‘Semen (or seminal fluid) is a fluid that is emitted from
the male genital tract and contains sperms that are
capable of fertilizing female ova. Structures involved in
production of semen are (Box 182:
Testes: Male gametes or spermatozoa (sperms) are
produced by testes; constitute 2-5% of semen volume.
+ Epididymis: After emerging from the testes, sperms
are stored in the epididymis where they mature;
potassium, sodium, and glycerylphosphoryicholine
(an energy source for sperms) are secreted by
epididymis.
+ Vas deferens: Sperms travel through the vas deferens
totheampulla which is another storage area. Ampulla
secretes ergothioneine (a yellowish fluid that reduces
chemicals) and fructose (source of nutrition for
sperms).
+ Seminal vesicles: During ejaculation, nutritive and
lubricating fluids secreted by seminal vesicles and
pprostateare added. Fluid secreted by seminal vesicles
consists of fructose (energy source for sperms), amino
acids, citric acid, phosphorous, potassium, and
prostaglandins, Seminal vesicles contribute 50% to
semen volume.
Prostate: Prostatic secretions comprise about 40% of
semen volume and consist of citric acid, acid
phosphatase, calcium, sodium, zinc, potassium,
proteolytic enzymes, and fibrolysin.
* Bulbourethral glands of Cowper secrete mucus.
| Box 15.1: Contributions to semen volume
+ Testes and epididymis: 10%
+ Seminal vesictes: 50%
+ Prostate: 40%
+ Cowper's glands: Small volume
‘Normal values forsemen analysis are shown in Tables
18. and 152.
INDICATIONS FOR SEMEN ANALYSIS
Availability of semen for examination allows direct
examination of male germ cells that is not possible with
female germ cells. Semen analysis requires skill and
should preferably be done in a specialized andrology
laboratory.
1. Investigation of infertility: Semen analysis is the first
‘step in the investigation of infertility. About 30% cases,
of infertility are due to problem with males.
2. To check the effectiveness of vasectomy by confir-
ming absence of sperm.
3, To support or disprove a denial of patemity on the
grounds of sterility.
4, To examine vaginal secretions or clothing stains for
the presence of semen in medicolegal cases.
5. For selection of donors for artificial insemination.
6, Forselection of assisted reproductive technology,eg.
in vitro fertilization, gamete intrafallopian transfer
technique.
COLLECTION OF SEMEN FOR
INVESTIGATION OF INFERTILITY
Semen specimen is collected after about 3 days of sexual
abstinence. Longer period of abstinence reduces motility
of sperms. If the period of abstinence is shorter than 3
days, sperm count is lower. The sample is obtained by
‘masturbation, collected in a clean, dry, sterile, and leak-
proof wide-mouthed plastic container, and brought to
the laboratory within 1 hour of collection. The entire
ejaculate is collected, as the first portion is the most
concentrated and contains the highest number of sperms.Essentials of Clinical Pathology
rable 151: Normal values of semen analysis (World Heath Organization, 1698)
1 ee |
22 mi
Volume
pH :
rm concentration
‘Total sperm count per ejaculate
Morphology
Vitality
‘White blood cells
Motility within 1 hour of ejaculation
= Class A.
+ Class A and B
‘Mixed antiglobuiln reaction (MAR) test
immunobead test
‘Table 15.2: Biochemical variables of
1. Total fructose (seminal vesicle marker)
2. Total zine (Prostate marker)
3, Total acid phosphatase (Prostate marker)
4, otal cite acid (Prostate marker)
5. a-glucosidase (Epididymis marker)
6. Camitine (Epididymis marker)
During transport to the laboratory, the specimen should
bbe kept as close to body temperature as possible (i.e. by
carrying it in an inside pocket). Ideally, the specimen
should be obtained rear the testing site in an adjoining
room. Condom collection is not recommended as it
contains spermicidal agent. Ejaculation after coitus
interruptus leads to the loss of the first portion of the
ejaculate that is most concentrated; therefore this method
should net be used for collection. Two semen specimens
should be examined that are collected 23 weeks apart;
if results are significantly different additional samples,
are required.
EXAMINATION OF SEMINAL FLUID
‘The tests that can be done on seminal fluid are shown in
Box 15.2. Tests commonly done in infertility are shown
in Box 153. The usual analysis consists of measurement
of semen volume, sperm count, sperm motility, and
‘sperm morphology.
Terminology in semen analysis is shown in Box 15.4.
721 81
220 milion’!
240 milion
320% sperms with normal morphology:
275% live
‘<1 million’!
225% rapidly progressive
on progressive
0% mote aperme with adherent particles *
‘250% motile sperms with adherent particles:
‘semen analysis (World Helath Organization, 1992)
213 ymoViejaculate
224 ymol/ejacuiate
2200Ulejaculate
252 jmoliejaculate
+220 mUlejaculate
08-29 ymoliejaculate
Box 15.2: Tests done on seminal fluid
|
ae cue pair
reat |
[ee ad Rc
secon!
Pete ta recs
coeenn
Serer ssa tae
Biochemical analysis: Fructose, zinc, acid phosphatase,
camitine.
|¢ Sperm function tests: Postcoital test, cervical mucus,
penetration test, Hamster egg penetration assay, hypo-
‘osmotic swelling of agela, and computer-assisted semen
Box 15.3: Semen analysis for initial
investigation of infertility
+ Volume
+ pH
sscopic examination for ()) percentage of motile
‘spermatozoa, (1) sperm count, and (i) sperm morphology(ld to moderate: 5-20 milion/ml; severe: <5 milion/m!)
‘Azpospermia: Absence of sperms in seminal fluid i
‘Aspermia: Absence of ejaculate |
‘Asthenozoospermia: Reduced sperm motility; <50% of
‘sperms showing class (a) and ciats (b) type of motity
OR <25% sperms showing class (a) type of motility,
+ Teratozoospermia: Spermatozoa with reduced proportion
fren morpelogy (or nereased proportion efabremmal
rm)
|
t Leukocytospermia: >1 million white blood cells/ml of |
+ Oligoasthencteratezoospermia: All sperm variables, are
abnormal
+ Necrozoospermia: All sperms are non-motle or non-viable:
Physical Examination
Examination is carried out after liquefaction of semen
that occurs usually within 20-30 minutes of ejaculation.
1, Visual appearance: Normal semen is viscous and
‘opaque gray-white in appearance. After prolonged
abstinence, it appears slightly yellow.
‘Viscosity: Immediately following ejaculation, normal
semen is thick and viscous. It becomes liquefied
within 30 minutes by the action of proteolytic
enzymes secreted by prostate. Ifliquefaction does not
‘occur within 60 minutes, itis abnormal. The viscosity
of the sample is assessed by filling a pipette with
semen and allowing itto flow back into the container.
‘Normal semen will fall drop by drop. If droplets form
‘threads’ more than 2 cm long, then viscosity is
increased. Increased semen viscosity affects sperm
‘motility and leads to poor invasion of cervical mucus;
itresults from infection of seminal vesicles or prostate.
3. Volume: Volumeof ejaculated semen sample should
normally be > 2 ml. It is measured after the sample
has liquefied. Volume < 2.0 ml is abnormal, and is
associated with low sperm count.
4. pH: A drop of liquefied semen is spread on pH paper
(of pH range 6.4-8.0) and pH is recorded after 30
seconds. Normal pH is 7.2 to 8.0 after 1 hour of
ejaculation. The portion of semen contributed by
‘seminal vesicles is basic, while portion from prostate
is acidic. Low pH (< 7.0) with absence of sperms
Semen Andiyeie? FE
(azoospermia) suggests obstruction of ejaculatory
ducts or absence of vas deferens. Low pH is usually
associated with low semen volume (as most of the
‘volume is supplied by seminal vesicles).
Microscopic Examination
‘The most important test in semen analysis for infertility
{s microscopic examination of the semen.
‘Sperm Motility
‘The first laboratory assessment of sperm function in a
‘wet preparation is sperm motility (ability of the sperms
to move). Sperm motility is essential for penetration of
cervical mucus, traveling through the fallopian tube, and
‘penetrating the ovum. Only those sperms having rapidly
progressive motility are capable of penetrating ovum and
fertilizing it.
Principle: All motile and non-motile sperms are counted
in randomly chosen fields in a wet preparation under
40x objective. Result is expressed as a percentage of
‘motile spermatozoa observed.
Method: A drop of semen is’placed on-a glais slide,
covered with a coverslip that is then ringed with
petroleum jelly to prevent dehydration, arid examined
‘under 40x objective. Atleast 200 spermatozoa are counted
in several different microscopic fields. Result is expressed
as a percentage of (a) rapidly progressive spermatozoa
(moving fast forward in a straight line), (b) slowly
progressive spermatozoa (slow linear or non-linear, i.
crooked or curved movement), (c) non-progressive
‘spermatozoa (movernent of tails, but with no forward
progress), and (d) immotile spermatozoa (no movement
at all) (WHO critera). Sperms of grades (c) and (A) are
considered to be poorly motile (asthenospermia).
Normally, 2 25% of sperms show rapid progressive
‘motility, or250% of sperms show rapid progressive and
slow progressive motility.
If the proportion of motile spermatozoa is <50%, then
proportion of viable spernis should be determined by
‘examining an eosin preparation.
Sperm Viebilty or Vitality
Principle: A cell with intact cell membrane (a vital or
viable cell) will not take tip the eosin Y and will not be
stained, while a non-viable’or dead cell ‘will have
‘damaged cell membrane, will take up the dye, and willEssentials of Clinical Pathology
Fig. 15.1: Eosin-rigrosin stain. Dead sperms are stained
pink-red, while live sperms are stained white
be stained pink-red (Fig. 15.1). Another stain (e.g.
nigrosin) may be used to stain the background material,
The testis performed if motility is abnormal,
Method
1. Mixone drop of semen with 1 drop of eosin-nigrosin
solution and incubate for 30 seconds.
2. A'smear is made froma drop placed on a glass slide.
3. The smear is airadried and examined under oil-
immersion objective. White sperms are classified as
live or viable, and red sperms are classified as dead
‘ornon-viable, Atleast 200 spermatozoa are examined.
4, The result is expressed as a proportion of viable
sperms against non-viable as an integer percentage.
Seventy-five percent or more of sperms are normally
live or viable.
‘Sperm Count
Principle: The sperm count is done after liquefaction in a
counting chamber following dilution and the total
‘number of spermatozoa is reported in millions/ml (106/
ml).
Method
1. Semen is diluted 1:20 with sodium bicarbonate-
formalin diluting fluid (Take 1 ml liquefied semen in
‘a graduated tube and fill with diluting fluid to 20 ml
mark. Mix well)
2. A coverslip is placed over the improved Neubauer
counting chamber and the counting chamber is filled
with the well-mixed diluted semen sample using a
Pasteur pipette. The chamber is then placed in a
humid box for 10-15 minutes for toz08 to
settle,
3, Thechamberis placed on the microscope stage. Using
the 20x or 40x objective and iris diaphragm lowered
sufficiently to give sufficient contrast, number of
spermatozoa is counted in 4 large comer:squares.
Spermatozoa whose heads are touching left and
upper lines of the square should be considered as
‘belonging’ to that square.
4. Sperm count per ml is calculated as follows:
‘Sperms counted xcontction factor
fordilution,
Numberof \%. jVolumeof==*
squarescounted , lequare,
yy
‘Sperms counted » 20
ro aa oe
Spermcount = 1000
= Sperms counted 50,000
5. Normal sperm count is 220 million/ml (Le. 2 20 x
10/ml). Sperm count < 20 million/m may be
associated with infertility in males,
‘Sperm Morphology
‘A smear is prepared by spreading a drop of seminal fluid
con a glass slide, stained, and percentages of normal and
abnormal forms of spermatozoa are counted. The
staining techniques used are Papanicolaou, eosin-
nigrosin, hematoxylin-eosin, and Rose Bengal-toluidine
blue stain. Atleast 200 spermatozoa shoitld be counted
under oil immersion. Percentages of riormial and
‘abnormal spermatozoa should be recorded.
Normal. morphology: A spermatozoon consists of three
main components: head, neck, and tail. Tal is further
subdivided into midpiece, main (principle) piece, and
‘end piece (Fig. 15.2 and Box 155).
Head is pear-shaped. Most of the head is occupied
by the nucleus which has condensed chromatin and few
‘areas of dispersed chromatin (called nuclear vacuoles).
‘The anterior 2/3rds of the nucleus is surrounded by
‘crosomal cap. Acrosomal cap isa flattened membrane-
bound vesicle containing glycoproteins and’ enzymes.
These enzymes are required for separation of cells of
corona radiata and dissolution of zona pellucida of ovum
during fertilization.| End piece
Fig. 15.2: Morphology of spermatozoa
Neck is a very short segment that connects the head
and the tail. Centriole in the neck gives rise to axoneme
of the flagellum. Axoneme consists of 20 microtubules
(arranged as a central pair surrounded by 9 peripheral
doublets) and is surrounded by condensed fibrous rings.
‘Middle piece is the first part of the tail and consists
of central axoneme surrounded by coarse longitudinal
fibers. These are surrounded by elongated mitochondria
that provide energy for movement of tail.
Principle or main piece constitutes most of the tail
and is composed of axoneme that is surrounded by 9
coarse fibers. This central core is surrounded by many
circularly arranged fibrous ribs.
Endpiece is the short tapering part composed of only
axoneme.
‘Normally, 2:30% of spermatozoa should show normal
morphology (WHO, 1999). The defects in morphology
that are associated with infertility in males include
defective mid-piece (causes reduced motility), an
incomplete or absent acrosome (causes inability to
penetrate the ovum), and giant head (defective DNA.
condensation).
Semen Analysis flag
[hen $88: Norma! orvmonnelega |
|+ Total length of sperm; About 60-1.
| Head:
= Length: 3-5 5.
= Width: 23
= Thickness: 1.5
Neo: Length: 0.3
Middle piece:
= Length: 36 .
= Wath: 1.0
Principal piece:
=. Length: 40-50
= Width: 05
End piece: 4-6
“Abnormal morphology (Fig. 153): WHO morphological
classification of human spermatozoa (1999) is given
below:
1. Normal sperm
2 Defects in head:
+ Large heads
‘Small heads H
Tapered heads
Pyriform heads
Round heads
“Amorphous heads
‘Vacuolated heads (> 20% of the head area occu-
pied by vacuoles)
+ Small acrosomes (occupying < 40% of head area)
‘= Double heads
3. Defects in neck:
‘+ Bent neck and tail forming an angle >90° to the
long axis of head
4. Defects in middle piece:
«Asymmetric insertion of midpiece into head
Thick or irregular midpiece
+ Abnormally thin midpiece
5. Defects in tail:
Bent tails
Short tails 3
Coiled tails
Irregular tails
Multiple tails
Tails with irregular width
Pin heads: Not to be counted
. Cytoplasmicdroplets
‘+ >'1/3ed the size of the sperm head
8. Precursor cells: Considered abnormal
2Essentials of Clinical Pathology
1 \—rerosome 2
Middle ace"
Principal piece
End piece
Fig. 18.
hhead, (11) Pin
Round Cells
Round cells on microscopic examination may be white
blood cells or immature sperm cells. Special stain
(peroxidase or Papanicolaou) is required to differentiate
between them. White blood cells >1 million/m indicate
presence of infection. Presence of large number of
immature sperm cells indicates spermatogenesis,
dysfunction at the testicular level.
Immunologic Analysis
Antisperm Antibodies
‘The role of antisperm antibodies in causation of male
infertility is controversial. The immunological tests done
fon seminal fluid include mixed antiglobulin reaction
(MAR test) and immunobead test. i
The antibodies against sperms immobilize or kill
them, thus preventing their passage through the cervix
to the ovum. The antibodies can be tested in the serum,
seminal fluid, or cervical mucus. If the antibodies are
ppresentbound to the head of the sperm, they will prevent
the penetration of the egg by the sperm. Ifantibodies are
bound to the tail of the sperm, they will retard motility.
(12) Round head without acrosome and thick midpiece, (13) Coiled tall, and (14) Double tail
‘Abnormal morphological sperm forms: (1) Normal sperm, (2) Large head, (3) Small head, (4) Tapered hea
Pynitorm head, (8) Round head, (7) Amorphous head, (8) Vacuoles in head, (9) Round head without acrosome, (
) Double
‘a. SpermMAR™ test: This test can detect IgG and IgA
antibodies against sperm surface in semen sample.
Indirect SpermMAR™ IgG test,a drop each of semen
(fresh and unwashed), IgG-coated latex particles, and
anti-human imm« in are mixed together on
a glass slide. At least 200 motile spermatozoa are
examined. If the spermatozoa have antibodies on
their surface, antihuman immunoglobulin will bind
IgG-coated latex particles tolgGon the surface of the
spermatozoa; this will cause attachment of latex
particles to spermatozoa, and motile, swimming
sperms with attached particles will be seen. If the
spermatozoa donot have antibodies on their surface,
they will be seen swimming without attached
particles; the latex particles will show clumping due
to binding of their IgG to antihuman immuno-
globuli
Indirect SpermMAR™ Iga test,a drop each of fresh
unwashed semen and of IgA-coated latex particles,
are mixed on a glass slide. The latex particles will
bind to spermatozoa if spermatozoa are coated with
IgA antibodies.
In indirect SpermMAR™ tests, fluid without
‘spermatozoa (e.g, serum) is tested for the presence ofantisperm antibodies. First, antibodies are bound to
donor spermatozoa which are.then mixed with the
fluid to be analyzed. These antibodies are then
detected as described above for direct tests.
‘Atleast 200 motile spermatozoa should be counted.
If 550% of spermatozoa show attached latex particles,
immunological problem is likely.
. Immunobead test: Antibodies bound to the surface of
the spermatozoa can be detected by antibodies
attached to immunobeads (plastic particles with
attached anti-human immunoglobulin that may be
either IgG, IgA,or IgM). Percentage of motile
spermatozoa with attached two or more immuno-
‘beads are counted amongst 200 motile spermatozoa.
Finding of >50% spermatozoa with attached beads is
abnormal.
Biochemical Analysis of Semen
Biochemical markers (Table 15.2) can be measured in
semen to test the secretions of accessory structures. These
include fructose (seminal vesicles), zine, citric acid or acid
phosphatase (prostate), and a-glucosidase or carnitine
(epididymis).
Test for Fructose
Resorcinol method is used for detection of fructose. In
this test, 5 ml of resorcinol reagent (50 mg resorcinol
dissolved in33 ml concentrated hydrochloric acid;dilute
up to 100 ml with distilled water) is added to 0.5 ml of
seminal fluid. The mixtures heated and brought to boil,
If fructose is present,a red-colored precipitate is formed
within 30 seconds,
‘Absence of fructose indicates obstruction proximal
to seminal vesicles (obstructed or absent vas deferens)
‘or lack of seminal vesicles. Ina case of azoospermia, if
fructose is absent, it is due to the obstruction of
ejaculatory ducts or absence of vas deferens, and if
resent, azoospermiais due to failure of testes to produce
sperm.
Sperm Function Tests or Functional Assays
‘These tests are available only in specialized andrology
laboratories. The tests are not standardized thus making
interpretation dificult. If used singly, a sperm function
test may not be helpful in fertility assessment. They are
more predictive if used in combination,
Semen Analysis, ff
Posteoital (Sims-Huhner) Test
Thisis the examination ofthe cervical mucus after coitus
land assesses the ability of the sperm to penetrate the
cervical mucus. The quality of the cervical mucus varies
during the menstrual cycle, becoming more abundant
and fluid at the time of ovulation (due to effect of
estrogen); this facilitates penetration of the mucus by the
spermatozoa. Progesterone in the secretory phase
increases viscosity of the mucus. Therefore cervical
mucus testing is scheduled just before ovulation
(determined by basal body temperature records or
follicular sizing by ultrasonography). Postcoital test is
the traditional method to detect the cervical factor in
infertility. Cervical mucus is aspirated with a syringe
shortly before the expected time of ovulation and 2-12
hours after intercourse.Gross and microscopic examina-
tions are carried out to assess the quality of cervical
‘mucus (elasticity and drying pattern) and to evaluate the
‘number and motility of sperms (Box 15). 1f2 10 motile
sperms are observed the test is considered as normal.
‘An abnormal test may result from: (a) poor quality of
cervical mucus due to wrong judgment of ovulation,
cervictis or treatment with antioestrogens (e.g. Clomid),
and (b) absence of motile sperms due to ineffective
technique of coitus, lack of ejaculation, poor semen
quality, use of coital lubricants that damage the sperm,
or presence of antisperm antibodies. Antisperm
antibodies cause immotile sperms, or agglutination or
clumping of sperms; they may be present in either
partner. If cervical factor is present, intrauterine
insemination is the popular treatment. The value of the
ppostcoital testis disputed in the medical literature,
This test can be carried out if semen analysis is
‘normal,and the female partner is ovulating and fallopian
tubes are not blocked. It is also done if antisperm
Box 15.8: Interpretation of postroltal test
* Normal: Sperms are normal in amount and moving forward
| in the mucus; mucus stretches atleast 2 inches (5 cm)|
| and dries in a fern-ike manner.
+ Abnormat Absence of sperms or large number of sperms!
‘are dead or sperms are clumped; cervical mucus cannet
‘stretch 2 inches (5 em) or does not dry in a, fer-ikeBL] Essentials of Clinical Pathology
antibodies are suspected and male partner refuses semen
analysis,
Cervical Mucus Penetration Test
In,this test, greatest distance traveled by the sperm in
seminal fluid placed and incubated in a capillary tube
containing bovine mucus is measured. Majority of fertile
men show score >30 mm, while most infertile men show
scores <20 mm.
Hamster Egg Penetration Assay
Hamster oocytes are enzymatically treated to remove the
‘cuter layers (that inhibit cross-species fertilization). They
are then incubated with sperms and observed for
Penetration rate. Itcan be reported as (a) Number of eggs
Penetrated (penetration rate <15% indicates low fertility),
ras (b) Number of sperm penetrations per egg (Normal
>5). This test detects sperm motility, binding to oocyte,
and penetration of oocyte. There is a high incidence of
false-negative results,
Hypo-osmotic Swelling of Flagella
This test assesses the functional integrity of the plasma
membrane of the sperm by observing curling of flagella
in hypo-osmotic conditions.
Computer-assisted Semen Analysis
Computer software measures various characteristics of
the spermatozoa; however, its role in predicting fertility
potential is not confirmed.
EXAMINATION FOR THE PRESENCE OF
SEMEN IN MEDICOLEGAL CASES
This includes examination of material obtained from
vagina, stains from clothing, skin, hair, or other body
parts for semen. This is carried out in cases of alleged
Tape or sexual assault,
Collection of Sample
‘+ Vagina: Direct aspiration or saline lavage
* Clothing: When scanned with ultraviolet light, semen
Produces green white fluorescence. A small piece (1
1) of clothing from stained portion is soaked in 1-2
mlof physiologic saline for | hour. A similar piece of
clothing distant from the stain is also soaked in saline
asa control
Tests
|. Microscopic examination for sperms: Presence of motile
sperms in vaginal fluid indicates interval of
<8 hours. Smears prepared from collected samples
arestained and examined for the presence of sperms.
2. Acid phosphatase: Acid phosphatase is determined on
vaginal or clothing samples. Due to the high level of
‘acid phosphatase in semen, its presence indicates
recent sexual intercourse. Level of 250 U/sample is
considered as positive evidence of semen.
3. Determination of blood group substances: When semen
is positively identified in vaginal fluid or other
sample, test can be carried out for the presence of
blood group substances in the same sample. The
‘secretor’ individuals (80% individuals are secretors)
will secrete the blood group substances in body fluids,
including semen.
4. Florence test: This test detects the presence of choline
found in high concentration in semen. To several
drops of sample, add equal volume of reagent (iodine
2.54 g, potassium iodide 1.65 g distilled water 30 ml);
in positive test rhombic or needle-like crystals of
periodide of choline form. False-positive tests can
occur due tohigh choline content of some otherbody
fluids.
EXAMINATION OF SEMEN TO CHECK THE
EFFECTIVENESS OF VASECTOMY
The aim of post-vasectomy semen analysis is to detect
the presence or absence of spermatozoa. The routine
follow-up consists of semen analysis starting 12 weeks
(or 15 ejaculations) after surgery. If two successive semen
samples arenegative for sperms, the semen is considered
as free of sperm. A follow-up semen examination at 6
months is advocated by some to rule out spontaneous
reconnection.
BIBLIOGRAPHY
1. Hirsh A. Male subfertiity. BMJ 2003;327:669-72
2. Phadke AM. Clinical Atlas of Spermn Morphology. New
Delht Jaypee Brothers Medical Publishers (P) Lid. 2007.
3. World Health Organization, Laboratory Manual forthe
Examination of Human Semgn and Sperm-Cervical
Mucus interactions 3rd edition. Cambridge: Cambridge
University Press, 1992.
4. World Health Organization. WHO laboratory manual
for the examination of human semen and sperm
‘cervical mucus interaction. 4th edition. UK:
‘New York NY: Published on behalfof the World Health
Organization (by] Cambridge University Press; 1999,