Technique of Semen collection
The methodology and standard values are presented according to WHO recommendations (WHO, 2010). A complete collection of the semen is fundamental for a correct analysis. To confirm pathological findings, two semen analyses at least 7 days apart are needed due to high inter-individual variations. Two days of sexual abstinence are recommended before the collection of the specimen.
After collection, the semen is stored in an incubator at 37 degrees Celsius. After 30–60 minutes, the consistency and appearance of the sperm is examined. After complete liquefaction, the volume and the pH are measured. In a first microscopic examination, the morphology and mobility of spermatozoa is assessed and the dilution for the sperm count is determined. After dilution of the semen and counting of the spermatozoa concentration, a smear specimen of semen is made for staining. The morphology of sperm is examined with the stained semen specimen. The WHO recommends the Papanicolaou, the Shorr or the Diff-Quick staining. Depending of the findings and clinical information, special investigations are initiated (vitality testing, inflammation and immunological tests, measurement of fructose) or the semen can be used for cryopreservation.
Normal Findings and Differential Diagnosis of Semen Analysis
Gross Appearance of Semen:
Immediately after ejaculation, the semen is usually a coagulum of various consistencies. The semen liquefies within 5–25 min at room temperature or in an incubator, some specimens may take longer and make the semen difficult to analyze. Normal liquid semen has a homogeneous, gray-opaque color. It is less opaque, if the sperm concentration is low. Some diseases cause a change in semen color: red-brown (hematospermia) or yellow (jaundice, intake of vitamins or drugs).
Consistency of the Semen
Normal semen liquefies within 60 min, after that time the length of the falling drops from the pipette tip should be shorter than 2 cm. Inadequate liquefaction can distort further investigation, with repeated pipetting the semen is homogenized and mixed.
The normal semen volume should be > 1.5 ml. The WHO recommends to determine the volume by weighing the vials before and after sperm collection. The specific weight of semen is approximated with 1 g per ml. Differential diagnosis of reduced semen volume:
- Incomplete semen collection
- Retrograde ejaculation
- Obstruction of the ejaculatory ducts
pH of the Semen:
The normal pH of semen is 7.2 to 8.0. For pH determination, the sperm must be liqued, the range of the indicator paper should be between pH 6–10.
An acidic pH of semen (with azoospermia and reduced semen volume) is caused by prostatic secrets only and an indicator for obstruction of the ejaculatory ducts or malformations of the seminal tract (e.g. missing seminal vesicles). A basic pH of >8.0 can be caused by an infection.
Microscopic Examination of the fresh Specimen
A well-mixed drop of semen (10 μl) is assessed on a slide under a cover glass (22 × 22 mm) with 200–400X magnification.
Agglutination of Motile Spermatozoa:
Agglutination of motile spermatozoa may be head-to-head, head-to-tail or tail-to-tail. Agglutination of spermatozoa suggests the existence of anti-sperm antibodies. The agglutination of mobile spermatozoa to each other has to be separated from non-specific adherence of immobile spermatozoa and from the adherence of motile spermatozoa to epithelial cells or debris. The extent and nature of agglutination should be judged with the following graduation:
- Grade 1 (isolated): <10 spermatozoa per agglutinate, many free mobile sperm
- Grade 2 (moderate): 10–50 spermatozoa per agglutinate, many free mobile sperm
- Grade 3 (strong): agglutinates with >50 spermatozoa, only few free mobile spermatozoa
- Grade 4 (complete): completely agglutinated spermatozoa, no mobile free spermatozoa visible
Next to spermatozoa the semen may also show epithelial cells and round cells, which can be leukocytes or immature germ cells. The further differentiation of round cells is made by staining with the peroxidase: peroxidase-positive cells are leukocytes.
Assessment of sperm motility:
The motility of sperm is best assessed immediately after liquefaction of the semen. The thickness of the liquid layer between the slide and cover glass should be 20 microns, this correlates with a well-mixed drop of sperm (10 μl) under a cover glass of 22 × 22 mm. The motility should be examined with 200–400X magnification: 200 spermatozoa are assessed for motility and classified using the following graduation:
- Progressive motility (PR): active linear or slightly curved forward movement.
- Non-progressive motility (NP): no forward movement or circular forward movement
- Immotility (IM): no visible movement
At least two sets of 200 spermatozoa on two slides should classified using the above mentioned graduation, the results should be comparable. The results of both tests are averaged and given in percent. Reference values for motility: >40% motile sperm (PR + NP), >32% progressive motility.
Asthenozoospermia is the medical term for reduced sperm motility: the percentage of progressively motile sperm is below 32%. Causes of asthenozoospermia are insufficient liquefaction, autoantibodies, inflammation and disorders of the sperm tails. Causes of false-negative asthenozoospermia are cold sperm, old sperm or sperm collection with contamination (e.g. soap).
Assessment of vitality:
If over 40% immobile spermatozoa are seen, a vitality test with eosin staining should be done. The dye enters dead sperm within 1 minute, this leads to red sperm heads in microscopy. Sperm without dye is considered vital. 200 spermatozoa should be classified, the standard value for vital spermatozoa is >58%. As an internal control, the proportion of dead sperm should not be higher than the immobile sperm proportion. A percentage of dead sperm over 42% is called necrozoospermia and is often caused by epididymal disease or inflammation.
Total sperm count correlates with time to pregnancy and pregnancy rate. After determining the sperm concentration using the hemocytometer (two sets of 200 sperm should be counted), the sperm concentration is used to calculate the total sperm count with the help of the semen volume. Reference values: >15 × 106 spermatozoa per ml or >39 × 106 total sperm count.
Oligozoospermia (oligospermia) refers to semen with a reduced sperm concentration: <15 × 106 per ml or <39 × 106 total sperm count.
Spermatozoa cannot be observed in a fresh semen sample. However, spermatozoa can be found after centrifugation and microscopy of the sperm pellet.
Azoospermia refers to semen without spermatozoa. Azoospermia should only be diagnosed after centrifugation of the ejaculate and microscopy of the sperm pellet.
The assessment of sperm morphology should be done with 1000 × magnification and (e.g.) Giemsa stain of dried smear specimen of sperm. Criteria for the classification of normal and pathological morphology please refer to table criteria of sperm morphology.
Table: Criteria of normal and abnormal sperm morphology (WHO, 2010) .
||Regular oval shape, well-defined acrosome region without vacuoles, acrosome size 40–70% of the head volume
||Too big, too small, too thin and long, pear-shaped, round, amorphous, with acrosome vacuoles (>2 or more than 20%), post-acrosomal vacuoles, too small or too large acrosomes.
||Narrow, regular, about as long as the head. The main axis of the head and middle piece should be in line. Cytoplasmatic droplets of the midpiece should be <30% of the head size.
||Asymmetric connection to the head, middle piece irregularly, too thick, bent or too thin. Cytoplasmatic droplets >30%.
||The tail should be thinner than the midpiece, the caliber should be uniform and the length about 10 times the length of the head. The tail may be curved, but without abrupt kinks.
||Too short, multiple tails, kinks, irregular thickness, spiral-shaped.
Morphologically normal spermatozoa are normally less than 25%. Teratozoospermia refers to semen with less than 4% morphologically normal spermatozoa. Optionally, the location of the defect can be specified: % head defects, % midpiece defects, % sperms with cytoplasmatic droplets and % tail defects.
Tests for Inflammation:
Evidence for infection of the seminal ducts provide the determination of peroxidase-positive cells (normal <1 × 106 cells per ml), elastase (normal <250 ng/ml), a positive sperm culture or evidence of bacterial prostatitis.
Pyospermia is a high number of white blood cells in the sperm (>106 peroxidase-positive cells per ml). A urinary tract infection should be ruled out. Significant bacteriospermia: if >103 bakteria/ml sperm are found
Anti-sperm antibodies cause the agglutination of spermatozoa, which is seen in fresh semen preparation (see above), this indicates further immunological studies. Anti-sperm antibodies (ASA) can be detected with the MAR test (mixed antiglobulin reaction test) or Immunobeads test. Detailed description of the procedures please refer to the current WHO manual (WHO, 2010).
Fructose in Semen:
The normal concentration of fructose in semen is >13 μmol/l in the seminal plasma (supernatant after centrifugation of liquid semen). Decreased levels are expected in obstruction of the ejaculatory ducts.
Index: 1–9 A B C D E F G H I J K L M N O P Q R S T U V W X Y Z
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- World Health Organisation
Cooper, T. G. (ed.)
- WHO laboratory manual for the examination and processing of human semen
Press, Geneva, Switzerland, 2010