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Andrology Clinical Laboratory

Frequently Asked Questions

My doctor ordered a complete semen analysis, what does it tell me?

Routine semen testing is the first and the most important step in the evaluation of the infertile male. It provides important information about the number and quality of sperm in the seminal ejaculate. It includes both macroscopic and microscopic tests.



What will the macroscopic examination of semen reveal?

The macroscopic tests provide information about the seminal ejaculate quality such as:

  • color
  • ejaculate volume
  • liquefaction time, i.e. time for sample to change from a gelatinous coagulum to a liquid state (20 minutes is normal)
  • pH of the sample (normal, acidic or alkaline)



What is the normal color of the ejaculate?

The seminal ejaculate is normally opaque in color.



Why is the color of the ejaculate important?

It is a good indicator of any underlying problem. For example, a yellowish color may indicate jaundice and a slightly pinkish color may be an indication of hematuria.



What is the definition of low semen volume?

A semen volume below 1.5mL is defined as low. Common causes of low semen volume are collection problems, partial retrograde ejaculation and androgen deficiency or obstruction of the ejaculatory duct.



What does semen pH indicate?

A semen pH below 7.0 in a sample with low volume, low or no sperm in the ejaculate, indicates ejaculatory duct obstruction.



What will the microscopic examination of semen include?

The microscopic examination will include:

  • sperm concentration
  • motility
  • normal morphology
  • presence of round cells



When is sperm count considered low?

According to the World Health Organization's (WHO) 5th edition manual (2010), <15 x106 /mL is defined as a low sperm count.



What is the cut-off for low motility?

The cut-off for sperm motility is below 25%. A special test called the viability test is recommended as the next step.



What are the sperm viability tests?

The Eosin-Nigrosin test helps identify the living from non-living sperm. Hypo-osmotic swelling test is another test which reveals the same information.

(Eosin-Nigrosin staining - pictured on right. Live sperm stained white, dead sperm stained pink.)



Why is it important to know the number of round cells?

The number of round cells is important as they may tell us if the round cells are white blood cells. These cells are indicators of any underlying infection.



Are there any particular types of white blood cells that are important?

Yes. White blood cells, called granulocytes, are responsible for infection as they can damage healthy spermatozoa. If the number of round cells in the seminal ejaculate is ≥1x106 /mL, a special test is conducted to verify if these round cells are white blood cells.



Could you tell me more about this test?

This test is called the Endtz test or peroxidase test. It stains the granulocytes dark brown.
 



What if my Endtz test is positive?

If the Endtz test is positive, we will inform your physician. If the value is ≥1.0 x106 white blood cells/mL of the seminal ejaculate, the condition is called leukocytospermia.



Is leukocytospermia important?

Yes. Your physician may recommend antibiotics and ask for repeat semen testing to see if the infection is resolved.



What are the additional tests performed apart from sperm count and motility?

Sperm clumping seen under the microscope is an important observation. This may indicate the presence of anti-sperm antibodies. Additional testing is necessary to confirm the presence of antibodies.



What is anti-sperm antibody test?

In the sperm antibody test (Direct MAR Screen), fresh semen containing live motile sperm is mixed with IgA and IgG-coated latex beads on a glass slide. As the sperm swim through the beads, beads bind on to the sperm surface if antibodies are present.



What is the significance of the anti-sperm antibody test?

Anti-sperm antibodies are thought to coat the sperm surface and thereby impair sperm motility or interfere with the actual fertilization process.



What are the advanced sperm function tests?

Sometimes a simple routine semen analysis may not be adequate to provide information of other underlying causes of infertility. The doctor may recommend an advanced test called a semen profile.



Could you tell me more about the semen profile?

Semen profile comprises three tests in addition to the routine semen analysis. These are measurement of reactive oxygen species, measurement of total antioxidant capacity and measurement of DNA fragmentation.



What are reactive oxygen species?

Reactive oxygen species are also called free radicals. These are unstable molecules or free radicals of oxygen or nitrogen. They are highly reactive and can damage the sperm when produced in large amounts.



Can you give me some examples of reactive oxygen species?

These are oxygen compounds such as hydroxyl radical, superoxide anion, hypochlorite or hydrogen peroxide. The peroxyl radical is the more common reactive nitrogen species.



Why are reactive oxygen species harmful to the sperm?

These are unstable molecules and in order to stabilize they seek an extra electron from the neighboring sperm. In doing so they cause damage to the sperm protein, sperm lipids and sperm DNA damage. As a result the sperm may have poor motility, or they may have sperm DNA fragmentation.



Why is it important to know the levels of reactive oxygen species?

It is important because it can affect the ability of the spermatozoa to fertilize the egg and affect subsequent stages of pregnancy.



What is the source or reactive oxygen species in the seminal ejaculate?

Both abnormal, immature and morphologically abnormal spermatozoa can produce very high amounts of reactive oxygen species. In addition significantly higher amounts of reactive oxygen species can be produced by white cells in the ejaculate.



When can the test be ordered?

The test can be ordered when there are:

  1. High numbers of round cells in semen due to infection,
  2. Infertile men with normal semen parameters
  3. Clinical varicocele
  4. Spinal cord injury
  5. No clinical cause i.e. unexplained infertility
  6. Idiopathic infertility



How do we measure reactive oxygen species?

Reactive oxygen species can be measured using a chemical probe called luminol. It reacts with the free radicals present in the ejaculate and produces a signal. This can be measured by an instrument called a luminometer.

Figure: Instrument to measure the formation of reactive oxygen species. It can be connected to computer and a monitor to visualize the production free radicals.


What is the Turn around Time for the test?

It is usually 1-2 days.



What are the normal levels of reactive oxygen species?

Levels below 92 RLU/sec/x106 sperm are considered normal.



What is the clinical utility of the test?

The test is used as a diagnostic test to screen patients who may have other underlying cause of infertility.



What can I do to lower the levels of reactive oxygen species?

If there is an underlying infection, your doctor may prescribe you some antibiotics to resolve the infection. Also multivitamins, fresh fruits and vegetables packed with antioxidants are also helpful in neutralizing high levels of reactive oxygen species.



My doctor has ordered the total antioxidant capacity test, what is it?

The fluid portion in the ejaculate called the seminal plasma is a rich source of free radical fighting compounds called antioxidants. Living organisms have developed a complex antioxidant system to counteract the effects of ROS and reduce damage.



Are there different types of antioxidants?

Yes. The antioxidant system of living organisms includes macromolecules such as albumin, ceruloplasmin, and ferritin; and an array of small molecules, including ascorbic acid, a-tocopherol, B-carotene, reduced glutathione, uric acid, and bilirubin. In addition there are other compounds called enzymes such as superoxide dismutase, catalase, and glutathione peroxidase. Vitamins such as vitamin A, C and E are also rich in antioxidants.



What is total antioxidant activity or capacity?

The sum of endogenous and food-derived antioxidants represents the total antioxidant activity of the extracellular fluid.



Why is it important to know the total antioxidant capacity?

The overall antioxidant capacity may give more relevant biological information compared to that obtained by the measurement of individual components, as it considers the cumulative effect of all antioxidants present in the seminal plasma.



How is total antioxidant capacity measured?

This test utilizes clear seminal fluid obtained after spinning a semen specimen that has been stored at -20°C. A colorimetric test called the total antioxidant assay is used to measure the levels of total antioxidants (enzymatic, nonenzymatic and macromolecules) in the seminal plasma. The intensity of the color is inversely proportional to the amount of antioxidants in the sample. Results are expressed as micromoles of trolox.



What are normal levels of total antioxidant capacity?

TAC levels greater than 1790 micromoles of trolox are considered normal. Values lower than this may indicate the inability of the antioxidants to effectively neutralize the formation of reactive oxygen species.



My result shows TAC values lower than the reference value, how I can increase this level.

Consuming diets rich in antioxidants such as fruits, green vegetables, bright colored fruits and vegetables (carrots, squash, broccoli, tomato, strawberries, and blueberries) may be helpful.



How long does it take for the results to be in the system?

The Turn around Time: for the test is about 2-3 weeks.



What is oxidative stress?

A balance between the amounts of reactive oxygen species produced and the antioxidants available is important. A shift in this balance resulting in a decrease in the available antioxidants or increase in free radicals is unfavorable, with the imbalance resulting in oxidative stress.



Why oxidative stress is important?

Data suggest that antioxidant concentrations are significantly lower in patients who have increased levels of reactive oxygen species and this may cause oxidative stress.



What is the clinical utility of the test?

Low TAC values indicate poor antioxidant reserve and inability of antioxidants to combat oxidative stress.



What is sperm DNA Damage test?

Intact genetic material, or DNA, is important for healthy fertilization. A morphologically normal looking motile sperm can have DNA damage, which can result in impaired fertilization, miscarriage or subsequent complications associated with pregnancy. Your doctor may order this test to evaluate the quality of sperm.



How is the test done?

After the sample is examined for routine semen analysis, a portion of the sample containing about 2.5x106 sperm is frozen. Samples are batched and analyzed using a sophisticated tool called flow cytometry. About 10,000 cells are examined. It is a rapid, objective and a robust method for analyzing the sample. A special stain is used and it binds to the areas in the DNA that have nicks or are damaged. The instrument can detect these breaks in the DNA and the percentage of sperm with DNA fragmentation can be easily calculated.

Figure: Schematics of the TUNEL staining. Single or double stranded DNA breaks are stained by the TdT enzyme and measured by flow cytometry.
Figure: A typical DNA plot showing the extent of DNA damage as measured by flow cytometry. A: semen specimen showing spermatozoa with low DNA damage (Normal) and B: sample with significantly high DNA damage (Abnormal).


Is the extent of DNA damage in patients higher than in the healthy men who have fathered a child?

Typically healthy men who have normal semen parameters tend to have significantly lower percentage of DNA damage. This damage can vary significantly in patients. The extent of the damage may depend on the underlying cause of male infertility. The figure below shows the extent of DNA damage in healthy controls and patients presenting for infertility at our infertility clinic.

Figure: A plot showing A: distribution of samples from donors and patients with different range of DNA damage from 0->40% and B: percentage DNA damage in donors and infertile men. Note: All donors had DNA damage below the cut-off.


What is the Turn around Time for this test?

The Turn around Time for this test is 2-3 weeks.



What levels are considered normal?

Value <12% DNA damage is considered normal.



What is the clinical utility of the DNA test?

This test can be offered to infertile patients who are idiopathic or have severe oxidative stress-related abnormal semen quality. High DNA damage in infertile men can lead to poor ART outcomes and increased miscarriage rates. Based on the extent of DNA damage found, certain assisted reproductive techniques may be recommended.



Do you provide reference lab services?

The lab offers DNA testing services to physicians outside Cleveland clinic as well as clinicians in other states. After initial semen analysis is conducted the remainder sample can be aliquoted and batched. The samples can be shipped overnight on dry ice.



 
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