Sperm Information and Courses from MediaLab, Inc.

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.

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

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.

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.

This course will give you an overview of the methods involved in performing a semen analysis. Semen analysis may be performed for one of several reasons. One of these is evaluation to assess male fertility. Infertility is a problem for approximately 1 in 7 couples who attempt a first pregnancy. In almost half of these cases (~50%) the cause of infertility can be traced, at least in part, to an abnormality in the male. Examination of sperm is the first step in evaluating male infertility. Semen analysis can also be used to: confirm the absence of sperm in post vasectomy patients; confirm the presence of sperm after vasectomy reversal; and to determine the presence of sperm for certain legal purposes, such as rape.

There are a number of abnormalities of sperm morphology. Abnormal heads can include enlarged head, double head, round head, constricted head, amorphous head, pinhead, and acute tapering forms. There are also heads with abnormal numbers of vacuoles. Midpiece abnormalities include distended and thin midpiece regions. Abnormal tails include short tails, double, triple or multiple tails, coiled tails, broken tails, or absent tail. Cytoplasmic droplets are also seen in some specimens. These are large regions of cytoplasm just below the head assumed to represent failure of complete sperm maturation or a sign of either toxicity or oxidation. There have also been reports that cytoplasmic droplets may be artifacts from the fixation and staining for morphology analysis.

The sperm in the center has both an enlarged head and a split tail.

The sperm in the center of this field is an example of a pinhead. The head is oblong in shape and much smaller than the normal sperm.

A double-headed sperm is seen in the center of this slide. The lower of these two sperm heads has a good acrosome.

A sperm with a tapered head is seen in the center of this field. The head, which is larger than a normal head, is somewhat rounded and tapers toward the tail.

Normal sperm morphology requires that the head tail and midsections of the sperm be normal. The heads of normal mature sperm are slightly oval in shape and measure approximately 4.0 to 5.5µm in length and 2.5-3.5µm at the widest part. The acrosomal region should be 40-70% of the head area. Normal midsections are tapered. They do not bulge out or contain extra cytoplasm. Sperm have a long tail which is slightly thicker near the head than at the end. Sperm tails measure about 50µm. Normal sperm are seen on the right (a) front view, and (b) lateral view.

Appearance of sperm with abnormal tails: a: short tail b: double tail c: multiple tail d: coiled tail wound around the head

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.

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.

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.

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.

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).

A sperm count is considered normal if it is over 20 million sperm/ml. Although lower counts are considered abnormal by World Health Organization (WHO) standards (see the WHO manual, 1999), it is sometimes possible for men with significantly reduced counts to father a pregnancy.

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.

To verify the absence of sperm after vasectomy, two successive negative specimens are ideally required. If no sperm are seen on the initial undiluted count, the specimen should be centrifuged to concentrate any cellular material, and re-examined.

The picture on the right shows the counting chamber of the Neubauer hemacytometer. This counting method is used to count many types of cells. To use this chamber for counting sperm the specimen will usually need to be diluted. Proper loading of the hemacytometer is also important for accurate sperm counts to be obtained.

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.

Once the microscopic examination is completed, it is important to decide whether the results are normal or abnormal. Correlation involves comparing the microscopic findings with the macroscopic findings. If the results are consistent with each other, the urinalysis may be reported. If a discrepancy exists, the microscopic results cannot be reported. The findings that do not correlate must be repeated. The following table illustrates results which may be found together in a urinalysis. Microscopic Macroscopic Casts (may be accompanied by mucous) Possible positive protein reaction White Blood Cells (bacteria may accompany WBCs in microscopic) Possible positive protein reaction Possible alkaline pH (fresh) Possible cloudy urine Red Blood Cells Possible positive blood reaction Possible positive protein reaction Possible negative blood reaction (if only a few RBCs are seen) Possible cloudy urine Possible red or brown urine Bacteria (may be accompanied by WBCs) Possible alkaline pH (fresh) Possible positive protein reaction Possible cloudy urine Possible positive nitrite reaction Yeast (may be accompanied by WBCs) Possible glucose Possible cloudy urine Crystals Should suggest approximate pH Possible cloudy urine Possible high sp. gravity Trichomonas Possible cloudy urine due to increased WBCs and mucous Report the microscopic findings if they correlate with the macroscopic. Report common crystals if requested or when an unusual number of one type is present. Do not report abnormal crystals unless confirmed by further tests and pathologist. Do not report sperm.

Sperm may be present in urine sediment. Sperm have a characteristic oval body with a long thin tail and are 50 microns in length.