DR.P.M. GOPINATHMD DGO FMMC FICS FICOG MBA

DR.P.M. GOPINATHMD DGO FMMC FICS FICOG MBA

Vice president, Fertility Preservation Society (I)

Former Director/ic of ISO & Govt KG Hospital,Chennai

Practicing OG and ART for the past 35 years

Director & Senior ConsultantSRM Institutes For Medical science

Vadapalani, Chennai

Director & Senior ConsultantSRM Institutes For Medical science

Vadapalani, Chennai

© Oozoa Biomedical Inc, April 2005

FERTILITY vs STERILITY

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 testsHypo-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 TestsSperm –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 oocyte-sperm 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 TestsThe 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 TECHNIQUESROS 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 testsDNA 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

• 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.

Comet

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 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.

Sperm Chromatin Structure Assay

© Oozoa Biomedical Inc, April 2005

RESEARCH & NEW TECHNIQUESSperm DNA Damage

Sperm Chromatin Structure Assay: SCSA™

© SCSA Diagnostics Inc, Brookings, SD, USA

Evenson et al. (1980) Science 240:1131-1133

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 testsIndirect 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 testsHyaluronic 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 testsSperm 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 pellucida- induced acrosome reaction of zona pellucida-bound 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.