| Semen is produced in which of the following organs? | View Page |
| Which of the following are indications for a semen examination? | View Page |
| A semen sample should generally be received at the testing site within _____________ hours of collection. | View Page |
| Normal semen volume is a volume greater than which of the following? | View Page |
| Normal pH of semen is a value GREATER than which of the following? | View Page |
| Liquefaction of semen normally takes place within: | View Page |
| A sperm counts of 19,000,000 sperm per ml is BELOW WHO criteria for normal semen. | View Page |
| The percentage of motile sperm in a normal semen sample should be at least: | View Page |
| Materials Needed The following is a list of materials needed for semen analysis. Laboratories will differ slightly in the equipment used. Use of this equipment will be described further in the later pages of this course.
Materials needed include:graduated test tube or serological pipets with safety bulb to measure volumepH paper in neutral to basic range (e.g. 7.2-8.8)counting chamber and/or automated counting machineglass slides and coverslips for wet mount if motility and sperm count are to be assessed separatelyhand counterif dilution is donediluting fluid calibrated automatic pipetspositive pressure pipets and glass boreslight microscope with phase contrast objectives for sperm count and bright field objectives for morphology assessmentglass slides and fixative for morphology slidesset-up for performing Papanicolaou or other morphology stainingEvery laboratory should also have a copy of the "WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction", published on behalf of the WHO by Cambridge University Press. The fourth edition was published in 1999. | View Page |
| Semen is a substance secreted by the male reproductive organs. It is composed of spermatozoa in a semi-viscous fluid. | View Page |
| Which of the following organs are normally involved in the production of semen? | View Page |
| Examination of semen is important because: | View Page |
| Importance of Semen Analysis Semen analysis is important for several reasons:To determine whether compromised semen parameters contribute to infertilityTo confirm that vasectomy has reduced sperm count to zeroTo confirm the presence of sperm following reversal of vasectomyTo confirm or refute the presence of sperm in cases where rape is suspected or paternity is in question | View Page |
| Limits of Semen Analysis Semen analysis can provide important information related to the function of the male reproductive system but, even when results are within normal limits, it does not ensure that a male is fertile.
A normal semen analysis result does not mean that all causes of male infertility have been ruled out. One reason for this is that there can be considerable differences between one semen analysis result and another in a single individual.
On the other hand, an abnormal result does not always mean that a couple cannot conceive a pregnancy. Men with suboptimal sperm counts have been known to father children. Also, infection, trauma, stress, febrile illness and medications can cause temporary subfertility.
For all of these reasons multiple specimens are recommended for a complete analysis of the semen.
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| Semen Semen is a substance produced by the male reproductive organs. It is composed of spermatozoa in a semi-viscous fluid. Structures within the male reproductive tract that are involved in the production of semen include:testes and epididymisprostateseminal vesiclesbulbourethral gland | View Page |
| Composition of Semen Semen is produced as a combination of secretions from the different regions of the male reproductive tract. Each fraction differs in chemical composition and function. The combination of these fractions during ejaculation results in the optimal environment for transporting sperm to the endocervical mucus in the female. Spermatozoa are produced in the testes. They mature in the epididymis. The testes also produce testosterone and inhibin.Fluid from the seminal vesicles accounts for approximately 70% of semen volume. The seminal vesicles are the source of fructose in semen. Fructose is used by the spermatozoa as an energy source.The prostate gland supplies about 20% of the volume of semen. Its fluids include acid phosphatase and proteolytic enzymes that lead to coagulation and subsequent liquefaction of semen. The prostate also contains most of the IgA found in semen.The bulbourethral gland produces mucoproteins that make up about 5% of the volume of semen. | View Page |
| Other cells in semen: Round cells and red cells In addition to the sperm in the center of this field, a round cell is seen at center right (1 o’clock) and red cells at center bottom (6 o'clock) and left (9 o’clock). Red cells have a round, smooth appearance when compared to the grainy appearance of the round cells.
Round cells may be either immature sperm or white cells. | View Page |
| Other cells in semen: Epithelial cells Epithelial cells are frequently found in semen. Two such cells are seen overlapping in the center of this field.
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| Other cells in semen: Round cells and red cells (continued) Several sperm can be seen in this field. A sperm head is pointing toward a red cell slightly above and to the left of center. A second red cell is slightly below center to the left of the five sperm. Round cells can be seen in the upper center and another just above the first at the very top of the screen. | View Page |
| White cells in semen Round cells in semen are of two types: immature sperm and white blood cells. To determine the percentage of white blood cells (specifically granulocytes) a special leukocyte screening test must be done. This test involves staining for the peroxidase enzyme present in the granulocytes.The 1999 WHO manual contains a protocol for doing this test (Appendix III). There is also at least one test kit on the market for this assessment (Leukoscreen: Bioscreen, Inc.).Laboratories with particular expertise in doing CBC and assessing granulocytes in stained blood smears may be able to do a differential count by this method rather than using a biochemical test for leukocyte screening. | View Page |
| Morphology of sperm Describing the morphology of the sperm in a semen specimen is an essential part of the microscopic examination. The presence of abnormal forms along with low counts and/or poor motility contributes to a poor prognosis in infertility cases. There are several different methods for determining morphology. The most common are the WHO III (WHO III manual, 1992)assessment and the Strict Morphology method found in the WHO IV manual (1999).
A specimen is considered normal if 30% or more of the sperm are normal morphology according to WHO III criteria. If strict morphology criteria are used then the specimen is considered normal if it has 14% or more normal forms. | View Page |
| Staining and fixation for sperm morphology To examine sperm morphology a semen smear is prepared on a clean glass slide, much like making a blood smear. It is important that the sperm be spread evenly on this slide and that the concentration be such that individual sperm can be clearly viewed. Too many sperm per slide makes evaluation difficult. Too few, makes it hard to find enough sperm for an adequate count.The examination of morphology is made using one of several commonly used stains. These include: Papanicolaou stainDiff QuikShorr stainDetails of these staining methods are available in the WHO IV reference manual.Two slides are prepared and 100 sperm are counted per slide using a bright field 40X or 100X objective. | View Page |
| Which of the following statements are true concerning the appearance of a semen? | View Page |
| Which of the following statements are true for the viscosity of semen? | View Page |
| Volume Normal semen volume is 2.0 ml or more.Volume is measured by pouring the semen into a graduated cylinder or by very carefully drawing the specimen into an appropriately sized pipet. With either of these methods it is important to avoid foaming or bubbling of the semen as these can effect the sperm count and motility. | View Page |
| Fructose Fructose makes up 99% of the reducing sugar present in semen. This sugar is produced in the seminal vesicles and its absence may indicate an obstruction proximal to these glands.
Although a fructose test is NOT part of a routine semen analysis, the clinician may want to measure this in cases of azoospermia. In azoospermia secondary to obstruction of the ejaculatory ducts or absence of the vas deferens, fructose is usually absent. When azoospermia is caused by failure of the testes to produce sperm, fructose is present. Measuring fructose levels can thus help the clinician determine the cause of azoospermia, although measurement of pH is often more useful in this regard.
The procedure for determining the amount of fructose in semen involves heating semen in a strong acid in the presence of resorcinol. Fructose gives a red color to this solution when present. | View Page |
| Viscosity Most semen is somewhat viscous. Liquefaction should be complete before viscosity is assessed.
Semen viscosity can be determined by trying to draw the specimen into a wide bore pipette. Normal semen can be dropped from a pipette in single droplets. Some laboratories report viscosity on a scale from 0-4. Others report the results as "non-viscous", "slightly viscous", "very viscous" and so forth.
A specimen that is more viscous than normal after liquefaction may have reduced sperm motility. During sexual intercourse, hyperviscosity can prevent the sperm from reaching the cervix. | View Page |
| pH The pH of normal fresh semen is 7.0 or greater. Acid conditions can lead to reduced sperm motility and viability.Secretions of the different glands of the male reproductive tract contain fluids of different pH. The portion of semen that originates in the seminal vesicles is basic, that of the prostate gland is acidic. If the pH is low it could mean that there is an obstruction in the ejaculatory duct below the level of the seminal vesicles or it could signal absence of the vas deferens. Low pH is often associated with low volume since both basic pH and much of semen volume are products of the seminal vesicles. Measuring pH can thus help a clinician determine the cause of azoospermia (absence of sperm in semen).The pH is measured using pH testing strips. A drop of semen is placed on a pH strip and allowed to develop full color. Final color is compared to a standard. For the most accurate results, use test strips with a sensitivity in the basic range, for example from 6.0 to 10.0. | View Page |
| Summary of macroscopic evaluation In summary, the macroscopic examination of semen can provide important information. Changes in semen liquefaction parameters, viscosity, volume, pH may provide clues to the clinician about causes of infertility or abnormalities of the reproductive tract. | View Page |
| Macroscopic examination of semen The macroscopic examination of semen includes the following observations and determinations:liquefactionappearancevolumeviscositypH | View Page |
| Appearance of semen The color of semen may vary. Normal values are white, yellowish, or gray.
Normally the consistency ranges from clear to cloudy.
A pink or reddish discoloration suggests the presence of red blood cells, an abnormal finding. | View Page |
| Normal volume for a semen specimen is: | View Page |
| Liquefaction Immediately following ejaculation, semen is in a gel-like condition.Liquefaction, or resolution of the gel-like consistency, is expected within 15 minutes. If liquefaction does not occur within 60 minutes you should note this on the report sheet.Occasionally a specimen does not liquefy. If this occurs, mechanical mixing or enzyme treatment may be necessary in order for the sperm count, motility analysis and other microscopic aspects of semen analysis to be performed. | View Page |
| Viability Viability is a measure of the percentage of cells that are alive. Since motile cells are inherently viable a viability assessment may not be necessary when motility is high. Most laboratories set a minimum motility after which a viability will also be performed.
To assess viability, place a drop of semen on a slide. Add an equal volume of a vital stain such as trypan blue. Cover with a coverslip. Allow color to develop for several minutes, but not more than 5 minutes. Count 100 cells (both motile and non-motile cells) on each of 2 slides. During the count differentiate between the white cells (living) and blue cells (non-living). | View Page |
| Collection (continued) Other aspects of specimen collection that must be considered are the temperature of the specimen and the time needed to transport it to the laboratory.Ideally, the specimen should be collected in a room at the testing site.If on-site collection is not possible, the specimen should be kept at body temperature (37°C) from the time of collection until it arrives at the laboratory. This can be facilitated by holding the container close to the body, for example by carrying it in an inside pocket.Semen should arrive at the laboratory as soon as possible after collection, preferably within one hour.Lubricants should not be used for collection unless absolutely necessary as most lubricants are toxic to sperm. If lubricant must be used then non-toxic forms such as KY jelly or cooking oil should be the only options.
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| Collection Accurate semen analysis results require appropriate sample collection. Patients must receive detailed directions for proper specimen collection and transport. Directions should be in writing. Specific instructions should include: The period of abstinence prior to collection should be between 2 and 5 days.The entire specimen must be collected because the different portions have varying concentrations of spermatozoa.An appropriate collection container must be used.Each laboratory should designate an appropriate, wide mouth, collection container.Each lot of collection containers should be tested to ensure that it is non-toxic to sperm.Alternative collection containers should be discouraged because their level of toxicity is unknown.Use of condoms for collection should be discouraged particularly when the purpose of the semen analysis is to test for fertility. Some condoms are toxic to sperm. Collection in condoms often results in inaccurate results for semen volume and other parameters. | View Page |
| Condoms are a good way to collect semen specimens for fertility analyses. | View Page |
| Which of the following statements are TRUE for semen specimen collection and transport? | View Page |
| Information to be obtained from patient At the time of semen collection the patient should provide the following information that will be reported as part of the final report:The time of collectionDays of abstinenceLocation at which specimen was collected: clinic or homeDifficulties during collection (e.g. spillage)Difficulties during transport (e.g. exposure to cold temperatures)Information on collection method (e.g. masturbation, withdrawal)Names of medications that he is takingA sample supplemental information collection form is shown below:
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| Testing collection containers In order to test collection containers for sperm collection, the sperm must be held in the container for several hours to ensure that neither the numbers nor motility are adversely affected. Numbers will decline if the sperm adhere to the container. Motility will decline if the container is toxic.
One method of testing involves removing sperm from semen. The specimen would be centrifuged and the sperm pellet diluted in a small volume of culture medium containing an energy source and at least 0.5% of a protein, such as serum albumin. The processed sperm specimen would be placed in the container to be tested. Total count and motility of the sperm would be tested at the start of incubation and 24 hours later. The container is non-toxic if the motility at the end of 24 hours is no less than 50% of the original value.
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| High viscosity If the specimen is more viscous than normal, it may be difficult to dilute it or to load it onto counting chambers in the undiluted condition. In this rare situation the semen may need to be manipulated to reduce the viscosity before a count is done. One method to do this is to repeatedly pipet the specimen up and down with an equal volume of culture medium. Care must be taken to avoid foaming. Other methods include enzyme digestion, for example with bromelain at a concentration of 1 gm / liter, or addition of a small amount of emulsifier, such as Alevare or chymotrypsin.
Any manipulation of this type must be recorded on the report sheet. Calculation of the number of sperm per milliliter will also have to be corrected for any dilution. | View Page |
| Microscopic Examination of Semen Microscopic examination of semen includes assessment of: sperm concentration percent motility percent viability cellular elements other than spermatozoa sperm morphology Sperm counting will be covered in this section. Assessment of other cellular components and of morphology will be covered in the next sections. For assessing count, motility, viability and other cellular components your laboratory will require a phase contrast microscope with 10x oculars and objectives up to 20x. For assessing morphology you will need bright field objectives up to 40x and 100x (oil immersion). | View Page |
| Sperm counting methods Sperm can be counted either manually or by automated methods. Although automated counting has some advantages for assessment of motility parameters, manual counting is still performed by most laboratories.
There are several manual counting methods available for semen. These include:Neubauer hemacytometerMakler chamberCellVu (Millennium Sciences, Inc)MicroCell (Conception Technologies)
The Makler, CellVu, and MicroCell methods have the advantage of requiring no dilution of the semen. Since semen is viscous, accurate dilution can be problematic. These methods also allow counting of motile and non-motile sperm at the same time and thus avoid the need for separate assessment via wet mount. Each laboratory should determine the best most reproducible method for their own situation, equipment, and expertise. | View Page |
| Diluting a specimen for counting on a hemacytometer Following liquefaction (20-30 minutes), mix the sample manually by swirling the container several times. Thorough mixing is essential for accurate counting. Calibrated automatic pipettes are used to prepare a dilution. Because of the viscosity of semen, the semen should be added to the diluent using a positive pressure pipettor.
The dilution often used for routine sperm counts is 1:20 but the actual dilution factor will vary depending on the total sperm count. For high concentration specimens a greater dilution will be necessary. For low concentrations an undiluted or minimally diluted specimen may be required. The appropriate dilution is determined by estimating the concentration needed to do a count of at least 100 cells per side of the loaded hemacytometer.
The diluent that may be used for sperm counts on a hemacytometer can be as follows: 5 gm of sodium bicarbonate in 100 ml of distilled water, plus 1ml of formalin (neutral). | View Page |
| Other counting chambers Some professionals believe that sperm counts done by hemacytometer are not accurate because of the need to dilute the viscous semen prior to counting. There are several other counting methods available to assess sperm concentration.The advantages of the following methods are: the specimen does not have to be diluted motile and non-motile sperm can both be counted avoiding the need for wet mount evaluation of motile cells. Note that counting moving sperm can be difficult and takes significant practice to avoid error. For each of these methods accurate counts are best obtained when at least 100 sperm per replicate are counted. Makler (Zygotek Systems, Inc.). An undiluted sample is placed on the chamber and covered with the coverglass. Ten squares on the grid contain 0.000001ml. CellVu (Millennium Sciences, Inc). Two sides of a special slide are loaded with a drop of undiluted semen. Coverslips with special grids are placed on top of the sperm according to manufacturer's directions. Sperm on both sides are counted. MicroCell (Conception Technologies) has two chambers on a single, disposable slide. A special eyepiece with a grid is needed for counting. | View Page |
| Which of the following is NOT part of the microscopic examination performed on semen? | View Page |
| Motility Motility of a normal semen sample is 50% or greater.Sperm motility is important because sperm must be moving in order to penetrate the cervical mucus, travel to the fallopian tube, and fertilize ova.Accurate motility evaluation requires that the temperature be standardized. Some laboratories read motility at 37°C while others routinely report motility at room temperature. The temperature of the assessment should be specified in the final report. | View Page |
| Requirements for the microscopic examination of semen For assessing count, motility, viability and other cellular components your laboratory will require a microscope with 10x oculars and phase contrast objectives up to 20x. You will also need hand counters.For assessing morphology you will need bright field objectives of 40x and/or 100x (oil immersion).You will also need counting chambers, glass slides and coverslips and a method for staining sperm for morphology assessment. | View Page |
| Assessing sperm motion parameters In addition to determining the percent motility, the laboratory must evaluate the quality of the movement. The process of rating motility may vary somewhat from one laboratory to another. Normal motile sperm should have strong forward progressive motion.
Automated sperm analyzers commercially available and specifically designed to evaluate semen can add specific information about motility parameters. In addition to determining percent motility, they calculate the speed at which the sperm are swimming in microns/second.
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