Semen Analysis, Graff's Textbook of Urinalysis and Body Fluids, Atlas of Semen Analysis
When a man and a woman have difficulty conceiving a child, one or both of the pair may be infertile. Approximately 40% of infertility cases are due to disorders of the male reproductive system. A semen analy- sis may disclose one of these male disorders or rule out male infertility. The most common parameters evaluated during a semen analysis include semen volume, viscosity, pH, and sperm concentration, motility, viability, and morphology. Other indications for performing a semen analysis include determining the effectiveness of a vasectomy, rapecase forensic studies, sperm donor evaluation, and paternity cases.
SEMEN COMPOSITION
Semen consists of several fluids produced in various male reproductive organs. The slightly alkaline fluid from semi- nal vesicles comprises over half the volume of semen and contains citric acid, flavins, fructose, and potassium. These substances provide nutritional support for spermatozoa. Spermatozoa are formed in the testis and are stored in the epididymis and vasa deferentia. The process of spermatozoa formation is under control of various hormones, testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH). The prostate gland contributes a slightly acidic fluid containing acid phosphatase, citric acid, and proteolytic enzymes. These substances account for about 20% of the semen’s volume. The remaining reproductive organs, bulbourethral glands, epididymis, urethral glands, and vasa deferentia, contribute little additional volume to the semen. Figure 12-1 illustrates the male reproductive system. Upon ejaculation, the fluids from all of these sources form the mixture, semen.
Figure 12-1. Detail of the male reproductive system.
SPERM FORMATION
Spermatogenesis is the formation of spermatozoa in the Sertoli cells of the seminiferous tubules of the testis. Further maturation of sperm occurs in the epididymis. This approximately 74-day process involves several phases: spermatocytogenesis, meiosis, and spermiogenesis. Spermato-cytogenesis is a two-step phase in which spermatogonia undergo mitotic division and maturation into spermato- cytes. Meiosis is the specific type of cell division that results in haploid gamete cells. Spermiogenesis is the phase in which the gamete cell develops a flagellum and transforms from a spermatid into a spermatozoon. Figure 12-2 illustrates the process of spermatogenesis, whereas Figure 12-3 illustrates the stages of human spermatid transformation into spermatozoon
Figure 12-2. The process of spermatogenesis in the seminiferous tubules
SPECIMEN COLLECTION AND HANDLING
The preferred method of semen collection is by masturbation. This procedure ensures the opportunity to collect the entire ejaculate. Collection should be performed after a 48- to 72-hour continence (abstinence from sexual activity) to provide a specimen containing the most accurate sperm count and viability.
A private, comfortable room should be provided for specimen collection that allows for quick delivery of the specimen to the laboratory. Written and verbal instructions for the procedure should be provided. Specimen collection containers should be clean glass or plastic and have a wide opening. Specimens should not be collected in a condom as these often contain spermicidal compounds and lubricants that may interfere with laboratory tests
If the specimen must be transported from a site distant to the laboratory, it must be kept near body temperature and extremes in temperature must be avoided. Shortly after ejaculation, the semen coagulates because of the action of a clotting enzyme, formed in the prostate, on a fibrinogenlike precursor substance that is produced by the seminal vesicles. Liquefaction occurs within 30–60 minutes.
Ideally, the specimen should arrive in the laboratory as soon after collection as possible so that an accurate liquefaction time may be recorded. The specimen should be labeled with all patient information and time of collection. In addition, the patient should be asked whether any part of the specimen was lost during collection. This information is important to note because the highest concentration of sperm is usually found in the first part of the ejaculate.
Figure 12-3. Final stages of spermatogenesis.
(A) Immature spermatid with round shaped nucleus,
early signs of acrosome and tail development;
(B) Immature spermatid with formation of the acro-
some, nuclear condensation developing tail piece;
(C) Spermatid with acrosome covering the nucl
developing middle piece, and shrinking cytoplasmic
membrane; (D) Mature spermatozooneus,
MACROSCOPIC EXAMINATION
Liquefaction. Once the specimen arrives in the laboratory, it is observed for liquefaction time. If coagulation did not occur, it should be reported. A noncoagulating semen in cases of azospermia may indicate a congenital bilateral absence of the vas deferens and seminal vesicles. If delivery of the specimen to the laboratory took longer than 30 minutes after collection, the specimen may already be liquefied, and there is no opportunity to notice whether it coagulated properly. Normal liquefaction occurs between 30 and 60 minutes. Liquefaction times beyond 60 minutes are considered abnormal. Specimens that do not liquefy must be treated with amylase or bromelin to break up mucus in order to obtain accurate sperm counts.5 The addition of alpha-amylase solution to semen will not alter motility.
■ Appearance. Semen is opaque and can exhibit several normal colors. Typical colors include gray, white, and light yellow. The higher the flavin concentration of semen, the darker the yellow color may be. A deep yel- low color has been associated with certain drugs.
Brown or red-colored semen may contain blood. A highly turbid semen specimen usually contains leukocytes and may indicate a reproductive tract infection or inflammation.
■ Volume. Semen volume is measured by using a serological pipette, or small graduated cylinder. Volume is recorded in milliliters to one decimal place (0.1 mL). Normal semen volume ranges from 2 to 5 mL for a complete ejaculate. Volumes both lower and higher than this range have been associated with infertility.
■ Viscosity. Viscosity may be assessed while measuring specimen volume or when pipetting the specimen for other tests. Normal semen is slightly viscous and
Figure 12-4. Normal semen viscosity test.
dispenses drop by drop. Increased viscosity is demonstrated by the formation of a string of fluid as the specimen is dispensed from a pipette.5 Figure 12-4 demonstrates normal semen viscosity. Semen with abnormal viscosity may be watery.
MICROSCOPIC EXAMINATION
SPERM CONCENTRATION
Automated methods for counting sperm are available and discussed in Chapter 15. Most laboratories use manual hemocytometer counting techniques as outlined in Chapter 8. A manual dilution of 1:20 using distilled water to immobilize sperm may be used. A platelet Unopette dilution of 1:100 is used by some laboratories as an alternative to manual dilutions with a volume displacement pipette.
Professional judgment should be used when determining the area to count on the hemocytometer. The center square millimeter may be sufficient for accurate counts when the sperm concentration is high. Otherwise, it may be necessary to count the four outside square millimeters or even one entire side of nine square millimeters for accurate counts when the sperm concentration is low.
Following the rules for hemocytometer counting needs professional judgment as well, because sperm do not always lie entirely inside or outside the counting area. What usu- ally works best is to determine the placement of the sperm heads on the hemocytometer grid rather than the tails. Figure 12-5 illustrates the inclusion criteria of counting sperm with heads that touch the upper and left borders of the counting grid and exclusion of sperm with heads touch- ing the bottom and right borders
Figure 12-5. Inclusion criteria of counting cells. Count cells
(sperm heads, not tails) that touch the upper and left boarders
of the counting grid. Do not count cells (sperm heads, not tails)
that touch the lower and right boarders of the counting grid.
Count only complete sperm.
When using a Neubauer hemocytometer, the simplified formula allows for rapid calculation of sperm concentration: C = NxDx10/A, where C is the concentration, N is the number of sperm counted, D is the dilution factor, and A is the area in square millimeters (not number of squares).
For example, if the number of sperm counted in nine square millimeters on a 1:100 dilution is 25, the calculation is (25x100x10)/9 = 2778/mm3. Sperm concentration is often reported in number per cubic centimeter (cc) or milliliters (mL). Therefore, multiplying by 1000 is necessary to convert the count to the correct unit. In this example, the final count is 2,778,000/cc
.
Normal sperm concentrations have been reported to range between 20 and 250 million per milliliter. Oligospermia is a sperm count less than 20 million per milliliter. Azoospermia is the complete absence of sperm. Sperm counts less than normal may be due to chromosomal disorders, ductal obstruction, drugs, gonadotropin deficiency, hyalinization of the seminiferous tubules, maturation arrest, pituitary disorders, radiation, renal failure, and Sertoli-cell-only syndrome. Hormone tests, discussed later in this chapter, may help differentiate among the various causes of azoospermia.
Fertility, however, is possible at counts as low as 1 million sperm per milliliter. Of greater importance in the analysis of semen for fertility evaluation are other microscopic tests. Tests that have a greater bearing on fertility include morphology, motility, penetration, and viability.
MOTILITY
Fertilization of an ovum is dependent on the ability of sperm to reach and unite with it. Motility should be evaluated within 1 hour of specimen collection, because motility will decrease over time. One way to evaluate sperm motility
Figure 12-6. Wet mount of semen. Many sperm are present (450x)
is to place a small drop of liquefied semen on a prewarmed slide and coverslipped. Observation of sperm movement is best performed on high dry (45x). Some laboratories prefer to use phase contrast microscopy while evaluating sperm motility2; however, bright light microscopy with the condenser turned down is adequate. Figure 12-6 shows the appearance of sperm on a wet mount for the evaluation of motility. Figure 12-7 shows a semen wet mount that includes a red blood cell and white blood cell
The movement of sperm is evaluated and may be subjec- tively estimated or counted into three categories. These cat- egories may be called high-motile, low-motile, and nonmotile; or progressive, nonprogressive, and nonmotile. Some laboratories may use as many as five categories: nonmotile, nonprogressive, slow nonlinear progression, moder- ate linear progression, and strong linear progression. Some laboratories report the percent of sperm in each category, whereas others report only the percent of motile sperm. At least 80% of the sperm demonstrate some forward progress in a normal semen sample.
An alternate method used by some fertility clinics is to evaluate sperm motility from a video recording that is
Figure 12-7. Wet mount of semen. Several sperm can be seen
along with a red blood cells (R) and a white blood cell (W) (450x)
played back with a grid overlaying the monitor. This method also provides for reevaluation should a motility result come into question. More recent use of technology for sperm evaluation includes the use of high-resolution video photography in combination with computer programs that can calculate velocity, linear progression, and motility efficiency and measure patterns of sperm motion.
Motility can be effected by temperature and other factors, such as the presence of antisperm antibodies. Therefore, a viability test should be performed, especially if a high number of nonmotile sperm are present.
AGGLUTINATION
Agglutination may be observed while evaluating a wet mount of semen for sperm motility. A few clumps of sperm or sperm sticking to mucus or other cells can normally be seen in a semen sample. However, true agglutination is present if sperm are distinctly clumped head to head or tail to tail, which may indicate the presence of antisperm antibodies. Both IgG and IgA antibodies have been found in the semen of some men with reduced fertility whose sperm demonstrate agglutination. Confirmation with immunologic tests can help determine the specific type of antibody.
These tests are discussed later in this chapter.
VIABILITY
Determining whether nonmotile sperm are viable or nonviable is important in establishing a cause for infertility in males. The membranes of dead sperm are damaged and can easily take up eosin stain. The membranes of viable sperm remain intact and do not allow eosin stain to penetrate, leaving the sperm colorless (they will appear white). Eosin stain can be used alone or in conjunction with nigrosin stain. Nirgrosin provides a dark background against which the red-colored dead sperm and the white or colorless sperm can be visualized. Figure 12-8 shows the white appearance of the colorless viable sperm, whereas Figure 12-9 shows the red-colored staining of nonviable sperm. At least 100 sperm heads are counted into two categories: red dead and white viable. The percent of viable sperm is reported. Viability and motility do not always correlate. Sperm that are nonmotile may be alive but may have defects of the tailpiece. However, the proportion of motile sperm should not be higher than the proportion of viable sperm. Dead sperm cannot demonstrate motility. Normally, 75% of sperm are viable.
PENETRATION
Even though sperm may be viable and motile, a couple can still be experiencing male infertility problems if the sperm are incapable of penetrating through cervical mucus. Some
Figures 12-8. Viable sperm do not take up the eosin stain
and remain colorless, thus appearing white (eosin/nigrosin stain 1000x).
physicians consider penetration to be the most important parameter to evaluate in the investigation of infertility.
A postcoital cervical mucus specimen is observed by the physician in his office. The presence of many motile sperm contained in this specimen is evidence for normal penetration ability.
A procedure for evaluating sperm penetration that is used in the laboratory setting involves the use of bovine cervical mucus. Bovine mucus is commercially available and is contained frozen in flat glass capillary tubes, which are scored at one end. These tubes are thawed upright with the scored end up to assist with the elimination of air bubbles from the test area. Once thawed and opened at the score marks, the opened ends of the tubes are placed in a sample cup that contains 0.2 mL of fresh semen. This set-up is allowed to incu- bate at room temperature for 90 minutes. Placing the pene- tration test set-up inside a closed cabinet helps keep it free
Figures 12-9. Nonviable sperm take up the eosin stain and appear
various shades of red (eosin/nigrosin stain 1000x)
from drafts that may alter its temperature. After incubation, the capillary tubes are removed from the specimen, placed on a ruled slide, and observed microscopically. The distance obtained by the vanguard sperm (the sperm that traveled the greatest distance) is recorded for both tubes and the average calculated. Normal sperm should be able to penetrate bovine cervical mucus to at least a distance of 30 mm
SPERM MORPHOLOGY
Sperm morphology is evaluated by preparing a stained smear of semen and counting and categorizing all forms of sperm seen. The smear may be made by placing a drop of semen on a slide, placing another slide on top, and pulling them apart in opposite directions. The smear may be fixed with a cytology fixative and then stained with Papanicolaou stain. Giemsa or Wright stain may also be used. Sperm morphologies are classified by counting 100–200 sperm using oil immersion. Values for the minimum number of normal sperm vary according to individual laboratories’ evaluation criteria. Minimum normal forms for sperm morphology may be 30 to 70%.
Normal Sperm
A normal spermatozoon has a flattened oval head and an elongated tailpiece. The head is about 4–5 m in length and 2–3 m in width and contains a nucleus that comprises 65% of the head. The acrosomal cap may be visible on the stained smear and contains enzymes that assist the sperm’s penetra- tion of the ovum. The head appears oval when viewed from the front and appears pyriform when viewed from the side. The side view may be mistaken for an abnormal form by inexperienced observers. The tailpiece is about 50–55 m in length and varies in thickness from 1 to 0.0.1 m (neck to tip). Four distinct regions comprise the tailpiece: neckpiece, midpiece, mainpiece, and endpiece. A cellular membrane, plasma lemma, surrounds the entire spermatozoon. Figure 12-10 illustrates the features of a normal spermatozoon. Figures 12-11–12-13 (page 274) show normal sperm stained with Papanicolaou stain. Notice the appearance of sperm observed with a side view.
Abnormal Sperm Morphology
Abnormal sperm morphology occurs as an anomaly of either the head or the tailpiece, or both. Head anomalies include acrosomal abnormalities, constricted heads, double-headed or double-nucleated heads, enlarged or pinheads, nuclear abnormalities, and vacuolation. Tailpiece anomalies include coiled tailpiece, cytoplasmic extrusion mass, lengthened or bent neckpiece, midpiece abnormalities, multiple tails, and variation in tail length. In addition, immature forms of sperm may be present.Figures 12-14–12-25 (page 274–277) illustrate some of the more
Figure 12-10. Features of a normal spermatozoon.
common sperm morphology anomalies. Figure 12-26 shows various immature spermatids that may be seen in semen samples.
Other Cells and Microscopic Findings
Semen may contain cells other than spermatozoa. Immature cells may be present in semen due to premature exfoliation from the seminiferous tubules. In addition, greater than 2% immature spermatozoa may be present during
Figure 12-11. Normal sperm (Papanicolaou stain, 1000x).
Figure 12-12. Normal sperm. The arrow points to a sperm
observed with a side view (Papanicolaou stain, 1000x).
Figure 12-13. Normal sperm, side view (Papanicolaou stain, 1000x).
Figure 12-14. Double-headed sperm. Notice the excessive
cytoplasmic membrane in image C (Papanicolaou stain, 1000x).
Figure 12-15. Double-tailed sperm. Arrows point to both tails
(Papanicolaou stain, 1000x).
testicular stress, after a viral infection, and as a result of heavy alcohol consumption. Immature spermatozoa may resemble leukocytes and must be properly identified to avoid misdiagnosis of infection. Urethral epithelial cells and white blood cells are usually present in low numbers and can be seen during the hemocytometer count and on morphology smears. An increased number of neutrophils indicates an infection or inflammatory process. Red blood cells are not normally present in semen and should be reported if seen. In addition, bacteria are not a normal finding in seminal fluid and should also be reported
CHEMICAL ANALYSIS
■ pH. The pH of semen should be measured within an hour of collection because semen can become either more acidic (lactic acid production with high sperm
Figure 12-16. Coiled-tailed sperm. Tails may coil completely
around the head as seen in image A (Papanicolaou stain, 1000x).
Figure 12-17. Flat-headed sperm. One normal sperm is seen among those
with flat heads, indicating the absence of the acrosomal cap (Papanicolaou stain, 1000x).
Figure 12-18. Various sperm head sizes. A. Normal sperm. B.
Large head. C. Small or pinhead (Papanicolaou stain, 1000x).
Figure 12-19. Normal sperm shown with sperm at arrow that has a constricted
(or pinched) head and excessive cytoplasmic membrane (Papanicolaou stain, 1000x).
counts) or more alkaline (loss of CO2 over time) as the specimen ages. Nitrozine paper is the simplest way to measure semen pH. The pH of fresh semen normally ranges from 7.2 to 7.8.5 Acidic semen pH may be seen in congenital aplasia of vasa deferentia and seminal vesicles, while a male reproductive tract infection produces an alkaline pH.
■ Acid Phosphatase. Semen acid phophatase is used to evaluate the secretory function of the prostate. Normal levels of acid phosphatase are equal or greater than 200 units per ejaculate. In addition, determining the presence of acid phosphatase in vaginal fluid, skin washings, or clothing helps establish validity of alleged sex- ual assault.
■ Fructose. Fructose provides energy for spermatozoa. Semen fructose is produced by the seminal vesicles, with normal levels being equal or greater than 13 mol
Figure 12-20. Various sperm anomalies. A. Normal sperm. B.
Sperm with constricted (or pinched) head and excessive cytoplasmic
membrane. C. Sperm with coiled tailpiece (Papanicolaou stain, 1000x)
Figure 12-21. These sperm both have bent neck pieces.
One has a normal head, whereas the other is a pinhead (Papanicolaou stain, 1000x).
per ejaculate and comprises 99% of reducing sugar found in semen. A low semen fructose level indicates the presence of ejaculatory duct obstruction or abnormalities in the vas deferens and is accompanied by azoospermia (absence of sperm). Low semen fructose levels have been found to correlate with androgen deficiency and decreased testosterone levels
Figure 12-22. These sperm have round heads rather than oval
(Papanicolaou stain, 1000x)
Figure 12-23. These sperm all have tapered heads rather than oval.
Image B also shows excessive cytoplasmic membrane and image C
has a coiled tail (Papanicolaou stain, 1000x)
Figure 12-24. The heads of these sperm all contain vacuoles
(Papanicolaou stain, 1000x).
Figure 12-25. The necks of these sperm have excessive
cytoplasmic membrane remaining (Papanicolaou stain, 1000x).
■ Hormones. Measuring the level of various hormones is helpful in differentiating among causes of azoospermia. These hormones include testosterone, LH, and FSH. Hyalinization of the seminiferous tubules is accompanied by a decreased to normal
Figure 12-26. Immature spermatids (Papanicolaou stain, 1000x)
testosterone level with an increase in both LH and FSH. Gonadotropin deficiency demonstrates de- creased levels of all three of these hormones. In Seroli-cell-only syndrome, the testosterone and LH levels are normal while FSH is increased. These hormone levels are normal if the cause of azoospermia is ductal obstruction or maturation arrest
IMMUNOLOGY
Autoimmune antibodies to sperm can form if trauma or infection causes a breakdown of the barrier between sperm and blood. These antibodies are present in both serum and semen. Women can develop isoantibodies to their husbands’ sperm. These antibodies may be individual specific or may be reactive to all human spermatozoa. Immunologic testing for antisperm antibodies can be performed as a confirmation when agglutination of sperm is present.
Several methods currently exist to test for antisperm antibodies. The Kibrick method involves incubating fresh, liquefied semen with serum from the male or serum from his female partner. Agglutination is observed macroscopically. The Isojima method tests for sperm-immobilizing antibody. Comparison is made between sperm motility of fresh, liquefied semen and that of semen incubated with either rabbit or guinea pig complement. A sperm immobi-lization value is calculated by dividing the percent of motile sperm in the fresh specimen by the percent of motile sperm in the incubated sample. A value of 2 indicates the presence of antibodies. Immunobead assays are used to detect the presence of sperm antibodies on the sur- face of sperm. These assays can determine whether anti- sperm antibodies are directed against head, midpiece, or tail and whether the antibodies are IgA, IgG, or IgM. In addition, the immunobead assay method allows for calculating the proportion of sperm in an ejaculate that is antibody bound. Enzyme-linked immunosorbent assay (ELISA) techniques can be used to detect antibodies to prostasomes (prostate-secreted organelles that adhere to spermatozoa).
MICROBIOLOGY
Urogenital infections, caused by various microorganisms, are responsible for about 15% cases of male infertility.
Microorganisms that may lead to antisperm antibody production include Ureaplasma urealyticum, Mycoplasma hominis, Chlamydia trachomatis, Herpes simplex.6 Urogenital infections with Candida albicans impair sperm motility by agglutinating with spermatozoa heads. Each of these microorgan- isms has specific media and growth requirements that are beyond the scope of this book
REFERENCES
Lillian A. Mundt and Kristy Shanahan, Graff's Textbook of Urinalysis and Body Fluids, Second Edition 2011
COMMENTS