SEMEN ANALYSIS
Semen
A mixture of seminal fluid and cells
• Seminal fluid contains:
• Prostatic fluid
• Epididymal plasma
• Seminal vesicle fluid
• The cells are:
• Spermatozoa
• Germ line cells
• Leukocytes
• Epithelial cells
• Occasional red cells
Formation of the sperm
• Formed in the seminiferous
tubules
• Start as spermatogonia
• The transformation of round germ
cell to sperm cell occurs during
passage to the centre of the
seminiferous tubule
Sperm production
Spermiogenesis:
differentiation of the
round spermatid into a
spermatozoon
This is the process in
which sperm
morphology is largely
determined
• At ejaculation, the sperm are transported out of
the vas and mix with accessory gland
secretions:
• prostatic fluid
. pH slightly acidic to neutral
. contains citric acid and zinc)
• seminal vesicle fluid
. pH strongly alkaline
. contains fructose)
Head
Mid- • The human sperm 70 µm long
piece
• The nucleus is in the head – contains
the 23 chromosomes
Tail
• binds to the egg at fertilization
• Midpiece: the energy for motility is
generated
• Tail: motility
INDICATIONS
• Diagnosis of infertility, function of the accessory
glands
• Prognosis for fertility
• Identify treatment options:
• surgical treatment – Vasectomy success
• medical treatment
• assisted conception treatment
• Medicolegal cases
Causes of Male Infertility
• Abnormality in sperm production
• Abnormality in sperm function
• Obstruction in the ductal system
Semen collection
Masturbation, directing the semen into a clean sample cup. Do
not use a lubricant which can kill sperms – Preferred
Coitus interruptus - withdrawing the penis from the partner just
before ejaculating follow by ejaculating into a clean sample cup.
Coitus - by using a condom. A special (silicon) condom that
does not contain any substance that kills sperm (spermicide).
After ejaculation, carefully remove the condom from the penis.
Tie a knot in the open end of the condom and place it in a
container that can be sealed in case the condom leaks or breaks.
Ordinary condoms should not be used since they usually contain
spermicides
Assisted ejaculation – electro-ejaculation used in paralegics
• Abstinence days 2-7
Semen Collection • Collect the entire
sample into the wide
mouth sterile
container, 70% of
sperms is in the first
part of the ejaculate
• Keep the sample at
body temperature, no
sunlight
• Deliver the sample
within one hour of
ejaculation
Macroscopic Examination
Volume Normal: 1.4 ml per ejaculation
Low volume (<1ml) – a block, retrograde ejaculation,
infection or lack of androgen.
Low semen volume cannot neutralize vaginal acidity
High semen volume dilute sperms/ active infection
pH Normal: =/>7.2 (alkaline)
Acidic pH (<7.0) in a low volume & density sample indicates
–congenital bilateral absence of vas deferens (in which
seminal vesicles are also poorly developed) and ejaculatory
duct obstruction
Macroscopic Examination
Appearance Normal: Whitish to grey opalescent
Yellow (urine, jaundice)
Pink/Reddish/Brown (RBCs)
Liquefaction Normal: 15–30 minutes after collection
Immediately after ejaculation, the semen is normally a semisolid coagulated
mass. At room temperature, the semen usually begins to liquefy (become
thinner) within a few minutes and completely liquefies within 15 minutes.
Within 30 minutes it becomes more
homogeneous and watery.
Lumpy >60 min – sperms may be trapped in unliquefied jelly; maybe sign of
prostatic infection, lack of prostatic protease
Viscosity Normal Smooth and watery
Fresh semen is fairly viscid and the viscosity can be estimated by gently
aspirating semen into a wide-bore plastic disposable pipette, allowing the
semen to drop by gravity. Normal semen falls drop by drop and if viscosity is
abnormal, the drop will form a thread more than 2 cm long. Normal viscosity is
important, since increase in viscosity affects sperm motility.
Abnormal –, thick with long threads
High viscosity impede sperm movements
MICROSCOPIC EXAMINATION :
Sperm count; Normal finding 15 millions/ml.
Clinical conditions :
1. A reduced sperm count- Oligozoospermia.
2.Complete absence- Azoospermia.
Causes :
Mumps orchitis, prostatitis, Hypopituitarism,
hypogonadism
• Sperm vitality: Normally 50 to 56 % live forms are observed. (conducted if
<40% motility)
Vitality of the spermatozoa is estimated by identifying those with an intact
cell membrane using eosin-nigrosin stain
• Total sperm count
Sperm count is carried out in an improved Neubauer chamber using a
Thoma pipette in a dilution of 1 in 20 (as for total leukocyte count).
Vitality Assessment
1. Eosin-nigrosin (dead sperm stain pink/red)
2. Eosin (1%) (dead sperm stain pink/red)
3. Trypan (0.4%) blue (dead sperm stain blue)
4. Hypo-osmotic swelling test (HOS) (live sperm shows tail
curling
• Test 1, 2 and 3 for diagnostic uses.
Usually 1:1 ratio of semen to dye mixture, mix well and smear
onto a slide. Read immediately at x40 objective, count 200
sperms
• Test 4 is use to choose live (immotile) sperm for ICSI
Dead sperms will not react in HOS while live sperm will take
up fluids causing their tails to curl within 5 min and stabilize at
30 min. Therefore viable sperms may be selected for ICSI [Lin
et al, 1998; Cayan et al, 2001].
Vitality Assessment
Dead sperms are stained Live sperms shows curling
pink/red tails
Motility : Normal findings 60~90%
Total motility less than 42% associated with
infertility.
-Aesthenozoospermia
Abnormal forms : 0~20% normal findings.
Clinical Conditions :
More than 20% abnormal forms associated with
infertility.
-Teratozoospermia
Morphology assessment
✓ Sperms must be stained for an accurate assessment
✓ Strict criteria /Kruger /Tygerberg
✓ The sperm head is oval, smooth-symmetrical outline, a length of 3-5 um and a
width of 2-3um.
✓ The head should have a well defined acrosome area of 40-70% and vacuoles
(=/<2) that occupies ~ 20% head area.
✓ The mid-piece must be straight and slender, 0.5 um in width and 7-8um long,
straightly aligned to the head.
✓ The tail must be straight and 45-50 um long.
✓ To be classified as normal, the sperm must be normal in all portions (head,
mid-piece, tail).
✓ Atleast 400 sperms must be scored on randomly chosen fields.
Normal Forms (%) = normal sperms / the total number of sperms
evaluated x 100.
• Classification of abnormal sperm morphology (Pap stain/Diff Quik stain)
Head defects:
• – Abnormal size: Large or small head
• – Abnormal shape: Pointed/tapered, round, pyriform, amorphous
• – Acrosomal defects
• - Vacuolated acrosomal region: More than two vacuoles or >20% of the head area
occupied by vacuoles. Presence of vacuoles in the post-acrosomal region.
• - Abnormality of acrosomal areas: Small or large acrosomal areas (<40% or >70% of the
head area)
• – Abnormal number: Double-headed
Neck and midpiece defects:
• – Sharply bent neck
• – Asymmetrical insertion of the midpiece into the head
• – Thick or thin or irregular midpiece
Tail (principal) piece defects:
• short or bent or coiled
• double tail.
Fructose test (Resorcinol test)
• Determines androgen deficiency or ejaculatory obstruction to semen.
In both conditions, the seminal fructose is low.
• Normal – 150-600 mg/dl
• Resorcinol test (Qualitative test)
5ml of dilute HCl +1 ml of semen + 5 mg of fresh resorcinol – Boil
Appearance of red colour – Fructose present which can be measured
by spectrophotometer.
Pus cells : Normally 1 to 2/hpf
Increased number of pus cells indicative of
infection of seminal vesicle.
RBC’s : Normally not seen.
If present indicates T.B. of seminal vesicles,
rupture of blood vessels.
Sometimes semen may contain infection by
parasite - Trichomonas.
Azoospermia
• secondary testicular failure
• Klinefelter’s (1 in 500) • congenital vas obstruction
• retrograde ejaculation
• Hypogonadotropic- hypogonadism
• endocrine causes (prolactinemia, low
• Ductal obstruction (absence of the Vas deferens)
• testosterone)
Oligospermia Abnormal volume
• Anatomic defects • Retrograde ejaculation
• Infection
• Exogenous (e.g. heat)
• Ejaculatory failure
• Varicocele
• vasectomy (should be 0 after 3-6 months)
• primary testicular failure (Klinefelters)
Parameters (WHO criteria 2021)
Parameter Lower Reference Limit
Semen volume (ml) 1.4
Sperm concentration (106/ml) 15
Total sperm number (106/ejaculate) 39
Progressive motility (PR, %) 30
Total motility (PR +NP, %) 42
Vitality (live sperms, %) 54
Sperm morphology (NF, %) 4
pH* >/=7.2
Leucocyte* (106/ml) <1
MAR/Immunobead test* (%) <50
Parameters agreed on consensus
Terminologies of Semen Analysis
➢ Oligospermia – sperm concentration <15 million/ml
➢ Asthenozoospermia – <42% grade (PR+NP) or < 30 PR%
➢ Teratozoospermia – <4% spermatozoa
➢ OAT =Oligo-astheno-teratozoospermia
➢ Azoospermia – no spermatozoa in semen
➢ Polyzoospermia – ++ high sperm concentration, >200M/ml
➢ Hypospermia – semen volume < 1.4 ml
➢ Hyperspermia – semen volume > 4.5 ml
➢ Aspermia – no semen volume
➢ Pyospermia – leukocytes present in semen, >1M/ml
➢ Hematospermia – red blood cell present in semen
➢ Necrozoospermia – “dead” sperm
Normal Sperm Morphology
Abnormal Morphology
Abnormal Morphology
1) Azoospermia
2)Klinefelter’s syndrome
Hypogonadotropic- hypogonadism
3) Prostatic infection/ prostatitis- decrease in hyaluronidase causing
high viscosity
1) Oligospermia
2) Bouin’s fluid- picric acid, formaldehyde and acetic acid.
3) Head defects: large head, small head, double head,vacuolated head.
Neck defects: bent neck, thick and thin insertion, asymmetrical.
Tail defects: Coiled, bent, double tail.
Semen analysis: A 30-year-old male with married life of 5 years has no children
SEMEN ANALYSIS SHOWED:
• Volume – 0.5 ml
• pH – 8.5
• Liquefaction time – 30 mins
• Sperm concentration – 10 million/ml
• Sperm morphology – 60% normal morphology
• Sperm motility – 50% showed forward motility
• Fructose level – 10 mg/ 100 ml of ejaculate
• QUESTIONS:
1. What is your diagnosis?
2. Mention the indications for this test
3. Mention other investigations that can be done in this case
1. Oligozoospermia with low volume and low fructose level
2. Infertility, successfulness of vasectomy, prognosis of fertility
treatment, MLCs
3. Transrectal/ Scrotal USG (varicocele,tumors) , Genetic tests for
abnormal genes/congenital obstruction/karyotyping/cystic fibrosis,
hormone levels (FSH), test for antibodies, infections
