SEMEN ANALYSIS

• 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.
CONTRIBUTIONS TO SEMEN
VOLUME

Testes and epididymis – 10%

Seminal vesicles – 50%
Prostate – 40%
Cowper's glands – small volume
 Reflects sperm
production by
the testes
Spermatozoa
Cellular Leucocytes
Immature
Germ Cells
Semen Epididymis
Seminal
Non Vesicles
Cellular Bulbourethral
Reflects the Prostate
secretory
activity of the
glands
 INDICATIONS
• Investigation of infertility: Semen analysis is the
first step in the investigation of infertility.
• To check the effectiveness of vasectomy.
• To support or disprove a denial of paternity on
the grounds of sterility.
• To examine vaginal secretions or clothing stains
for the presence of semen in medico legal cases.
• For selection of donors for artificial insemination.
 EQUIPMENTS AND REAGENTS REQUIRED

 Semen diluting fluid:

• Sodium bicarbonate
• Neutral formalin
• Distilled water

 Eosin and nigrosin stain

Wide mouthed sterile
container
 PH paper strip
Pipette, graduated tube,
glass slide, cover slip
Neubauer’s counting
chamber
 SEMEN
• Collected in a private room near
COLLECTION
laboratory
• After 2-7 days of abstinence
• In a sterile container
• If collected at home - transported to the
laboratory within 1 hour, kept between
20 -37 C temperature.
• Entire ejaculate should be collected.
• Condom collection is not recommended.
 Semen analysis involves the following
steps with time constraints
 In the first 5 minutes: Liquefaction
 Between 30 and 60 minutes:
• Assessing liquefaction and appearance of
the semen.
• Measuring semen volume & Ph.
• Preparing a wet preparation for assessing
microscopic appearance, sperm motility
and the dilution required for assessing
sperm number.
 Making semen smears for assessing sperm
morphology.
 Making semen dilutions for assessing
sperm concentration.
 Performing the mixed antiglobulin reaction
(MAR) test and immunobead test.
 Biochemical markers assay.
 Macroscopic
examination
1. Appearance and color:
• Grey - Normal
• Opaque - High sperm concentration
• Transparent - Low sperm
concentration
• Reddish brown - Hematospermia
• Yellow - jaundice or taking vitamin
tablets
 Macroscopic examination

2. Liquefaction : 15-60min (usually

in 15 minutes) at 37 degree Celsius.
• homogenous, watery
• >60 min is abnormal
– Chronic prostatitis
– Seminal vesiculitis
3.Viscosity - Resistance to
flow
• Estimated by Glass rod into the liquified
sample and observing the length of the
thread that forms upon withdrawal of the
rod
• Thread length >2cm : high viscosity
• Accessory gland dysfunction(prostatitis,
seminal vesiculitis)
4. Semen pH :
• Reflects the balance between the pH values
of the different accessory gland secretions
Alkaline seminal vesicular secretion vs acidic prostatic
secretion.
• Measured after liquefaction, preferably after
30 min to < 1 hr
• Consensus value of 7.2 as a lower threshold
value
5. Semen volume :
Reference volume : >1.4 mL
 High semen volume :
-May reflect active exudation in cases of
active inflammation of the accessory organs.
 Low semen volume :
- Ejaculatory duct obstruction
MICROSCOPIC EXAMINATION

• Sperm number
• Motility
• Vitality
• Morphology
• Proportion of white cells
 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 (10^6/ml).
Semen is diluted 1:20 with sodium bicarbonate formalin diluting
fluid

Coverslip is placed over the improved Neubauer chamber and the

counting chamber is filled with diluted semen sample

Number of spermatozoa is counted in 4 large corner squares.

Sperm count per ml is calculated as follows:
Total number of Sperms counted
X 1000
Sperm count =
Area X Dilution factor X Depth factor

N X 20 X 10 X 1000

4X1X1
= Sperms counted × 50, 000

 Normal sperm concentration is ≥ 15 million/ml .

 SPERM
MOTILITY
• Sperm motility is essential for penetration of
cervical mucus, traveling through the fallopian
tube, penetrating the ovum.
• Principle: All motile and non-motile sperms are
counted in randomly chosen fields in a wet
preparation.
• Result is expressed as a percentage of motile
spermatozoa.
 A drop of semen is placed on a glass slide

covered with a coverslip that is then ringed with petroleum jelly to

prevent dehydration

200 spermatozoa are counted

Result is expressed as a percentage of
(a) Rapidly progressive spermatozoa ( 25 µm/s) –
moving actively, either linearly or in a large circle,
covering a distance, from the starting point to the end
point, of at least 25 µm (or ½ tail length) in one
second.

(b) Slowly progressive spermatozoa - moving actively,

either linearly or in a large circle, covering a distance,
from the starting point to the end point, of 5 to < 25
µm (or at least one head length to less than ½ tail
length) in one second.
(c) Non-progressive spermatozoa - all other patterns
of active tail movements with an absence of
progression – i.e. swimming in small circles,
displacement of head less than 5 µm (one head
length).
(d) Immotile spermatozoa (no movement at all)
Total motility-PR+NP
The lower reference limit for
 Total motility (PR + NP) is 42%
 Progressive motility (PR) is 30%
SPERM VITALITY
• Assesses the cell membrane integrity of the
spermatozoa - % of live spermatozoa
• Especially important for samples with sperm motility
<42% PR sperms.
• Clinically important to know whether immotile
spermatozoa are alive or dead
• Large proportion of viable but immotile cells may
be indicative of structural defects in the
flagellum[Kartagener syndrome –genetic defect in cilia].
 Eosin Nigrosin smear
Lower reference limit for viability - 54 %
 SPERM
MORPHOLOGY
• A spermatozoon consists of three
main components:
Head
Neck
Tail
• Tail is further subdivided into
midpiece, main (principle) piece,
and end piece
 STAINING
• METHODS
Stains used:
– Papanicolaou stain
– Shorr stain
– Diff-Quick stain
• Fixative- methanol/ethanol
based
The head :
• smooth, regularly contoured & generally oval in shape
• Well-defined acrosomal region comprising 40–70%
• The acrosomal region should contain no large vacuoles, & not
>2 small vacuoles, which should not occupy >20% of the sperm
head.
• The post-acrosomal region should not contain any vacuoles.
The midpiece- should be
slender, regular and about the
same length as the sperm head.
The major axis of the midpiece
should be aligned with the
major axis of the sperm head.
Excess residual cytoplasm
(ERC)
Defective spermatogenic
process
Irregular stained cytoplasm,
one third or more of the sperm
head size, often associated with
defective midpieces
The principal piece :
should have a uniform calibre
along its length, be thinner
than the midpiece, & be
approximately 45 µm long
(about 10 times the head
length).
Endpiece is the short tapering
part.
 Analysing a sperm morphology smear

• Evaluate at least 200 spermatozoa.

• The lower reference limit for normal forms is 4%
 Biochemical
•
•
analysis
TEST FOR FRUCTOSE :
4.5 ml of resorcinol reagent (50 mg
resorcinol dissolved in 33 ml
concentrated hydrochloric acid; dilute up
to 100 ml with distilled water) is added
to 0.5 ml of seminal fluid.
• The mixture is heated and brought to
boil. If fructose is present, a red-colored
precipitate is formed within 30 seconds
 Terminologies
• ASPERMIA- absence of ejaculate
• AZOOSPERMIA- absence of spermatozoa in
ejaculate
• OLIGOSPERMIA- <15million sperm/ml semen
• ASTHENOSPERMIA- reduced motility
• NECROSPERMIA- dead sperms
• TERATOSPERMIA- abnormal morphology
• GLOBOSPERMIA- rounded heads, lag
acrosomal cap
Assessment of kidney
function
 Introduction
• The glomerular filtration rate (GFR) is directly related to
the number of functioning nephrons in the kidney, and
this number declines in all forms of progressive kidney
disease.
• The accumulation of nitrogenous waste products and
other uraemic toxins is inversely related to GFR.
• Integrity of the glomerular filtration barrier can also be
compromized in kidney disease, and albuminuria (or
proteinuria) has historically been regarded as the cardinal
sign of kidney disease.
 Introduction
• Changes in GFR as reflected by changes in
blood creatinine concentration, are the basis
for the diagnosis of acute kidney injury.
• GFR is widely accepted as the best overall
measure of kidney function
• GFR and albuminuria to categorize patients in
management of chronic kidney disease
Glomerular filtration rate
 Gfr calculation
• The amount of S filtered at the glomerulus equals
GFR multiplied by plasma concentration of S (PS)
• S= GFR x PS
• The amount of S excreted equals the urine
concentration of S (US) multiplied by the urinary
flow rate
• filtered S = excreted S (US)
• GFR x PS(plasma) = US(urine) x V(volume excreted)
• GFR = (US x V)/PS
Measurement of GFR using endogenous
substances
 Introduction
• Creatinine and certain low molecular weight
proteins (e.g. cystatin C, b-trace protein) have
been used as endogenous markers of GFR.
• Use of urea in this context is of limited value
• Serum urea measurement is occasionally
useful in situations where there is a suspicion
that the creatinine result is misleading (e.g.
muscle-wasting disorders) and no alternative
methods are available to assess GFR .
 Creatinine
• The most widely used endogenous marker of GFR
• It is freely filtered at the glomerulus, and its concentration is
inversely related to GFR
• It is affected by a variety of non-renal influences and has poor
sensitivity for CKD.
• Reduced muscle mass can result in serum creatinine concentrations
within the reference range even when kidney function is
impaired(old people).
• Most laboratories use modifications of the Jaffe method to
measure creatinine, although more specific and accurate enzymatic
and isotope dilution-mass spectrometry methods are available.
Limitations of serum creatinine
concentration as a marker of GFR
 Non-renal
influences
• GENDER: Male individuals have relatively high serum creatinine for
the same GFR level
• ETHNICITY: African-Caribbean individuals have relatively high
serum creatinine for the same GFR level
• RECENT DIETARY INTAKE: Cooked meat and fish contain creatinine
• MUSCLE MASS: limitation in patients with muscle-wasting disorders
or amputees
• EXTRA-RENAL CLEARANCE: Becomes more significant in patients
with CKD because of degradation as a result of bacterial
overgrowth in the small intestine
• DRUGS: In Vivo Effect
 Clinical utility
• POOR SENSITIVITY FOR CKD: Does not
detect patients with stage 2 CKD and fails
to identify many patients with stage 3
CKD
• NOT USEFUL IN ACUTE KIDNEY INJURY:
There is a temporal delay between
change in GFR and the resulting change
in serum creatinine concentration
Creatinine clearance
• This requires a timed urine collection, which is inconvenient and
introduces inaccuracies.
• Day-to-day coefficient of variation for repeated measures of
creatinine clearance exceeds 25%.
• Tubular secretion further undermines the theoretical value of
creatinine
• As GFR falls, the tubular secretion of creatinine rises
disproportionately, and creatinine clearance values can reach
nearly twice those for the true GFR.
• Creatinine clearance provides at best only a crude index of GFR.
Estimated GFR
 Estimated GFR
• Derived using serum creatinine
corrected for some or all of
gender, body size, race and age.
These produce a better estimate
of GFR than serum creatinine
alone.
lower molecular weight proteins
 lower molecular
weight proteins
• Lower molecular weight (<30 kDa)
proteins are relatively freely filtered at
the glomerulus and either reabsorbed
(and metabolized) in the proximal tubule
or excreted into the urine.
• As they are entirely eliminated from the
circulation, they have the potential to be
used as GFR markers.
 Cystatin C
• Cystatin C is a low molecular weight (12.8 kDa) cysteine protease
inhibitor synthesized by all nucleated cells.
• It offers a more sensitive and specific means of detecting CKD than
serum creatinine.
• GFR-prediction equations based on cystatin C have also been proposed,
including equations that incorporate both cystatin C and creatinine.
• These equations show only modest improvements in accuracy of GFR
estimation compared with creatinine-based equations
• There is increasing evidence that use of cystatin C outperforms
creatinine in risk stratification.
• A further advantage is that it is less influenced by age, dietary intake and
race.
 Proteinuria
• Proteinuria is not just a consequence of, but also directly
contributes to, progression of kidney disease.
• Albumin constitutes approximately 25% of total urinary protein.
• At higher levels of proteinuria, the relative contribution of
albumin progressively increases, and when loss exceeds 1
g/day, more than 90% is albumin.
• ‘Microalbuminuria’ is a misleading historical term referring to
the loss of albumin in the urine in amounts that are abnormally
increased but below the limit of detection of conventional
urine reagent strip (‘dipstick’) tests
 Proteinuria
• Higher molecular weight proteins, the size of albumin and
larger, are mostly retained within the circulation by the
glomerular filter.
• Lower molecular weight proteins are more freely filtered,
reabsorbed by proximal tubular cells and then catabolized
within the tubular cells.
• Any increase in the filtered load (glomerular damage, increased
glomerular vascular permeability, increased circulating
concentration of low molecular weight proteins) or decrease in
reabsorptive capacity (tubular damage) can result in
proteinuria.
 Proteinuria
• If significant non-albumin proteinuria is suspected,
specific assays for tubular proteins should be used
(e.g. alpha1-microglobulin, monoclonal heavy or
light chains (Bence Jones protein)).
• Total protein assays are insensitive in the detection
of isolated tubular proteinuria.
• Proteinuria is commonly classified as glomerular,
tubular or overflow proteinuria
Characterization of proteinuria
 Glomerular
• Increased glomerular
permeability (e.g. from immune
complex deposition)
• Progressively increasing loss of
higher molecular weight proteins
as permeability increases (e.g.
albumin, immunoglobulin G)
 Tubular
1. Proximal tubular damage: Decreased tubular reabsorptive
capacity and/or release of intracellular components (e.g.
caused by nephrotoxic drugs or heavy metals, anoxia)
2. Decreased nephron number from progressive CKD: Increased
filtered load per nephron
3. Distal tubular damage: Tamme Horsfall glycoprotein & π-
Glutathione-S-transferase are seen.
• In proximal tubular damage & Decreased nephron number
from progressive CKD, predominantly lower molecular
weight proteins and enzymuria are seen.
 Overflow
• Due to increased plasma
concentration of relatively freely
filtered protein. (Multiple
myeloma, Rhabdomyolysis)
• Bence Jones protein, Myoglobin
are seen
 Sample collection
and interpretation
• Historically, the 24-hour urine sample was regarded as the
definitive means of demonstrating proteinuria. This is an
imperfect reference method, and measurement of the
albumin:creatinine ratio in a spot urine sample is now
accepted.
• Expressing the albumin concentration as a ratio to
creatinine enables correction for urinary dilution.
• An early morning urine sample is preferred because it
correlates well with 24-hour protein loss and is required to
exclude the diagnosis of orthostatic (postural) proteinuria.
 Sample collection
and interpretation
• Samples should be collected in the absence of urinary
tract infection or acute metabolic crises.
• Albumin:creatinine ratios <3.0 mg/mmol require no
further investigation
• For patients demonstrating ratios above, or equal to,
this cut-off, confirmation at least one further occasion
must be done(ideally within 1 month).
• Samples with increased albumin:creatinine ratios
have albuminuria.
 Sample collection
and interpretation
• In the international classification
of CKD, three categories of
albuminuria are recognized
• In the absence of reduced GFR,
CKD may still be identified in
some individuals by the presence
of albuminuria.
The Kidney Disease
Improving Global
Outcomes (KDIGO)
criteria

Prognosis of CKD
by GFR and
albuminuria
category