UNDERSTANDING SEMEN ANALYSIS

 

SEMEN

 
SourceVolumeCharacteristics
Urethral and bulbourethral glands0.1-0.2ccViscous, clear
Testes, epididymides,vasa deferentia0.1-0.2ccSperm present 
Prostate0.5-1.0ccAcidic,watery
Seminal vesicles1.0-3.0ccGelatinous, fructose positive
Complete ejaculate2.0-5.0ccLiquefies in 20-25min

 

The male factor infertility is most commonly defined as abnormalities in the number of sperm present, proportion of the motile and morphologically normal sperm. WHO has defined normal values for human ejaculate.

 

 Commonly used normal semen parameters 

VOLUME>2.0 ML
pH7.2-7.8
CONCENTRATION>20x106/ML
MOTILITY>50%
MORPHOLOGY>30% WITH NORMAL MORPHOLOGY
WBC< 1x106/ML
 

 

Semen analysis is not a test for fertility. Fertility determination is a couple-related phenomenon that requires the initiation of a pregnancy. The patient cannot be considered fertile based only on normal semen analysis. It was shown that 30% of all patients with normal semen analysis have abnormal sperm function.

Semen specimen are obtained by masturbation into a sterile wide-mouth container after 2-5 days of abstinence and analyzed within 1 hour of collection. Therefore, the patients should be strongly recommended to collect samples within clinic area. If intercourse is the only way to collect sample, special nonreactive condoms are available.

Typically two to three semen analyses are obtained over a 3 month period prior to making any final conclusion regarding baseline sperm quality or quantity. However, if the first semen analysis is normal, the repeat test is not required. Recent febrile illness or exposure to gonadotoxic agents may affect spermatogenesis for up to 3 months, therefore semen analysis has to be postponed.

Normal ejaculate volume is between 2 and 6 ml. 65%of the volume is from seminal vesicles, 30-35% is from the prostate and only 5% from the vasa. Low volume is associated with absence or decrease of seminal vesicle component of ejaculate( absence of SV, complete or partial obstruction of ejaculatory ducts) or retrograde ejaculation

Normal semen pH is 7.2-8.0. Prostatic secretion is acidic while seminal vesicle fluid is alkaline (seminal fructose is derived from seminal vesicles). Acidic ejaculate (pH<7.2) may be associated with blockage of seminal vesicles. Infection is usually associated with alkaline ejaculate (pH >8.0_ Azoospermia with low ejaculate volume, fructose negative and acidic may imply obstruction of the ejaculatory ducts. pH over 8.0 may indicate infection. The semen is initially in liquefied state but quickly coagulate by the action of protein kinase secreted by the seminal vesicles. Proteolytic enzymes from the prostate liquefy coagulum in 20-25 minutes. Abnormal liquefaction may be cased by prostatic abnormalities, e.g. prostatitis. Increased viscosity may affect sperm motility

Concentration: Concentration: evaluated in Mackler or Cell-VU chambers. Azoospermic specimen contains no sperm, oligospermic specimen reveals concentration of less than 20x106 and normospermic specimen contains more than 20x106.

 

Motility and forward progression: normally >50% of sperm in the specimen are motile. Forward progression describes how fast the motile sperm are moving (normal 2+ in the scale from 0 to 4)

 

0No movement
1Movement, none forward
1+Occasional movement of a few sperm
2Slow, undirected
2+Slow , directly forward movement
3-Fast, but undirected movement
3Fast, directed forward movement
3+Very fast forward movement
4Extremely fast forward movement
 

 

 

Morphology = shape of spermatozoa: Several techniques have been described to evaluate sperm morphology. Sperm are classified into normal-oval shaped, tapered, amorphous, duplicated and immature. Normal spermatozoid must have an oval form with smooth contour, acrosomal cap encompassing 40-70% of head, no abnormalities of midpiece, or tail and no cytoplasmic vacuoles of more than half of the sperm head. Head size is 5-6m M x 2.5-3.5m M. Any borderline sperm are counted as abnormal( amorphous, tapered,duplicated, immature, coiled tail, blunted tail, midpiece abnormalities). The predictive value of sperm morphology in determining pregnancy rates is low

a. WHO criteria: >30% normal forms ( 100 cells evaluated)

b.Strict criteria (higher predictive value in determining rates of pregnancy in IVF program) are based on the morphology of postcoital spermatozoa found at the level of the internal cervical os. 100 cells evaluated for only normal sperm (>14% normal forms). Men with fewer than 4% normal forms usually failed to fertilize without micromanipulation. Strict criteria for normal sperm morphology include:

Sperm head: Smooth oval configuration. Length-5-6 microns. Width:2.5-3.5 microns. Acrosome comprises 40-70% of the anterior sperm head

  Midpiece: Axially attached, 1.5 times the head length, £ 1m m in width

Tail: Straight, uniform, slightly thinner than the midpiece, uncoiled, ± 45m m long

 

White blood cells (WBC):

All semen samples have WBC in them. If greater than 1 million WBC per 1 ml are present, there is concern of infection. Generally leukocytospermia (WBC in the semen) affects 5-10% of the patient population, but can rise to 20% in certain patients groups. Semen has to be cultured for aerobic and anaerobic infection as well as Chlamydia and Mycoplasma. Additionally, leukocytes have to be differentiated from immature germ cells using immunohistochemical methods. WBC cells are deleterious because of their ability to stimulate the release of reactive oxygen species (ROS), thereby inhibiting sperm motility and sperm functionReactive oxygen species (ROS) are produced by polymorphonuclear cells .The three main ROS are superoxide anion, hydrogen peroxide, and the hydroxyl radical. On the other hand, seminal plasma contains a number of antioxidants that protect sperm from oxidative damage from exposure to ROS. Men who have higher concentrations of such antioxidants may be able to tolerate greater concentrations of seminal leukocytes. Despite an apparently abnormal threshold level for leukocytes within the semen, a wide range of conflicting evidence exists as to the significance of seminal leukocytes and infertility. The impact of this condition and its treatment on semen quality are extremely controversial

Viability. Viability tests are used in cases of low motility to determine the presence of live sperm vs. necrozoospermia. The eosin test is based on the fact that eosin is excluded by live cells which are not stained. The tail of only live spermatozoa is swelling in the hypoosmotic solution (Hypoosmotic swelling test)

Fructose (13 mmol or more per ejaculate): Fructose is androgen-dependent and is produced in the seminal vesicles. Fructose levels should be determined in any patient with azoospermia and especially in those whose ejaculate volume is less than 1 ml, suggesting seminal vesicle obstruction or atresia. Absence of fructose, low semen volume, and failure of the semen to coagulate indicate either congenital absence of the vas deferens and seminal vesicles or obstruction of the ejaculatory duct.

Semen analysis has comparatively limited predictive value for the ability of the individual to achieve pregnancy. Additionally, 10-20% of infertile couple will not have any abnormalities. In order to enhance the diagnostic power of semen analysis, new tests have been developed to identify functional defects and fertilizing potential of the sperm. The clinical data to support their use are not conclusive.

1Antisperm antibodies test. Sperm agglutination, reduced sperm motility, abnormal postcoital test are suspicious for the presence of antisperm antibodies. Several tests are presently available including Sperm Immobilization test, Sperm Agglutination tests, Indirect immunofluorescence test, Enzyme-Linked Immunosorbent Assay, Radiolabelled Antiglobulin Assay. Immunobead Rosette Test is one of the most informative and specific and can identified different antibody classes involved (IgG, IgA, IgM) and location on the sperm cell (head, body or tail)

2.CASAComputer Assisted Semen Analysis. Mostly for assessment of sperm concentration and specific patterns of sperm motility (velocity, linearity etc). The available clinical data show that the measurement obtained by CASA are correlated with conception in vivo and fertilization in vitro, but comprehensive quality control and quality assurance programs are necessary to ensure accuracy. The equipment is highly expensive.

3.Acrosome reaction. Absence of acrosome reaction implies poor prognosis for fertilization. The test for acrosome reaction is very expensive, labor intensive, subjective and not cost-effective since only 5% of infertile patients do not demonstrate an acrosome reaction.

4.Hamster egg penetration test to check sperm fusion ability. The diagnostic value is controversial because of difficulty in optimizing protocol. However, a zero test score may indicate a major impairment of sperm fusion capacity.

 5.In Hemizona test (to evaluate sperm zona-binding capacity) the two halves of human zona pellucida is incubated with patient's capacitated sperm and control fertile donor's sperm.

6.PCR-based detection of the pathogens in the semen in patients with asymptomatic genital infection.

7. Biochemical markers e.g. Creatine Kinase, Reactive Oxygen Species

 

Postejaculate urine analysis.

 

Retrograde ejaculation is commonly seen in patients with diabetes, after transurethral surgery, retroperitoneal lymph nodes dissection, and spinal cord injuries. When bladder neck coaptation is not complete, the seminal fluid may travel retrograde into the bladder during periurethral muscular contraction. Patient may present with low semen volume, low motility and sperm concentration.

Urinalysis performed immediately after ejaculation. Unspun specimen examined for sperm under the microscope. If sperm are present, specimen is processed further to evaluate concentration, motility and morphology. In certain situations sperm retrieved from the urine may be used for assisted reproduction.

 

 

Selected Bibliography

  1. Bar-Chama N. Lamb DJ. Evaluation of sperm function. What is available in the modern andrology laboratory?. Urologic Clinics of North America. 1994; 21(3):433-46
  2. Irvine DS. Aitken RJ. Seminal fluid analysis and sperm function testing. Endocrinology & Metabolism Clinics of North America. 1994;23(4):725-48
  3. Sigman M. Laboratory testing in the evaluation of male infertility. A rational approach. World Journal of Urology. 1993;11(2):96-101
  4. Bernstein D. Tyler JPP. Driscoll GL. A comparison of WHO and Tygerberg strict criteria for assessing humanspermatozoal morphology. Australian Journal of Medical Science1995; 16(3): 115-117.
  5. Irvine DS. Computer assisted semen analysis systems: Sperm motility assessment. Human Reproduction. 1995;10(SUPPL. 1): 53-59
  6. WHO Laboratory manual for the examination of Human semen and Sperm-Cervical mucus interaction. Cambridge University Press, 3rd edition, 1992

 

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