This document provides information about evaluating abnormalities in a semen analysis panel, including: - The indications, sample collection/transport procedures, and normal ranges for semen volume, pH, motility, concentration, morphology, and other tests. - How to interpret abnormalities in these parameters, such as low/high volume, pH, motility, oligospermia/azoospermia, teratozoospermia and their potential causes. - Quality control procedures like repeat testing, and transient defects that could affect initial semen analysis results. Four clinical cases are then presented to demonstrate applying this evaluation and interpretation of semen analysis results.

1. PA23.3
DESCRIBE AND INTERPRET THE
ABNORMALITIES IN A PANEL CONTAINING
SEMEN ANALYSIS
Dr IRA BHARADWAJ
MCI TEACHER ID
PAT 2300569

2. TEXTBOOK REFRENCES
• WHO 2010
• Basics of body fluid analysis for UG&PG
students: Dr. Akhil Bansal

3. SLO
• INDICATIONS FOR SEMEN ANALYSIS
• COLLECTION & TRANSPORT OF SAMPLE
• PHYSICAL EXAMINATION & ITS INTERPRETATION
• MICROSCOPIC EXAMINATION & ITS INTERPRETATION
• CHEMICAL EXAMINATION & ITS INTERPRETATION
• OTHER SPECIAL TESTS
• WHO CRITERIA 2010
• REPEAT SEMEN ANALYSIS & TRANSIENT DEFECTS
• QUALITY CONTROL
• CASE DISCUSSION [FOUR CASES]

4. INDICATIONS
• To investigate infertility
• To investigate genetic disorders like Klinefelter
syndrome
• To investigate inflammatory or neoplastic
diseases of genital tract
• Semen banking
• Medicolegal cases of rape/ alleged rape
• To evaluate effectiveness of vasectomy

5. SAMPLE COLLECTION
• Pt is instructed to collect the complete,
ejaculated specimen by masturbation,
following 2-7 days of sexual abstinence
• Sample is collected in a clean wide mouthed
glass or plastic container or in a properly
washed dry condom.
• Sterile collection is needed for microbiology
examination & assisted reproductive therapy
[ART]

6. SAMPLE TRANSPORT
• Specimen should be delivered as early as
possible to the laboratory
• And not later than one hour after collection
• Sample should be maintained between
20-37 *c

7. GROSS EXAMINATION
The following normal features are present:
• Color: translucent whitish, grey white or
yellowish
• Volume : between 2.5 to 5ml
• Viscosity : viscous and falls drop by drop
• Reaction: alkaline with pH of more than 7.2
• Liquefaction : liquefaction occurs because of
presence of fibrinolysin. Normally occurs
between within 10-30 minutes

8. SEMEN VOLUME & ITS SIGNIFICANCE
Low semen volume
• Collection problems like loss of a fraction of the
ejaculate & partial retrograde ejaculation
• Obstruction of the ejaculatory duct
• Congenital absence of the vas deferens,
• Poorly developed seminal vesicles
• Androgen deficiency
High semen volume
• Active inflammation

9. SEMEN pH & ITS SIGNIFICANCE
pH less than 7.0 with low volume and low
sperm count:
• Ejaculatory duct obstruction or
• Congenital absence of the vas deferens
• Poorly developed seminal vesicles
High pH values provide little clinically useful
information.

10. MICROSCOPIC EXAMINATION
Semen is examined microscopically for:
• Motility
• Count
• Morphology

11. MICROSCOPIC EXAMINATION
Motility
Method:
• Place a drop of liquefied semen on a clean glass
slide & cover with a cover slip
• Examine under the microscope – first low power
and then high power
• Assess at least 300 sperms
• Motile or non motile
• Progressive[PR] or non progressive motility[NP]

12. MICROSCOPIC EXAMINATION
Motility
Normal range:
• Within 1 hr – 70 – 90% motility
• 2 hrs – 40 - 70% motility
• 6 hours – 25 -50% motility
• The lower reference limit for total motility (PR
+ NP) is 40%
• The lower reference limit for progressive
motility (PR) is 32%

13. SPERM MOTILITY & ITS SIGNIFICANCE
• If motility is less than 50%; stain for viability [ eg
eosin] should be done to differentiate between
dead & viable non motile sperms
• Red dye accumulates in the head of dead sperms.
• Viable but immotile sperms are associated with
structural defects in the flagellum eg immotile
cilia syndrome [Kartagener syndrome-
bronchiectasis, situs inversus, sinusitis, infertility]
• Non-viable cells (necrozoospermia) may indicate
epididymal pathology

14. MICROSCOPIC EXAMINATION
sperm viability

15. SPERM MOTILITY & ITS SIGNIFICANCE
• Temperature - sperm motility value will be
inaccurately low if the semen sample gets cold.
• ASTHENOZOOSPERMIA – sperm motility less
than 40%
• Test should be repeated under ideal
conditions to rule out laboratory error

16. SPERM MOTILITY & ITS SIGNIFICANCE
Causes of asthenozoospermia:
• Abnormal spermatogenesis
• Epididymal sperm maturation defect
• Abnormalities in transport
• Varicocele

17. MICROSCOPIC EXAMINATION
sperm count [concentration]
• Manual method: Using Neubauer’s Chamber
& WBC pipette
• Normal range: 40-140 million/ml
• Oligospermia : < 15-20 million/ml
• Azoospermia: no sperms

18. MICROSCOPIC EXAMINATION
Sperm concentration & total number
The terms “total sperm number” and “sperm
concentration” are not synonymous.
• Sperm concentration refers to the number of
spermatozoa per unit volume of semen
• Total sperm number refers to the total
number of spermatozoa in the entire ejaculate
and is obtained by multiplying the sperm
concentration by the semen volume.

19. MICROSCOPIC EXAMINATION
Sperm concentration & total number
• Lower reference limit for sperm concentration
is 15 million spermatozoa per ml .
• Lower reference limit for total sperm number
is 39 million spermatozoa per ejaculate .

20. SPERM COUNT & ITS SIGNIFICANCE
AZOOSPERMIA is total absence of sperms. Common
causes are:
Pretesticular causes
• Deficient gonadotropin secretion by pituitary
Testicular causes
• Undescended testis
• Maldeveloped testis eg Klinefelter’s syndrome
• Severe testicular damage eg mumps, radiation
Post-testicular causes
• Ductal obstruction at any level eg ejaculatory duct

21. SPERM COUNT & ITS SIGNIFICANCE
OLIGOSPERMIA is sperm concentration less than
15 million per ml. Common causes are:
Pretesticular causes:
• Hormonal imbalance (testosterone, luteinizing
hormone (LH), follicle-stimulating hormone
(FSH), or prolactin excess
• Long term illness such as diabetes &
hypothyroidism
• Excess estrogen & corticosteroids

22. SPERM COUNT & ITS SIGNIFICANCE
Testicular causes:
• Orchitis
• Radiation treatment to the testicles
• Diseases that can cause shrinking (atrophy) of
the testicles (such as mumps).
Post –testicular causes:
• Varicocele

23. MICROSCOPIC EXAMINATION
MORPHOLOGY
• Prepare a thin smear of liquefied semen &
stain it with Romanowsky Stain, Pap Stain or H
& E Stain after fixing it in 95% ethanol
• Examine under oil immersion and look for
normal and abnormal form of sperms, RBCs,
WBCs & epithelial cells.
• Normally 60% sperms are of normal
morphology

24. MICROSCOPIC EXAMINATION
SPERM MORPHOLOGY
Spermatozoa is about 60 um in length, it consist of :
• Head &neck,
• Middle piece (midpiece),
• Tail and
• Endpiece, which is difficult to see with a light
microscope, so practically sperm consists of three parts
• Head and neck & midpiece and tail.
• For a spermatozoon to be considered normal, all three
parts should be normal.
• All borderline forms should be considered abnormal.

25. SPERM MORPHOLOGY

26. SPERM MORPHOLOGY & ITS SIGNIFICANCE
HEAD OF SPERM
• The head should be smooth, regularly contoured
and generally oval in shape.
• It measures 4-5um in length & 2.5-3.5 um in
diameter
• There should be a well-defined acrosomal region
comprising 40–70% of the head area
• Neck is short & connects head to midpiece
• Abnormalities are small, large, tapering & tear
drop shaped heads.
• Large vacuoles in the head are also abnormal

27. SPERM MORPHOLOGY & ITS SIGNIFICANCE
MIDPIECE OF SPERM
• The midpiece is 5-7um in length
• It 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.
• Residual cytoplasm is considered an anomaly
only when in excess, i.e. when it exceeds one
third of the sperm head size
• Abnormalities are thick, thin or bent midpiece
with asymmetric connection to head

28. SPERM MORPHOLOGY & ITS SIGNIFICANCE
TAIL OF SPERM
• The tail piece should have a uniform caliber
along its length,
• It should be thinner than the midpiece
• Approximately 45 um long (about 10 times the
head length).
• Abnormalities are short, multiple, spiral tails
• Kinked tail or tail of irregular thickness are also
abnormal.

29. MICROSCOPIC EXAMINATION
MORPHOLOGY
OTHER CELLS WHICH MAY BE PRESENT IN
SEMEN ARE:
• IMMATURE GERM CELLS – suggest some
defect of maturation
• LEUKOCYTES – suggest some inflammatory
disease of genital tract
• AGGLUTINATION OF SPERMS – suggest some
immunological cause eg autoantibodies

30. CHEMICAL EXAMINATION & ITS
SIGNIFICANCE
• Routinely tested chemical is Fructose
• Normal seminal fructose is 150-600 mg/dl
• It is low in androgen deficiency or ejaculatory
obstruction
• This test is used for seminal stain and vaginal
aspirate in medico legal cases

31. OTHER SPECIAL TESTS
IMMUNOLOGICAL ASSAYS
• Presence of antisperm antibody binding to head or
tail antigens suggest some defect of immunity
MICROBIOLOGICAL ASSAYS
• If WBC are present in large numbers, semen should
be cultured to rule out microbial infection
SPERM FUNCTION TESTS
• These tests assess the functional aspects of the
sperm like, abilities related to transport in female
genital tract & fertilization of ovum

32. WHO 2010
Parameter Lower Reference Limit
Semen volume (ml) 1.5
Sperm concentration (106/ml) 15
Total sperm number (106/ejaculate) 39
Progressive motility (PR, %) 32
Total motility (PR +NP, %) 40
Vitality (live sperms, %) 58
Sperm morphology 40% normal forms
pH >/=7.2
Leucocyte (106/ml) <1
Fructose 1.5-6.5 mg/ml

33. REPEAT SEMEN ANALYSIS
• Should be undertaken if any abnormalities are
present
• It is best to repeat SA after a period of 10
weeks (64-70 days), as this is the time taken
for a new batch of sperm to be generated by
the testes
• There are several causes of transient defects
in semen analysis

34. TRANSIENT DEFECTS IN SA
• Incorrect semen collection technique –
spillage, dirty container, long delay in
delivering sample
• History of recent illness like flu or high fever
may depress sperm counts
• Long period of abstinence, may lead to
increased abnormal sperm morphology and
decrease motility
• Short abstinence period may cause lower
semen volume and sperm count

35. QUALITY CONTROL IN SA
• Quality Assurance Program
– Standard Operating Procedures
– Laboratory Manual
– Documentation
– Sample ID and Tracking
• External QC
– Comparison of tests with an external source
• Internal QC
– Minimized variation by training
– Purchased QC samples with known values
– Video recordings for motility

36. CLINICAL CASE 1
A 27yr old male is being investigated as part of
infertility work up
Semen analysis report is as follows
• Appearance – clear
• Consistency – liquified in 20 mins
• Volume – 3ml
• pH – 7.5
• Fructose – 700mg/dl

37. CLINICAL CASE 1
Microscopy
• Sperm concentration – 8 million / ml
• Motility – 50%
• Morphology of sperms – normal
• Other cells – absent
Ans the following
• What is the total sperm count
• What do these findings suggest
• Name some common causes for this defect

38. CLINICAL CASE 2
A 27yr old male is being investigated as part of
infertility work up
Semen analysis report is as follows
• Appearance – turbid
• Consistency – liquified in 20 mins
• Volume – 6.5 ml
• pH – 7.5
• Fructose – 700mg/dl

39. CLINICAL CASE 2
Microscopy
• Sperm concentration – 18 million / ml
• Motility – 40%
• Morphology of sperms – normal forms 45%, abnormal
forms with big head, kinked tail are seen
• Other cells – neutrophilic leukocytes 25/HPF seen
Ans the following
• What is the total sperm count
• What do these findings suggest
• How will you confirm your diagnosis

40. CLINICAL CASE 3
A 27yr old male is being investigated as part of
infertility work up
Semen analysis report is as follows
• Appearance – greyish white
• Consistency – liquified in 20 mins
• Volume – 0.5 ml
• pH – 6.2
• Fructose – 50mg/dl

41. CLINICAL CASE 3
Microscopy
• Centrifuged smears do not show any sperms
Ans the following
• What do these findings suggest
• Enumerate some causes for this condition

42. CLINICAL CASE 4
A 27yr old male is being investigated as part of
infertility work up
Semen analysis report is as follows
• Appearance – clear
• Consistency – liquified in 20 mins
• Volume – 3ml
• pH – 7.5
• Fructose – 700mg/dl

43. CLINICAL CASE 4
Microscopy
• Sperm concentration – 48 million / ml
• Motility – 80%
• Morphology of sperms – normal
• Other cells – absent
Ans the following
• What is the total sperm count
• What do these findings suggest