SPERM ANALISIS

dr. Almaycano Ginting, M. Kes, M.Ked(Clin.Path), Sp.PK

Clinical Pathology Department
Faculty of Medicine
Universitas Sumatera Utara
2023
Laboratory evaluation of male reproductive function
• The evaluation of reproductive dysfunction in the male usually begins
with semen analysis because it is a cost-effective and relatively simple
procedure.
• In addition, if the results are normal, further evaluation is often
unnecessary.
• If semen analysis is abnormal, then hormone analyses are performed,
which include FSH, LH, and testosterone levels.
Definition of semen/ejaculate/sperm
• A mixture of spermatozoa submerged in testicular fluid, epididymis
and at the time of ejaculation mixed with the secretions of the glands:
prostate, seminal vesicles and bulbo urethralis
Semen analysis

• In addition to its use in the evaluation of reproductive dysfunction, particularly

infertility, semen analysis is used to select donors for therapeutic insemination and
to monitor the success of surgical procedures, such as varicocelectomy and
vasectomy. Semen analysis consists of microscopic and macroscopic components;
the latter include measuring physical (e.g., volume) and chemical (e.g., pH)
properties.
Semen analysis
Diagnostic terminology with respect to semen analysis:
• Azoospermia: no sperm in semen,
• Oligozoospermia: sperm concentration in semen < 20 x 106/mL,
• Severe oligozoospermia, < 5 x 106/mL,
• Astenozoospermia: sperm motility < 50%, and
• Teratozoospermia: normal sperm shape <30% (WHO)
Semen analysis

Macroscopic
Microscopis
Semen analysis
• Macroscopic:
✓ Liquefaction : < 60 minute
✓ Color : Yellowish white
✓Volume : N = 2,5-5 ml
✓ pH : 7,2-8,0
✓ Viscosity : Thick
Semen analysis
• Microscopic:
✓ Sperm count → 60-150 million/ ejaculate
✓ Motility → Active : > 50 %
Weak : < 30 %
Not moving : < 20 %
✓Morfology → Normal : >60%
Abnormal : <40%
 Anatomi of Sperm
• Head: oval in shape, acrosome covers 1/3 of it, 3-5
microns long, to 2/3 long.

• Midpiece: slender (< ½ head

width), 2x the length of the
head, and is in one arm line
the long axis of the head.
• Tail: a clear boundary, in the
form of a long line 9 x the
length of the head.
Sample Collection
• The patient should be instructed to collect semen after 2 to 5 days of sexual
abstinence to ensure that the sperm count will be at its highest and improve the
reliability of the test. Longer periods of abstinence usually result in a higher
sperm concentration but reduced sperm motility. For semen collection, the
laboratory should provide a sterile plastic (polypropylene) container with a screw
top. The semen specimen should be delivered to the laboratory within 1 hour of
collection and kept warm during transportation.
• A post ejaculate urine sample may be collected at this time if retrograde
ejaculation is suspected. Two specimens collected within 2-to 3-week intervals
should be used for evaluation. If they are markedly different, additional
specimens should be collected.
Sample Collection
• Ideally, semen specimens should be collected in the privacy of a room adjacent to
the laboratory, because some samples require sperm to be separated from
seminal plasma as soon as possible. Assisted reproductive technologies (ARTs),
such as in vitro fertilization (IVF), require that motile sperm be isolated from
seminal plasma within 1 hour of ejaculation to protect sperm from the inhibitory
effects of seminal plasma on fertilization.
• Semen should be obtained by masturbation; if circumstances preclude such
collection, special Silastic condoms should be made available for semen collection
with intercourse. Incomplete semen specimens should not be analyzed because
they can give inaccurate values.
• Silastic seminal fluid collection device
Examination
• Macroscopic examination should be performed after liquefaction, which usually occurs in less
than 20 minutes at room temperature. Failure to liquefy may indicate inadequate prostate
secretion.
• Semen should be thoroughly mixed before examination and its viscosity recorded.
• The volume of ejaculate can be measured by weighing the collection cup before and after
specimen collection. The appearance of a yellow hue in a semen specimen is associated with
pyospermia; a rust color is due to small bleedings in the seminal vesicle.
• The pH ranges from 7.2 to 7.8; however, it may be 8.0 or higher with acute infection in the
prostate, seminal vesicle, or epididymis. The pH will be 7.0 or lower if there is contamination with
urine, an obstruction in the ejaculatory ducts, or if the specimen consists of mainly prostatic fluid.
• Viscosity: normal : no more than 2 cm
• Smell: Normal: Typical like acacia flowers, Infection: fishy smell, rotten.
Examination
• Microscopic → sperm concentration, motility, morphology, and agglutination.
• Other cellular elements, such as polygonal cells of the urethral tract and round
cells such as spermatogenic cells and leukocytes, can also be observed when
sperm are counted in a hemocytometer or Makler chamber. Because sperm
motility and velocity are temperature dependent, these parameters must be
assessed on a microscope with a warm stage. At least four different fields of two
specimen aliquots should be counted and the mean of the eight separate readings
recorded.
• Total sperm count is then calculated by multiplying the dilution factor (normal
concentration range, 15–50 million/mL) by its volume (normal range, 2–5 mL).
Examination
• Total motility (normal range, 40% or above) Progressive motility (normal range, 32% or
above) is expressed as the percentage of sperm that move in a forward, linear motion. In
addition, forward movement is graded. Sperm that move rapidly in a straight line with
little yaw and lateral movement are grade 4 or, if they move more slowly, grade 3. Grade
2 sperm move even more slowly and with substantial yaw. Grade 1 sperm have no
forward progression. Zero progression denotes absence of any motility. If motility is less
than 30%, a viability stain of eosin Y with nigrosin as a counterstain is done.
• In bright-field microscopy, dead sperm will stain red, whereas live sperm will exclude the
dye and appear unstained. In samples with no visible sperm, such as postvasectomy
semen, the entire sample should be centrifuged and the pellet examined for intact or
damaged sperm fragments. The analysis should be repeated in 4 to 6 months.
Examination
• Agglutination occurs when motile sperm stick to one another in an
orientation that is reproducible within a given specimen, such as head
to head, tail to tail, midpiece to midpiece, or mixed ways, depending
on the specificity of sperm antibodies.
• Agglutination suggests an immunologic cause of infertility, and a
description of the type of agglutination should be recorded. This can
usually be distinguished from clumping due to bacterial infection or
tissue debris, which typically involves nonspecific orientation of the
sperm.
Examination
• Sperm morphology can predict fertility.
• In 2010, the World Health Organization (WHO) standardized the new normal values based on data from men
with proven fertility. These men were defined as men who were known to help their partners conceive in the
previous 12 months. Typically, more than 4% of the sperm in a semen specimen should exhibit normal
morphology.
• Kruger and colleagues (1988) developed strict criteria for normal sperm morphology. Morphologically
abnormal sperm usually have multiple defects; the average number of defects per sperm, designated as the
teratozoospermic index, is a significant predictor of sperm function both in vivo and in vitro. A strict
morphology score of greater than 4% of normal indicates excellent fertilizing capacity. Scores between 0%
and 3% predict probable inability to fertilize. Wide variability in the size of the acrosomal cap is the most
obvious characteristic of abnormal sperm.
• An acrosomal cap less than one-third of the head surface is considered abnormal, as are retention of a
cytoplasmic droplet greater than one-half of the head size and a tail less than 45 μm long. Of particular note is
the direct relationship between acrosome size and the frequency of fertilization or pregnancy.
sperm abnormalities
Types Sperma abnormalities types:

• Head defect
• Neck and midpiece defect
• Tail defect
• Execess residual cytoplasma
Refference
• Gandosoebrata R. 2016. Penuntun Laboratorium Klinik. 16th Edition. Jakarta: Dian
Rakyat. 171-5p.
• Mc Pherson dan Pincus. 2022. HENRY’S CLINICAL DIAGNOSIS AND MANAGEMENT BY LABORATORY
METHODS. 24th Edition. Philadelphia : Elsevier Inc, 429-430p
• Moeloek, N, Siri, Z. 2006. Analisis semen pada kasus infertility. Pendidikan Berkesinambungan Patologi
Klinik. 220-234p.
• Sherwood L. 2016. Fisiologi Manusia Dari Sel ke Sistem. 8th. Edition. Ong OH, Mahode AA, Rahmadani D.
Editor. Jakarta: Buku Kedokteran EGC. 782-803p
• WHO. 2010. WHO Laboratory Manual for The Examination and Processing of Human
Semen. 5th Edition. Switzerland: 7-44p