LEARNING OBJECTIVES
Upon completion of this article, the reader will be able to:
1. demonstrate a basic understanding of semen production.
2. describe the composition of seminal plasma, including key
components included in the first and last portions of the ejaculate.
3. guide a patient through the proper steps for preparing for a
semen analysis and for collecting and transporting the specimen to the
laboratory.
4. perform a comprehensive semen analysis acceptable for fertility
diagnosis that includes: (1) macroscopic evaluation; (2) assessment of
the minimum number of spermatozoa to reduce statistical counting error
for microscopic parameters; (3) correct calculations; (4) morphology
assessment using Strict Criteria; and (5) appropriate quality control.
5. recognize abnormal semen findings and be able to troubleshoot.
6. use correct terminology associated with the production of semen
and performance of a semen analysis.
Seminal plasma (semen) is composed of spermatozoa and glandular
fluids and is produced by the primary organ of the male reproductive
system, the testes and the accessory sex glands. This primary organ is a
dual gland that produces exocrine products (spermatozoa and testicular
fluid) and endocrine products (testosterone and inhibin). Testicular
function is controlled by the pituitary hormones, leutinizing hormone
(LH) and follicle stimulating hormone (FSH), under the influence of
gonadotropin releasing hormone (GnRH) from the hypothalamus. FSH acts on
Sertoli cells, located in the seminiferous tubules, to produce
androgen-binding protein (ABP) and inhibin. Both ABP and testosterone
produced by the testicular Leydig cells are required for
spermatogenesis, the process in which spermatozoa are formed in the
seminiferous tubules. Spermatogenic stem cells (2n) undergo
transformation and a series of meiotic cell divisions to produce haploid
(n) spermatids. The final step in the process, termed spermiogenesis, is
the conversion of the haploid spermand to a polarized, flagellated,
motile spermatozoon, typically referred to as a sperm. (1) During
spermatogenesis, which requires approximately ten weeks, the germinal
cells are supported and nourished by Sertoli cells (Figure 1). (2)
'Me Sertoli cells have tight junctions that form a blood testis
barrier providing the appropriate fluid environment for spermatogenesis.
Sperm are released into the epididymis where, over an approximate two
week period, they mature and gain the ability to become motile.
[FIGURE 1 OMITTED]
Upon sexual stimulation, the bulbourethral glands secrete a small
amount of fluid that lubricates the penis for sexual intercourse and
neutralizes the urethral contents that are toxic for spermatozoa. At
ejaculation, spermatozoa stored in the epididymis are released along
with a small amount of testicular fluid. Secretions from the accessory
glands are then added: first, from the prostate gland followed by a
second fluid from the seminal vesicles. Prostatic fluid is acidic,
contributing to the pH of semen, and is rich in zinc and citric acid.
This fluid also provides enzymes, including fibrinolysin, for
liquefaction of the coagulum that forms at ejaculation. Seminal vesicle
fluid is alkaline, rich in flavin that is responsible for semen color
and contains fructose for nourishment as the male gamete travels through
the female reproductive track to the site of fertilization in the
oviduct. Seminal vesicle fluid contains enzymes responsible for semen
coagulation and contributes approximately 70% of the total volume of
seminal plasma (semen).
At ejaculation, the mixture of spermatozoa and glandular secretions
(semen) empties through the penile urethra. The semen clot that is
formed at ejaculation liquefies minutes later in response to enzymes
from the prostate. (1) In vivo semen is ejaculated into the vagina and
spermatozoa travel through the female reproductive track to the site of
fertilization in the Fallopian tube. Sperm can only traverse the female
reproductive track when the cervical mucus during mid-cycle of the
menstrual cycle is receptive. As the sperm pass through the female
reproductive track, they undergo capacitation, which is a series of
biological and biochemical changes necessary for successful
fertilization. Fertilization occurs at mid-cycle in the female when a
mature oocyte is ovulated and competent sperm are present in the
ampullar region of the Fallopian tube. The sperm must penetrate the
cumulus cells that surround the oocyte in order to recognize receptors
on the zona pellucida (ZP), the outer membrane of the oocyte. The sperm
binds to the ZP and undergoes the acrosome reaction, releasing enzymes
that allow penetration of zona pellucida. The sperm plasma membrane then
fuses with the vitelline membrane of the oocyte, the two gametes fuse,
and the sperm enters the oocyte for fertilization.
A dysfunction or abnormality in the production of testicular
products, or in the glands that contribute products to semen, may lead
to pathology, including infertility. Inability of the sperm to traverse
the female reproductive track and penetrate the oocyte also leads to
infertility. Many of these abnormalities and/or deficiencies will be
evident in the semen analysis. A comprehensive semen analysis, performed
by a qualified laboratorian using appropriate quality control, on a
specimen that has been properly collected, provides important diagnostic
information. (3) Semen analysis is the first and most important test
performed for the evaluation of male fertility. Infertility is often
defined as not being able to become pregnant after one year of
unprotected intercourse. The 2002 National Survey of Family Growth
reported that seven percent of married couples in which the woman was of
reproductive age (2.1 million couples) reported that they had not used
contraception for 12 months and the woman had not become pregnant. (4)
When a diagnosis of male factor infertility is made, knowledge of semen
components and values of the associated parameters is essential for
successful interpretation and treatment. For example, a specimen with a
low sperm concentration and/or motility can be processed to select for
the motile sperm fraction. The non-motile and dead sperm along with
harmful debris are discarded and the enriched motile fraction is used
for partner insemination, thus increasing the chances of fertilization.
PATIENT PREPARATION AND SPECIMEN COLLECTION
In order for the patient to properly prepare for the semen
analysis, he must interact with laboratory personnel or the medical
practitioner ordering the test. Patients must be provided with
information that instructs them on: (1) preparation for the test; (2)
specimen collection and transport; (3) documentation regarding medical
history, preparation, specimen collection and transport; and (4)
directions to the testing laboratory directions to the testing
laboratory along with contact information.
a) Patients should be instructed to abstain from any type of
ejaculation for three days (a two to five day range is acceptable)
before collecting the sample for evaluation. A shorter abstinence period
may result in a decreased sperm concentration and a longer abstinence
may yield a higher concentration, but a decreased motility.
b) Patients should be directed to wash their genitals using only
water (certain soaps are toxic to spermatozoa) before collecting the
specimen by masturbation into a sterile container provided by the
laboratory (seminal pouches are available at the request of the
practitioner for patients who cannot collect by masturbation). The time
of collection should be recorded on the collection container and/or a
patient information form that is also provided by the laboratory.
Ideally, the specimen is collected at the laboratory setting, but if
this is not feasible, the patient should be instructed to maintain the
specimen at or near body temperature (e.g., in shirt pocket) for
delivery to the laboratory since both heat and cold adversely affect
semen quality. If collected off-site, the specimen should reach the
laboratory within 30 minutes of collection and must be delivered to the
laboratory within 60 minutes of collection in order for the motility
evaluation to be valid.
c) The patient should complete the patient information form and
report days of abstinence, method of collection, and whether any part of
the ejaculate was lost. If the first portion containing the spermatozoa
and prostatic secretions is lost, the coagulum will fail to liquefy, the
pH will be increased, and the sperm concentration will be decreased. If
the last part of the ejaculate that consists of seminal vesicle fluid is
missing, the coagulum will not formed, the pH will be decreased, the
volume will be decreased, and the sperm concentration will be falsely
elevated due to the loss of the diluting effect of the seminal vesicle
fluid. Current medications should be recorded and illnesses or fevers
within the past three months should be noted since increased temperature
negatively impacts sperm production and quality.
ACCESSION AND PATIENT REPORTING
The specimen must be delivered directly to laboratory personnel who
will denote time of specimen receipt and take responsibility for
documenting chain of custody prior to specimen receipt and during
testing and reporting of results. The technologist receiving the
specimen should review the patient information form to determine if the
specimen is acceptable for processing or if additional information is
needed. Since two specimens that are collected one to three weeks apart
should be evaluated, the patient should be reminded of any future
appointments.
SPECIMEN HANDLING
The semen specimen should be maintained at 37[degrees]C during
evaluation. The sample must be thoroughly mixed prior to each
macroscopic and microscopic assessment.
Notes regarding reference ranges and quality control
When performing the comprehensive semen analysis, the technologist
should be able to correlate abnormal parameter values with possible
causes and be able to troubleshoot when necessary. Table 1 summarizes
possible causes for semen parameters that fall outside reference ranges,
ways the abnormal finding may influence other semen parameters, and
associated troubleshooting. The values listed are based on the 4th
edition of the WHO Laboratory Manual, published in 1999 for the
evaluation of semen and sperm-cervical mucus interaction. The 5th
edition, to be published in 2007, will report reference ranges for sperm
concentration based on semen quality from men who impregnated their
partners with a time to pregnancy of up to 12 months (personal
correspondence with Trevor G Cooper PhD, chair, WHO Manual Editorial
Committee, August, 2006).
MACROSCOPIC EXAMINATION (1)
The macroscopic portion of the test evaluates coagulum formation,
liquefaction, volume, viscosity, appearance, and pH. If microbiology
cultures are ordered, the specimen must be treated aseptically and
plated immediately after volume is assessed.
Coagulum formation and liquefaction
Coagulum is the clot that forms in response to enzymes from the
seminal vesicles. Liquefaction is due to enzymes in the prostatic fluid.
The semen analysis can be performed once the clot liquefies.
Procedure: The semen specimen is swirled in the collection
container to determine if the coagulum has liquefied. A liquefied sample
will take the shape of the container.
Reference range: Coagulum liquefies within 15-30 minutes at
37[degrees]C or within one hour at room temperature.
Comments: In some patients the coagulum liquefies very soon after
ejaculation and is therefore not detected in the laboratory. If
formation is not detected and the practitioner needs this information,
the patient can be asked to self-report on this parameter.
Volume
Procedure: Volume is assessed using a sterile 5-mL or 10-mL pipet
and recorded to the nearest 0.1 mL.
Reference range: [greater than or equal to] 2 mL; a volume of <
1 mL is referred to as oligospermia.
Comments: If volume is < 0.8 mL, the specimen may be diluted 1:1
using Sperm wash medium (Hepes based Human Tubal Fluid supplemented with
protein or Dulbeccos' medium; Irvine Scientific, Irvine CA). If the
specimen is diluted, the sperm concentration must be doubled prior to
reporting results.
Viscosity
Viscosity refers to the fluid nature of semen.
Procedure: Viscosity is assessed after the volume has been measured
by aspirating the sample into the pipet and allowing semen to drop back
into the container by gravity. The length of any thread that forms is
measured to the nearest 0.1 cm.
Reference range: Thread should be [less than or equal to] 2 cm.
Grade as slightly viscous (thick), moderately viscous (difficult to
pipet) or extremely viscous (cannot pipet).
Comments: Viscosity may indicate a high mucus content. Increased
viscosity has been reported to be higher in men with oligoasthenospermia
(decreased motility and decreased concentration) and has been associated
with antibodies to spermatozoa. (6,7,8)
Appearance
Appearance refers to the color and consistency of semen.
Procedure: Note the exact appearance of the specimen.
Reference range: homogeneous, opalescent, and grayish-white in
color
Comments: Colors other than grayish-white are associated with
certain pathologies, contamination, and use of certain medications
(Table 1).
pH
pH refers to the alkalinity of semen, which buffers against the
acid pH of the vagina harmful to spermatozoa.
Procedure: Alkalinity is assed by placing a drop of semen on a pH
paper with a range of 6.0-8.0 and reading against the calibrated strip
after 30 seconds. The pH is recorded to the nearest 0.1.
Reference range: [greater than or equal to] 7.2 (WHO Laboratory
Manual, 1999); for clinical purposes, however, a pH below 7.6 or above
8.6 may be considered abnormal.
Comments: Have available pH paper in the range of 4.0-10.0 for
specimens with values that fall outside the 6.0-8.0 range.
Quality control (QC): Standard commercial controls (Fisher
Scientific International Inc., Pittsburgh, PA) should be used for pH,
but quality control materials are not available for other macroscopic
semen parameters. Photos or videos of normal and abnormal materials are
recommended for training laboratorians and for comparison with specimens
being evaluated (Semen Analysis Training Tool, Reproductive Educational
Resources, Ltd., Lexington KY).
MICROSCOPIC EXAMINATION (1)
The microscopic analysis includes an assessment of semen quality,
motility, vitality, sperm concentration, total sperm count, morphology,
and round cells. Microscopic analyses for all unstained preparations of
fresh semen (assessment of semen quality, motility, and vitality) should
be made under high power (40x) using a phase contrast microscope or
lowering the condenser on an ordinary light microscope.
Ideally all preparations for assessment of motility should be
performed using a microscope with a stage warmed to 37[degrees]C. If
this tool is not available, specimens should be placed in a 37[degrees]C
incubator and the prepared slides incubated on 37[degrees]C warming tray
prior to reading. Because motility is affected by temperature, slides
should be read immediately following incubation on the warming tray.
Assessment of quality
Assessment of quality is performed before evaluating specific
microscopic parameters to estimate motility, concentration,
agglutination, clumping, bacteria, and cells. This will determine if
additional parameters need to be assessed and proper dilutions to be
made for counts.
Procedure: A 10 [micro]l aliquot of semen is placed on a microscope
slide, covered with a 22 x 22-mm coverslip, and incubated for one minute
before viewing. The depth of preparation under the coverslip is 20.7
[micro]m. The diameter of the microscopic field is calculated by
dividing the diameter of the aperture ocular (e.g., 10) by the
magnification power of the objective (e.g., 40). The field area is
calculated using the formula area = [pi][r.sup.2] once the diameter has
been calculated using a stage micrometer. (2) Using a standard 14-mm
ocular and a 40 x objective, the microscopic field is 0.0996 [mm.sup.2]
and the number of spermatozoa in the field will approximate the sperm
concentration in millions/mL. For example, if an average of 50
spermatozoa were counted, the concentration would be expected to be
approximately 50 million spermatozoa/mL. The newer, wide-field oculars
are 20 mm and the field area is 0.1963 [mm.sup.2] with the number of
spermatozoa in the field being approximately one-half the sperm
concentration. For example, using a 20 mm ocular, the 50 spermatozoa
counted in the microscopic field would result in a concentration
estimate of 25 million spermatozoa/mL. Once the number of sperm is
approximated, the estimate is used to set up proper dilutions for
counting. Also estimated in the initial assessment are percent motility,
clumping and agglutination, and cellular elements that are not
spermatozoa, including leukocytes, erythrocytes, and microorganisms.
Debris, bacteria, epithelial cells, and erythrocytes detected on the
initial assessment are reported as none, slight, moderate, or excessive.
If agglutination or round cells are noted on the initial assessment,
additional testing is warranted and is described below.
Motility
Motility determines the number of sperm that are motile and rates
their speed of progression. This assay should be determined within 30
minutes after collection and must be evaluated no later than one hour
post-ejaculation.
Procedure: Motility is assessed by placing 10 [micro]L of
well-mixed semen on a microscope slide and adding a 22 x 22 mm
coverslip. Preparations should be prepared in duplicate. Each slide is
evaluated by counting sperm in ten random fields away from the edge of
the coverslip. Because a minimum of 200 spermatozoa must be counted to
reduce statistical counting error, it may be necessary to count
additional fields for specimens with significantly decreased
concentrations. Four categories of sperm motility are scored based on
their speed of progression:
A = rapid progressive sperm have a speed of progression that is
[greater than or equal to] 25 [micro]m/s at 37[degrees]C (approximately
equal to half a sperm tail length or five sperm head lengths)
B = sluggish progressive sperm move at a rate 5-25 [micro]/s at
37[degrees]C
C = non-progressive sperm move at a rate of < 5 [micro]m/s at
37[degrees]C, but do not make forward progress; the sperm may move in
circles or "twitch" in place
D = immotile sperm do not exhibit movement
The motile sperm should be counted first, followed by the
non-progressive population, and finally the immotile sperm. Sperm may
enter and leave the field during the motility evaluation. Those sperm
that enter after the count is initiated should not be counted and the
evaluator should not be concerned about sperm that have left the field
after having been counted. The percentage for each of the four
categories is tallied and the evaluation is repeated on a duplicate
preparation. Categories A and B are totaled to equal the percent of
progressive motile sperm. The following formula is used to determine if
the difference between duplicates is greater than that expected in 95%
(confidence interval) of samples due to counting error alone:
Y = [+ or -] 1.96 [square root of X] when:
X = sum of the two progressive motile percentages (A+B) from the
duplicate slides
1.96 = Z value for 95% confidence interval
In order to be accepted, the difference between the two counts must
be less than the calculated value of Y (Table 2). (9)
If the difference between the counts is not greater than expected
for counting error alone the values for the duplicate preparations are
averaged and the percent progressive motile (A+B) is reported. If the
difference occurring between the counts is greater than that due to
counting error alone, the assessment must be repeated.
QC Video depicting the types and rates of progression should be
used to ensure consistent reading among laboratory personnel (Semen
Analysis Training Tool, Reproductive Educational Resources, Ltd.,
Lexington KY). For ongoing QC, videos of sperm motility may be taped
in-house or purchased commercially (Fertility Solutions, Cleveland OH).
Aliquots of frozen semen with known post-thaw motility may be used for
QC. These controls may be cryopreserved onsite and stored in liquid
nitrogen. Motility controls also are available commercially. Conception
Technologies (San Diego CA) markets frozen human semen with normal and
low values for motility and kinematic assay and for sperm concentration.
Reference range: [greater than or equal to] 50% (rapid progressive
and sluggish progressive).
Comments: (1) Graphs and tables may be generated for determining
acceptable counts between duplicates by determining points on a graph
based on the formula Y=1.96 [square root of x] ; (2) Not all commercial
quality control materials for semen analysis are FDA approved; (3) The
4th edition of the WHO laboratory manual for the examination of human
semen and sperm-cervical mucus interaction reports the reference range
for motility as [greater than or equal to] 50% (rapid and sluggish
progressive) or 25% rapid progressive. (5)
Vitality
Vitality staining is performed to determine if immotile sperm are
alive or dead. Sperm that are alive and have an intact membrane will be
able to extrude a vital dye whereas dead sperm will not be able to
extrude the dye and will pick up the color of the stain. There are
various methods for assessing vitality. The vital stain, eosin Y, may be
added directly to semen and read immediately as described below or it
may be mixed with nigrosin and air dried before reading.
Procedure-Eosin: Five microliters (5 [micro]L) of 0.5% Eosin Y
(Sigma-Aldrich, St. Louis MO) is placed on each of two glass slides and
25 [micro]L of well-mixed semen is added to the stain and mixed using
the tip of the pipet. A 22 x 22 mm coverslip is placed on the mixture
and incubated at 37[degrees]C for one minute before reading. Live
spermatozoa will be able to extrude the stain and appear colorless while
the dead sperm will take in the eosin Y and stain red.
Procedure-Eosin-nigrosin: One drop (50 [micro]L) of one percent is
mixed with 50 [micro]L of semen. Following a 30 second incubation, 150
[micro]L of ten percent nigrosin solution is added, and mixed. A thin
smear is made within 30 seconds of adding the nigrosin and allowed to
air dry. Live sperm will appear white and the dead sperm will be stained
red against a dark background.
Reading: For both the eosin and the eosin-nigrosin preparations,
slides are examined under oil immersion (1000x) with a light microscope.
Using a laboratory tally counter, spermatozoa are counted in ten random
fields away from the edge of the coverslip, counting at least 200 sperm.
Each sperm is classified as either alive or dead. The percentage of live
and dead sperm is calculated. The evaluation is repeated on a duplicate
slide and the values are averaged and the average reported. As with the
motility assessment, the counts must be repeated if not within 95%
confidence limits.
QC: Frozen specimens with predetermined vitality values may be used
for checking the quality of the stain and for assessments. Quality
control slides are commercially available for the Eosin-nigrosin stain
(Fertility Solutions, Inc., Cleveland OH).
Reference range: [greater than or equal to] 75% alive.
Comments: (1) The vitality stain is a check on the motility because
the total number of live sperm (% alive) should exceed the number of
sperm that are motile (total motile) since there will be a population of
sperm that are alive but immotile; (2) The term viability is often
erroneously used to refer to the vital staining described; (3) Vitality
refers to life or energy whereas viability means feasibility or
capability; and (4) The 0.5% Eosin Y stain is prepared with a aqueous
sodium chloride solution and the 1% Eosin Y stain for the Eosin-nigrosin
assay is prepared by diluting with distilled water. (5)
[FIGURE 2 OMITTED]
Agglutination
Agglutination refers to the specific attachment of motile sperm to
each other. Agglutination is different from clumping which involves
immotile sperm associated with debris and perhaps other cell types. Any
agglutination may signify anti-sperm antibodies and is therefore
significant. The assay is performed only when agglutination is noted on
the initial assessment of quality.
Procedure: The preparation for counting is the same as that for
motility: 10 [micro]L of well-mixed semen is placed on a microscope
slide and a 22 x 22 mm coverslip is added. Motile agglutinated sperm and
motile non-agglutinated sperm are estimated in ten random fields away
from the edge of the coverslip, counting at least 200 spermatozoa.
Motile sperm agglutinate in specific patterns, including head-to-head,
tail-to-tail, head-to-tail and midpiece-to-midpiece (Figure 2).
Agglutination is estimated to the nearest five percent.
QC: Frozen semen samples that show agglutination or fresh semen
exposed to antisperm antibody stock.
Reference range: Any agglutination is considered significant.
Comments: (1) Agglutination is associated with immunologic
infertility and infection. Escherichia coli can colonize the prostate
and induce production of IgA which may cause agglutination of sperm;
(1,6) (2) Agglutination must not be confused with clumping of non-motile
sperm associated with debris in a non-specific fashion; (1) (3) Antibody
testing should be recommended to the physician when agglutination is
present.
Concentration
Concentration refers to the number of spermatozoa per milliliter of
semen. The term "sperm count" is frequently and erroneously
used when referring to the concentration. Sperm count is NOT the same as
sperm concentration; the total sperm count is the total number of
spermatozoa in the entire ejaculate (concentration x semen volume).
To determine the concentration of sperm, a minimum of 200
spermatozoa should be counted in the central grid on each side of a
Neubauer hemacytometer, counting a total of five squares within the
grid. The volume of five squares is calculated by multiplying the area
(.2 [mm.sup.2]) x chamber depth (0.1 mm) which is .02 [mm.sup.3] = .02
[micro]L= 20 nL. Approximately 200 spermatozoa in five squares are equal
to 10cells/nl (see Figure 3). The dilution is therefore made based on
the formula:
# cells/nl in the initial assessment / 10 cells/nL
Example: If a concentration of 50 x [10.sup.6] was estimated based
on counting 50 spermatozoa/field on the initial assessment, the dilution
factor would be 50/10 or 5 and the dilution would be 1 in 5. The
dilution can be made using a convenient volume (e.g., 10 [micro]L semen
+ 40 [micro]L sperm diluent). Alternatively, the volume of semen can be
calculated:
x [micro]L @a 50 cells/nl = 50 [micro]L (or any convenient volume)
x 10 cells/nL
x = 10 [micro]L semen (added to 40 [micro]L diluent).
[FIGURE 3 OMITTED]
See Table 3.
Dilutions should be made in duplicate, mixed, and an aliquot of
approximately 10 [micro]L from each should be loaded on a side of a
Neubauer hemacytometer. The hemacytometer is placed in a moist chamber
for 10 to 15 minutes to allow the sperm to settle. The hemacytometer is
viewed using 400x magnification, the central grid is located and sperm
are counted in five squares within the central grid. Sperm on two of the
four borders (left and top) should be counted and the established
pattern followed by all laboratorians. The number of sperm on both sides
of the chamber are counted and averaged. If the counts are not within
95% confidence levels the dilutions should be mixed and the counts
repeated. If the rejected counts are still not acceptable, new dilutions
should be made and counted. If the concentration is decreased, the
undiluted specimen may be assessed or concentrated by centrifugation.
(1) The sperm concentration is calculated using the formula:
Sperm concentration = # spermatozoa x dilution / depth x area x
(1000)
The number of spermatozoa is from the chamber count average (e.g.,
50 x [10.sup.6] was estimated and an average of 208 x [10.sup.6]
spermatozoa were counted). Dilution means, for example, one in five
expressed as five. Depth of chamber is a constant of 0.1 mm. Area for
five squares counted is 0.2 [mm.sup.2]. Depth x area is expressed in
[mm.sup.3], making it necessary to multiply by 1000 to express the
concentration in millions per milliliter.
Concentration = 208 x 5/0.1 x 0.2 [mm.sup.2] x 1000 = 52 x
[10.sup.6] spermatozoa/mL
QC: Commercial beads that are similar in size to human sperm
(Hamilton-Thorne Research, Beverly MA) may be used for quality control.
Conception Technologies (San Diego CA) markets frozen human semen with
normal and low values for kinematic assay for sperm concentration and
concentrations of stabilized human sperm (Fertility Solutions, Inc.,
Cleveland OH) are available in different concentrations. Refrigerated or
frozen semen with predetermined sperm concentration may be used, but the
refrigerated aliquots should not be stored for more than one month.
Reference range: [greater than or equal to] 20 x [10.sup.6]/mL
(Comment 2).
Comments: (1) Recent reports suggest that semen concentrations are
declining; (10-12) (2) Although alternate methods of evaluation for
semen concentration are available including the Makler chamber
(MidAtlantic Diagnostics, Mt. Laurel NJ) and the disposable Cell-Vu
chambers (Fisher Scientific, Pittsburg PA), the hemacytometer remains
the gold standard. Data from several studies that are being reanalyzed
for semen quality for reference ranges for the 5th edition of the WHO
included weighting factors to account for different techniques to
establish semen quality. Samples that were not counted using
hemacytometer chambers were not included (personal correspondence with
Trevor G Cooper PhD, chair, WHO Manual Editorial Committee, August
2006).
Total sperm count
Total sperm count refers to the total number of spermatozoa in the
ejaculate.
Procedure: Calculate the total sperm count using the formula: Sperm
concentration in millions/mL x the semen volume in mL
Reference range: [greater than or equal to] 40 x [10.sup.6]
spermatozoa.
Total motile sperm count
Total motile sperm count refers to the total number of motile
spermatozoa in the total ejaculate and is a value sometimes requested by
fertility specialists ordering the semen analysis.
Procedure: Calculate the total motile sperm count using the
formula:
Total sperm count x percent motile sperm
Reference range: [greater than or equal to] 20 x [10.sup.6]
spermatozoa.
Morphology
Morphology is the assessment of the size and shape of spermatozoa.
A considerable degree of morphological variability exists in human
spermatozoa. A large number of defective sperm may be observed in a
normal specimen. The morphology is important because it correlates with
in vivo and in vitro fertilizing capability as well as in vitro sperm
function tests. Morphology is assessed using the Strict Criteria as
recommended in the 4th edition (1999) of the WHO manual (5) and will be
recommended in the 5th edition WHO manual in progress for fertility
evaluations. (personal correspondence with Trevor G Cooper PhD, chair,
WHO Manual Editorial Committee, August 2006).
Procedure (staining, classification, and differential)
Staining: Slides for morphology assessment should be prepared using
fresh, well-mixed semen, but slides can be batched for staining and,
once stained, the reading can be delayed. Four slides should be
prepared: two for duplicate staining and two for reserve. A drop of
semen should be placed on each of two slides and a second slide placed
over the slide containing the semen drop with edges overlapping the
bottom slide. The semen is allowed to spread and the slides are pulled
apart and allowed to air dry. Slides should be placed in an ether:
ethanol fixative for 30 minutes prior to staining. The Papanicolaou
stain is recommended for clarity of reading. Permount is added while the
clearing agent, CitriSoly (Fisher Scientific International, Inc.
Pittsburgh PA) is still wet on the stained slides and the slides are
allowed to dry overnight in a 37[degrees] incubator. The morphology
should be read using oil immersion x 1000, examining 200 spermatozoa in
the area of the slide where sperm are evenly distributed. Stained sperm
using the Papanicolaou method should exhibit a standard pattern with the
acrosomal region of the head staining a light blue and the postacrosomal
region staining a dark blue. The midpiece may stain blue or red;
staining red is not considered abnormal unless the midpiece is distended
or abnormal. The tail stains blue and the cytoplasmic droplets stain
green.
Classifying spermatozoa: Sperm should be classified as normal or
with defects (abnormal). Dimensions that define a normal spermatozoon
are based on sperm that have been fixed and stained using the
Papanicolaou method. To be considered normal, a spermatozoon must meet
Strict Criteria guidelines for both shape and size, which is determined
by measuring with a calibrated ocular micrometer. The sperm must be oval
in shape with a length between 4.0 and 5.0 [micro]m and a width between
2.5 and 3.5 [micro]m. Spermatozoa having different dimensions are
considered abnormal, regardless of shape. The length-to-width radio
should be 1.50 to 1.75, the acrosome should cover the anterior head and
should comprise 40% to 70% of the head area. The midpiece should be one
pm in width and 1.5 times the length of the head. The tail should be
thinner than the midpiece, approximately 45 [micro]m long and should be
uniform in shape.
[FIGURE 4 OMITTED]
There are four categories of defects within the abnormals: (1)
head; (2) neck/midpiece; (3) tail; and (4) cytoplasmic droplets. An
abnormal sperm may therefore have from one to four defects. Head defects
may be attributed to abnormal size or abnormal shape. The acrosome may
be absent or abnormal and the head may be amorphous, tapering, pyriform,
double, or have a flat side/base. If the head is oval and meets all size
requirements but tapers slightly, it is still considered normal.
Vacuoles that occupy more than 20% of the head are considered a head
defect. Heads that have surface irregularities or a flattened side or
base may be difficult to assess and may be considered as either normal
or abnormal depending on the severity of the defect. Some spermatozoa
also exhibit surface irregularities. Neck/midpiece defects include thin
or bent midpiece, noninserted tail, and abaxial implantation. Abnormal
tails may be short, multiple, or have irregular widths. Tails may be
broken, coiled, or have hairpin loops. Cytoplasmic droplets are usually
located on the midpiece and are considered abnormal only if they are
greater in size than one third of the normal head.. In order to perform
the morphology differential, the reader must be able to distinguish
normal and abnormal sperm and to identify the various defects associated
with those that are abnormal.
Performing the differential. The morphology differential may be
performed as a basic or complete evaluation. When performing either the
basic or the complete differential, 200 spermatozoa are evaluated and
each spermatozoon is identified as either normal or abnormal. When a
basic morphology is requested, the sperm are evaluated as being either
normal or abnormal and the specific defects are not recorded. When a
complete morphology is performed, the type of defect (or defects) is
recorded for each spermatozoon evaluated. A hand tally and a
differential counter are used when performing the complete morphology
evaluation because each sperm evaluated contributes to two separate but
simultaneous counts. The hand tally is used to count the requisite 200
spermatozoa and a differential counter with a key labeled
"normal" and keys marked for the four types of defects (head
defect, neck/midpiece defect, tail defect and cytoplasmic droplet) are
used to track the defects. A normal spermatozoon receives one mark on
the hand tally and one mark on the differential counter under
"normal". A sperm with a single defect receives one mark on
the hand tally and one mark on the differential counter under the type
of defect (e.g., head). An abnormal sperm with more than one defect
would receive one mark on the hand tally and two marks on the
differential counter under the types of defects noted (e.g., head and
neck/midpiece) (Figure 4).
Since abnormal sperm will receive one to four tallies on the
differential counter for each count on the hand tally, this total will
exceed 200. Sperm lying on the side are not included in the morphology
differential nor are sperm without heads, with pinpoint heads, or
without tails. Conjoined sperm are included in the count with each sperm
counted separately and each defect tailed separately, including fused
regions of the spermatozoa. An abundance of any particular defect such
as more than 20% of the population with absent or abnormal acrosomes
should be reported. The percent of normal sperm is reported for both the
basic and complete evaluations and the teratozoospermia index (TZI) is
also reported as part of the complete evaluation. TZI is the average
number of defects per spermatozoon and is a predictor of sperm function
both in vivo and in vitro. The TZI is calculated by dividing the total
number of defects divided by the total number of abnormal sperm.
QC Prestained morphology slides or illustrations (Reproductive
Educational Resources, Ltd.) depicting various defects should be
available for comparison; frozen semen with known morphology should be
fixed and stained to check the quality of staining, and blank control
slides should be passed through the staining process to ensure that
there is no carry-over from previous specimens.
Reference range: > 14% positively associated with in vitro
fertilization rate; < 14% normal forms associated with decreased in
vitro fertilization rate.
Strict Criteria morphology: Strict morphology: normal > 14%
(threshold for assisted reproduction)
TZI. The TZI should be [less than or equal to] 1.6.
Comments: (1) Multicenter population-based studies, using Strict
Criteria assessments have been performed and should be reported in the
5th edition of the WHO Manual; (2) A TZI that is [greater than or equal
to] 1.6 is associated with lower pregnancy rates in untreated couples.
Round cells
Round cells may be either leukocytes or sperm precursors. The total
number of round cells should not exceed 5 x [10.sup.6]/mL of semen. If
more than 5 x [10.sup.6] round cells/mL are found, staining is indicated
to determine whether the cells are leukocytes or sperm precursors.
Procedure: Round cells are tracked when performing either the basic
or complete morphology. One key on the differential counter is
designated for round cells. As the 200 spermatozoa are counted, each
round cell encountered is noted. Only an occasional round cell may be
seen. The number of round cells/mL of semen is calculated using the
formula:
Round cells/100 sperm x semen concentration / 100
Comments: If more than 5 x [10.sup.6] round cells/mL are found,
staining is indicated to determine if the cells are leucokytes or sperm
precursors. Peroxidase staining (LeucoScreen, Conception Technologies,
San Diego CA) or immunocytochemistry will detect leukocytes and the
Bryan-Leishman stain is helpful for identifying sperm precursors. Semen
should not contain more than one million white cells per mL of semen.
Proficiency testing
The College of American Pathologists (CAP, Chicago IL) has
proficiency testing for sperm motility, vitality, concentration, and
morphology. The American Association of Bioanalysts has CAP-approved
proficiency testing for sperm vitality, concentration and morphology.
[FIGURE 5 OMITTED]
Additional testing
Based on the results for repeated semen analyses, a complete
physical, assessment of hormones and/or invasive testing (e.g.,
testicular biopsy) may be indicated. Additional testing on semen also
may be warranted. Anti-sperm antibody testing is indicated when
agglutination or a low motility (may indicate sperm immobilizing
antibodies) are noted on semen analysis. Semen analysis results may
suggest the need for assays for accessory gland function. Fructose
serves as a marker for the seminal vesicles and enzymes are markers for
the prostate gland (acid phosphatase) and the epididymis ([alpha]
glucosidase). The hypo-osmotic swelling test is an alternative to the
vitality test, and there are various assays to predict sperm function.
Most sperm function assays are preformed in andrology reference
laboratories and some are considered research assays and have not been
approved for diagnostic testing. (3)
Treatments for male factor infertility
Treatment for male factor infertility is dependent on the final
diagnosis. Hormone therapy (e.g., low FSH) or surgery (e.g., varicocele
vein repair) may resolve the problem or laboratory interventions may be
applicable. Intrauterine insemination may be performed to negate hostile
cervical mucus or to allow for sperm concentration to select the motile
fraction. Assisted reproductive technology (ART) procedures are
indicated for semen with significantly abnormal parameters or for
infertility of unknown etiology. Intracytoplasmic sperm injection
(ICSI), the process of injecting a single sperm into the oocyte,
circumvents many sperm problems, including very low sperm concentration
and/or motility and morphology (Figure 5). ICSI has a high success rate
for male infertility and is now a routine ART procedure. Use of donor
sperm is the only option in cases of absolute azoospermia.
ACKNOWLEDGEMENTS: The author would like to thank Professor Philip
Campbell and Dr. Karen McDowell for editing.
ABBREVIATIONS: 2n = diploid number of chromosomes; ABP =
androgen-binding protein; ART = assisted reproductive technology; CAP =
College of American Pathologists; FDA = Food and Drug Administration;
FSH = follicle stimulating hormone; GnRH = gonadotropin releasing
hormone; ICSI = intracytoplasmic sperm injection; LH = leutinizing
hormone; n = haploid number of chromosomes; QC = quality control; TZI =
teratozoospermia index; VTS = viscosity treatment system; WHO = World
Health Organization; Y = yellowish cast of eosin dye; ZP = zona
pellucida.
INDEX TERMS: infertility; male factor infertility; semen analysis;
seminal plasma; Strict Criteria sperm morphology.
REFERENCES
(1.) Baker DJ, Witmyer JF. Semen analysis training tool CD-Rom.
Lexington, KY Reproductive Educational Resources, Ltd.; 2000.
(2.) Mortimer D. Practical laboratory andrology. New York, NY:
Oxford University Press; 1994
(3.) California study finds labs with trained personnel do more
accurate testing. ASCP News. July 1997.
(4.) National Survey of Family Growth. Center for Health
Statistics, Centers for Disease Prevention and Control; 2002. Available
from http://www.cdc.gov/nchs/nsfg.htm. Accessed???
(5.) WHO Laboratory manual for the examination of human semen and
sperm-cervical mucus interaction. 4'. ed. New York, NY Cambridge
University Press; 1999.
(6.) Mendeluk FL, Flecha G, Castello PR, Bregni C. Factors involved
in the biochemical etiology of human seminal plasma hyperviscosity. J
Androl 2000;21:262-7.
(7.) Lin MC, Tsai TC, Yang YS. Measurement of viscosity of human
semen with a rotational viscometer. J Formos Med Assoc 1992;4:419-23.
(8.) Moulik S, Kamala G, Hinduja 1, Shahani S. Presence of sperm
antibodies and association with viscosity of semen. Hum Reprod
1989;4:290-1.
(9.) Daniel, WW Biostatistics: a foundation of analysis in the
health sciences, 41h ed. New York: John Wiley & Sons; 1987.
(10.) Crissman JW, Cooke PS, Hess, RA, Marty MS, Liberacki AB.
Postulated human sperm count decline may involve historic elimination of
juvenile iodine deficiency: a new hypothesis with experimental evidence
in the rat. Toxicol Sci 2000;53:400-10.
(11.) Dindyal S, The sperm count has been decreasing steadily in
Western industrialized countries: is there an endocrine basis for this
decrease? Internet J Urol 2004:2-13.
(12.) Swan SH, Elkin EP, Fenster L. The question of declining sperm
density revisited: An analysis of 101 studies published 1934-1996.
Environ Health Perspect 2000;108:961-6.
(13.) Stephen EH, Chandra A. Declining estimates of infertility in
the United States: 1982-2002. Fertil Steril 2006;96:516-23.
Doris J Baker PhD HCLD(ABB) MT(ASCP) CLS(NCA) is professor, Center
of Excellence in Reproductive Sciences, and director, Division of
Clinical & Reproductive Sciences, University of Kentucky, Lexington
KY.
Address for correspondence: Doris J Baker PhD HCLD(ABB) MT(ASCP)
CLS(NCA) Professor, Center of Excellence in Reproductive Sciences, and
Director, Division of Clinical &Reproductive Sciences, University of
Kentucky, 126E CTW Building, 900 South Limestone, Lexington
KY40536-0200. (859) 323-1100 ext. 80854, (859) 323-8957 (fax).
dbake0@uky.edu.
Doris J Baker PhD HCLD(ABB) MT(ASCP) CLS(NCA) is the Focus: Seminal
Plasma editor.
Table 1. The semen examination
Test Finding Possible cause
Coagulum No coagulum - Liquefied prior to lab
examination
- Lost second part of the ejaculate
- Dysfunction of the seminal
vesicles
Fails to - Lost first part of the ejaculate
liquefy - Dysfunction of the prostate
Volume Low volume - Obstruction or blockage
- Seminal vesicle aplasia
- Congenital absence of the seminal
vesicles
- Retrograde ejaculation
- Loss of first part of the
ejaculate
- Infection
Viscosity Viscous - May indicate high mucus content
specimen - Associated with antibodies
to sperm
Appearance Specimen - Infection
tinged with - Trauma
red indicates - Malignancy of the testes
fresh blood - Prostate cancer
and brownish
color
indicates the
presence of
old blood
Appearance Greenish - Infection
specimens - Use of certain medications
- Bacterial contamination
White-yellow - Infection
color - Urine contamination
- Prolonged abstinence
Bright yellow - Presence of bilirubin
color - Use of certain medications
Other colors - Use of certain medications
Mucous - Incomplete liquefaction
threads
Many clumps - May indicate antibodies to sperm
Watery - Low sperm concentration
- Absence of sperm
Odor Pungent or - Collection in unacceptable
not inducted foul container
in the - Infection
analysis but - Prolonged abstinence
may be noted - Urine contamination
by patient
pH < 7.2 - Disorder such as blockage of
the seminal vesicles
- Chronic prostatitis
- Delayed reading
pH > 8.0 - Microbial contamination
- Infection
- Disorder of the prostate
(pH > 8.6 is indicative of acute
prostatitis)
Motility Decreased - Exposure to cold
- Incomplete liquefaction
- Improper collection container
- Clumping
- Exposure to toxins such as urine
- Physiological causes
- Kartagener syndrome (motility
decreased due to ciliary
immobility in some of the sperm
population
Vitality Decreased - Prolonged abstinence
- Necrozoospermia
- Kartagener syndrome
- Exposure to urine
Agglutination Present - Anti-sperm antibodies
- Infection
Concentration Decreased - Loss of the first part of the
ejaculate
- Short abstinence period
- Anatomical defect
- Physiological problem
- Recent fever
- Medications
Increased - Loss of the second part of the
ejaculate
Prolonged abstinence
Morphology Abnormal - Anatomical defects
- Physiological problems
- Infection
- Varicocele vein
- Scrotal heating
- Frequent ejaculations
Round cells Increased Infection or inflammation
white cells
Increased Physiological or reproductive
sperm problem
precursors
Test Finding Troubleshooting
Coagulum No coagulum Ask patient to self-report
if practitioner needs this
information
Check patient information form
to determine if second fraction
of ejaculate was lost
Fails to Check patient information form
liquefy to determine if first fraction of
ejaculate was lost
Encourage liquefaction by going
from the least aggressive to the
most aggressive treatment: extend
incubation time, followed by
repeatedly pipetting specimen
using a 10-mL pipette and, as last
resort, enzymatically treat with
150 USP/mL chymotrypsin (Sigma
Diagnostics, St. Louis MO) or
Semen VTS proteolytic enzyme
(Conception Technologies,
San Diego CA.)
Volume Low volume Check patient information form to
determine if second fraction was
lost
May need to dilute the specimen
Viscosity Viscous Proceed with the least aggressive
specimen to the most aggressive treatment:
extend incubation time, followed
by repeatedly pipetting specimen
using a 10-mL pipet; as last resort
enzymatically treat with 150 USP/
mL of chymotrypsin or Semen
VTS proteolytic or Semen VTS
proteolytic enzyme
Appearance Specimen Anticipate other abnormal
tinged with parameters
red indicates
fresh blood
and brownish
color
indicates the
presence of
old blood
Appearance Greenish Check time specimen received
specimens to ensure that processing was timely
Check patient information form
to determine use of medications
White-yellow Check patient information form
color for abstinence period
Bright yellow Check patient information form
color to determine use of medications
Other colors Check patient information form
to determine use of medications
Be alert to other abnormal findings
Mucous Be alert to other abnormal findings
threads May need to treat to liquefy
Many clumps Be alert to other abnormal findings
Watery Be alert to other abnormal findings
Odor Pungent or Check patient information form
not inducted foul to determine abstinence period
in the
analysis but Check patient information form
may be noted to determine use of medications
by patient
pH < 7.2 Check patient information form
to determine if the second fraction
of the ejaculate was lost
pH > 8.0 Check time to determine if
processing was delayed
Check patient information form
to see if first part of specimen
was lost
Motility Decreased Review transport, accession
and time motility was performed
Vitality Decreased Check patient information form
to confirm abstinence
Agglutination Present If more than 10% of sperm are
agglutinated, motility should be
assessed on the free spermatozoa
only
Concentration Decreased Check patient information form
to determine if the first part
of the ejaculate was lost
May need to concentrate the
specimen to evaluate semen
parameters
Increased Check the patient information form
to determine if second fraction of
the ejaculate was lost
Morphology Abnormal Check patient information form
to determine date of last ejaculate
(may see immature sperm due
to frequent emissions)
Round cells Increased Check patient information form
white cells to see if patient had recent fever
Check initial assessment to
determine if bacteria were present
Increased If more than 5 X [10.sup.6] round
sperm cells/mL recommend peroxidase
precursors staining to confirm leucocytes
Test Finding Effect on other parameters
Coagulum No coagulum If due to loss of the second part
of the ejaculate, parameters,
especially concentration, may be
concentration skewed; will be
increased; pH will be decreased
Fails to If due to loss of the first part of
liquefy the ejaculate, parameters,
especially concentration, may be
skewed; concentration will be
decreased; pH will be increased
Volume Low volume If due to loss of the second part
of the ejaculate, parameters,
especially concentration, may be
skewed; concentration will be
increased; pH will be decreased
If due to retrograde ejaculation,
sperm will not be present but would
be present in urine
Viscosity Viscous May have lot of debris or abnormal
specimen sperm in the specimen
High viscosity may be associated
with decreased sperm concentration
and decreased motility
Appearance Specimen If due to infection, may see
tinged with increased bacteria and/or white
red indicates blood cells; microbiology cultures
fresh blood may be positive
and brownish
color
indicates the
presence of
old blood
Appearance Greenish If due to infection, may see
specimens increased bacteria and/or white
blood cells microbiology cultures
may be positive
If due to contamination, bacteria,
but not white blood cells, may be
present
White-yellow If due to infection, may see
color increased bacteria and/or white
blood cells; microbiology cultures
may be positive
If due to prolonged abstinence,
sperm concentration may be
increased and motility decreased
If due to urine contamination, toxic
effects will result in decreased
motility and vitality; viscosity and
decreased due to the diluting
effects of urine
Bright yellow
color
Other colors
Mucous Interference with assessment of
threads motility and concentration
Many clumps Interference with assessment of
motility and concentration
Watery Decreased sperm concentration
Odor Pungent or If due to infection, may see
not inducted foul increased bacteria and/or white
in the blood cells; microbiology cultures
analysis but may be positive
may be noted If due to prolonged abstinence,
by patient sperm concentration may be increased
and motility decreased
If due to urine contamination, toxic
effects will result in decreased
motility and vitality; viscosity and
sperm concentration will be
decreased due to the diluting
effects of urine
pH < 7.2 If pH is acidic and the volume
is decreased, this indicates
blockage of the seminal vesticles
pH > 8.0 If due to infection, may see
increased bacteria and/or white
blood cells; microbiology cultures
may be positive
If due to microbial contamination,
may see increased numbers of
bacteria, but not white cells
Motility Decreased Immotile sperm due to Kartagener
syndrome stain as live sperm on
vitality stain
Vitality Decreased If due to prolonged abstinence,
sperm concentration may be
increased, motility and vitality
decreased, and pungent odor may be
present
Immotile sperm due to the Kartagener
syndrome stain as live sperm on
vitality stain
If due to urine concentration, toxic
effects will result in decreased
motility and vitality; viscosity and
sperm concentration will be
decreased due to the diluting
effects of urine
Agglutination Present May interfere with testing for
motility and concentration
Concentration Decreased If due to the loss of the first part
of the ejaculate, parameters,
especially concentration, may be
skewed; concentration will be
decreased; pH will be increased
Increased If due to the loss of the second
part of the ejaculate, parameters,
especially concentration, may be
skewed; concentration will be
increased; pH will be decreased
If due to prolonged abstinence,
motility may be decreased
Morphology Abnormal If due to infection, may see
increased bacteria and/or white
blood cells; microbiology cultures
may be positive
Round cells Increased If due to infection/inflammation,
white cells may see increased bacteria and/or
white blood cells; microbiology
cultures may be positive
Increased
sperm
precursors
Table 2. Sperm motility
Slide 1
Field A: rapid B: sluggish C: non- D: immotile
progressive progressive progressive
1 22 8 0 20
2 25 10 0 22
3 19 9 0 26
4 20 10 0 20
5
6
7
8
9
10
Total 218 94 0 211
Total progressive motile sperm (A+B) = 312;
A+B/A+B+C+D = 312/523 = 60% motile
Slide 2
Field A: rapid B: sluggish C: non- D: immotile
progressive progressive progressive
1 29 8 1 25
2 17 11 0 28
3 22 12 0 19
4
5
6
7
8
9
10
Total 240 88 1 198
Total progressive motile sperm (A+B) = 328 (A+B) = 328
A+B/A+B+C+D = 312/ 511 = 61.1 % motile
Y = 1.96X [square root of X]
328-312 = 1.96 [square root of 328] + 312
17 = 19.6 X [square root of 640]
17 = 19.6 X 25.3
19.6X = 25.3-17
19.6X = 21.8
The difference must be < 21.8; 17 is < 21.8, so the
differences between duplicate slides is acceptable
Table 3. Objective: count approximately 200 spermatozoa
in five squares, which is 10 cells/nl
Examples of Cells to be Dilution Dilution
estimates from counted calculation volume *
initial microscopic
assessment
10 X [10.sup.6]/mL 10/nL 10/10 = 1 1 in 2
50 X [10.sup.6]/mL 10/nL 10/50 = 5 1 in 5
100 X [10.sup.6]/mL 10/nL 10/100 = 10 1 in 10
Examples of Semen Diluent
estimates from volume *
initial microscopic
assessment
10 X [10.sup.6]/mL 20 [micro]L 20 [micro]L **
50 X [10.sup.6]/mL 10 [micro]L 40 [micro]L
100 X [10.sup.6]/mL 10 [micro]L 90 [micro]L
* Determine convenient volume
** A total of 40 pl is prepared since a total of 20 pl of
the mixture is required to fill the two sides of the
hemacytometer chamber Sperm diluent described in Reference 1