Semen composition and analysis (animal, human), related tests

What is semen?
Semen is composed of spermatozoa (sperm), produced in the seminiferous epithelium of the testis, and seminal plasma, the components of which are secreted by the excurrent duct system and accessory sex glands. When a spermatozoon is released from the seminiferous epithelium the major structural elements are in place, but additional changes are induced by exposure to sequential milieus provided by the epididymis and mixture with fluids from the accessory sex glands at ejaculation. Typical spermatozoa of the rat, human and stallion are shown in Fig. 1 and the important elements of a spermatozoon are depicted in Fig. 2. A partial list of spermatozoal attributes essential for fertility is presented in Table 1. Collectively, these attributes depend on the normal development and function of the genomic package, the mitochondria, dense fibers and microtubular elements of the axoneme, the acrosome and enzymes therein, and the multi-compartmentalized plasma membrane.

Seminal plasma is the fluid portion of an ejaculum, but only one of several distinctly different fluids to which sperm are exposed. Spermatozoa are transmitted from the seminiferous epithelium in a fluid milieu, and the solutes therein are removed and replaced within the efferent ducts and epididymis. Ultimately, sperm in cauda epididymal fluid are conveyed through the vas deferens at the time of ejaculation and mixed with fluids from the accessory sex glands, namely the prostate gland, vesicular glands (i.e., seminal vesicles), and bulbourethral glands. Some species have a complete array of accessory sex glands, including the above three types. Other species lack the bulbourethral glands or vesicular glands. Certain proteins and other molecules in the secretion of one or more accessory sex glands are identical to some components of cauda epididymal fluid, or even blood plasma, and others are unique products of that gland. Thus, seminal plasma includes a broad spectrum of chemical constituents contributed by the epididymis and the accessory sex glands.

The relative contributions of the epididymis or different accessory sex glands to the seminal plasma of a given ejaculum are dependent on many factors including the interval of sexual abstinence, duration of foreplay, pathophysiological processes in the male, and the species. Because semen is a mixture of spermatozoa and fluids moved by emission from the cauda epididymidis and vas deferens with fluids from the accessory sex glands, the sperm to fluid ratio is quite variable. The more important attribute is the total number of normal sperm in an ejaculum rather than the concentration of sperm per unit volume. For similar reasons, in analyses of constituents of seminal plasma, the total amount of a component should be considered in parallel with its concentration. A human might ejaculate 40-300 million spermatozoa, not greatly different from the number ejaculated by a rabbit (100-300 million), but substantially less than a dog (0.2-2 billion) or horse (5-25 billion).

What is the goal of seminal analysis?
For a clinician, evaluation of seminal quality is Iinked with a desire to predict potential fertility, identify causes of infertility, or detect changes in potential fertility. The clinician is concerned with minimal requirements to achieve fertilization or contraception. For an epidemiologist or toxicologist, seminal evaluations are the basis of assessing hazards in the workplace, environmental factors, or risk assessments relating to drugs and chemicals. Detection of a significant probability of reduced fertility in a population is more important than accurate prediction of fertility for an individual. For the animal breeder, the primary goal is to determine which male(s) will be the most fertile of genetically superior sires. Evaluations of sperm quality and estimation of potential fertility are the basis for management decisions which might lead to production of several hundred thousand offspring from an individual sire. For each application, the implied goal is to predict accurately the potential fertility of a seminal sample from an individual male.

Unfortunately, this goal is not easily achieved. Success in predicting fertility is limited by features of spermatozoa, the process of fertilization, and approaches used for evaluation in vitro of seminal quality. Also, spermatozoal attributes necessary for fertilization will depend on the methodology used to join the gametes, i.e., copulation or in vitro fertilization; on prior history of the sperm, i.e., freshly ejaculated sperm or frozen-thawed sperm; and on female factors, i.e., age or uterine and tubal environments.

Sometimes the conclusion from a seminal analysis is obvious. When the semen analysis reveals azoospermia, no progressively motile spermatozoa, or a high proportion of morphologically abnormal spermatozoa, the fertilizing potential of the individual is poor. In most cases the challenge is more complex. The goal of a clinician or animal breeder is to predict correctly that a given male probably will be infertile or will be reasonably fertile, relative to the average value for males of that race or species, or that a given seminal sample will provide fertility similar to that previously obtained with other samples from the same male. As contrasted to lack of fertilizing capability, considered above, accurate prediction of high fertilizing capability is extremely difficult, or impossible, because a spermatozoon must retain function of each of a number of essential attributes (see Table 1) to be capable of fertilizing an oocyte. It follows that a number of spermatozoa in a sample could be incapable of fertilizing an oocyte, each for a different reason. Limitations of current approaches for evaluation of seminal quality provide great opportunity for individuals intrigued by investigating male reproductive function.

How is semen evaluated?
Traditionally, evaluations of seminal quality, regardless of species, include measurement of seminal volume, determination of spermatozoal concentration and, by multiplication (volume x concentration), calculation of the total number of spermatozoa in an ejaculum. This provides quantitative information which, with knowledge of the interval since the previous ejaculations by that male, and with information on testicular volume, is an indication of the capability of that individual's testes to produce sperm. Absence of spermatozoa in an ejaculate could be evidence of retrograde ejaculation, blockage of excurrent ducts, or testicular failure. There is no cut-off for the total number of sperm in an ejaculum below which fertilizing potential is reduced or eliminated. Males of most species, but less so for humans, typically ejaculate a number of sperm far in excess of that necessary for maximum fertilizing potential when deposited in the vagina or uterus by copulation. For many species, <1% of the number of sperm in a typical ejaculation will result in maximum fertility when sperm are deposited by artificial insemination, provided the sperm are of good quality.

Quality traditionally is considered in terms of the percentages of progressively motile sperm or morphologically normal sperm. Until recently, both were subjective evaluations and influenced by substantial observer bias. Despite these problems, the visual assessment of sperm motility and morphology is the standard method used by most clinical andrology laboratories. Computerized image analysis systems are now available for determining both percentage of motile sperm and the distribution profiles for velocity or other kinematic attributes of individual cells. Relatively simple imaging systems for objective evaluation of sperm morphology are being introduced, but have not yet gained wide acceptance.

Functional tests are used to further define quality of a seminal sample in an infertility practice or research laboratory. These include capability of the spermatozoa to undergo an acrosome reaction (spontaneously or stimulated), penetrate into a heterologous oocyte or bind and penetrate into homologous zona pellucida, swim through cervical mucus, undergo motility hyperactivation, or simply swim rapidly away from a population of immotile or slow sperm. Often one or more of these tests is supplemented by immunological tests to determine if the spermatozoa or seminal plasma contains auto-antibodies associated with reduced fertility. In a research setting, one might perform more detailed analyses for the amounts of certain enzymes normally present in sperm, or analyze the presence and surface distribution of glycoproteins thought to be involved in the fertilization process. Better predictions may be possible after image or flow cytometric analysis of permeability of the sperm plasma membrane, mitochondrial function, surface properties of the plasma membrane, and/or denaturation of nuclear proteins. Finally, it is increasingly obvious that peroxidation of lipids of the plasma and acrosomal membranes of spermatozoa is associated with decreased quality. The extent of lipid peroxidation can be quantified. Many of these analyses provide only a mean value for the population of sperm, rather than a distribution of values for the individual cells. Unfortunately, information is needed on how many cells "pass'' for the full set of essential attributes.

Applications of semen analysis
Infertility occurs in approximately 15% of all human couples. In general, 30% of these couples have a predominant male factor, 30% have a predominant female factor, and the remainder have factors in both or no demonstrable cause. Semen analysis is the first step taken to establish a diagnosis of male factor infertility, and is performed in the initial screening tests of an infertile couple. Because of large day to day variation in the quality of the semen from an individual, at least two, and preferably three, semen analyses at least a week apart are usually performed to evaluate the male partner of an infertile couple. In general, an analysis of human semen is regarded as normal if:

1) ejaculate volume is >/= 2 ml,
2) sperm concentration >/= 20 million/ml,
3) >/= 50% of the sperm are progressively motile, and
4) >/= 30% of the sperm are morphologically normal (WHO, 1992).

These assessments are performed in an andrology laboratory, usually by visual examination using a light microscope. The diagnosis is based on the semen analysis together with information from a physical examination and medical history. If a patient has azoospermia (no sperm) or very severe oligozoospermia (less than 5 million/ml), endocrine status is evaluated by measurements of serum concentrations of follicle-stimulating hormone, luteinizing hormone and testosterone. This helps in diagnosis of the underlying etiology and assessment of prognosis.

For >70% of the patients with >2-3 abnormal semen analyses, no specific cause of abnormal testicular function can be identified. With these patients, specialized tests of sperm function (Table 2) focusing upon sperm surface proteins, autoantibodies against sperm, acrosome reaction, zona-free hamster oocyte penetration, human zona pellucida penetration and binding, and other functions may be required. Through a combination of these tests, more specific sites of dysfunction causing abnormality of the spermatozoa may be identified, and appropriate therapy planned. For instance, if investigations revealed that most spermatozoa in an ejaculum are unable to bind to the zona pellucida, the appropriate advice to the couple would be in vitro fertilization by subzonal injection of spermatozoa or direct intracytoplasmic injection of a spermatozoon into each oocyte. With these new andrologic techniques, fertilization and subsequent pregnancies have occurred in couples where the male partner has very severe sperm dysfunction.

In veterinary medicine and animal breeding, there are two general types of semen evaluation. The first is by a clinician evaluating a male for breeding soundness and potential fertility. Typically, testicular size is measured and a single seminal sample evaluated. Animals whose testes are substantially smaller than average values for males of the same breed and age are rejected, as are males whose semen contains <80% morphologically normal sperm or <50% progressively motile sperm. Failure to meet these criteria does not mean that the male is sterile, but rather that there is a reasonable probability the male will not be highly fertile.

The second type of evaluation is used in a facility housing males, such as bulls or boars, for wide-spread commercial distribution of their spermatozoa, or a facility where dogs, stallions or males of other species are brought to enable collection and cryopreservation of a limited number of doses for artificial insemination. To enable cryopreservation, the semen is mixed with an "extender'', a salt solution containing egg-yolk or milk proteins and sugars, and 4-12% glycerol, which is an essential cryoprotectant. Determination of the total number of sperm in the ejaculum is crucial to enable extension of the semen to a concentration which provides the requisite number of sperm in each insemination dose, and is linked with evaluations of sperm quality before processing. The extended semen is then sealed in a series of plastic containers which, for most species, are shaped like a drinking straw and contain 0.25, 0.5 or 4.0 ml; each straw is one insemination dose. Representative straws of cryopreserved semen are thawed and the cells evaluated immediately, and after several hours of incubation at 37 C, to establish the percentage of progressively motile sperm, their velocity, and often the percentage of sperm with a normal-appearing acrosome. Similar approaches also are utilized by individuals involved in preservation of sperm from humans or sperm from exotic animals, ranging from antelopes to zebras.

What of the future?
It is likely that approaches for seminal analysis in a clinical setting will remain similar to those in use today, with primary reliance on the manual counting of the number of spermatozoa and visual estimation of the percentage of motile sperm and the percentage of abnormal sperm. As a secondary screening, these classic tests may be augmented by binding or enzyme-linked assays measuring one or more attributes of the plasma membrane or a sperm enzyme. More importantly, it is likely that some secondary and most tertiary laboratories will have access to instruments which characterize multiple attributes on several thousand individual sperm representing the population. Flow cytometers now serve this purpose. New imaging instruments and techniques likely will be developed to evaluate motion and morphology of individual sperm in a wet preparation concurrently with multiple probe assessment of biochemical attributes. Such analyses would add data for 3 to 5 functional attributes to those for 2 or 4 selected attributes of sperm motion and morphology. These newer tests may be able to replace some of the biological tests currently being used such as the zona-free hamster oocyte penetration and human zona pellucida tests which are imprecise, time consuming, technically demanding and expensive. With appropriate selection of independent attributes essential for sperm to have fertilizing capability, improved prediction of fertility should be possible.

Suggested Reading

Amann RP. Can the fertility potential of a seminal sample be predicted accurately? J Androl 1989;10:89-98.

Amann RP, Hammerstedt RH. In vitro evaluation of sperm quality: An opinion. J Androl 1993;14:397-406.

Davis RO, Katz DF. Operational standards for CASA instruments. J Androl 1993;14:385-394.
Wang C, Swerdloff RS. Evaluation of testicular function. Bailliere's Clin Endocrinol Metab 1992;405-434.

WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. Cambridge, England: Cambridge University Press; 1992.