AUBF Lecture Module 8 Chapter 10: SEMINALYSIS

SEMEN ANALYSIS

- fluid from males

- contains semen, necessary for reproduction
- FOUR FRACTIONS OF SEMEN:
» Testes and epididymis: sperm cell itself; sperm cells- majority found on the very first
part of semen (pre-cum)
» Seminal vesicles: causes the semen to become alkaline
 Important to become an alkaline to neutralize the acidity of the pH within the
vagina
» Prostate: acidity of semen
» Bulbourethral glands
- Major contribution of semen comes from the seminal vesicles and prostrate
- SEMEN COMPOSITION:
 Seminal fluid: 60-70%
 Prostate fluid: 20-30%
 Spermatozoa: 5%
 Bulbourethral glands: 5%
- The mixing of all four fractions during ejaculation is essential for the production of a
normal semen specimen

EXAMINATION OF SEMEN:

1. For fertility testing

2. Evaluate the effectiveness of vasectomy – 2 weeks to 1 month post vasectomy (no
sperm or viable sperm in semen sample)
3. Investigation of sexual assault cases – rape cases
> Surface of female genitalia [acid phosphates (prostatic fluid from semen) and prostate
specific antigen (from prostatic fluid)] = sexual contact

Seminal fluid and sperm – only a component of semen

SPERMATOZOA

- produced in the seminiferous tubules of the testes

- They mature and are stored in the epididymis
- spermatozoa and fluid from the epididymis contribute about 5% of the semen volume

SEMINAL VESICLES

- produce the majority of the fluid present in the semen (70%)

- the fluid contains high content of fructose (carbohydrate being utilize for energy
purposes) that the spermatozoa readily metabolize
- spermatozoa do not become motile until they are exposed to the fluid from the seminal
vesicle
 AUBF Lecture Module 8 Chapter 10: SEMINALYSIS

PROSTATE GLAND

- 20 to 30% of the semen volume is acidic fluid

- the fluid contains high concentrations of acid phosphates, citric acid, zinc and proteolytic
enzymes responsible for both the coagulation and liquefaction of the semen following
ejaculation

BULBOURETHRAL GLANDS

- contribute about 5% of fluid volume in the form of a thick, alkaline mucus that helps
neutralize acidity from the prostate secretions and the vaginal acidity.

SPECIMEN HANDLING

- majority of the sperm are contained in the first portion of the ejaculate (pre-cum)
- should be abstinence 3 days and not longer than 5 days
» Prolonged abstinence – higher volumes and decreased motility (bc long abstinence
means more dead sperm cells); longer liquefaction time
» Short abstinence – low volume, low sperm count
- fertility testing: 2-3 samples within 2-week interval
» 2 samples with abnormal results = infertility
- provide an area where collection is possible; not normal comfort room but it should
resemble the environment at home to let the patient comfortable and not affect the
quality of the specimen
- laboratory should provide warm sterile glass or plastic containers
- specimen should be kept at room temperature and deliver to the laboratory within 1 hr
of collection
- fresh specimen is clotted and should be liquefy within 30-60 min after collection
(colloidal suspension; more liquid)
- specimen awaiting analysis should be kept at 37C
- specimen should be collected by masturbation. If not possible, only non-lubricant
polymeric silicone (silastic) condoms should be used
- specimen are potential reservoirs of HIV and hepa virus
- specimen are discarded as biohazardous wastes
» biohazardous waste: yellow
» non-hazardous solid waste: black
» non-hazardous biodegradable: green

3 METHODS OF COLLECTION

1. Induce ejaculation by masturbation: most preferred

2. Coitus interruptus (withdrawal): sexual act, not preferred bc the pre-cum is already lost
which contains most number of sperm cells; very low sperm count
3. Use of condoms: not preferred; condoms should be free from lubricants, which are
spermicidal (kill the sperm cells)
 AUBF Lecture Module 8 Chapter 10: SEMINALYSIS

SEMEN ANALYSIS

NORMAL VALUES FOR SEMEN ANALYSIS

Volume 2-5 ml
(extended period of abstinence = greater
volume
Viscosity pours in droplets (already liquefied)
(aspirate in disposable pipette, 2cm)
(bigger than 2cm = more viscous)
pH 7.2-8.0
(greater pH = there is infection within male
genitalie)
(less than 7.2 = there is a greater
contribution from prostate, indicate poorly
developed seminal vesicles)
Sperm concentration >20 millions
Motility >50% within 1hr
Quality >2.0
Morphology >14% normal forms (strict criteria)
= there are more parameters to consider

>30% normal forms (routine criteria)

WBC <1.0 million/ml
(greater = infection or inflammation)

APPEARANCE

- Gray-white color, translucent, musty or bleach like odor

- Increased turbidity = presence of WBC and infection within the male genital tract
- WBCs must be differentiated from immature sperm (spermatids)
- Leukocyte esterase reagent strip = useful to screen the presence of WBCs
- Red color = presence of RBC and abnormal; indicate that there is a breakage in
barrier of testes and blood vessel
- Yellow color – urine contamination; prolonged abstinence and medications
- urine is toxic to the sperm = affecting the evaluation of motility

VOLUME

- Can be measured by pouring it into graduated cylinder calibrated 0.1 ml increments

- Increased volume = extended abstinence
- Decreased volume = infertility and may indicate improper functioning of semen
producing organs (seminal vesicles of prostate glands)
- Incomplete liquefied = clumped and highly viscous; affect the motility of the sample
 AUBF Lecture Module 8 Chapter 10: SEMINALYSIS

- Normal semen specimen should be easily drawn from the pipette and from droplets that
do not appear clumped of string when discharged from the pipette
- Ratings of 0 (watery) to 4 (gel-like)

pH

- Increased pH = infection within male reproductive tract

- Decreased pH – increased prostatic fluid or decrease in the contribution of seminal
vesicles
- Semen for pH testing can be applied to the pH pad of a urinalysis reagent strip and the
color compared to the manufacturer’s chart
- Dedicated pH testing paper can also be used
- Urine reagent – pH, WBC, RBC

SPERM CONCENTRATION

- perfomed using the Newbauer counting chamber

- counted in the same manner as CSF count, that is by diluting the specimen and counting
the cells in the Newbauer chamber
- the most commonly used dilution is 1:20 prepared using a mechanical rather than a
Thoma pipette (positive displacement pipette)
- Diluting = immobilize sperm
- Traditional diluting fluid = sodium bicarbonate and formalin, which immobilize and
preserve the cells; however good results can also be achieved using tap water (chilled
water)
- The Makler counting chamber provides a method for counting undiluted specimen using
a counting chamber with 1mm2 grid divided into 100 squares engraved in the cover
plate
- Sperm = immobilized by heating part of the specimen prior to charging the chamber
- Sperm motility – using the unheated portion of the chamber
- Using the Newbauer, sperms are usually counted in the four corner and center squares
of the large center square
- Both sides are loaded and counted and the counts should agree within 10%

Ex. 230 + 10%

230 x .10 = 23

230 – 23 = 207

230 + 23 = 253

Range between 207 -253

- Only fully developed sperms should be counted

 AUBF Lecture Module 8 Chapter 10: SEMINALYSIS

- Immature sperms and WBCs are often referred to as ‘round cells’ must not be included
- The presence of round cells may be significant and they may need to be identified and
counted separately
- Counting chamber
» Only small number of sperm cells = 4 corner large squares
» Routine procedure – 5 medium squares of central large square

FORMULAS:
𝒔𝒑𝒆𝒓𝒎𝒔 𝒄𝒐𝒖𝒏𝒕𝒆𝒅 𝒙 𝟐𝟓 𝒙 𝟏𝟎 𝒙 𝒅𝒊𝒍𝒖𝒕𝒊𝒐𝒏 𝒇𝒂𝒄𝒕𝒐𝒓
𝑺𝒑𝒆𝒓𝒎 𝒄𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏/𝒖𝑳 =
# 𝒐𝒇 𝒎𝒆𝒅𝒊𝒖𝒎 𝒔𝒒𝒖𝒂𝒓𝒆𝒔 𝒄𝒐𝒖𝒏𝒕𝒆𝒅 𝒊𝒏

𝒔𝒑𝒆𝒓𝒎𝒔 𝒄𝒐𝒖𝒏𝒕𝒆𝒅 𝒙 𝟏𝟎 𝒙 𝒅𝒊𝒍𝒖𝒕𝒊𝒐𝒏 𝒇𝒂𝒄𝒕𝒐𝒓

𝑺𝒑𝒆𝒓𝒎 𝒄𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏/𝒖𝑳 =
# 𝒐𝒇 𝒍𝒂𝒓𝒈𝒆 𝒔𝒒𝒖𝒂𝒓𝒆𝒔 𝒄𝒐𝒖𝒏𝒕𝒆𝒅 𝒊𝒏

𝑺𝒑𝒆𝒓𝒎 𝒄𝒐𝒖𝒏𝒕/𝒆𝒋𝒂𝒄𝒖𝒍𝒂𝒕𝒆 = 𝒔𝒑𝒆𝒓𝒎 𝒄𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏/𝒎𝑳 𝒙 𝒗𝒐𝒍𝒖𝒎𝒆 𝒐𝒇𝒔𝒂𝒎𝒑𝒍𝒆

Convert microliter to millilitre – multiply 1000

PROBLEMS:

343 sperm cells were counted in 5 medium squares of the central large square on one side of
the INCC. 253 sperm cells were counted on the other. Compute for sperm concentration and
sperm count if the volume of the sample was 3.5mL.

253 x .10 = 25

253 – 25 = 228 253 + 25 = 278

343 > 278

= invalid count

343 sperm cells were counted in 5 medium squares of the central large square on one side of
the INCC. 325 sperm cells were counted on the other side. Compute for sperm concentration
and sperm count if the volume of the sample was 3.5mL.

325 x .10 = 32

325 – 32 = 293 325 + 32 = 357

343 < 357

 AUBF Lecture Module 8 Chapter 10: SEMINALYSIS

𝒔𝒑𝒆𝒓𝒎𝒔 𝒄𝒐𝒖𝒏𝒕𝒆𝒅 𝒙 𝟐𝟓 𝒙 𝟏𝟎 𝒙 𝒅𝒊𝒍𝒖𝒕𝒊𝒐𝒏 𝒇𝒂𝒄𝒕𝒐𝒓

𝑺𝒑𝒆𝒓𝒎 𝒄𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏/𝒖𝑳 =
# 𝒐𝒇 𝒎𝒆𝒅𝒊𝒖𝒎 𝒔𝒒𝒖𝒂𝒓𝒆𝒔 𝒄𝒐𝒖𝒏𝒕𝒆𝒅 𝒊𝒏
(𝟑𝟒𝟑 + 𝟑𝟐𝟓) 𝒙 𝟐𝟓 𝒙 𝟏𝟎 𝒙 𝟐𝟎
𝑺𝒑𝒆𝒓𝒎 𝒄𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏/𝒖𝑳 =
𝟏𝟎
(𝟑𝟒𝟑 + 𝟑𝟐𝟓) 𝒙 𝟐𝟓 𝒙 𝟏𝟎 𝒙 𝟐𝟎
𝑺𝒑𝒆𝒓𝒎 𝒄𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏/𝒖𝑳 =
𝟏𝟎
𝑺𝒑𝒆𝒓𝒎 𝒄𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏/𝒖𝑳 = 𝟑𝟑𝟒 𝟎𝟎𝟎/𝒖𝑳

𝑺𝒑𝒆𝒓𝒎 𝒄𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏/𝒖𝑳 = 𝟑𝟑𝟒 𝟎𝟎𝟎 𝟎𝟎𝟎/𝒎𝑳

𝑺𝒑𝒆𝒓𝒎 𝒄𝒐𝒖𝒏𝒕/𝒆𝒋𝒂𝒄𝒖𝒍𝒂𝒕𝒆 = 𝒔𝒑𝒆𝒓𝒎 𝒄𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏/𝒎𝑳 𝒙 𝒐𝒍𝒖𝒎𝒆 𝒐𝒇𝒔𝒂𝒎𝒑𝒍𝒆

𝑺𝒑𝒆𝒓𝒎 𝒄𝒐𝒖𝒏𝒕/𝒆𝒋𝒂𝒄𝒖𝒍𝒂𝒕𝒆 = 𝟑𝟑𝟒 𝟎𝟎𝟎 𝟎𝟎𝟎 𝒙 𝟑. 𝟓

𝑺𝒑𝒆𝒓𝒎 𝒄𝒐𝒖𝒏𝒕/𝒆𝒋𝒂𝒄𝒖𝒍𝒂𝒕𝒆 = 𝟏 𝟏𝟔𝟗 𝟎𝟎𝟎 𝟎𝟎𝟎 / 𝒆𝒋𝒂𝒄𝒖𝒍𝒂𝒕𝒆

SPERM MOTILITY

- progressive movement and motility = critical for fertility because once presented to the
cervix, the sperm must propel themselves through the cervical mucosa to the uterus,
fallopian tubes and ovum
- assessment of sperm motility should be performed in a well-mixed, liquefied specimen
within 1 hr of semen collection’
- SPERM MOTILITY GRADING

GRADE CRITERIA
4.0 Rapid, straight-line motility
3.0 Slower speed, some lateral movement
2.0 Slow forward progression, noticeable lateral movement
1.0 No forward progression
0 No movement

- Greater than 50% show grade 2 3 4 motility; less than that would be abnormal

SPERM MORPHOLOGY

- Head and tail appearance

- Acrosomal cap in head, 1 half of head
- Head abnormality = poor ovum penetration
- Tail abnormality – poor mortility
- Normal sperm – oval-shaped head approximately 5 um long and 3 um wide and a long,
flagellar tail approximately 45 um long
- Head and tail are connected by neck and the middle piece, which contains mitochondria
that provide energy for flagellar tail motion
- Should have 30% normal motility (relaxed/routine criteria)
 AUBF Lecture Module 8 Chapter 10: SEMINALYSIS

- Acrosomal cap = with enzyme

- Sperm morphology is evaluated from a thinly smeared, stained slide under oil immersion
- Staining can be performed using Wright’s, Giemsa, or Papanicolaou Stain (Pap stain)
- Air-dried slides are stable for 24 hr
- Atleast 200 sperm should be evaluated and the percentage of abnormal sperm reported
(atleast 60 is normal in appearance to be considered normal)

ADDITIONAL TESTING

SPERM VIABILITY

- Decreases sperm viability may be suspected when a specimen has a normal sperm
concentration with markedly decreased motility (sperms are already dead)
- Viability is evaluated by mixing the specimen with eosin-nigrosin stain, preparing a
smear, and counting the no. of dead cells in 100 sperm
- Living cells – bluish white color
- Dead cells – red against a purple background
» stains the dead cells bc the living cells has an intact cell membrane
- Normal viability – 75% living cells, bluish white sperm in a purple background

SEMINAL FLUID FRUCTOSE

- specimens can be screened for the presence of fructose using Resorcinol test
- a normal quantitative level of fructose is equal to or greater than 13 umol/ejaculate
- specimens for fructose levels should be tested within 2 hrs or frozen to prevent
fructolysis
- Fructose = carbohydrate for energy needs

ANTI SPERM ANTIBODIES

- can be present in both men and women

- may be detected in semen, cervical mucosa, or serum and are considered possible
causes of infertility
- male anti-sperm antibodies = more frequently encountered
- when the blood-testes-barrier is disrupted, as can occur following surgery, vasectomy,
reversal, trauma, and infection, the antigens of the sperms produce an immune
response that damages the sperm
- present following an infection of genital tract
- (+) Ab in male partner: clumps of sperm during routine analysis
- (+) Ab in female partner: normal semen analysis accompanied by continued infertility.
Mixing the semen with the cervical mucosa and observing agglutination of semen with
cervical mucosa or serum of female
 AUBF Lecture Module 8 Chapter 10: SEMINALYSIS

TWO FREQUENTLY USED TESTS

- MAR Test
» Used to detect the presence of IgG antibodies
» (+) microscopically visible clumps of sperms and particles or cells
» Less than 10% of the motile sperm attached to the particles is considered normal
- Immunobead Test
» More specific procedure
» Detect the presence of IgG, IgM, and IgA and will demonstrate what area of the
sperm is affected
» Presence of beads on less than 20% of the sperm is normal

ADDITIONAL TESTING FOR ABNORMAL SEMEN ANALYSIS

ABNORMAL RESULT POSSIBLE ABNORMALITY TEST
Decreased motility with Viability Eosin – nigrosin stain
normal count
Decreased count Lack of seminal vesicle Fructose level
support medium
Decreased motility with Male anti-sperm antibodies MAR & Immunobead test
clumping Sperm agglutination with
male serum
Normal analysis with Female anti-sperm antibodies Immunobead test
continued infertility Sperm agglutination with
female serum

MICROBIAL AND CHEMICAL TESTING

- Presence of more than 1 million leukocytes/mm = infection within the reproductive

frequently the prostate
- Routine aerobic and anaerobic cultures and tests for Chlamydia trachomatis,
Mycoplasma homilis, and Ureaplasma urealyticum are most frequently perfomed
» Infection = production of anti-sperm antibodies
- Additional chemical testing performed on semen may include determination of leves of
neutral a-glucosidase, zinc, citric acid, and acid phosphates.
- Decreased fructose = lack of seminal fluid
- Decreased neutral a-glucosidase = disorder of epididymis
- Decreased zinc, citrate, and acid phosphates = lack of prostatic fluid

NORMAL SEMEN CHEMICAL VALUES

Neutral a-glucosidase > 20mu/ ejaculate
Zinc > 2.4 umol/ ejaculate
Citric acid > 52 umol/ ejaculate
Acid Phosphatase > 200 u/ ejaculate
 AUBF Lecture Module 8 Chapter 10: SEMINALYSIS

POST VASECTOMY SEMEN ANALYSIS

- Presence or absence of spermatozoa

- Specimens are routinely tested at months interval, beginning at 2 months post
vasectomy and continuing until consecutive monthly specimens shows no spermatozoa
- Examination of wet preparation using phase microscopy for the presence of motile and
non-motile sperm
- A negative wet preparation is followed by viability testing

SPERM FUNCTION TEST

TEST DESCRIPTION
Hamster egg penetration Sperms are incubated with species-
nonspecific hamster eggs and penetration is
observed microscopically
Cervical mucus penetration Observation of sperm penetration ability of
partner’s mid cycle cervical mucus
Hypo-osmotic swelling Sperm exposed to low sodium concentrations
are evaluated for membrane integrity and
sperm viability
In vitro acrosome reaction Evaluation of the acrosome to produce
enzymes essential for ovum penetration