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How is normal sperm detected?
A recent history of proven fertility (having achieved a pregnancy) or a history of fertilization at In Vitro Fertilization are powerful indications that the sperm can act (fertilize an egg) normally.
The semen analysis is the best lab test that we currently have available for the evaluation of sperm. The three most commonly used parameters of the test are the sperm concentration (how many sperm are present per unit volume), the morphology (percentage of the sperm with a normal shape) and the motility (percentage of sperm that are moving, occasionally with some further characterization of the quality of motion).
A single isolated poor quality semen analysis is not necessarily ominous. The same man may produce two samples within the same week under ideal conditions for analysis and the quality of the sperm can be very different. Research has also demonstrated that the same sperm sample tested at multiple labs yields large variations in all of the parameters including the concentration. Therefore, the initial evaluation of the semen should most likely include two independently timed semen analyses (within 1-2 weeks) if the initial analysis is abnormal. If the initial semen analysis is normal then there is no reason to repeat it.
Spermatogenesis, spermiogenesis and the storage of sperm in the epididymis occur over relatively long periods of time. The sperm produced by ejaculation today started its course of development about 3 months ago. Thus, if the repeat semen analysis confirms an abnormal result then a transient insult (such as a viral infection or fever) can be essentially ruled out (as the cause of the abnormality) by checking a semen sample a few months later (to allow submission of a “new sample” created under different conditions). If the initial analysis is very abnormal (such as azoospermia or severe oligospermia) then treatment options do not need to be delayed in order to reevaluate in 3 months.
Once there is a relatively consistent set of semen analyses, the question of interpretation becomes important. Unfortunately, there are no universally accepted reference ranges for semen analysis. In Monmouth and Ocean counties of New Jersey, I am familiar with the values considered normal on semen analysis at 7 medical centers. These normal ranges vary considerably from one another. Thus, if the same semen sample readings are reported by 2 different local medical centers one center may report the analysis as normal while the other might report it as abnormal. This can lead to confusion and frustration. I prefer to use the World Health Organization (WHO) guidelines and reference ranges for normality since the American Society also supports them for Reproductive Medicine.
The semen analysis can provide a great deal of information. It should be routinely ordered during the evaluation for all infertile couples.
The collection of the sample is important. Basic instructions include:
- Call the office to schedule the semen analysis to avoid delays in evaluating the sample.
- Sexual abstinence for at least 2 days but not longer than 7 days before obtaining the sample.
- Obtain the sample by manual masturbation only. There should be no vaginal, oral or anal contact with the penis since this may result in a poor result. Avoid using lubricants since they may be toxic to sperm.
- Collect the entire semen specimen in a warm, clean, wide mouthed, plastic or glass container. It is very important to obtain the whole specimen. Also, please check to be sure the top is properly fastened to prevent spillage.
- Label the specimen with both names (of partners), the period of abstinence, the date, and the time of collection.
- The specimen must arrive at the lab within about 1 hour of collection and should be protected from temperature extremes (cold or warm)
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The semen analysis supported by the WHO includes the following normal ranges:
1) Volume:
Normal is 2-5 mL. Less than 2 mL might be due to incomplete collection. Historically, a low volume has been thought to be associated with decreased numbers of sperm able to swim into the cervical mucus. This has recently been questioned. A large volume of sperm may result in low concentration (since the total number of ejaculated sperm are in a larger volume). Thus, there might be decreased numbers of sperm able to move into the cervical mucus at the sperm-mucus interface.
2) PH:
Normal is 7.2-7.8, which is alkaline. A higher pH is associated with an infection in the prostate. The pH of the vaginal tract is low (acidic), about 3-4, while the pH of the pre-ovulatory cervical mucus is generally greater than 7. Sperm in the vagina does not last longer than 1-2 hours. In the pre-ovulatory mucus sperm can often survive for 2 or more days since the pH and other mucus characteristics are friendly to sperm.
3) Concentration:
Greater than 20 million per milliliter (mL) is normal. There have been several recent articles claiming to document a decrease in the sperm counts of men. This normal value was determined in the early 1950s and has not been changed since that time. Many experts in male factor infertility now believe that only very low concentrations, such as 5-10 million per milliliter, accurately reflect a decrease in concentration that is important for fertility. Since the low normal volume is 2 milliliters, a normal number of sperm per ejaculate is 40 million sperm (20 million per milliliter times 2 milliliters).
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4) Motility and progression:
The WHO divides motility into nonmotile, nonprogressive but moving, slow but linear or nonlinear, and rapid linear movement. Greater than 50% of sperm showing either slow but linear, nonlinear or rapid linear movement is normal. Also, greater than 25% showing rapid linear movement is normal. Poor motility may be able to be enhanced using chemical agents similar in structure and function to caffeine.
5) Morphology:
Greater than 30% normal forms is normal. Until a few years ago, the WHO required 50% normal forms to be considered normal. A “strict morphology” which excludes any sperm with even minor abnormalities from being considered normal was developed by a researcher named Thinus F. Kruger from South Africa while working with the infertility group at the Jones Institute for Reproductive Medicine in Norfolk Virginia. Using the strict morphology if greater than 14% normal forms are identified then this is associated with a normal fertilization rate (70-80%) at In Vitro Fertilization, if 4-14% normal forms are identified there is a proportionate decrease in fertilization, and if less than 4% normal forms are identified then there is only a poor (7-8%) fertilization rate at IVF. Although advocates of the strict morphology claim it to have good predictive value in terms of fertilization at IVF, it has not been widely accepted by the infertility community as a standard test (at least in 2002).
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6) Round cells or white blood cells (WBCs):
A count of greater than 1 million per milliliter is abnormal. It is not possible to distinguish immature sperm from WBCs without staining the cells, therefore, most labs report out the concentration of “round cells” with the understanding that the identity of these cells has not been determined. If these cells represent WBCs and if these cells are persistently elevated in concentration, then it suggests an infection. The usual location of these asymptomatic infections is the prostate gland. These infections often are difficult to treat since the blood supply to the prostate is poor, so antibiotics are usually given for several weeks.
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7) Agglutination of sperm (when motile sperm stick to one another), appearance of the semen (color, time to liquefaction, presence of streaks or grains), consistency (an estimate of viscosity), and antibody testing
are other components of the WHO’s basic semen analysis.
Sperm Function Tests:
A good sperm function test with high positive predictive value and low negative predictive value would be useful. Unfortunately, there are no really good sperm function tests available. Semen analysis is not a sperm function test. The semen analysis tells you that there is what seems to be a reasonable number of sperm, the sperm appear to be moving well and they are normally shaped. The strict morphology of Kruger approaches a sperm function test but has not been widely accepted as such.
A sperm function test would be able to identify sperm that is able to “do its job,” which in a reproductive sense is to fertilize an egg. A great looking sperm that appears to move well on semen analysis may not fertilize an egg while another less attractive sperm may reliably be able to accomplish fertilization. A sperm function test would use fertilization or pregnancy to judge outcome.
The sperm penetration assay (SPA) has been proposed as a sperm function test, and in fact, was widely accepted until recently. The SPA determines the frequency with which a “sample sperm” penetrates hamster eggs compared to the frequency of fertilization with “known fertile sperm.” To allow the eggs to be fertilized by another species’ sperm the outer shell (zona pellucida) of the egg is removed by chemical digestion prior to the test. Additionally, the sperm must undergo the process of capacitation that enables it to undergo the acrosome reaction.
In the standard SPA, 16% of men with no fertilization of hamster eggs have been reported to achieve human pregnancies. This high false negative rate is one factor that has led to the decline in popularity of this test. In an attempt to reduce the false negative rate sample sperm and donor sperm are often pre-treated with one of a number of chemical agents (follicular fluid, test yolk buffer, calcium ionophore) that enhance capacitation. Ongoing problems with the test such as large changes in assay results following small changes in assay conditions have limited its acceptance.
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Less popular sperm function tests have included:
(1) The hemizona assay test:
Human egg “shells” or zona pellucidae are divided into halves and the binding of sample sperm is compared to the binding of known fertile sperm on zona from the same egg. The almost complete lack of available fresh human eggs for experimentation has largely been responsible for the lack of acceptance of this test
(2) The in vitro sperm penetration of mucus test:
Sperm is placed adjacent to mucus on a microscope slide and the progression into the mucus is monitored. Bovine (cow) cervical mucus is available and has been proposed to allow standardization. This test became more popular as a means of assessing abnormal post coital tests prior to the wide acceptance of intrauterine inseminations
(3) Sperm motility using sperm quality analyzers:
Electro-optical techniques and computer assisted techniques of assessing sperm motility have been reported to correlate to fertilization rates at IVF. Lateral head displacement is thought to be a good prognostic indicator. The general lack of standardization and interpretation of these interesting tests has restricted their use largely to experimental settings
(4) The hypoosmotic swelling test:
Placement of sample sperm into a solution (of fructose or sodium citrate) of low concentration (hypoosmotic) results in the swelling and coiling of the sperm’s tail as it takes up water. Due to the lack of standardization and predictive value of this test it has remained of low clinical value
(5) Seminal ATP concentration:
The adenosine triphosphate level in sperm has been proposed as a discriminator of fertile sperm. A large multicenter WHO study failed to find a reasonable predictive value for semen ATP concentrations when there was no recognized female factor and the sperm concentration was greater than 20 million per milliliter
(6) Acrosome reaction:
The acrosome reaction occurs at or near the egg’s shell (zona pellucida) and examination of sperm samples to determine the percentage of sperm having undergone this reaction has been suggested as a screen for sperm function. In reports correlating IVF fertilization with the initiation of the acrosome reaction only very small differences in the percentage of acrosome reacted sperm was seen between groups with differing fertilization rates. This lack of predictive value and the expensive nature of this specialized test have limited its use clinically
(7) Acrosin measurements:
Acrosin is a proteolytic enzyme within the acrosome of the sperm head and is possibly important in aiding the sperm through the zona pellucida. A low level of acrosin has been correlated with deminished fertility. Research on this test is active and hopefully will yield a useful test in the next few years.
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