Advances in andrology and assisted reproductive technology (ART), as well as increased concern about fertility, particularly among couples choosing to have children later in life, have led to a greater emphasis on semen analysis. Patients who have abnormal results on routine semen analysis in the clinical laboratory are frequently referred to specialized andrology laboratories for additional testing to determine the need for in vitro fertilization (IVF). Clinical laboratory technicians may also be employed in andrology laboratories to perform routine and specialized testing.
Aside from fertility testing, the clinical laboratory also conducts postvasectomy semen analysis and forensic analyses to determine the presence of semen.
Physiology
The testes, epididymis, seminal vesicles, prostate gland, and bulbourethral glands all contribute to the composition of sperm. The composition of each fraction varies, and the mixing of all four fractions during ejaculation is required for the production of a normal semen specimen.
| Spermatozoa | 5% |
| Seminal fluid | 60% to 70% |
| Prostate fluid | 20% to 30% |
| Bulbourethral glands | 5% |
The testes are paired glands in the scrotum that contain sperm-secreting seminiferous tubules. The scrotum’s external location contributes to a lower scrotum temperature, which is ideal for sperm development. Germ cells for spermatozoa production are found in the epithelial cells of the seminiferous tubules. Sertoli cells are specialized cells that provide support and nutrients to germ cells during mitosis and meiosis (spermatogenesis). When spermatogenesis is finished, immature (nonmotile) sperm enter the epididymis. Sperm mature and develop flagella in the epididymis. The entire process takes about 90 days. The sperm remain in the epididymis until ejaculation, when they are propelled to the ejaculatory ducts via the ductus deferens (vas deferens).
The ejaculatory ducts receive both sperm and fluid from the seminal vesicles from the ductus deferens. The seminal vesicles produce the majority of the fluid found in sperm (60-70%), and this fluid serves as a transport medium for the sperm. The fluid has a high fructose and flavin concentration. The fructose is metabolized by spermatozoa to provide the energy for the flagella to propel them through the female reproductive tract. Sperm do not show motility in the absence of fructose in the semen analysis. Flavin is to blame for the gray appearance of sperm. Various proteins secreted by seminal vesicles play a role in ejaculate coagulation.
The muscular prostate gland, which is located just below the bladder, surrounds the upper urethra and helps to propel sperm through the urethra through contractions during ejaculation. The acidic fluid produced by the prostate gland accounts for approximately 20% to 30% of the total volume of sperm. The milky acidic fluid contains high levels of acid phosphatase, citric acid, zinc, and proteolytic enzymes, which are responsible for the coagulation and liquefaction of the sperm after ejaculation.
The bulbourethral glands, which are located beneath the prostate, contribute approximately 5% of the fluid volume in the form of a thick, alkaline mucus that helps to neutralize acidity from the prostate secretions and the vagina. It is critical for semen to be alkaline in order to neutralize the vaginal acidity caused by normal bacterial vaginal flora. Sperm motility would be reduced in the absence of this neutralization.
| Structure | Function |
| Seminiferous tubules of testes | Spermatogenesis |
| Epididymis | Sperm maturation |
| Ductus deferens | Propel sperm to ejaculatory ducts |
| Seminal vesicles | Provide nutrients for sperm and fluid |
| Prostate gland | Provide enzymes and proteins for coagulation and liquefaction |
| Bulbourethral glands | Add alkaline mucus to neutralize prostatic acid and vaginal acidity |
Specimen Collection
Because the composition of the semen fractions varies, proper collection of a complete specimen is required for accurate male fertility evaluation. Because the majority of the sperm is found in the first portion of the ejaculate, complete collection is required for accurate testing of both fertility and postvasectomy specimens. When a portion of the first portion of the ejaculate is missing, the sperm count drops, the pH rises falsely, and the specimen does not liquefy. When a portion of the last ejaculate is missing, the semen volume is reduced, the sperm count is falsely increased, the pH is falsely decreased, and the specimen will not clot. Patients should be given specific instructions for collecting specimens.
Specimens are collected after at least 2 days and no more than 7 days of sexual abstinence. Specimens collected after a period of abstinence have higher volumes and lower motility. The World Health Organization (WHO) recommends that two or three samples be collected no less than seven days apart and no more than three weeks apart when performing fertility testing, with two abnormal samples considered significant. Warm sterile glass or plastic containers should be provided to the patient by the laboratory. Whenever possible, the specimen is collected in a laboratory-provided room. If this is not possible, the specimen should be kept at room temperature and delivered to the laboratory within 1 hour of being collected. The patient’s name and birth date, the period of sexual abstinence, the completeness of the sample, difficulties with collection, and the times of specimen collection and specimen receipt must all be recorded by laboratory personnel. Specimens that are awaiting analysis should be stored at 37°C. Masturbation should be used to collect specimens. Only nonlubricant-containing rubber or polyurethane condoms should be used if this is not possible. Normal condoms are not permitted because they contain spermicides.
Coitus interruptus is not a reliable method of collecting sperm because the first portion of the ejaculate, which contains the most spermatozoa, may be lost, and the low pH of the vaginal fluid may affect sperm motility.
Specimen Handling
Because all sperm specimens are potential reservoirs for HIV and hepatitis viruses, standard precautions must be taken at all times during analysis. Specimens are thrown away as biohazardous waste. When performing sperm culture or processing the specimen for bioassay, intra-uterine insemination (IUI), or in vitro fertilization, sterile materials and techniques must be used (IVF).
Semen Analysis
Semen analysis for fertility evaluation includes both macroscopic and microscopic examination. Appearance, volume, viscosity, pH, sperm concentration and count, motility, and morphology are just a few of the parameters reported.
I. Appearance
Normal semen is gray-white in color, appears translucent, and has a distinct musty odor. When the sperm concentration is extremely low, the specimen may appear nearly clear. White turbidity increases in the presence of white blood cells (WBCs) and infection in the reproductive tract. Specimen culturing is performed if necessary before proceeding with the semen analysis. WBCs must be distinguished from immature sperm during microscopic examination (spermatids). To screen for the presence of WBCs, the leukocyte esterase reagent strip test may be useful. Variable amounts of red coloration are abnormal and are associated with the presence of red blood cells (RBCs). Urine contamination, specimen collection after a period of abstinence, and medications can all cause yellow coloration. Urine is toxic to sperm, affecting motility evaluation.
| Volume | 2 to 5 mL |
| Viscosity | Pours in droplets |
| pH | 7.2 to 8.0 |
| Sperm concentration | >20 million/mL |
| Sperm count | >40 million/ejaculate |
| Motility | >50% within 1 h |
| Quality | >2.0 or a, b, c |
| Morphology | >14% normal forms (strict criteria) >30% normal forms (routine criteria) |
| Round cells | <1.0 million/mL |
II. Liquefaction
A fresh semen specimen is clotted and should liquefy within 30 to 60 minutes of collection; thus, recording the time of collection is critical for evaluating sperm liquefaction. Failure to liquefy within 60 minutes may be caused by a lack of prostatic enzymes and should be reported. The analysis of the specimen cannot begin until the specimen has liquefied. If the specimen has not liquefied after 2 hours, an equal volume of physiologic Dulbecco’s phosphate-buffered saline or proteolytic enzymes such as alphachymotrypsin or bromelain may be added to induce liquefaction and allow the remainder of the analysis to proceed. Because these treatments have the potential to affect biochemical tests, sperm motility, and sperm morphology, their use must be documented. When calculating sperm concentration, the dilution of semen with bromelain must be taken into account. In liquefied semen specimens, jelly-like granules (gelatinous bodies) may be present but have no clinical significance. Mucus strands, if present, may obstruct semen analysis.
III. Volume
The volume of normal semen ranges between 2 and 5 mL. Pouring the specimen into a clean graduated cylinder calibrated in 0.1-mL increments allows it to be measured. Following long periods of abstinence, there may be an increase in volume. Reduced volume is more commonly associated with infertility and may indicate improper functioning of one of the semen producing organs, specifically the seminal vesicles. Incomplete specimen collection must also be taken into account.
IV. Viscosity
Specimen viscosity refers to the fluid’s consistency and is related to specimen liquefaction. Specimens that have not been completely liquefied are clumped and viscous. The normal sperm specimen should be easily drawn into a pipette and form small discrete droplets that do not appear clumped or stringy when falling from the pipette by gravity. Droplets that form threads longer than 2 cm in length are considered highly viscous and marked as abnormal. The viscosity report can be given a rating ranging from 0 (watery) to 4 (gel-like). Viscosity is also classified as low, normal, or high. Increased viscosity and incomplete liquefaction make testing for sperm motility, concentration, antisperm antibody detection, and biochemical markers difficult.
V. pH
The pH of sperm reflects the balance of pH values from acidic prostatic secretion and alkaline seminal vesicles secretion. Due to the loss of CO2, the pH should be measured within 1 hour of ejaculation. The normal pH of sperm is alkaline, ranging from 7.2 to 8.0. A rise in pH indicates an infection in the reproductive tract. Increased prostatic fluid, ejaculatory duct obstruction, or poorly developed seminal vesicles may be associated with a lower pH. Semen can be tested for pH by applying it to the pH pad of a urinalysis reagent strip and comparing the color to the manufacturer’s chart. A special pH testing paper can also be used.
Sperm Concentration and Sperm Count
Despite the fact that fertilization is accomplished by a single spermatozoon, the number of sperm present in a sperm specimen is a valid measure of fertility. Various factors, such as the number of days of sexual abstinence prior to collection, infection, or stress, can all affect sperm concentration; therefore, more than one sperm specimen should be evaluated for infertility studies. Typically, reference values for sperm concentration range from 20 to 250 million sperm per milliliter; concentrations between 10 and 20 million per milliliter are considered borderline. By multiplying the sperm concentration by the specimen volume, the total sperm count for the ejaculate can be calculated. Total sperm counts of 40 million or more per ejaculate are considered normal (20 million per milliliter 2 mL).
The Neubauer counting chamber is commonly used in clinical laboratories to perform sperm concentration. Sperm are counted in the same way that cells in cerebrospinal fluid are counted, by diluting the specimen and counting the cells in the Neubauer chamber. The amount of dilution used and the number of squares counted differ between laboratories.
The most common dilution is 1:20, which is prepared with a mechanical (positive-displacement) pipette. Dilution of the sperm is required because it immobilizes the sperm prior to counting. The traditional diluting fluid contains sodium bicarbonate and formalin, which immobilize and preserve the cells; however, saline and distilled water can also produce good results.
Sperm are typically counted using the Neubauer hemocytometer in the four corner and center squares of the large center square, similar to a manual RBC count. Both sides of the hemocytometer are loaded and allowed to settle for 3 to 5 minutes before counting, with the counts agreeing within 10%. In the calculation, the average of the two counts is used. If the counts do not agree, the dilution and counts are both repeated. Counts are done with either phase or bright-field microscopy. When using bright-field microscopy, the addition of a stain, such as crystal violet, to the diluting fluid aids in visualization.
Only sperm that has fully developed should be counted. Immature sperm and WBCs, also known as “round” cells, must be excluded. Their presence, on the other hand, can be significant, and they may need to be identified and counted separately. The diluting fluid contains a stain that aids in the differentiation of immature sperm cells (spermatids) from leukocytes, and they can be counted in the same way as mature sperm. A count of more than 1 million leukocytes per milliliter is associated with reproductive organ inflammation or infection, which can lead to infertility.
The presence of more than one million spermatids per milliliter indicates that spermatogenesis has been disrupted. This could be caused by viral infections, toxic chemical exposure, or genetic disorders.
A. Calculating Sperm Concentration and Sperm Count
The sperm concentration is calculated based on the dilution used as well as the size and number of squares counted. Using the 1:20 dilution and counting the five squares (RBCs) in the large center square, the number of sperm can be multiplied by 1,000,000 (add 6 zeros) to get the sperm concentration per milliliter. In contrast to blood cell counts, sperm concentration is reported in millions per milliliter rather than microliters. The basic formula for cell counts can also be used to calculate sperm concentration. Because this formula gives the number of cells per microliter, it must be multiplied by 1000 to get the number of sperm per milliliter. The total sperm count is computed by multiplying the number of sperm per milliliter by the volume of the specimen.
Examples
- Using a 1:20 dilution, an average of 60 sperm are counted in the five RBC counting squares on both sides of the hemocytometer. Calculate the sperm concentration per milliliter and the total sperm count in a specimen with a volume of 4 mL.
60 sperm counted × 1,000,000 = 60,000,000 sperm/mL
60,000,000 sperm/mL × 4 mL = 240,000,000 sperm/ ejaculate
- In a 1:20 dilution, 600 sperm are counted in two WBC counting squares. Calculate the sperm concentration per milliliter and the total sperm count in a specimen with a volume of 2 µ L.
Several methods have been developed that do not require specimen dilution and use specially designed and disposable counting chambers. When these methods were compared to the standard Neubauer counting chamber method, they showed poor correlation with the Neubauer method as well as among themselves. According to the WHO, “the validity of these alternative counting chambers must be established by checking chamber dimensions, comparing results with the improved Neubauer hemocytometer method, and achieving satisfactory performance as demonstrated by an external quality control program.”
B. Sperm Motility
Because sperm must propel themselves through the cervical mucosa to the uterus, fallopian tubes, and ovum once presented to the cervix, the presence of sperm capable of forward, progressive movement is critical for fertility. Clinical laboratory reporting of sperm motility has traditionally been a subjective evaluation performed by examining an undiluted specimen and determining the percentage of motile sperm and the quality of the motility.
Sperm motility should be assessed within 1 hour of specimen collection using a well-mixed, liquefied sperm specimen. The practice of examining sperm motility at timed intervals over an extended period of time has been shown to be ineffective. To ensure consistency in reporting, laboratories should use a calibrated positive-displacement pipette to place a consistent amount of semen on a slide under the same size cover slip, such as 10 L under a 22 22 mm cover slip, and allow it to settle for 1 minute. To ensure accuracy, this procedure should be repeated twice. After evaluating approximately 20 high-power fields, the percentage of sperm showing actual forward movement can be estimated. An alternative method is to examine 200 sperm per slide and manually count the percentages of the various motile categories using a cell counter. Motility is measured in terms of both speed and direction. Grading can be done on a scale of 0 to 4, with 4 representing rapid, straight-line movement and 0 representing no movement. After one hour, a minimum motility of 50% with a rating of 2.0 is considered normal.
The WHO employs a four-point scale: a, b, c, and d. According to the interpretation, 50 percent or more of the sperm in categories a, b, and c should be motile within 1 hour, and 25 percent or more should show progressive motility (a and b).
| Grade | WHO Criteria | Sperm Motility Action |
| 4.0 | a | Rapid, straight-line motility |
| 3.0 | b | Slower speed, some lateral movement |
| 2.0 | b | Slow forward progression, noticeable lateral movement |
| 1.0 | c | No forward progression |
| 0 | d | No movement |
Because of the difficulty in standardized reporting, the WHO Laboratory Manual for the Examination and Processing of Human Semen currently recommends a simpler system for grading motility that excludes speed. Motility is classified into three types: progressive motility (PM), nonprogressive motility (NP), and immotility (IM). Total motility (PM and NP) or progressive motility must be specified (PM).
| Progressive motility (PM) | Sperm moving linearly or in a large circle |
| Nonprogressive motility (NP) | Sperm moving with an absence of progression |
| Immotility (IM) | No movement |
A high percentage of immobile sperm and clumps of sperm necessitates additional testing to determine sperm vitality or the presence of sperm agglutinins.
Instrumentation capable of performing computer-assisted semen analysis (CASA) has been developed in recent years.
CASA allows for the objective determination of sperm velocity and trajectory (direction of motion). The analysis also includes sperm concentration and morphology. CASA instrumentation is currently found primarily in laboratories that specialize in andrology and analyze a large volume of sperm.
C. Sperm Morphology
Infertility is caused by the presence of a normal number of nonmotile sperm, just as it is caused by the presence of sperm that are morphologically incapable of fertilization.
The structure of the head, neckpiece, midpiece, and tail of the sperm is evaluated. Head morphology abnormalities are linked to poor ovum penetration, whereas neckpiece, midpiece, and tail abnormalities affect motility.
Normal sperm has an oval-shaped head that is approximately 5 m long and 3 m wide, as well as a long, flagellar tail that is approximately 45 m long. The enzyme-containing acrosomal cap at the tip of the head is essential for ovum penetration. The acrosomal cap should cover approximately half of the head and two-thirds of the sperm nucleus. The neckpiece connects the head, tail, and midpiece. The midpiece is about 7.0 m long and the thickest part of the tail because it is surrounded by a mitochondrial sheath that produces the energy needed by the tail for motility.
Sperm motility can be assessed at either room temperature or 37°C. When assessing motility at 37°C, the sample should be incubated at that temperature, and the preparation should be done with prewarmed slides and cover slips.
Sperm morphology is assessed using a thinly smeared, stained slide immersed in oil. Smears are created by placing about 10 L of semen near the frosted end of a clean microscope slide. Place a second slide with a clean, smooth edge at a 45° angle in front of the semen drop and draw the slide back to the edge of the semen drop, allowing the semen to spread across the end. When the semen is evenly distributed across the spreader slide, gently pull it forward with a continuous movement across the first slide to produce a smear. Staining can be done with Wright’s, Giemsa, Shorr, or Papanicolaou stain, depending on laboratory preference. Slides that have been air-dried are stable for 24 hours. A minimum of 200 sperm should be tested, and the percentage of abnormal sperm should be reported. Double heads, giant and amorphous heads, pinheads, tapered heads, and constricted heads are all common abnormalities in head structure. Sperm tails with abnormalities are frequently doubled, coiled, or bent. An abnormally long neckpiece may cause the sperm head to bend backward, impairing motility.
Measurement of the head, neck, and tail size; measurement of acrosome size; and evaluation for the presence of vacuoles are additional parameters in evaluating sperm morphology. The inclusion of these parameters is known as Kruger’s strict criteria. The use of a stage micrometer or morphometry is required for strict criteria evaluation. At the moment, sperm morphology evaluation using strict criteria is not routinely performed in clinical laboratories, but it is recommended by the WHO. Strict criterion evaluation is an essential component of assisted reproduction evaluations.
Normal values for sperm morphology vary depending on the evaluation method used, ranging from greater than 30% normal forms when using routine criteria to greater than 14% normal forms when using strict criteria.
Calculating Round Cells
During the morphology examination, round cells (immature sperm and leukocytes) can be differentiated and counted. Peroxidase-positive granulocytes are the most common type of leukocyte in sperm and can be distinguished from spermatogenic cells and lymphocytes with a peroxidase stain. The amount per milliliter can be calculated by counting the number of spermatids or leukocytes seen in conjunction with 100 mature sperm and using the following formula, where N is the number of spermatids or neutrophils counted per 100 mature sperm and S is the sperm concentration in millions per milliliter:
This method can be used when counting cannot be performed during the hemocytometer count and to verify hemocytometer counts.
More than 1 million WBCs per milliliter of ejaculate indicates an inflammatory condition associated with infection and poor sperm quality, which may impair sperm motility and DNA integrity.
