LE9 AUBF

SY 2021-2022
1st SEMESTER
BS-MLS
PROF. ETHEL MARIE MANGADA, RMT

SEMEN

OUTLINE
I SEMEN
A Physiology
B Composition
C Collection
D Appearance
E Volume and Viscosity
F pH
G Sperm concentration & count
H Motility
II SPERM MORPHOLOGY
III SPERM VIABILITY
IV ANTISPERM ANTIBODIES
V MICROBIAL & CHEMISTRY TESTING
VI SCREEN DETECTION
VII POSTVASECTOMY ANALYSIS
VIII SEMEN FUNCTION TEST
IX OTHER TEST
X SEMEN ANALYSIS QUALITY CONTROL

SEMEN o damage in prostate results in non-production of

 Purpose of semen analysis – in Andrology lab acidic components necessary for liquefaction
 Main concern: and coagulation of semen
o Determine if the male is sterile or not, capable of o damage/absence of one component affects
impregnation the viability of the sperm cell and its travel to the
o Determine success of vasectomy ovum
 Vasectomy includes cutting of vas deferens
as not to deliver/ejaculate sperm together  Spermatozoa
with the rest of the semen composition as not o Produced in seminiferous tubules of testes
to cause pregnancy to female partners. o Mature and stored in the epididymis
 Checking the presence of sperm after 2 o Contribute 5% of volume
months of vasectomy. Detection of sperm  The sperm cells, once mature, are released in
indicates unsuccessful vasectomy. vas deferens meeting in the seminal vessels
o Help solving forensic cases – forensic analysis and are combining with the seminal fluids.
 e.g alleged rape case
 Seminal vessels
SEMEN PHYSIOLOGY o Majority of fluid (60%)
Semen consists of four components contributed o Produce seminal fluid which provide fructose for
separately by the ff: sperm metabolism
o Testes and epididymis  Seminal fluid has an alkaline component
 Testes – where sperm cells are produce  Fructose is necessary for the motility of the
 Epididymis – sperm cell undergoes sperm
maturation  no fructose – no energy to utilized towards
o Seminal vessels – secrete seminal fluid that will the ovum.
combine with the sperm cell and enter in the; o Sperm are not motile without this fluid
o Prostate gland – where acid components of o Provides alkaline environment for the sperm
semen is added
o Bulbourethral glands – fluid from bulbourethral Note: purpose of seminal vessels and Bulbourethral
gland provides alkaline mucus gland is the same. This is to produce fluids/mucus that
provides the alkalinity of the semen.
 Normal specimen must have all four components  seminal vessels produce seminal fluids
o e.g problem in epididymis –more likely there will  bulbourethral gland produce alkaline mucus
be problem in number and appearance of Seminal fluid and alkaline mucus produced to
sperm cell neutralize the acidity of the prostate fluid as well as
o problem in seminal vessels – there will be problem the vagina of the female during coitus/sexual
in producing seminal fluid necessary for the intercourse. Why neutralize? Acidity of prostate gland
sperm cells to thrive and vaginal alone can affect motility of sperm cell.

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COMPOSITION OF SEMEN
 Spermatozoa 5%
 Seminal Fluid 60%–70%
 Prostate Fluid 20%–30%
 Bulbourethral Glands fluid 5%
 Sperm together with seminal fluid enter to:

Prostate gland
 Produce Acidic fluid or prostatic fluid(20%–30%)
 Prostatic fluid contains acid phosphatase, citric acid,
zinc, and proteolytic enzymes
 Problem in prostate gland results to
diminished prostatic fluid component
 Enzymes coagulate semen prior to ejaculation and
cause liquefaction after ejaculation

Bulbourethral glands
 Thick, alkaline fluid (5%) to neutralize acid from
prostate and acid pH of vagina; otherwise, no
sperm motility
 Same purpose with seminal fluid
 Thick alkaline fluid – wash out urethra which is
the passage way of sperm to make the
environment detrimental.

SPECIMEN COLLECTION
 Must be a complete specimen
 Majority of sperm is in first part of ejaculate
 Detailed instructions to patients
 2–3 days abstinence; no longer than 5 days (more
sperm but non motile)
 Laboratory provides containers and ideally a
room for collection
 Home: deliver to laboratory within 1 hour; keep
specimen warm

METHODS OF COLLECTION
SUMMARY OF SEMEN PRODUCTION  Self-production or masturbation
o BEST because it prevents contamination
SEMINIFEROUS  Coitus interruptus
SPERMATOGENESIS o withdrawal method
TUBULES
Epididymis Sperm maturation o one of the disadvantage is sometimes the boy
Propel sperm to ejaculatory ducts doesn,t know that meron ng nailabas
Ductus *Peristalsis- the muscle movement will  Vaginal vault aspiration
deferens cause the release of sperm and - Aspiration of seminal fluid from the vaginal vault
other seminal fluids after coitus
Provide nutrients (fructose) for sperm o While he male patient is having coitus with his
Seminal partner dun siya mageejaculate sa loob ni
and fluid
vesicles partner after that the fluid will be collected from
*Provide alkalinity of prostate fluid
the vaginal vault(so eto yung gagamitin natin)
Provide enzymes and proteins for
o DISADVANTAGE: squamous epithelial cells
coagulation and liquefaction
coming from the vaginal wall that can cause
Prostate gland *Acidic because it contains acid
interference in viewing the sperm cells
phosphatase, citric acid, zinc
 Condom method
and proteolytic enzyme
o Only nonlubricant-containing rubber or
Add alkaline mucus to
Bulbourethral polyurethane condoms should be used.
neutralize prostatic acid and
glands o the patient will masturbate and dun na siya
vaginal acidity
maglalabas sa condom but make sure that the
condom in nonlubricant to avoid causing motility

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in the sperm cells and if it is a usual condom can  When the pH is very very low or acidic, it may
usually contain spermicides that can be indicate increased amount of prostrate/prostatic fluid
detrimental in the sperm cell. added to the semen (provides acidity of semen)
 We can check the pH of semen with the pH paper or
SPECIMEN COLLECTION we can use the pH pad of reagent strip that is used
 Record time of collection and time of receipt (make for urine
sure that you collected it not more than 1 hour)
o should store it at room temperature that is 37 SPERM CONCENTRATION AND COUNT
degrees celcius or you can place it sa baba ng  Concentration = number sperm/mL
armpit(physiologic body temp)  Count = number of sperm per ejaculate
 Clotted specimen should liquefy in 30–60minutes  Normal concentration = 20 – 160 million/mL
(normal)  When the concentration is between or within 10 – 20
o Problems in the liquefaction is when it liquify after million, it is considered borderline (not high, not low)
2 hrs then there will be the a problem in the o It may be an indication that the patient is infertile
seminal fluid or has very low sperm concentration
 No lubricated, antispermicidal condoms  Normal count = Normal concentration x volume of
 Semen specimens are reservoirs for hepatitis the collected specimen/semen from the patient
 and HIV o >40 million/ejaculate, lower values may indicate
 Discard as biohazardous waste infertility among males
 Concentration performed in Neubauer chamber,
NOTE!!! same as red blood cell count – 5 small center
 We should use gloves when processing to avoid squares
infection and for precautionary measure and if the  Diluting fluid = sodium bicarbonate and formalin,
semen needs to be culture to determine if the must immobilize sperm
patient is suffering from an infection we should first o We dilute fluid to immobilize sperms cells
perform the culture before we perform other test in o Prior to performing sperm concentration and
the semen analysis like testing the concentration of count, you should have already performed the
the morphology of the concentration and other test that evaluates motility if required in the
methods that may cause contamination. analysis.
o Dilution: 1 to 20
APPEARANCE  Count both sides of the chamber, sides must agree
 Normal is gray-white(pearly white), translucent within 10% and then use the average
 White turbidity = infection: culture  With this method, (example) if an average of 60
 Red = blood cells, abnormal sperm are counted, multiply the average by
 Yellow = urine, prolonged abstinence, medications 1,000,000
 Urine is toxic to sperm: no motility o 6 – Normal
 Clots remaining after 1 hour: wait for liquefaction
before analyzing
o To facilitate liquefaction add alphakimotrypsin

VOLUME AND VISCOSITY

 Normal: 2 – 5 mL( may be increased when there,s
prolonged abstinence but of course the motility and
viability is affected0
 Measure in a graduated cylinder
 Decreased = infertility, incomplete collection
 Viscosity: droplets with thin threads ( usually
continous and not stringy or not appear clumping)
from a pipette are normal
 Rate 0 (watery) to 4 (gel-like) or low, normal, high

NOTE!!!
 The volume, viscosity, liquefaction time and semen is
part of macroscopic exam
 Semen analysis involve two testing: Macroscopic and
microscopic examination

pH
 Normal pH of semen is alkaline so it ranges from 7.2 to  Must multiply sperm per μL by 1000 to reach
8.0  sperm/mL when using a different number of squares
 When the semen has the pH of over 8.0, it may  Sperm count
indicate infection = over alkalinity 60,000,000 sperm concentration x 3 mL
volume = 180,000,000 sperm count

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 Instrumentation has been developed; used o thickest part of the tail because it is surrounded
 primarily in fertility clinics by a mitochondrial sheath that produces the
(*count only sperms that are fully developed, don’t energy required by the tail for motility
count the sperms consisting head only)  Tail
 AZOOSPERMIA- complete or total absence of o long flagellum for movement
spermatozoa seen in (ex. Patient who undergone o 45 µm long
vasectomy)  Abnormalities:
 NECROSPERMIA- presence of sperm cells whether o Head= associated with poor ovum penetration
completely dead or immobile o Neckpiece, midpiece, and tail= affect motility.
 OLIGOSPERMIA- deficiency in the number of sperm  Observe on thin smear under oil immersion
cells or presence of few motile cells seen in  Procedure:
o Smears are made by placing approximately 10
SPERM MOTILITY µL of semen near the frosted end of a clean
 Need to have sperm with forward, progressive microscope slide. Place a second slide with a
movement clean, smooth edge in front of the semen drop at
 Examine within 1 hour; evaluate undiluted on glass a 45° angle and draw the slide back to the edge
slide with cover slip (keep sperm cells motile, para of the drop of semen, allowing the semen to
madetermine grading ng kanilang motility) spread across the end. When the semen is evenly
 Estimate percentage with progressive, forward distributed across the spreader slide, lightly pull
motion in 20 fields the spreader slide forward with a continuous
 Grading scale movement across the first slide to produce a
o 0 to 4 smear.
o 4: rapid straight-line motility  Staining:
o 3: slower speed, some lateral movement o Wright’s stain
o 2: slow forward movement, more lateral o Giemsa
o 1: no forward progression o Shorr ; or
o 0: no movement o Papanicolaou stain (lab preference)
 Using the American scale:  Air-dried slides are stable for 24 hours.
4= A  Count at least 200 and report number of abnormal
3&2= B  Strict criteria measures, acrosome, tail, neck, and
1= C midpiece recommended by World Health
0=D Organization (WHO)
 Normal: 50%; rated as 2  Abnormal head:
 Instrumentation available o double heads, giant and amorphous heads,
pinheads, tapered heads, and constricted heads
CASA- COMPUTER ASSISTED SEMEN ANALYSIS o abnormally long neckpiece may cause the
 Provides objective determination of both sperm sperm head to bend backward and interfere
velocity and trajectory (direction of motion) with motility
 Sperm concentration is also included in the  Abnormal tail:
analysis o doubled, coiled, or bent
 Normal: >30% normal
SPERM MORPHOLOGY  Strict: > 14% normal
 Critical to fertilization
o Just as the presence of a normal number of
sperm that are nonmotile produces infertility, the
presence of sperm that are morphologically
incapable of fertilization also results in infertility
 Evaluate head, neck, midpiece, tail
 Head:
o oval with acrosomal cap (Contains enzymes for
ovum penetration) at end and covering half of
the head
o acrosomal cap should encompass
approximately half of the head and cover
approximately two thirds of the sperm nucleus.
o 5 µm long and 3 µm wide and a long
 Neckpiece:
o attaches head to midpiece and tail; midpiece
surrounded by sheathe of mitochondria for tail
movement
 Midpiece
o 7.0 µm long and

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TECHNICAL TIP:
 Sperm motility can be evaluated at room
temperature or 37°C. When assessing motility at 37°C,
the sample should be incubated at this temperature
and the preparation made with prewarmed slides
and cover slips.

 We have here some pictures showing you now the

abnormal morphologies.
 So this are actually H&E stains smears.
 Refer to the left picture. So if you can see here. This
one has an amorphous head (Amorphous ung head
niya, kita niyo ung shape niya ganun ganun lang )
 Refer to the right picture. And this one naman
showing you now a double head sperm (ayan
double head siya tapos ung ulo nga actually ito
parang tapered siya so abnormal)

SPERM MORPHOLOGY (CONT’D)

 We have here a picture showing you now the

different morphology of the sperm. (Abnormal and
normal).
 Referring to first pic is the normal cell. And the rest are
the abnormal variations.
 Number 1 is we have double head. (So tignan mo
dalawa ung ulo niya, So ano ngayon kung dalawa
ung ulo) What will happen? There will be what?
Problem dun sa ating motility. And even the direction
(ung isang ulo dito gusto pumunta tapos ung isa dito)
So may mga, mas madaming lateral movements.
 And then next, we have your giant head. Whats the  Refer to left picture. This one naman, you can see
problem with these? Motility and as well as your here double tail. (So meron siyang double tail)
penetration.  Refer to right picture. And this one naman, here we
 And then we also have your amorphous head. (so can see bent neck. (So alam niyo guys sabi nila pag
kung makikita niyo ung amorphous head, wala di ka daw lumingon pinanggalingan mo, di ka
siyang observable nuclues, as well as acrosomal makakarating sa paroroonan mo. Pero this one is an
cap). So anong problema natin? The penetration can exemption kasi lumingon siya sa pinanggalingan
be a problem. niya, malamang di na siya makakarating dun sa
 And then we also have here your, pinhead. pupuntahan niya. CHAROT)
(Napakaliit nung ulo niya)  Those are the abnormal morphologies of our cells.
 Tapered head (So ito kasing normal, something oval
and this one naman is masyado siyang pointy ung
tapered head natin) So there can be problems with
motility and as well as penetration. So with your
constricted heads.
 Double tail ganun din yon. Like your double heads.
There will be a problem in the direction of your
movement as well as your coiled tail.
 And finally the last one, we have your spermatid (So
no tail, malamang-samalamang di yan makaka swim
papunta sa target niya)
 This picture showing the normal and the abnormal
morphology of our sperm cell.
SPERM MORPHOLOGY (CONT’D)
SPERM MORPHOLOGY (CONT’D)  Round bodies

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o White blood cells (WBCs) and spermatids

(immature sperm)
o Differentiate on morphology smear
o Count number of each separately in100 cells

SPERM VIABILITY
 Eosin-nigrosin stain
o Also called BLOOM’S TEST
 this test determines cells that are still living and those
that are already dead
 presence of a lot of dead cells indicates infertility
among men
 We also have to compute for our sperm morphology.  Dead cells stain red; normal are blue-white
Under sperm morphology are your round bodies. (So  Count number/100 cells
guys, kapag kasi inobserve natin ung cells natin,  Abnormal: if there is more dead cells than living cells
other than of course seeing normal sperm cells.  Normal: 75% living
Makakakita tayo nung mga solo ulo lang o kaya  Corresponds to motility
minsan mga WBCs). We call this your round bodies. o Living cells are motile while dead cells are
(Diba naalala niyo sa sperm count, minsan sila ung stagnant (not moving)
problema natin. Sa sperm count, kina count lang
natin ung mga may butot and ung walang ulo and
the WBCs are not counted).
 This cells are actually also counted, and they are
differentiated now. Same as other morphologies,
using now ung smear natin.
 We count the number of each separately in hundred
cells. (So paano yon) Ilang spermatids/sperm na ulo
ung walang tail ang na count natin sa 100 na cells. O
kaya naman ilang WBC ang nakita natin in 100 cells
na nacount natin. So are you going to compute
that?
 Concentration is equal to number of the
spermatids/number of WBCs that we have counted
over 100.
 Normal values is it should be: < 1,000,000. Bakit? SEMINAL FRUCTOSE TESTS
Kapag nakakita kasi tayo ng greater than 1 million na  Seminal Fluid Fructose
WBCs it may indicate infection. O kaya greater than  When there is a low sperm count there is a problem
1 million na spermatids ibig sabihin there’s problem with the production of components of our seminal
with what? Spermatogenesis. fluid
 How are we going to compute it: Number of o Seminal fluid provides fructose
spermatids times the concentration. So kunwari ung o Fructose provides motility for our sperm cells, so if
number nung nakita mo na cell in 100 sperm cells ay absence of fructose in our seminal fluid then it
52 times the concentration example that is about 20 cause problems in the viability of our sperm cells
million for example. So you just have to multiply it so if  Resorcinol test
that’s more than 1 million after dividing it by 100. That o for screening for the presence of fructose
is considered abnormal. But if the concentration o add 1 ml of semen with 9 ml of the reagent then
lower than 1 million it’s considered normal. boil it, if there is a red-orange color, it indicates
 So uulitin ko ano ang ibig sabihin kapag there’s that fructose is present
greater than 1 million WBCs it may indicate infection.  Positive: orange-red color
Kapag ka naman greater than 1 million spermatids it  Normal number of fructose: 13 umol/ejaculate
may indicate what? Indicates problem in your  Spectrophotometry is used to test fructose
spermatogenesis. That’s for your sperm morphology  Specimens: should be tested within 2 hours or frozen
round bodies computation. Again when we say
round bodies we mean what? Presence of your ANTISPERM ANTIBODIES
WBCs o kaya yung mga spermatids natin.  Present in both men and women
 Male more common: surgery, vasectomy reversal,
trauma

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 Sperm normally do not encounter the immune o You need to perform culture first before
system, so body considers them foreign performing other tests.
 Damaged sperm create female antibodies o WBC value is computed through concentration
 Suspect male antibodies when clumps of sperm are of round bodies computation:
seen; female no clumping
 Female: mix female serum with sperm and check for
agglutination 
 Immunoassays: males
 Mixed agglutination reaction (MAR) test
o Incubate sperm with antihuman globulin (AHG)  𝑒 𝑔:
and IgG-coated latex particles o no of round bodies= 50
o AHG combines with particles and o Sperm concentration= 4,000,000 (4
antibodycoated sperm forming clumps million/mL)
 Normal <10% motile sperm attach to particles
5 𝑥4
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑅𝑜𝑢𝑛𝑑 𝐵𝑜𝑑𝑖𝑒𝑠
ANTISPERM ANTIBODIES (CONT’D)
 Immunobead Test 2
o Demonstrates antibodies to head, neck,
midpiece, and tail
 Advantage feature: we can detect what 𝟐 𝟎𝟎𝟎 𝟎𝟎𝟎
type of antibody is present in the male ∗∗ 𝑉𝑎𝑙𝑢𝑒 𝑖𝑠 𝑔𝑟𝑒𝑎𝑡𝑒𝑟 𝑡ℎ𝑎𝑛 𝑚𝑖𝑙𝑙𝑖𝑜𝑛
patient 𝑡ℎ𝑢𝑠 𝑖𝑛𝑑𝑖𝑐𝑎𝑡𝑖𝑛𝑔 𝑝𝑟𝑜𝑠𝑡𝑎𝑡𝑒 𝑖𝑛𝑓𝑒𝑐𝑡𝑖𝑜𝑛
(Where is the antibody directed—on the
head, neck, midpiece or tail).
o Beads are coated with antihuman globulin  Chemistry tests: neutral α-glucosidase, zinc, citric
 Used to attack the antibody where it was acid, acid phosphatase
attached o Components being measured are components
o Microscopic examination shows where on sperm of prostatic fluids that provides acidity and
antibodies are attacking enables liquefaction and coagulation of semen.
 The portion of the sperm cell with antibodies o Proteolytic enzymes located in prostatic fluids are
attached to latex particles, it means that it the ones allowing the semen to coagulate and
was the type of antibodies the patient has. liquify.
 Eg. If you observe it in sperm tail area, the  ↓ Neutral α -glucosidase = there’s problem in
antibodies the patients have are directed epididymis
against the tail of the sperm cell. o It has something to do with the maturation of
 If you can see it in the sperm head, it was sperm.
antibodies directed against the sperm head.  ↓ zinc, citric acid, acid phosphatase =↓ prostatic fluid
o Tail = movement; head = penetration o Indicate problems in prostatic fluid or in prostate
 Indications: glands itself.
 antibody directed to tails may affect
movement—there is problem in the NORMAL SEMEN CHEMICAL VALUES
sperm motility. Neutral a-glucosidase ≥20 mU/ejaculate
 Antibody directed to head affects the Zinc ≥2.4 µmol/ejaculate
penetration of sperm cell in the egg cell. Citric acid ≥52 µmol/ejaculate
o Normal: beads on <20% of sperm
Acid phosphatase ≥200 Units/ejaculate
 Eg. If you have 100 sperms and only 18 or 19
sperm has attached beads, it was still
considered normal, but values higher than SEMEN DETECTION
that, like 50 sperms with antibodies attached  Semen present in specimen?
with the latex particles in their tail portion, o Specimen acid phosphatase content
then we can say that there is a significant o Detection of seminal plasma glycoprotein, p30
amount of antibodies directed against the  Observe microscopically for sperm
tail (movement is affected). o Enhance viewing with xylene; using phase
 Values higher than 20% of the total sperm microscopy
count containing beads is considered o Xylene: a clearing agent that enhance the
significant. refractive index of specimen
 DNA analysis (ABO blood grouping)
MICROBIAL AND CHEMISTRY TESTING o Usually used if semen is not present
 >1 million WBCs usually indicates prostate infection: o E.g: in rape cases wherein, there are remnants of
culture and test for Mycoplasma hominis, Chlamydia blood (even if it was already washed out), Blood
trachomatis, Ureaplasma urealyticum is done to will be isolated using elution technique and be
determine what type of organism is causing the determined of its blood cells and undergo DNA
infection.

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nalysis. This determines if the blood came from viability. However, when the cells have
the victim or from the suspect. impaired membrane integrity and no sperm
viability, even you put a hypotonic solution;
POSTVASECTOMY ANALYSIS the tail will not swell and remain flagellar.
 Only concern is presence of sperm  In vitro acrosome reaction - Evaluation of the
o It expected that a patient underwent vasectomy acrosome to produce enzymes essential for ovum
for him to prevent impregnating her partner. If penetration
there is presence of sperm in the patient, it
means that the vasectomy is unsuccessful. OTHER TESTS
 Takes several months for all sperm to be gone, based  Spinbarkeit test
on time and ejaculations o For female to determine if the they are fertile, to
 Begin in 2 months; continue until 2 months are know when to intercourse for a sure pregnancy.
negative o Test for the tenacity of mucus
o We perform this to determine if there is presence  Sims Huhner test
of sperm after surgery of the patient. We do it in o Post-coital test
a monthly interval.  Usually performed 4-6 hours after coitus
 Wet preparation under phase; if negative, centrifuge o Test for the ability of sperm cells to penetrate the
for 10 minutes, examine again cervical mucosa
o Using a phase contrast microscope is easy to see
the target because it gives better visualization SEMEN ANALYSIS QUALITY CONTROL
than bright field microscope, such when material  CLIA rates semen analysis as high complexity
is unstained, and we have chances to miss the o The result depends in the observation of medical
sperm cell probably there in sperm. technologist. We have differences in the result,
o Remember: to make sure that we have collected because of the subjectivity of the 1 performing
complete specimen. Why? If it’s incomplete, a 1 the test.
sperm that you want to see is you can’t see  Commercial controls and training materials are
because it is with other specimen that is not available
passed in the laboratory, and you reported that o Increased interest in fertility testing has promoted
there is no sperm cell. the development of quality control materials and
 Only one sperm is required for fertilization in-depth training programs
o Missing that just single sperm would mean  College of American Pathologists (CAP) offers
jeopardy to your patient and his family. proficiency testing

SEMEN FUNCTION TEST

 Hamster egg penetration - Sperm are incubated with
speciesnonspecific hamster eggs and penetration is
observed microscopically REFERENCES:
o Hamster egg is look like human ova. We used 15-
20 hamster eggs. Notes from the discussion by Prof. Ethel Marie
o Result: if >50 of the eggs is penetrated, we can Mangada, RMT
say that the sperm have fertilization capability. If
<50, the sperm has limited capacity of Cagayan State University PowerPoint presentation
penetration.
 Cervical mucus penetration - Observation of sperm
penetration ability of partner’s midcycle cervical
mucus
o We usually use spinbarkeit mucus, a mucus we
see in a female patient during times when they
are fertile.
 Hypo-osmotic swelling - Sperm exposed to low-
sodium concentrations are evaluated for membrane
integrity and sperm viability
o Sodium citrate dihydrate plus fructose, a
hypotonic solution , and we combine it (Sodium
citrate dihydrate plus fructose 1mL + 0.1Ml of
semen). Next we incubated it at 37C for 30-60
mins. After that we are going to add the semen
in the slide, and we are going to observe at least
hundred cells.
 Normal result: at least ≥ 58% has swelling tail.
** it means when the cells are normal, and
add at a hypotonic solution, the tail will swell
and there is membrane integrity and sperm

APOSTOL, ALLAUIGAN, ATAL, BARANGAN, BAYER, DIAZ,

8
FLORIA, LIM, PANALIGAN, RICAMORA