Semen – Part 1 – Semen examination, Semen analysis, Semen Count Procedure
- This is preferred if the sample is collected by the patient in the lab.
- 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.
- Don’t use condoms particularly with spermicide.
- The specimen should be maintained at 37 °C during transport if brought from home and should be examined within 3 hours of collection.
- The sterile container is needed and the sample should be collected at 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.
Purpose of the test (Indications)
- This is the workup of the infertile couple.
- This is done to evaluate the quality of sperms.
- To confirm the efficacy of vasectomy (postvasectomy).
- For forensic studies.
- Definition of semen: This is the secretion of prostate glands, two seminal vesicles, two Cowper glands and testes serve as the vehicle for sperms transport and create the alkaline medium, also provides nutrition. This serves as the medium which promotes the sperm’s motility and survival. This is called semen.
- Spermatogenesis occurs in the seminiferous tubules of the testes. These are matured in the epididymis.
- These are stimulated by testosterone.
- The concentration of the testosterone in the testes is 100 times more than the peripheral blood.
- This high concentration of testosterone is needed for spermatogenesis.
- LH stimulates the Leydig’s cells to produce testosterone.
- These are lined with 46 chromosome germ cells called spermatogonia.
- These cells undergo mitotic division and ultimately give rise to primary spermatocytes.
- These are stimulated by testosterone.
- Secondary spermatocytes give rise to spermatids and these will differentiate into sperm or spermatozoa containing 23 chromosomes.
- Semen production depends upon the function of the testis.
- Hypothalamus producing 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.
- In addition, the Sertoli cells secrete inhibin, which inhibits FSH secretion from the pituitary gland.
- LH stimulates the Leydig cells which produce the testosterone. Testosterone then stimulates the seminiferous tubules to produce sperm.
- Spermatogenesis (in the seminiferous tubules) takes approximately 3 months.
- Maturation of the spermatogonia to mature spermatocytes takes approximately 75 days, through the epididymis.
- In the epididymis the motility is acquired, it takes another 14 days.
- Spermatocytes develop from the Sertoli cells in the seminiferous tubules. They get matured in the epididymis and are ready for fertilization.
- In primary gonadal failure:
- FSH is raised.
- LH is raised.
- In secondary gonadal failure:
- FSH is decreased.
- LH is decreased.
- Aspermia (azoospermia) When there is no sperm.
- Oligospermia when the count is < 20 million/mL.
- Asthenospermia is low sperm motility.
- Necrospermia Normal count of the sperm but are non-motile.
- Hemospermia when there are abundant RBCs.
Semen analysis includes:
- Volume. Check when the sample is fresh.
- Sperm count. This can be done when the semen in completely liquified.
- Motility. This should be checked when the sample is fresh and repeat when it is liquefied.
- Morphology. This can be better appreciated if the smear is stained.
- volume is 2 to 6 ml.
- Color is gray to white or opalescent.
- Liquefaction time 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 has usually prostatic secretion.
- 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 is alive.
- Sperm motility > 50 % at one hour. This is moderate to the 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 one.
- Normally >70% sperm is 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 sperms 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 be taken up by the dead sperms only.
- Clumping or agglutination of the sperms should be reported which indicates antibodies.
- Fructose level is 150 to 600 mg/dL.
- Low or nil fructose indicates an obstruction in the ejaculatory duct.
- 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.
- Volume = 2 to 5 mL
- Liquefaction time = 20 to 30 minutes afte 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% normal shaped
- The color is brown to red. Or may yellow, and turbid.
- Liquefaction time is >60 minutes.
- If the volume is <1.5 ml. This may <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.
|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 ejaculate||≥70%|
|After 3 hours of ejaculate||≥60%|
|After 4 hours of ejaculate||>50%||<35%|
Sperm count method:
- Sperm count solution
- Formalin = 1 mL
- Sodium bicarbonate = 5 grams
- Distal water = 100 mL
- Mix well above ingredients.
- Take 0.02 ml semen
- Take 0.38 mL above solution (diluting fluid).
- Mix well above semen and diluting solution.
- Now load the Neubauer chamber.
- Count the large two squares.
- Important that move the microscope up and down to count the sperm at a different level.
- Calculation: count in the large two square x100,000 = count /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:
Forensic value of semen analysis:
- Acid phosphatase is in high concentration in the semen.
- It is suggested that swab from the suspected case of rape is taken, preserve it in 2.5 mL of a protective broth and store 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 thaw at 2 to 4 °C for 24 hours before the assay.
- Creatinine phosphokinase (CPK) is a high level in the semen. This can also be used for medicolegal purposes.
- 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 the interval of 3 weeks apart.