Seminal Fluid Analysis

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Seminal Fluid Analysis

Learning Objectives

Key Terms1⁎ *

Seminal fluid, or semen, is a complex body fluid used to transport sperm or spermatozoa. It is analyzed routinely to evaluate infertility and to follow up after a vasectomy to ensure its effectiveness. Other reasons for analysis include the evaluation of semen quality for donation purposes and forensic applications (e.g., DNA analysis, detection of semen). Familiarity with the male reproductive tract and its various functions facilitates understanding of the physical, microscopic, and biochemical abnormalities that can occur in semen.


Semen is composed primarily of secretions from the testes, epididymis, seminal vesicles, and prostate gland, with a small amount derived from the bulbourethral glands. The biochemical composition of semen is complex. Although the specific functions of some components (e.g., fructose) are known, the functions of others (e.g., prostaglandins) remain uncertain. The testes are paired glands suspended in the scrotum and located outside the body (Fig. 12.1). Their external location allows for the lower organ temperature necessary for sperm formation.

The testes perform both an exocrine function by the secretion of sperm and an endocrine function by the secretion of testosterone. These two functions are interdependent and are regulated by two pituitary hormones: follicle-stimulating hormone and luteinizing hormone. The cells responsible for these two functions are distinctly different. Sperm production is regulated by Sertoli cells in the seminiferous tubules, whereas production and secretion of the male sex hormone, testosterone, is the responsibility of the interstitial cells of Leydig, which are located in the interstitium of the testes, between the seminiferous tubules.

Sertoli cells of the seminiferous tubular epithelium have several functions. Because of their tight interconnections, they essentially form a barrier that separates the epithelium into two distinct compartments: the basal compartment (i.e., germ cell layer) and the adluminal compartment (i.e., epithelium nearest the tubular lumen). As this barrier, or gatekeeper, they limit the movement of chemical substances from the blood into the tubular lumen—playing a role in supplying nutrients, hormones, and other substances necessary for normal spermatogenesis. They also control the movement of spermatocytes from the germ cell layer into the adluminal compartment. Last, they continuously produce a fluid that carries the newly produced immotile sperm into the lumen of the seminiferous tubules and on to the epididymis.

The epithelium of the numerous coiled seminiferous tubules consists of Sertoli and germ cells. The undifferentiated germ cells (spermatogonia) continuously undergo mitotic division to produce more germ cells. At the same time, some of them move slowly toward the tubular lumen, changing in size and undergoing meiotic (reduction) division until they form spermatids. Fig. 12.2 depicts spermatogenesis in the seminiferous tubular epithelium with all stages of spermatogenesis depicted. Spermatogonia (germ cells) evolve into spermatocytes and then spermatids. With nuclear modification and cellular restructuring, spermatids ultimately differentiate into immotile sperm.

When Sertoli cells release sperm into the lumen of the seminiferous tubules, they are nonmotile and still immature. Luminal fluid from Sertoli cells carries the sperm into the tubular network of the epididymis, where they undergo final maturation and become motile. The epididymis also adds carnitine and acetylcarnitine to the lumen fluid. Although the exact function of these chemicals remains to be elucidated, abnormal levels of them have been associated with infertility. Other functions of the epididymis include the concentration of sperm by the absorption of lumen fluid and their storage until ejaculation. After a vasectomy, the epididymis is the site of leukocyte infiltration and phagocytization of accumulated sperm.

The epididymis ultimately forms a single duct that joins the vas deferens. The vas deferens is a thick-walled muscular tube that transports sperm from the epididymis to the ejaculatory duct, and the dilated end of the vas deferens is located inferior to the bladder. Secretions from the seminal vesicles are added at the ejaculatory duct. Both ejaculatory ducts then pass through the prostate gland and empty into the prostatic urethra along with secretions from the prostate. All structures preceding the prostate gland are paired (e.g., two ejaculatory ducts, two seminal vesicles, two testes).

The seminal vesicles and the prostate gland are considered accessory glands of the male reproductive system and are testosterone dependent. They produce and store fluids that provide the principal transport medium for sperm. Seminal vesicle fluid accounts for approximately 70% of the ejaculate and is high in flavin. Flavin imparts the characteristic gray or opalescent appearance to semen and is responsible for its green-white fluorescence under ultraviolet light.1 Another characteristic of seminal vesicle fluid is its high concentration of fructose, believed to serve as a nutrient for spermatozoa. The various proteins secreted by the seminal vesicles play a role in coagulation of the ejaculate, whereas the function of prostaglandins remains under investigation. (Prostaglandins were originally thought to be a prostatic gland secretion, hence their misnaming.)

Prostatic fluid secretions account for approximately 25% of the ejaculate volume. The principal components of this milky, slightly acidic fluid are citric acid; enzymes, particularly acid phosphatase and proteolytic enzymes; proteins; and zinc. Semen is unique in its high concentration of the enzyme acid phosphatase, hence acid phosphatase activity can be used to positively identify the presence of this body fluid. Proteins and some enzymes in prostatic secretions play a role in coagulation of the ejaculate, whereas the proteolytic enzymes are responsible for its liquefaction. Zinc is primarily added to semen by the prostate gland; however, the testes and sperm also contribute zinc. Semen zinc levels can be used to evaluate prostate function; a decreased level is associated with prostate gland disorders.

In summary, semen is a highly complex transport medium for sperm. The paired seminal vesicles and the single prostate gland are the major fluid contributors to semen. Sperm produced by the testes are matured and concentrated in the epididymis and make up only a small percentage of an ejaculate. Dilution of sperm by the relatively large volume of seminal fluid at ejaculation enhances sperm motility. Without adequate dilution, sperm motility is significantly reduced. The entire process of spermatogenesis and maturation (i.e., from primary spermatocyte to mature motile spermatozoon) takes approximately 90 days.

Specimen Collection

Because sperm concentration in normal seminal fluid can vary significantly, two or more samples should be analyzed to evaluate male fertility. Specimen collections should take place within a 3-month period and at least 7 days apart. Sexual abstinence for at least 2 days (48 hours), but not exceeding 7 days, should precede the collection. The patient collects the specimen through masturbation, and the entire ejaculate is collected in a clean, wide-mouth sterile plastic or glass container. Although some plastic containers are toxic to spermatozoa, others are not. Sterile urine specimen or similar containers are often satisfactory, but the laboratory must evaluate them before their use.2 The collection container should be kept at room temperature or warmed (to approximately body temperature) before the collection to avoid the possibility of cold shock to the sperm. The container can be warmed easily by holding it next to the patient’s body or under the arm for several minutes before the collection. This technique can also be used to control the temperature of specimens being transported in cold climates. Specimen containers and request forms must be labeled with the patient’s name, the period of sexual abstinence, and the date and time of specimen collection. The time of actual specimen collection is crucial in evaluating liquefaction and sperm motility.

During specimen collection, lubricants and ordinary condoms should not be used because they have spermicidal properties. For patients unable to collect a specimen through masturbation, special nonspermicidal (e.g., Silastic) condoms can be provided for specimen collection.

The collection of seminal fluid requires sensitivity and professionalism. Written and verbal instructions should be provided to the patient, as well as a comfortable and private room near the laboratory. If the specimen is to be collected elsewhere and delivered to the laboratory, clearly written instructions regarding specimen transport conditions must be provided. Specimens must be received in the laboratory within 1 hour after the collection, and they must be protected from extreme temperatures, that is, maintained at 20°C to 40°C.3 If these criteria are not met, the specimen will not be satisfactory for sperm function tests and an abnormally low sperm motility can result. Because the ejaculate differs in its composition, only complete collections are acceptable for analysis. Patient instructions must state this clearly, and patients should be asked whether any portion of the specimen was not collected. When a portion of the initial ejaculate is not collected, the sperm concentration will be falsely decreased and, owing to a reduction in prostate secretions, the pH is falsely increased and the coagulum will fail to liquefy. Conversely, when the last portion of an ejaculate is missing (primarily seminal vesicle fluid), the semen volume will be decreased, the sperm concentration falsely increased, the pH falsely decreased, and a coagulum will not form.

As with all body fluids, seminal fluid represents a potential biohazard and must be handled accordingly. Because seminal fluid can contain infectious agents such as hepatitis virus, human immunodeficiency virus, herpes virus, and others, all personnel must adhere to standard precautions (see Chapter 1) when handling these specimens.

Physical Examination


Normal semen is gray-white and opalescent in appearance. A brown or red hue may indicate the presence of blood, whereas yellow coloration has been associated with certain drugs. If large numbers of leukocytes are present, the semen appears more turbid with less translucence. When the specimen appears almost clear, the sperm concentration is usually low. Mucus clumps or strands can be present. Semen has a distinctive odor that is sometimes described as musty. Although infections in the male reproductive tract can modify this odor, a change is rarely noted or reported. Table 12.1 (and Appendix C) summarizes the semen characteristics (physical, microscopic, and chemical parameters) associated with fertility.

Table 12.1

Semen Characteristics Associated With Fertility
Parameter Reference Intervala Lower Reference Limitb
Physical Examination
Appearance Gray-white, opalescent, opaque  
Volume 2-5  mL 1.5  mL (1.4-1.7)
Viscosity/liquefaction Discrete droplets (watery) within 60 minutes  
Microscopic Examination
Motility 50% or more with moderate to rapid linear (forward) progression 40% (38-42)
Concentration 20 to 250 × 106 sperm per mL 15 × 106 sperm per mL
Morphology 14% or more have normal morphology 4% normal forms
Vitality 75% or more are alive 58% (55-63)
Leukocytes Less than 1 × 106 per mL  
Chemical Examination
pH 7.2-7.8 ≥7.2
Acid phosphatase (total) ≥200 U per ejaculate at 37°C (p-nitrophenylphosphate)  
Citric acid (total) ≥52 μmol per ejaculate  
Fructose (total) ≥13 μmol per ejaculate ≥13 μmol per ejaculate
Zinc (total) ≥2.4 μmol per ejaculate ≥2.4 μmol per ejaculate


aBased on the strict criteria evaluation recommended by the World Health Organization WHO (1999) for assessing sperm morphology for fertility purposes.

bThe one-sided 5th centile lower reference limit recommended by the WHO (2010) for assessing semen characteristics.

Semen is a homogeneous, viscous fluid that immediately coagulates after ejaculation to form a coagulum. Within 30  minutes, the coagulum liquefies (becomes watery). The actual time of specimen collection must be known to evaluate liquefaction. Although liquefaction can take longer, any delay beyond 60 minutes is considered abnormal and must be noted. Because complete liquefaction is necessary to perform analysis, semen specimens that do not liquefy completely can be chemically treated (see Appendix D, “Semen Pretreatment Solution”). After normal liquefaction, undissolved gel-like granules or particles can be present in the specimen, with a small amount considered normal.


The physical and microscopic analyses of seminal fluid should take place immediately after liquefaction or within 1 hour after collection (for specimens collected away from the laboratory). Specimen volume is measured to one decimal place (0.1 mL) using a sterile serologic pipette (5.0 mL or 10.0 mL). If a semen culture for bacteria is requested, the volume measurement should be performed first using sterile technique. Normally, a complete ejaculate collection recovers 2 to 5 mL of seminal fluid. Volumes less than and greater than this range are considered abnormal and have been associated with infertility.


After complete liquefaction, the viscosity of the semen is evaluated using a Pasteur pipette and observing the droplets that form when the fluid is allowed to fall by gravity. A normal specimen is watery and forms into discrete droplets. Abnormal viscosity or fluid thickness is indicated by the formation of a string or thread greater than 2 cm in length.3 A high content of mucus can increase the viscosity. Other conditions associated with increased viscosity include the production of antisperm antibodies and oligoasthenospermia (i.e., decreased concentration and motility of sperm).47

Grading viscosity varies among laboratories. Numeric terms can be used, with 0 indicating a normal, watery (i.e., forms discrete drops) specimen and 4 indicating a specimen with gel-like consistency.8 An alternate reporting format uses descriptive terms, such as normal, slightly viscous (thick), moderately viscous, and extremely viscous (unable to be aspirated into the pipette).

Microscopic Examination

As in other laboratory areas, standardization of procedures and techniques is necessary to enhance the precision and reproducibility of semen analysis. Once achieved, this standardization enables intralaboratory and interlaboratory comparisons of data. Appropriate quality control measures must also be in place whenever applicable. The World Health Organization (WHO) publication WHO Laboratory Manual for the Examination and Processing of Human Semen is an excellent and necessary reference for any laboratory performing semen analysis.3 Microscopic examination includes the determination of sperm motility, concentration, morphology, and viability; the concentration of other cells present; and the presence of sperm agglutination. Some laboratories use a single stain for the evaluation of several parameters, such as eosin-nigrosin stain for sperm vitality, morphology, and the identification of other cells, whereas others use different stains that specifically enhance each parameter to aid in the identification and evaluation of sperm and other cells.


Motility is one of the most important characteristics of sperm because immotile sperm, even in high concentrations, are unable to reach and fertilize an ovum. Traditionally, the evaluation of sperm motility has been assessed subjectively by experienced technologists. Today, computerized systems that use electro-optical techniques or videography have been developed for semen evaluation. This advanced technology enables objective evaluation of sperm motility and morphology; however, the cost of the equipment precludes many laboratories from acquiring it.

Without an automated system, sperm motility is evaluated subjectively and semiquantitatively using phase-contrast microscopy (brightfield microscopy can also be used with appropriate condenser adjustments). After complete liquefaction, the semen sample is mixed well to ensure homogeneity. A consistent volume of each specimen is evaluated by pipetting a fixed volume (e.g., 10 or 20 μL) of semen onto a microscope slide using a calibrated positive-displacement pipette. The sample is covered with a coverslip of predetermined size (e.g., 18 × 18 mm), and the slide is allowed to settle for about 1 minute before evaluation. To enhance the accuracy and precision of results, wet mounts of each sample should be prepared and evaluated in duplicate.

Because sperm motility is affected adversely by temperature, some laboratories control the temperature of the microscope slide at 37°C using an air curtain incubator.8 Others perform the analysis at room temperature (i.e., 22 ± 2°C).

Initially, each wet mount is screened to ensure uniformity in sperm movement throughout the preparation. Next, sperm motility is graded subjectively from 0 to 4 under ×200 (or ×400) magnification. Table 12.2 shows typical grading criteria used to evaluate sperm motility. Some laboratories use a manual cell counter and evaluate the motility characteristics in 100 sperm, whereas others grade the sperm encountered in 6 to 10 high-power fields (×400).

Table 12.2

Sperm Motility Grading Criteria
0 Immotile
1 Motile, without forward progression
2 Motile, with slow nonlinear or meandering progression
3 Motile, with moderate linear (forward) progression
4 Motile, with strong linear (forward) progression

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Oct 18, 2022 | Posted by in PATHOLOGY & LABORATORY MEDICINE | Comments Off on Seminal Fluid Analysis
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