutes at 37 °C.
- The volume normally is 1.5 to 5 mL.
- pH varies between 7.7 to 8. Below, 7.0 usually has prostatic secretion.
- The sperm count is 20 to 250 million/mL.
- Normally a total count of more than 80 million/ml. But now, it is considered as > 20 million /mL as fertile semen.
- Live spermatozoa of more than 50%.
- >65% of the sperm does not take stain, and these are alive.
- Sperm motility >50% at one hour. This is a moderate to strong forward motion.
- Place the semen drop on the prewarmed slide at 37 °C.
- Examine under the 40x and find the motile sperm.
- Evaluate the sperm actively motile and sluggish.
- Normally >70% of sperms are motile after one hour when the sperms are kept at 37 °C.
- The progressive motility score is 3 to 4.
- Sperm morphology should be >30%. Normally normal forms are >70%, and immature forms are <4%.
- Can stain the slide with the Papanicolaou method.
- Count at least 200 sperm under oil emersion.
- Abnormal forms are <30%.
- Note immature sperms as well.
- Also, note RBCs and polys.
- WBC is 4 to 6/HPF.
- Epithelial cells are few.
- RBCs are not found.
- Aggregation may be seen, but very few.
- Crystals are amorphous phosphate.
- Stain the slide with eosine, which will only be taken up by the dead sperm.
- Clumping or agglutination of the sperms should be reported, which indicates antibodies.
- The fructose level is 150 to 600 mg/dL.
- Low or nil fructose indicates an obstruction in the ejaculatory duct.
- The spermatozoa utilize fructose.
- Acid phosphatase = 100 to 300 mg/100 ml.
- Citric acid = >3 mg/ml.
- Zinc = >75 µg/ml.
- Magnesium = >70 µg/ml.
- Inositol = >1 mg/ml.
- Glucosidase = >20 mU/ ejaculate.
- Carnitine = >250 µg/ml.
Source 2 Normal semen
- Volume = 2 to 5 mL
- Liquefaction time = 20 to 30 minutes after the collection.
- pH = 7.12 to 8.0
- Sperm count = 50 to 200 million/mL
- Sperm motility = 60 to 80% actively motile.
- Sperm morphology = 70 to 90% normally shaped.
Normal semen (another source):
|Characteristic features||Values per ejaculate|
|The volume of semen per ejaculate||>2 mL|
|Sperm density||>20 million/mL|
|Sperm count||>40 million/mL|
|Morphology||>30% normal morphology|
|pH||7.2 to 8.0|
|Color (appearance)||Gray-white to yellow|
|Time of liquefaction||Within 40 minutes|
|Fructose level||>1200 µg/mL|
|Acid phosphatase||100 to 300 mg/mL|
|Citric acid||>3 mg/mL|
|Prostaglandins (PGE1 and PGE2)||30 to 200 µg/mL|
|Glucosidase||>20 mU per ejaculate|
- The color is brown to red. Or maybe yellow and turbid.
- Liquefaction time is >60 minutes.
- Suppose the volume is <1.5 ml. This may be <2 ml or >5 ml.
- pH is <7.2.
- total count <20 million /mL.
- live sperm <35 %.
- Motility <50 % after 2 hours.
- The progressive motility score is 0 to 1.
- Morphology >30 % abnormal forms.
- Fructose is decreased.
Difference between normal and abnormal semen:
|Parameter||Normal semen||Pathological semen|
|Liquefaction||Complete in 10 to 30 minutes||Delayed|
|pH||7.2 to 7.8|
|Volume||2 to 6 mL||<1.5 mL|
|Sperm density x106/mL||>20||<10|
|Total sperm count x106/mL/per ejaculate||>80||<20|
|Progressive motility score (after 2 to 4 hours of ejaculate)||3 to 4||0 to 1|
|One hour after ejaculating||≥70%|
|After 3 hours of ejaculating||≥60%|
|After 4 hours of ejaculating||>50%||<35%|
The first method to count Sperms:
Sperm count solution:
- Formalin = 1 mL
- Sodium bicarbonate = 5 grams
- Distal water = 100 mL
- Mix well the above ingredients.
- Add all the above ingredients into 100 mL distal water.
Procedure for counting the sperms:
- Take 0.02 ml of semen
- Take 0.38 mL above the solution (diluting fluid).
- Mix well above semen and diluting solution.
- Now load the Neubauer chamber.
- Count the two large squares.
- Important that move the microscope up and down to count the sperm at different levels.
- Count in the large two square x100,000 = count in a million/mL.
- Take 1 mL of semen, and mix it with sodium bicarbonate-formalin solution to make a total of 20 mL.
- Fill the Neubeaur chamber with diluted semen and wait 3 to 5 minutes. So that spermatozoa get settled.
- Now count two large squares (2 sq mm).
- Calculate the number of spermatozoa in 1 mL of fluid by multiplying the number by 100,000.
The second method to count sperms in the semen:
- Dilute the semen with water or above sodium bicarbonate solution.
- Take a 1:20 dilution of the semen.
- Count the five RBC counting squares (4 at the corner and one at the center).
- Calculations: suppose the sperms are 50 in all these 5 RBC squares.
- 50 x 1,000,000 = 50,000,000/mL (million/mL).
To calculate % motility:
- Count at least 200 motile or non-motile sperms in 5 different microscopic fields. % of the nonmotile sperm are calculated as follows:
- The ejaculated sperm in the semen remain viable for several days in the female genital tract.
- Fertilization can occur as long as the sperms are alive, and these can survive for 5 days.
- The sperm can be preserved for decades when the semen is freezed at -20 °C.
- Sperm viability is needed when the progressively motile sperms are less in number.
Method to assess the viability:
- Take one drop of the semen.
- Add an equal volume of vital stain (trypan blue).
- Cover with the cover glass.
- Keep for a few minutes, but not more than 5 minutes.
- Count 100 cells, including viable sperm (not stained) and dead sperm (take stain).
- This will give you the count of viable sperms.
Forensic value of semen analysis:
- Acid phosphatase is in high concentration in the semen.
- It is suggested that a swab from the suspected case of rape is taken, preserved in 2.5 mL of a protective broth, and stored at 4 °C or room temperature.
- A sample stored in this way will keep acid phosphatase activity for one month.
- In the sample with thymolphthalein as the substrate, it was found that vaginal acid phosphatase in noncoital women is <10 U/L.
- There is a noncoital level if the vaginal sample is taken after 4 days of intercourse.
- The sampling swab can be kept in 1 mL of normal saline, but these samples are freezed and thawed at 2 to 4 °C for 24 hours before the assay.
- Creatinine phosphokinase (CPK) is a high level in semen. This can also be used for medico-legal purposes.
Importance of the semen analysis:
- That one sample cannot confirm the infertile couple because of variation in the count of semen.
- To repeat one more sample. If possible, take the third sample at an interval of 3 weeks apart.
- In the case of vasectomy, no sperms should be seen in the ejaculate.
Workup for the infertile male:
- Get the report of the previous semen analysis.
- Advise antispermatozoal antibody.
- Advise luteinizing hormone (LH) and Follicle-stimulating hormone (FSH). These hormones will affect the pituitary gland’s gonadal function and spermatogenesis.
- Sims-Huhner test. In this test, a postcoital cervical mucus smear is taken. To see the sperm mobility penetration in the mucus and maintenance of the mobility.
Questions and answers:
Question 1: What is count in pathological semen?
In pathological semen, the count is <20 million/mL.
Question 2: What is the value of acid phosphatase in medicolegal case?
Semen is rich in acid phosphatase and in case of rape, take vaginal swab which will be positive for acid phosphatase.