DR.P.M. GOPINATH
MD DGO FMMC FICS FICOG MBA
Director & Senior Consultant
SRM Institutes For Medical science
Vadapalani, Chennai
Vice president, Fertility Preservation Society (I)
Former Director/ic of ISO & Govt KG Hospital,Chennai
Practicing OG and ART for the past 35 years
FERTILITY vs STERILITY
© Oozoa Biomedical Inc, April 2005
Lab Evaluation of Male Infertility
• Semen analysis is the cornerstone
• There are a subset of patients in which there is a
deficiency in one of the tasks necessary for
spermatozoa to reach and fertilize an ovum,
which is not detected by the standard semen
analysis
• This subset is estimated at 40–50 percent of
men presenting for subfertility.
• We will try to analyze the semen tests to evaluate
specific aspects of spermatozoa function.
Non-routine tests
Hypo-Osmotic Swelling Test(HOST)
- Based on the principle that viable spermatozoa have
intact membranes.
- Characteristics: Swelling of the cytoplasmic space
and curling of the sperm tail.
- Normal test results: >60% of spermatozoa with
swelled and curling tail is considered normal.
- Test may be used to aid selection of viable
spermatozoa for use in intra cytoplasmic sperm
injection, especially in those cases when only immotile
spermatozoa are available.
Non-Routine Tests
Sperm –Zona Penetration(Hemi zona Assay)
-Depends on the availability of human oocytes that have failed to fertilize
in vitro.
-Spermatozoa are analyzed by standard methods and selected by the
swim-up technique.
-Fertile donor’s sperm are used to verify the occurrence of normal oocytesperm binding of non-fertilized oocytes.
-After incubation , oocytes are washed in a culture medium and the no. of
spermatozoa binding to and penetrating into the zona pellucida and petri
vitelline space is assessed.
-The final result is expressed by a penetration index(HZI) = hemi zona
index) and the value considered as normal is above or equal to 35%.
HZI = Number of attached spermatozoa from the patient
Number of attached spermatozoa from fertile donor
Zona-Free Hamster Oocyte Test
• This was one of the most widely used accessory tests in the
evaluation of advanced sperm function in the yester years .
• The fusion between human sperm and the hamster oocyte
is nearly identical to that occurring with the human egg.
• Fusion with the vitelline membrane of the oocyte is
normally initiated by the healthy sperm's plasma
membrane that lies over a special section (equatorial
segment) of the sperm head in a sperm that has prepared
itself for normal fertilization.
• This test relies upon the ability of sperm that are tested in
the laboratory to undergo the necessary reactions to
fertilize an egg
• This test may be used as a screen to assist in determining
which men may need the assistance of "ICSI" fertilization by
the embryology team.
Non-Routine Tests
The Acrosome reaction test
-Acrosome reaction is triggered by the sperm-zona pellucida
binding and is an exocytotic secretory process.
-Proteolytic enzymes released during the acrosome reaction
facilitate both zona pellucida penetration and modification of the
sperm surface in preparation for the oocyte fusion.
-The test is performed by application of semen processing
techniques or addition of enzymes and hormones to the sperm
preparation media.
-Acrosomal status can be assessed by electron transmission
microscopy (expensive) or by fluorescence microscopy.
-This is a very labor-intensive test.
Acrosome Reaction
Reactive Oxygen Species
• Reactive oxygen species in the sperm result from the metabolism of
oxygen by the sperm cell.
• These species include superoxide anion, hydrogen peroxide and nitric
oxide.
• The detection of excess amounts of these chemicals in the sperm
combined with abnormally high concentrations of certain enzymes
may indicate that the involved sperm are immature, damaged or
abnormal.
• Excess levels of reactive oxygen species can produce oxidative
damage to important components of the sperm.
• Sperm normally maintain levels of "anti" oxidant systems to protect
the sperm cell.
• If the presence of high levels of oxidants are present, the cell's
defenses may be overwhelmed, and cellular damage may result.
• Chemiluminescent testing in the laboratory can detect excess levels
of reactive oxygen species.
RESEARCH & NEW TECHNIQUES
ROS and sperm
© Oozoa Biomedical Inc, April 2005
Zona Pellucida Binding Tests
• When a normal sperm binds to the zona pellucida of a healthy
egg, the sperm normally undergoes an "acrosome reaction"
that releases components that lead to normal fertilization.
• This process may be approximated in the laboratory by
exposing the sperm being tested to the dissected zona
pellucida of human eggs obtained from non-fertilized human
eggs.
• The zona pellucida of such an egg is split in half.
• Sperm from the man being tested are exposed to one half of
the split zona pellucida, while sperm from a man known to
have "normal-fertile" sperm are exposed to the other half.
• A comparison of the performance of the sperm of the two
men is carried out to ascertain the functional binding capacity
of sperm of the man being tested.
Anti-Sperm Antibodies
• Sperm antibodies in semen belong almost entirely to
two classes of antibodies; IgA and IgG.
• IgA antibodies are probably more important sources of
sperm problems than IgG.
• IgM antibodies, because of their large molecular size,
are rarely seen in semen.
• Of import, it has been found in the IVF laboratory that
unless 50% or more of the sperm have antibodies
coated on them, there is little chance that the sperms
function will be significantly impacted.
• That is, problems are rarely encountered from antibody
levels lower than 50%.
Non-routine tests
DNA fragmentation
- DNA damage positively correlated with poor semen
parameters , leucocytospermia and high reactive oxygen
species levels.
-Many studies suggest that sperm DNA fragmentation
could influence fertilization , embryo quality and
development, blastocyst achievement and pregnancy
rates and losses.
- Normal Comet Assay results range from 80-95%
-Couples are twice as likely to become pregnant with
regular IVF methods if the TUNNEL DNA fragmentation
index(DFI) is less than 30%
Sperm DNA fragmentation tests
• TUNNEL assay [Terminal deoxynucleotide transferase mediated dUDP nick-end labelling]
• COMET assay
• SCSA or sperm chromatin structure analysis
• DBD-FISH [DNA breakage detection-fluorescence insitu hybridization
• Sperm chromatin dispersion tests
Comet
• The Comet method is based on single cell electrophoresis
with migration of small DNA fragments into a gel and
formation of a “comet”.
• This method is also based on a microscopic assessment.
• Number of cells (usually 100) is far too low to satisfy the
need for good counting statistics.
Sperm Chromatin Structure Assay
• Conventional sperm parameters as measured on a routine semen analysis
had no bearing on success when ICSI was used.
• Many couples pursued egg donation after failed IVF attempts because the
husband's semen parameters were relatively normal and yet conception
hadn't occurred.
• Some of these same couples were still unable to conceive even with the
"better quality" donor eggs leaving both the doctors and the couples
frustrated and perplexed.
• Some couples then went on to use both egg donors and surrogates thinking
it was both an egg quality and implantation issue, again without success.
• The only commonality was the husband's sperm. In 2005, a relatively new
concept was introduced to clinical practice; sperm quality was dependent
on the amount of damage to the sperm DNA or DNA fragmentation.
• Simply put, DNA is arranged in a double helix or ladder configuration with
side rails and rungs.
• If the rungs are broken, then the ladder is unsteady and won't function
properly.
Sperm Chromatin Structure Assay
• Sperm DNA fragmentation has little or nothing to do with the
parameters that we measure on the routine semen analysis.
• It has little to do with the shape of the sperm or whether the sperm
are moving. It is a completely independent variable.
• Men with otherwise normal semen analyses can have a high degree
of DNA damage and men with what was called very poor sperm
quality can have very little DNA damage.
• More importantly what has also been demonstrated is that the
degree of DNA fragmentation correlates very highly with the inability
of the sperm to initiate a birth regardless of the technology used to
fertilize the egg such as insemination, IVF or ICSI.
• Sperm with high DNA fragmentation may fertilize an egg and embryo
development stops before implantation or may even initiate a
pregnancy but there is a significantly higher likelihood that it will
result in miscarriage.
RESEARCH & NEW TECHNIQUES
Sperm DNA Damage
Sperm Chromatin Structure Assay: SCSA™
© SCSA Diagnostics Inc, Brookings, SD, USA
Evenson et al. (1980) Science 240:1131-1133
© Oozoa Biomedical Inc, April 2005
SCD
• Ideal test should be:
-Objective (independent of technician)
-Precise
-Enables to obtain results which can be used to guide
couples to the most effective
treatment.
• The SCSA®-method and TUNEL are the only methods
which can be combined with flow cytometry and thus has
the precision and objectivity needed for clinical use.
Questions unanswered
• ART Failure with low DFI sperms
• High DFI in spite of good semen characteristics
• How could DFI be improved to become a
versatile tool for ART
In case of high DFI
• Find the cause and treat
– Varicocele
– MAGI
– Avoid exposure to toxins
• No cause found out
– Scrotal cooling
– Anti oxidants
– Life style modifications
Non-routine tests
Indirect methods for DNA damage assay
- Sperm chromatin integrity assays
-DNA intercalating dyes(acridine orange)
 Significant intraindividual variations exist for sperm chromatin
structure assays.
 There is a higher rate of DNA damage in ejaculated or
epididymal sperm than in intratesticular spermatozoa , hence
the use of intratesticular spermatozoa for men with high DNA
fragmentation index is recommended for ICSI according to some
authors.
 ASRM(2006) best summarizes the current viewpoint on DNA
integrity testing, concluding that there are not enough data to
make DNA testing routine in infertility testing and that
treatments have yet to prove their clinical value.
Non-routine tests
Hyaluronic acid binding by human sperm
- Novel technique of sperm evaluation
- Used for assessing sperm maturity and
function.
- Huszar .G
Sperm binding to hyaluronic acid were
mature, whereas non-binding sperm showed
signs of cytoplasmic maturity , judging on their
cytoplasmic and nuclear properties.
Non-routine tests
Sperm hyper activation
- Chan PJ et al. (1992):
Novel technique, proposed as a quality
control test for the sperm – zona penetration
assay.
- Liu et al. (2007):
Hyper activation of capacitated human
sperm highly correlated with zona pellucidainduced acrosome reaction of zona pellucidabound sperm.
Conclusions
• The conventional semen analysis is a critical first step in
the evaluation of male fertility, but provides limited
information about sperm function.
• In a subset of patients, specialized semen tests will be
needed to elucidate the etiology of subfertility.
• These tests are useful for determining specific defects of
human sperm physiology.
• Newer sperm function tests may eventually be useful
clinically.
• Their addition to potential to the routine semen analysis
could help in the diagnosis of what is currently classified
as unexplained infertility.