Understanding Semen Analysis

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

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).

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 function. Reactive 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.

1. Antisperm 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. CASA- Computer 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.

Why It Is Done

A semen analysis is done to determine whether:

• A man has a reproductive problem that is causing infertility

• A vasectomy has been successful

• The reversal of a vasectomy has been successful

How To Prepare

You may be asked to avoid any sexual activity that results in ejaculation for 2 to 5 days before a semen analysis. This helps ensure that your sperm count will be at its highest, and it improves the reliability of the test. If possible, do not avoid sexual activity for more than 1 to 2 weeks before this test, because a long period of sexual inactivity can result in less active sperm.

You may be asked to avoid drinking alcohol for a few days before the test.

Be sure to tell your health professional about any medications or herbal supplements you are taking.

How It Is Done

You will need to produce a semen sample, usually by ejaculating into a clean sample cup. You can do this in a private room or in a bathroom at your health professional's office or clinic. If you live close to your health professional's office or clinic, you may be able to collect the semen sample at home and then transport it to the office or clinic for testing.

• The most common way to collect semen is by masturbation, directing the semen into a clean sample cup.

• You can collect a semen sample during sex by withdrawing your penis from your partner just before ejaculating (coitus interruptus). You then ejaculate into a clean sample cup. This method can be used after a vasectomy to test for the presence of sperm, but other methods will likely be recommended if you are testing for infertility.

• You can also collect a semen sample during sex by using a condom. If you use a regular condom, you will need to wash it thoroughly before using it to remove any powder or lubricant on it that might kill sperm. You may also be given a special condom that does not contain any substance that kills sperm (spermicide). After you have ejaculated, carefully remove the condom from your penis. Tie a knot in the open end of the condom and place it in a container that can be sealed in case the condom leaks or breaks.

If you collect the semen sample at home, the sample must be received at the laboratory or clinic within 1 hour. Keep the sample out of direct sunlight and do not allow it to get cold or hot. If it is a cold day, carry the semen sample container against your body to keep it as close to body temperature as possible. Do not refrigerate the semen sample.

Since semen samples may vary from day to day, 2 or 3 different samples may be evaluated within a 3-month period for accurate testing.

A semen analysis to test the effectiveness of a vasectomy is usually done 6 weeks after the vasectomy.

How It Feels

Producing a semen sample does not cause any discomfort. However, you may feel embarrassed about the method used to collect it. If masturbation is against your religious beliefs, discuss alternate methods of collection with your health professional.

Risks

There are no risks associated with collecting a semen sample.

Results

A semen analysis measures the amount of semen a man produces and determines the number and quality of sperm in the semen sample. Results of a semen analysis are usually available within a day. Normal values may vary from lab to lab.

Semen Analysis Certain Conditions May Be

Associated with a Low or Absent Sperm Count

These conditions include orchitis, varicocele, Klinefelter syndrome, radiation treatment to the testicles, or diseases that can cause shrinking (atrophy) of the testicles (such as mumps).

If a low sperm count or a high percentage of sperm abnormalities are found, further testing may be done. Other tests may include measuring hormones, such as testosterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), or prolactin. A small sample (biopsy) of the testicles may be needed for further evaluation if the sperm count or motility is extremely.

What Affects the Test

Factors that can interfere with your test or the accuracy of the results include:

• Medicines, such as cimetidine (Tagamet), male and female hormones (testosterone, estrogen), sulfasalazine, nitrofurantoin, and some chemotherapy medicines.

• Caffeine, alcohol, cocaine, marijuana, and smoking tobacco.

• Herbal medicines, such as St. John's wort and high doses of echinacea.

• A semen sample that gets cold. The sperm motility value will be inaccurately low if the semen sample gets cold.

• Exposure to radiation, some chemicals (such as certain pesticides or spermicides), and prolonged heat exposure.

• An incomplete semen sample. This is more common if a sample is collected by methods other than masturbation.

• Not ejaculating for several days. This may affect the semen volume.

What To Think About

• A semen sample collected at home must be received at the laboratory or clinic within 1 hour. Keep the sample out of direct sunlight and do not allow it to get cold or hot. If it is a cold day, carry the semen sample container against your body to keep it as close to body temperature as possible. Do not refrigerate the semen sample.

• Consistently detecting sperm in the semen of a man who has had a vasectomy indicates that his surgery was not successful, and another form of birth control should be used to prevent pregnancy. A low number of sperm may be present in a semen sample taken initially after a vasectomy. However, sperm should not be present in subsequent samples.

• A man whose mother took the medicine diethylstilbestrol (DES) during her pregnancy with him has a greater-than-normal risk of being unable to father a child (infertile).

• Additional tests may include measuring hormone levels, such as testosterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), or prolactin. For more information, see the medical tests Testosterone, Luteinizing Hormone, Follicle-Stimulating Hormone, and Prolactin.

• Other fertility testing, including sperm penetration, the presence of antisperm antibodies, or analysis after sexual intercourse (postcoital), may be recommended for infertility problems. For more information, see the medical test Infertility Testing.