Sperm Function Tests are the keystones of evaluating functional condition of sperms. The fertility potential of a sperm will be decided not only with the number & motility but with the functional competence which is of utmost importance.

1. SPERM FUNCTION
TESTS:
WHAT, WHY, WHEN…?
Dr. Rahul Sen (Ph.D)

2. sperm is a male reproductive cell, which is motile.
It is 60µm long and 2-5µm wide and contains 23 set
of chromosomes

3. ATTRIBUTES OF NORMAL SPERMATOZOA
1-Plasma membrane
2-Outer acrosomal membrane
3-Acrosome
4-Inner acrosomal membrane
5-Nucleus
6-Proximal centriole
7-Rest of the distal centriole
8-Thick outer longitudinal
fibers
9-Mitochondrion
10-Axoneme
11-Anulus
12-Ring fibers
A-Head, B-Neck, C-Mid
piece, D-Principal piece, E-
End piece
• TO EXHIBIT A NORMAL STRUCTURE
• PERSISTS MOTILE MOVEMENTS TO PASS THROUGH REPRODUCTIVE TRACT BARRIERS
• PROPERLY PACKAGED AND STABLE DNA
• MAINTAIN FUNCTIONAL METABOLIC PATHWAYS (RECEPTOR PROTIENS,
ACROSOMAL ENZYMES, SURVIVAL PROTEINS TO MAINTAIN IN FEMALE TRACT )

4. SFTs are the keystones of male
infertility evaluation
“In vivo” and “in vitro” conditions
sperm functions are same
The fertility will be decided not
only with the number but also the
functional competence of sperms

5. SPERM FUNCTION TESTS
 Semen analysis is an essential component when
assessing male reproductive capacity
 Semen analysis incompletely provides a
comprehensive assessment of male fertility potential
 Sperm function tests adds important data regarding
sperm ability for fertilization and impact in post
embryo development
 Use of SFT in conjunction with semen analysis
contributes more efficient treatment strategy

6. SO, WHAT ARE SPERM FUNCTIONS?
 TO BE PRESENT IN AN EJACULATE ?
 TO BE IN A GOOD NUMBER?
 TO BE MOTILE ?
 HAVE A GOOD MOTILE SPEED?
NAAAAAAAAAHHHHHHHH……. 

7. SPERM FUNCTIONS ARE
 TO FERTILIZE AN OOCYTE
 DELIVER INTACT DNA INTO OOCYTE
 ACHIEVE HEALTHY PREGNANCY

8. Advent of sperm function tests
 Patient demand derived innovation
 Patient satisfactory treatment approaches
 Detailed assessment in Sperm assessments to rule
out cause
 Translation of benchtop to bedside

9. SPERM FUNCTIONS
BASIC FUNCTION IS TO FERTILIZE AN OVUM AND
CONTRIBUTES CHROMOSOMES
sperm functions including their potential for movement, cervical
mucus penetration,
capacitation, zona recognition, the acrosome reaction and
sperm-oocyte fusion

10. ZONA
PELLUCIDA
CUMULUS
CELLS
ISTHMUS (103)
UTERO-TUBAL
JUNCTION (104)
UTERUS (106)
CERVICAL
MUCUS (106)
VAGINA (107)
(107) – Sperm Number

11. ROUTINE SEMEN ANALYSIS:
GAP IN KNOWLEDGE

12. SEMEN EXAMINATION
Macroscopic parameters
• Volume
• Appearance
• Color
• Liquefaction
• Viscosity
• pH
Microscopic parameters
• Sperm concentration
• Sperm motility
• Morphology
• Leucocyte concentration
• agglutination

13. CRITERIA SECOND EDITION;
1987
THIRD EDITION;
1992
FOURTH
EDITION;
1999
FIFTH EDITION;
2010
Volume
(ml’s)
>or = 2 >or = 2 2.0 1.5
Total Sperm
(Mill in ejaculate) > or = 40 > or = 40 40 39
Sperm Count
(Mill/ml)
>or = 20 >or = 20 20 15
Total Motility (%) > or = 50 > or = 50 50 40
Progressive
Motility (%)
> or = 40 > or = 40 40 32
Sperm Vitality
Live spermatozoa
(%)
> or = 70 > or = 70 60 58
% Normal Forms
> or = 50 > or = 30 14 4
Lower Reference Values*

14. Diff-Quick test
Procedure
A- Fixative reagent (triarylmethane dye dissolved in methanol)
B- Staining solution 1 (eosinophilic xanthene)
C- Staining solution 2 (basophilic thiazine)
Utility
Shape correlates with male fertility
Shape correlates with DNA fragmentation
Shape correlates with pregnancy outcomes

15. Morphology
Manifestation of prospective fertilizing spermatozoa
METHODS
 Papanicolaou staining procedure
 Shorr staining procedure
 Rapid staining procedure

19. Motility
 Microscopic examination
 Computer assisted semen analyzers (CASA)
Advantages
High accuracy
Quantitative information on the kinematic parameters of
spermatozoa (forward progression and hyper activated
motility, characteristic of capacitated cells)
Utility
 Productivity of testis
 Patency of GT
 Substantial motility reveals severe infection and
inflammatory reactions in RT
 Abnormal sequence of ejaculation
 Disorders of sex glands
 Correlated with DNA fragmentation

20. Eosin and Nigrosin Procedure
1- Mix 50µl of semen with 2 drops of eosin
2- mix 3 drops of Nigrosin after 30 seconds
3- Prepare a slide after 30 seconds and leave it for dry
4- examine immediately after drying with oil immersion at 100x
Utility
 Progressive motility < 40%
LRL-58%

21. Hypo-Osmotic Swelling (HOS) test
Procedure
1- Incubate 1ml of HOS solution for 5-10 minutes
2- mix 0.1 ml of semen with HOS Solution and incubate
again for 30 minutes
3- Make a wet preparation with 10-15µls of incubated
sample
4- Examine at 40x
Utility
Discover active sperms
The sample can be reused

22. BIOCHEMICAL TESTS
 Poor-quality semen may result from testicular production
of abnormal spermatozoa, or from post-testicular
damage to spermatozoa in the epididymis or the
ejaculate from abnormal accessory gland secretions.
 Secretions from accessory glands can be measured to
assess gland function
 An infection can sometimes cause a decrease in the
secretion of these markers, but the total amount of
markers present may still be within the normal range.

23. BIOCHEMICAL TESTS
 The amount of zinc, citric acid (Möllering & Gruber, 1966) or acid
phosphatase (Heite & Wetterauer, 1979) in semen gives a reliable
measure of prostate gland secretion, and there are good
correlations between these markers.
 Secretory capacity of the seminal vesicles. Fructose in semen reflects
the secretory function of the seminal vesicles.
 Secretory capacity of the epididymis. L-Carnitine, GPC and neutral
–glucosidase are epididymal markers used clinically. Neutral -
glucosidase has been shown to be more specific and sensitive for
epididymal disorders than L-carnitine and GPC (Cooper et al.,
1990a).
 There are two isoforms of –glucosidase in the seminal plasma: the
major, neutral form originates solely from the epididymis, and the
minor, acidic form, mainly from the prostate.

24. MEASUREMENT OF ZINC
 Spectrophotometric Assay
 The volumes of semen and reagents can be
proportionally adjusted for spectrophotometers
using 3-ml or 1-ml cuvettes.
 The lower reference limit for zinc is 2.4 mol per
ejaculate (Cooper et al., 1991 and unpublished
data from TG Cooper).

25. MEASUREMENT OF FRUCTOSE
 Fructose is an energy source for sperm motility,
Azoospermia negative fructose results may indicate
absence of SV/VD or any obstruction
 Can be assessed via both ways qualitative &
quantitative
 Quantitatively,
 The lower reference limit for fructose is 13 mol per
ejaculate

26. MEASUREMENT OF FRUCTOSE
 Fructose testing should be considered for patients
with azoospermia, low volumes
 Determined by heating semen in a strong acid in the
presence of resorcinol. Fructose gives red color
upon heating.
 Low fructose in semen is characteristic of ejaculatory
duct obstruction, bilateral congenital absence of the
vas deferens

27. MEASUREMENT OF GLUCOSIDASE
 Seminal plasma contains both a neutral -glucosidase
isoenzyme, which originates in the epididymis, and
an acid isoenzyme contributed by the prostate.
 The lower reference limit for neutral -glucosidase is
20 mU per ejaculate

28. CASA
(COMPUTER AIDED SEMEN ANALYSIS)
 Until recently, it was not feasible to measure sperm
concentration by computer aided sperm analysis (CASA)
because of difficulties in distinguishing spermatozoa from
particulate debris (ESHRE, 1998).
 Several manufacturers produce CASA systems. These
machines are capable of measuring sperm motility and
kinematics, and some can also be used to estimate sperm
concentration.
 CASA, including assessment of motility, concentration and
morphology, has two advantages over manual methods: it
has high precision and it provides quantitative data on the
kinematic parameters of spermatozoa (forward progression,
hyperactive motility)

29. CASA
(COMPUTER AIDED SEMEN ANALYSIS)
 Some studies have suggested that CASA estimates
of concentration and movement characteristics of
progressively motile spermatozoa are significantly
related to fertilization rates in vitro and in vivo, as
well as to time to conception
 Many factors affect the performance of CASA
instruments, e.g. sample preparation, frame rate,
sperm concentration and counting-chamber depth

30. CASA terminology

31. CASA terminology

32. Sperm Antibodies
 Because mature spermatozoa are formed after
puberty, they can be recognized as foreign protein by
the male immune system. If there is a breach in this so-
called “blood–testis barrier,”
 These antibodies can then come into contact with the
sperm and may cause them to clump.
 There are several tests currently employed for detecting
the presence of sperm antibodies.
 The two most common are mixed agglutination
reaction (MAR) and the Immunobead binding test.

33. Mixed Agglutination Reaction (MAR)
 This test is performed by mixing semen, IgG- or IgA-
coated latex beads or red blood cells, and IgG or IgA
antiserum on a microscope slide. The slides are
incubated and observed at 400× magnification. At
least 200 sperm are counted. If antibodies are present,
the sperm will form clumps with the coated latex beads
or coated red blood cells. If antibodies are absent, the
sperm will swim freely.
 The WHO considers a level of binding of ≥50% to be
clinically significant. This test is used only for detection
of direct antibodies in men, and is not specific for the
location of bead attachment to the sperm.

34. The Immunobead Binding Test
 This test is performed by combining IgG- or IgA-coated
latex beads and washed sperm on a slide. The sperm
must be removed from the seminal plasma by washing
the sample with media plus bovine serum albumin (BSA).
The presence of human protein on the surface of the
sperm interferes with the binding of the immunobeads
to the sperm, and thus may mask a positive result. After
washing, the sperm is placed on a slide with IgG- or
IgA-coated latex beads and is read at 200× or 400×
magnification. If antibodies are present, the small
beads will attach directly to the sperm.
 The level of binding of ≥50% is commonly considered
to be clinically significant

35. SPERM ACROSOME
 The acrosome is an intracellular organelle, similar to a
lysosome, which forms a cap-like structure over the
apical portion of the sperm nucleus (36). The acrosome
contains multiple hydrolytic enzymes, including
hyaluronidase, neuraminidase, proacrosin,
phospholipase, and acid phosphatase, which, when
released, are thought to facilitate sperm passage
through the cumulus mass, and possibly the zona
pellucida.
 In fact, only acrosome-reacted sperm is capable of
penetrating the zona pellucida, binding to the
oolemma, and fusing with the oocyte

36. SPERM ACROSOME
 This has led to the necessity for the development of
biochemical markers for the acrosome reaction.
 Contemporary assays for the determination of
acrosomal status employ fluorescent plant lectins or
monoclonal antibodies, which can be detected much
more easily with fluorescence microscopy.
 one of the predominant enzymes that is present in
the acrosome is proacrosin. The enzymatic action of
acrosin is not necessarily correlated to the presence
of an intact acrosome

37. HEMI-ZONA BINDING ASSAY
 a significant correlation between tests of sperm–zona
pellucida binding and subsequent fertilization in ART
 the hemizona assay (HZA) employs sperm and
nonviable oocytes in an in vitro assessment of
fertilization
 This test assesses the ability of sperm to bind to the
zona itself. Although the HZA is relatively expensive,
labor intensive, and difficult to perform, there are some
data that suggest that the HZA may help to identify
individuals with a poor prognosis for success with ART

38. HEMIZONA BINDING ASSAY
 oocytes that failed to fertilize during an ART
procedure are bisected, and then sperm from a
proven fertile donor (500,000/mL) is added to one
hemizona, while sperm from the subject male is
added to the other hemizona. Following a four-hour
incubation, each hemizona is removed and pipetted
in order to dislodge loosely attached sperm. A
comparison or hemizona index (HZI) is then
calculated

39. HEMI-ZONA BINDING ASSAY
Unlike several other tests of sperm function,
a cutoff value
(35%)
has been identified as a predictor of IVF success.

40. MANNOSE BINDING ASSAY
 Another test has been developed in order to assess
the ability of sperm to bind to the zona.
 involves the recognition by a sperm surface receptor
of a specific complementary receptor on the surface
of the zona pellucida.
 This zona receptor appears to be a glycoprotein,
the predominant sugar moiety of which is mannose
 In vitro assays in which labeled probes of mannose
conjugated to albumin are co-incubated with semen
specimens allow for the differential staining of
sperm
 invites further study

41. PICSI- Physiological ICSI

42. PICSI- Physiological ICSI
 In the process of fertilization, the sperm bind
to the hyaluronic acid (HA) present in the
cumulus oophorus by HspA2 protein, with
hyaluronidase activity that induces acrosome
reaction.
 The PICSI device, a dish similar to ICSI dish,
contains microdots of hyaluronan hydrogel
which needs to be hydrated by media before
ICSI.
 Selectively binding of sperm to the culture
dish shows:
More viability,
Non DNA-fragmented, and
with lower rates of aneuploidies.

43. Selection of HA binding spermatozoa
Incubation, RT, 10
min
Add sperm to the hyaluronan
microdot
Gentle aspiration of a bound
spermatozoa
After 15 min, the proportion of hyaluronan-bound spermatozoa, that exhibited vigorous tail
beating, were assessed versus the unbound motile spermatozoa

44. Magnetic Activated Cell Sorting

45. ASSAYS FOR SPERM DNA INTEGRITY
 The most current area of investigation into sperm
function involves the assessment of sperm DNA
integrity
 Sperm chromatin has been demonstrated to be
packaged very differently from chromatin in
somatic cells.
 As there are many ways in which this DNA
organization or the sperm chromatin itself can be
damaged,

46. CAUSES FOR IMPAIRED DNA INTEGRITY

47. ASSAYS FOR SPERM DNA INTEGRITY
 SPERM KARYOTYPING
 SPERM FISH ANALYSIS
 SPERM DNA FRAGMENTATION TESTS

48. INDICATIONS FOR SPERM DNA ASSESSMENT
 Unexplained infertility
 Repeated ART Failures
 Recurrent Pregnancy Losses
 Prediction of ART outcomes
 Assessment of DNA/Genetic Integrity
- Post Chemotherapy
- Advanced Paternal Age

49. DETECTION OF SPERM DNA DAMAGE
Strand breaks in DNA
Strand breaks in DNA
Susceptibility to
Denaturation

50. OTHER TESTS FOR
DETECTION OF SPERM DNA DAMAGE
 Strand breaks in DNA (incorporation of probes at site of damage)
 DNA breakage detection FISH (DBD-FISH) (potential DNA damage)
 In situ nick translation (ISNT) (real DNA damage)
 8-Hydroxydeoxy-guanosine (8-OHdG) (real DNA damage)
 Susceptibility to denaturation in an acidic solution
 Halosperm test (potential DNA damage)
 Chromatin condensation
 Aniline blue stain
 Toluidine blue stain
 ChromomycinA3 stain

51. Sperm Chromatin Dispersion Assay
 sperm are immersed in an agarose matrix on a slide,
 treated with an acid solution to denature DNA that contains
breaks, and
 then treated with lysis buffer to remove membranes and
proteins.
 The agarose matrix allows working with unfixed sperm on a
slide in a suspension-like environment.
 Removal of nuclear proteins results in nucleoids with a
central core and a peripheral halo of dispersed DNA loops.
 Sperm nuclei with elevated DNA fragmentation produce
very small or no halos of DNA dispersion, whereas those
sperm with low levels of DNA fragmentation release their
DNA loops forming large halos.

52. Without
HALO
Small
HALO
With Proper
HALO
Degraded
HALO
Nucleoids/ Sperms
With Fragmented DNA
Nucleoids/ Sperms
With Non-Fragmented DNA
Sperm Chromatin Dispersion Assay

53. SPERM CHROMATIN STRUCTURE ASSAY
 The SCSA measures the level of DNA
fragmentation after denaturation by using
acridine orange staining and flow cytometry.
 Double-stranded (native) DNA stains green
and single stranded (denatured) DNA stains
red.
 SCSA also detects immature sperm that have
increased histones rather than protamines.

54. SPERM CHROMATIN STRUCTURE ASSAY
• SCSA is an accurate and
reproducible test for
measuring sperm DNA
damage
• Test requires a highly skilled
staff, expensive equipment
which limits its widespread
use

55. TUNEL Assay
(Terminal deoxytransferase-mediated dUTP nick end-labeling)
 TUNEL is a direct method to detect single and
double strand breaks in the DNA
 Detects DNA strand breaks resulting from apoptotic
signaling cascades
 TUNEL can be used in a clinical setting using
flowcytometer.

56. TUNEL Assay
(Terminal deoxytransferase-mediated dUTP nick end-labeling)
 This single-step staining method labels
DNA breaks with fluorescein
isothiocyanate (FITC)-dUTP followed
by flow cytometric analysis.
 TUNEL utilizes a template-
independent DNA polymerase called
TdT which non-preferentially adds
deoxyribonucleotides to 3´-hydroxyl
(OH) single- and double-stranded
DNA.
 dUTP is the substrate that is added
by the TdT enzyme to the free 3´-OH
break-ends of DNA

57. COMET Assay
 Comet assay (single cell gel electrophoresis) commonly
used to detect extent of DNA damage in spermatozoa
with exposure to an electric field under neutral or
alkaline conditions
 Damage is measured by measuring displacement
between genetic material of nucleus „comet head‟ and
resulting tail

58. COMET Assay
 Embedding of sperm in agarose gel
 Lysing of cells and decondensation of DNA
 Unwinding of DNA
 Separation and staining of DNA fragments
 Image and statistical analysis
 Quantifies actual damage in individual sperm
COMET images showing
1. Damaged
2. Undamaged DNA

59. UTILITY OF SDF TESTS
 SDF is mainly oxidative stress mediated
 Elevated SDF is associated with infertility, Poor ART
outcomes and recurrent miscarriages
 SDF gives different information than routine semen
analysis and have better prognostic value
≤ 15% DFI EXCELLENT TO GOOD SPERM DNA INTEGRITY
> 15% TO < 25% GOOD TO FAIR SPERM DNA INEGRITY
≥ 25% TO <50% FAIR TO POOR SPERM DNA INEGRITY
≥ 50% VERY POOR SPERM DNA INEGRITY

62. THE TECHNOLOGY AWAITED…
 PROTEOMICS
METABOLOMICS
EPIGENETICS

63. Utilities of SFTs
Diagnosis of infertility
Selection of preparation
Selection of insemination method
Selection of sperms for Micromanipulation
Reduce embryonic development abnormalities
Give an overview of overall outcome

64. Summary
Number
increases
value
decreases
Sperm
contributes
50%