The latest WHO recommendations,2010 are based on semen parameters from approximately 2000 fertile men, from eight countries and three continents, whose partners achieved pregnancy within 12 months of unprotected sexual intercourse. Pitfalls- huge shift in the lower reference values, one sided criteria. Reference limits shouldn’t be over-interpreted Interpret along with clinical history and physical examination.

1. SPERM ASSESSMENT – TRADITIONAL AND
NOVEL APPROACHES
Dr. Deepeka. T. S.
Fellow, Reproductive Medicine
CIMAR, KOCHI

2. Male factor infertility
Recognition of male factors has been
increasing over years.
Contributes directly or indirectly to 20-70%
of infertility in couples.

3. SEMEN ANALYSIS IS THE KEY INVESTIGATION IN
ASSESSING MALE FERTILITY
Overall
functionality of
sperm production
by testes
Secretory
activity of
accessory
glands
Patency of
genital tract

5. • The latest WHO recommendations,2010 are based on
semen parameters from approximately 2000 fertile men,
from eight countries and three continents, whose
partners achieved pregnancy within 12 months of
unprotected sexual intercourse.
• Pitfalls- huge shift in the lower reference values, one
sided criteria.
• Reference limits shouldn’t be over-interpreted
• Interpret along with clinical history and physical
examination.

6. SPERMATOGENESIS

7. Structure of a mature spermatozoon
• 5 μm length
• Haploid
nucleus
• Acrosomal cap
• Mictochondria
-ATP
• Flagellum

8. • Gene directing spermatogenesis -Y Chromosome
• Approximately 70 days to complete
• 12-21 days- transport – testes through epididymis to
ejaculatory duct.
• 100-200 million sperms/day
• 1 trillion-during normal
reproductive life span

9. TRADITIONAL APPROACHES TO SPERM
ASSESSMENT

10. Conventional semen analysis
• Collection
• Liquefaction
• Macroscopic examination
• Microscopic examination
• Adjunct test

11. Specimen collection for diagnostic
purposes
• Collection of semen
should ideally mimic the
in vivo situation.
• Under natural conditions-
ejaculation happens in a
sequential manner.

12. • During ejaculation the first semen fractions voided
are mainly sperm-rich prostatic fluids, whereas later
fractions are dominated by seminal vesicular fluid
(Björndahl & Kvist, 2003).
• Therefore, losing the first (sperm-rich) portion of
the ejaculate has more influence on the results of
semen analysis than does losing the last portion.
– WHO, 2010.

13. Semen quality
Sperm
production
Function of
accessory
glands
Abstention
time
Recent
febrile
illness
• There is some
evidence that the
quality of semen
specimens varies
depending on how
the ejaculate is
produced/method of
arousal.

14. Period of abstinence
• The sample should be collected after a minimum of 2
days and a maximum of 7 days of sexual abstinence.
If additional samples are required, the number of days of
sexual abstinence should be as constant as possible at
each visit.
WHO,2010.
• Ideally, 2-3 days of sexual abstinence.
Shorter abstinence period- ART procedures.

15. Collection at the laboratory
• A private room near the laboratory(limits the exposure of the
semen to fluctuations in temperature and to control the time
between collection and analysis).
• Obtained by masturbation and ejaculated into a clean, wide-
mouthed, non-toxic container made of glass or plastic.
• Without using lubricants or condoms with spermicidal agents.
• Clear instructions & documentation.
• The specimen container should be kept at ambient
temperature, between 20 °C and 37 °C.

16. Home collection
• In exceptional circumstances, such as a demonstrated inability
to produce a sample by masturbation.
• Clear instructions.
• Recording the time of semen production and deliver the
sample to the laboratory within 1 hour of collection.
• Documentation of loss of fractions/incomplete collection.
• During transport to the laboratory, the sample should be kept
between 20 °C and 37 °C.
• Inability to collection- coitus interruptus, non spermicidal
condoms(commercially available), not a reliable method.
WHO 2010.

17. Sample handling at laboratory
• Record the details- name, identification number, time
of collection, time received, date, place and method
of collection, period of abstinence, completion of
collection, time interval between collection and
examination.
• Universal precautions to avoid contamination.
• Analysis to be completed within 60 minutes.

18. Macroscopic Evaluation
• Should begin with a simple inspection soon after
liquefaction, preferably at 30 minutes, but no longer
than 1 hour after ejaculation, to prevent
dehydration or changes in temperature from
affecting semen quality.

19. Liquefaction
• Immediately after ejaculation, semen is typically a semisolid
gel like coagulated mass(cross-linking of seminal vesicle
proteins).
• Within a few minutes at room temperature, the semen usually
begins to liquefy (become thinner), becomes more
homogeneous and quite watery.
• The complete sample usually liquefies within 15 minutes at
room temperature, although rarely it may take up to 60
minutes or more.
• Continuous gentle mixing or rotation of the sample container.

20. Delayed liquefaction
• If liquefaction does not occur within 30 min, incubate for another
30 min at room temperature or in 37º c incubator.
• If semen fails to liquefy within 60 min- delayed liquefaction.
• Methods to liquefy
– addition of an equal volume of physiological medium (e.g.
Dulbecco’s phosphate-buffered saline
– repeated (6–10 times) gentle passage through a blunt gauge 18
or gauge 19 needle attached to a syringe.
– digestion by bromelain, a broad-specificity proteolytic enzyme
WHO 2010.

21. Appearance of the ejaculate
• A normal liquefied semen sample has
a homogeneous, grey-opalescent
appearance.
• It may appear less opaque if the sperm
concentration is very low.
• The colour may also be different, i.e.
red-brown when red blood cells are
present (haemospermia), or yellow in
a man with jaundice or taking certain
vitamins or drugs.

22. Semen viscosity
• Allowing the semen to drop by gravity.
• A normal sample leaves the pipette in small
discrete drops.
• If viscosity is abnormal, the drop will form a
thread more than 2 cm long.
• High viscosity – interferes with determination of
sperm motility, sperm concentration, detection
of antibody-coated spermatozoa.
• Hyperviscosity treated – physiological medium,
repeated pipetting, α-chymotrypsin and
incubate extra 10 min at 37º c

23. Semen volume
• Precise measurement of volume is essential in any
evaluation of semen, because it allows the total number
of spermatozoa and non-sperm cells in the ejaculate to
be calculated.
• Best measured by weighing the sample in the vessel in
which it is collected.
– Collect the sample in a pre-weighed, clean,
disposable container.
– Weigh the vessel with semen in it.
– Subtract the weight of the container.
– Calculate the volume from the sample weight,
assuming the density of semen to be 1 g/ml

24. • Alternatively, the volume can be measured directly.
– Collect the sample directly into a modified graduated glass measuring
cylinder with a wide mouth.
– These can be obtained commercially.
– Read the volume directly from the graduations (0.1 ml accuracy).
• Measuring volume by aspirating the sample from the specimen container
into a pipette or syringe, is not recommended, because not all the
sample will be retrieved and the volume will therefore be
underestimated.
• The volume lost can be between 0.3 and 0.9 ml
(Brazil et al., 2004a; Iwamoto et al., 2006; Cooper et al., 2007).

25. The lower reference limit for semen volume is
1.5 ml
Low semen volume
• ejaculatory duct obstruction.
• congenital bilateral absence of
the vas deferens (CBAVD)
• poorly developed seminal vesicles
• partial retrograde ejaculation
• androgen deficiency.
• loss of a fraction of the ejaculate,
short abstinence(false low)
High semen volume
• may reflect active exudation
in cases of active
inflammation of the
accessory organs.
de la Taille et al., 1998; Daudin et al., 2000; von
Eckardstein et al., 2000; Weiske et al., 2000

26. Semen pH
• The pH should be
measured after
liquefaction at a uniform
time, preferably after 30
minutes, but in any case
within 1 hour of
ejaculation since it is
influenced by the loss of
CO2 that occurs after
production.

27. WHO lower reference value- 7.2
• Higher than 8 is abnormal.
• If the pH is less than 7.0 in a semen sample with low
volume and low sperm numbers= ejaculatory duct
obstruction, CBAVD, poorly developed seminal
vesicles.

28. Initial microscopic investigation
• Gives information about quality of sperms as well as
presence of non-sperm cells.
• The liquefied sample is thoroughly mixed in the
original container and only about 50 µL is used for
evaluation.
• A phase-contrast microscope
• Involves scanning the preparation at a total
magnification of ×100.

29. Aggregation of spermatozoa
• The adherence either of immotile spermatozoa to each
other or of motile spermatozoa to mucus strands, non-
sperm cells or debris is considered to be nonspecific
aggregation.

30. Agglutination-Attachment of motile sperms to
each other forming clumps.

31. • The presence of agglutination is suggestive of the
presence of anti-sperm antibodies; further testing is
required.
• Severe agglutination can affect the assessment of
sperm motility and concentration.

32. Sperm motility
• Should be assessed as soon as possible after
liquefaction of the sample, preferably at 30 minutes,
but in any case within 1 hour, following ejaculation,
to limit the deleterious effects of dehydration, pH or
changes in temperature on motility.
• phase-contrast optics at ×200 or ×400 magnification

33. Categories of sperm movement
• Progressive motility (PR): spermatozoa moving actively,
either linearly or in a large circle, regardless of speed.
• Non-progressive motility (NP): all other patterns of
motility with an absence of progression, e.g. swimming in
small circles, the flagellar force hardly displacing the
head, or when only a flagellar beat can be observed.
• Immotility (IM): no movement.

34. • The previous edition of WHO manual recommended that
progressively motile spermatozoa should be categorized
as rapid or slow, with a speed of >25 m/sec at 37 °C as
“grade a” spermatozoa.
• However, it is difficult for technicians to define the
forward progression so accurately without bias.
(Cooper & Yeung, 2006).
• When discussing sperm motility, it is important to specify
total motility (PR + NP) or progressive motility (PR)

35. • Prepare a wet preparation approximately
20 m deep
• Evaluate at least 200 spermatozoa in a total
of at least five fields in each replicate, in
order to achieve an acceptably low
sampling error.
• Assess only intact spermatozoa (defined as
having a head and a tail;
• Scan and count quickly to avoid
overestimating.

37. Lower reference limit WHO, 2010.
• Total motility (PR + NP) is 40%
• Progressive motility (PR) is 32%
• The total number of progressively motile spermatozoa in the
ejaculate is of biological significance.
• This is obtained by multiplying the total number of spermatozoa in
the ejaculate by the percentage of progressively motile cells.
• Falsely low motility
– Non liquefaction
– Hyper-viscosity
– Delayed analysis.

38. Sperm concentration
• No of sperms per mL of ejaculate
• Assessed by using a hemocytometer.
• Laborious and time consuming.
• Proper mixing with phosphate buffered saline(PBS) should
be made.
• Assessing the samples within 10–15 minutes
• Counting at least 200 spermatozoa per replicate.

39. The improved Neubauer
haemocytometer

40. Sperm concentration, sperm count
• Sperm concentration- no of spermatozoa per ml
• Total sperm count- sperm concentration x volume
• Both are related to both time to pregnancy and
pregnancy rates and are predictors of conception
(Slama et al., 2002) (WHO,1996; Zinaman et al., 2000) (Bonde et al., 19980Larsen et al.,
2000).
When semen volume is small and fewer spermatozoa are counted than
recommended, the precision of the values obtained will be significantly
reduced.

41. • Lower reference limit- 15 × 10 6 spermatozoa per ml,
39 × 10 6 spermatozoa per ejaculate.
• Azoospermia- Absence of sperms in ejaculate in at least
2 samples on different days.
• The term azoospermia can only be used if no
spermatozoa are found in the sediment of a centrifuged
sample.
• Endocrine evaluation useful in oligo-zoospermia and
non-obstructive azoospermia.

42. Undifferentiated Round Cells
• Includes immature cells and leukocytes.
• >1 X 10 6/mL is abnormal.
• Immature germ cells indicate testicular damage.
• Leukocytospermia is suggestive of inflammation.
• Characterisation of leukocytes confirmed by
assessing peroxiadase acitivity with Endtz test.

43. Morphology
• Crucial, especially of ART.
• Complex and difficult parameter
• Abnormal morphology- abnormal spermatogenesis, sperm
maturation.
• Impact on chromatin condensation, acrosome reaction, sperm
motility.
• For morphological analysis, it is customary to prepare semen
smears that are air-dried before fixation and staining.
• Lower reference value- 4%. Teratozoospermia < 4% of
normal.

44. The concept of normal spermatozoa
• The human zona pellucida selects a subpopulation of
morphologically similar spermatozoa, but such “zona-
preferred” spermatozoa display a wider range of forms
(Liu et al., 1990; Garrett et al., 1997).
• The percentage of motile spermatozoa in semen from fathers
displaying “zona-preferred” morphology is also low (8–25%)
(Liu et al., 2003).
• The range of normal values for both fertile and infertile
men is likely to be 0–30%, with few samples exceeding
25% normal spermatozoa.
(Menkveld et al., 2001)

46. Staining methods
• The use of the Papanicolaou, Shorr
or Diff- Quik stain is
recommended.
• The head is stained pale blue in the
acrosomal region and dark blue in
the post-acrosomal region.
• The midpiece may show some red
staining and the tail is stained blue
or reddish.

47. • Slides stained using the Papanicolaou procedure can
be permanently mounted and stored for future use
in internal quality control programmes.
• If stored in the dark, they should be stable for
months or years.

48. Adjunct test for semen analysis

49. Vitality
• The evaluation of vitality differentiates live or dead
immotile sperms.
• Especially important for samples with less than about
40% progressively motile spermatozoa.
• Principle-The percentage of live spermatozoa is assessed
by identifying those with an intact cell membrane, from
dye exclusion or by hypotonic swelling.
• Lower reference limit- 58%.

50. Eosin-Nigrosin Staining

51. Hypo-0smotic swelling test
• Based on the principle
that only living cells
with intact membrane
will swell under hypo-
osmotic conditions –
150 mOsmol/L
• Doesn’t kill sperms, can
be used for ART.

52. Endtz test
• Indicated when undifferentiated round cell >1 X 10 6/mL
• To detect peroxidase within neutrophils
• Ortho-toluidene stain
• A concentration of leukocytes ≥1 x 10 6/mL- positive.
• Inflammation and infective etiology, elevated anti-
sperm antibody levels, and high levels of ROS.

53. Fructose test
• Fructose in semen is a marker for seminal vesicles
function.
• Usually performed to localize the level of obstructive
azoospermia in men with low ejaculate volume.
• Lower reference limit = 13 µmol.
• Low fructose, low semen volume and low pH=
obstructive azoospermia.
• Low fructose is characteristic of partial retrograde
ejaculation.

55. Computer-Assisted Sperm
Analysis(CASA)

56. Computer-Assisted Sperm
Analysis(CASA)
• Main objective- reducing the
subjectivity , possibility
decreasing the intra- and inter-
observer variability.
• Reduced the labor-intensive and
time consuming-nature of
standard techniques.
• Principle- etablishing a centroid,
evaluating cell motion based on
centroid trajectory.
• Bulk movements.

57. What semen parameters can be evaluated by
CASA?
• Sperm concentration, vitality, motility
• Very few systems – morphology- computer aided sperm
morphometric assessment(CASMA).
• Recent CASA systems have incorporated DNA
fluoroescent staining and fluorescent microscopy.
• The precision of CASA decreases outside an optimum
range of sperm concentration between 2 and 50 x
106/mL.

58. Advantages of CASA
• Decreases subjectivity
• More rapid analysis of larger sample with reduced
sampling error.
• Introduction of novel concepts- kinematics – may
reveals overall quality of sperms
• Images , recorded pictures are helpful in
standardization and quality assurance.

59. Disdvantages of CASA
• Limited number of parameters
• Doubtful cost-effectiveness
• Equipment maintenance, training of personnel
• Biological factors like hyperviscosity, sperm
agglutination hinders CASA interpretation.
• Technical errors, lack of internal quality control
• Lack of accuracy

60. Applicability of CASA
• A significant association between CASA analysis of sperm
concentration and progressive motility with fertilization
rates and time to conception.
• Only small number of studies provide the evidence.
• Many laboratories have included CASA as control for
comparison to standard technical analysis rather than a
stand-alone replacement technique.
• Many studies are needed on CASA using large samples.

61. Home test
• Commercially available
• Colour change antibody
reaction
• Positive when sperm
concentration is at lest
20 x10 6/mL

62. Novel Approaches for Sperm Assessment

63. • Enthusiasm in male factor infertility has decreased since
the introduction of ICSI.
• Abnormal semen parameter cannot adequately predict
male fertility potential
• A significant proportion of men with apparently normal
semen parameter are classified as idiopathic male
infertility.
• The attempt to detect abnormal sperm function is one of
the major challenges to fertility experts.

64. Do they better discriminate
infertile and fertile men ?
DNA
damage
assessment
acrosome
reaction
test
Free
radical
assessme
nt

65. FREE RADICALS

66. Oxidative Stress(OS)
Free
radicals(ROS,
RNS)
Antioxidant
capacity

67. Methods to measure Reactive Oxygen Species
• Chemiluminescence – most common –
this method is based on production of
light through reaction between a probe
and ROS.
• Flow cytometry – non fluorescent
probes.
• Thiobarbituric Acid Reactive
Substances(TBARS)
• Nitroblue Tetrazolium (NBT)

70. ROS-TAC Score
• Overall level of oxidative stress by balance between
pro-oxidants and anti-oxidants.
• ROS-TAC score has higher predictive value than ROS
or TAC alone.
• Infertile men with higher ROS-TAC score are more
prone to initiate pregnancies than those with low
scores.

71. Mioxyx system – Oxidation-reduction
potential(ORP)
• ORP values higher than the
established reference values
are indicative of oxidative
stress.
• ORP valueof 2.59 mV/million
sperm/mL is a good predictor
of oligozoospermia.
• Can be use for fresh and
frozen samples.
• Results available in 2 min

72. Clinical applicability of ROS/TAC/ORP
• Elevated ROS were detected in semen of 25-40% of
infertile men.
• Some infertile men demonstrate 30-43 % decrease in
TAC.
• Increased ROS levels correlated with abnormal sperm
parameters and DNA fragmentation, leukocyte
concentration and incidence of apoptotic/necrotic
sperms.
• Elevated ROS – increased time to natural conception,
impaired IVF pregnancy rates, recurrent miscarriages.

73. • Oral antioxidant therapy, varicocelecotmy and life style
modifications can be attempted and response assessed.
• Modifications of media by applying antioxidants to reduce OS.
• Analysing ROS and TAC can be complementary to conventional
semen analysis in ART treatment and oral antioxidant can be
used before ART.
• ORP with its high sensitivity, specificity, accuracy and low
intra-inter observer variability has the potential in
distinguishing infertile and fertile men, additional research is
needed.

74. Sperm DNA damage
• Sperm DNA integrity is vital for fertilization and
trasmission of genetic material to the offspring.
• The DNA of mammalian sperm is the mostly known
compact eukaryotic DNA(packaged six times more tightly
than the somatic cells).
• DNA damage can occur not only during spermatogenesis,
but epididymal transit and collection for ART.
• Single stranded breaks and double stranded breaks are
commonly termed DNA fragmentation.

76. Methods to measure sperm DNA damage
1. Terminal deoxynucleotidyl transferase-mediated
dUTPnick end labeling assay (TUNEL)
– based dUTP binding to the 3-OH break ends of single
and double stranded DNA breaks.
– Fluorescent signal = percent of DNA fragmentation.
– cut-off values- 17 %
– High specificity (92%), high PPV(91%)
– Available as commercial kit

77. 2. Comet assay
embedding the certain number of sperm into the agar and lysing their cellular
proteins and membranes using detergent and high density salt solutions
exposed to electrophoresis
DNA fragments move towards postively charged anode.
evaluation of the comet image by fluorescence microscope or cytometer

78. • Length of comet tail and degree of fluorescence
• Prediction of fertilization rates with 93.3% specificity.
• DNA fragmentation >40 % reflects a 9.5 times higher
risk of poor fertilization.
T.R.Dias et al

79. 3. Sperm Chromatic Dispersion(SCD)
Single-cell agarose gel
electrophoresis
Acid/alkaline denaturation
Bright field or fluorescent
microscope

80. SCD-DFI ≤ 25 %

81. Sperm chromatin structure assay
• Principle DNA damaged sperm are more vulnerable to
heat or acid denaturation compared to the intact sperm.
• The denatured DNA damaged sperm are stained red
while the intact sperm turn into green when exposed to
acid and acridine orange stain.
• The stained sperm are analyzed by flow cytometry.
• The greatest advantage -large number of cells could be
evaluated

82. • DFI ≥30 % - high risk of low
blastocyst rates, failure to initiate
ongoing pregnancy
• DFI ≥15 % - lower fertilization
rates, ICSI recommended.
T.R.Dias et al

83. Clinical value of sperm DNA damage evaluation
• High rates of DNA fragmentaiton were correlated with decreased
semen quality.
• Sperm DNA damage is significantly increased in idiopathic and male
factor infertility.
• Sperm DNA fragmentation is associated with longer time for
natural conception, idiopathic infertility, recurrent IUI and IVF
failures and spontaneous miscarriage.
• Clinical utility proposed –
– Infertile men with normal semen analysis
– Recurrent spontaneous abortion
– To determine most suitable ART

84. Assessment of sperm fertilization
capacity

85. Acrosome reaction test
• Invitro acrosome reaction
dysfuction can be assessed.
• Artificial acrosome reaction
induced by calcium inophore A
23187.
• Fluorescent labelled lectins and
monoclonal antibodies-used to
asses the acrosomal
membranes and contents.
• 5-30 % reacted sperms means
higher fertility potential.
T.R.Dias et al

86. Sperm mitochondrial activity
• A fluorescent probe is used to distinguish between
sperm with poorly and highly functional
mitochondria using flow cytometry.
• Only method available for assessment of sperm
mitrochondria.
• A recent study- DNA FRAG testing + sperm
mitochondrial membrane potential could be stronger
predictors of natural conception.

87. Antisperm antibodies(ASA)
• Among all male infertility cases, 10 % are associated with ASA.
• Indication – agglutination in conventional SA.
• Mixed antiglobulin reaction(MAR)
• Immunobead test(IBT)
• Clinically significant when ≥50 % are coated , when sperms are
unable to penetrate pre-ovulatory cervical mucus or sperms
demonstrated impaired fertilizing capacity.
• Controversy about predicting ART outcome.

88. Future perspectives
• Digital holography microscopy- morphology and
motility characterisation, using three-dimensional
images to delineate normal and abnormal sperm.
• Study of semen using genomics, proteomics,
metabolomics -new insight into biochemical basis of
defective semen, could be a new tool for diagnostic
and therapeutic purposes.

89. Concluding remarks
• A properly conducted standardized semen analysis
technique is of utmost importance for diagnosis and
evaluation of infertile men.
• Introduction of CASA aims to overcome limitations of
conventional SA, however, with questionable efficiency,
lack of standard guidelines, inconsistency between
different equipments and high cost.
• The necessity to over come the limitations of routine
semen analysis is quite clear.

90. • Sperm function test can be valuable complement to
conventional semen analysis to evaluate fertility,
counsel the couple as well as predict reproductive
outcome by natural conception or ART.
• Sperm DNA assessment may have role in idiopathic
infertility, recurrent IUI and IVF failures and
spontaneous miscarriage in choosing the best
method of ART.

92. References
• WHO laboratory manual for the Examination and processing
of human semen(FIFTH EDITION)
• Dias T.R., Cho CL., Agarwal A. (2019) Sperm Assessment:
Traditional Approaches and Their Indicative Value. In: Nagy Z.,
Varghese A., Agarwal A. (eds) In Vitro Fertilization. Springer,
Cham. https://doi.org/10.1007/978-3-319-43011-9_22
• Dias T.R., Cho CL., Agarwal A. (2019) Sperm Assessment: Novel
Approaches and Their Indicative Value. In: Nagy Z., Varghese
A., Agarwal A. (eds) In Vitro Fertilization. Springer, Cham.
https://doi.org/10.1007/978-3-319-43011-9_23