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Sperm Evaluation | Sperm Tests | Advanced Tests | Azoospermia & Related Syndromes | Donor Sperm Bank | |
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Additional, Advanced Sperm Testing Procedures Many of the following tests are found useful at The Fertility Institutes in our investigations of males who have demonstrated "problem" results on earlier semen analysis. These tests are not performed routinely in the initial testing of sperm, but are employed to help further understand the nature and extent of a possible sperm or semen "problem".
This fairly simple and relatively inexpensive test is based on the semipermeability (ability to allow fluid or elements to pass through it's membrane) of the intact sperm cell. A live sperm cell will "swell" when placed in a solution that is "hypo-osmotic", expanding the cell's volume. This test is easy to score and provides valuable information on the integrity and behavior of the cell membrane of the sperm tail. Zona-free hamster oocyte test In the past, this was one of the most widely used accessory tests in the evaluation of advanced sperm function. The fusion between human sperm and the hamster oocyte (egg) is nearly identical to that occuring 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. Because the sperm prepare themselves in a slightly different manner in the laboratory than in the body, false negatives (fail in the hamster test but normally fertilize the partner's human egg) have been reported. This test may be used as a screen to asist in determining which men may need the assistance of "ICSI" fertilization by our embryology team. Due to the very high fertilization success rates routinely seen with assisted fertilization (ICSI), a standard move to ICSI has replaced the costly hamster evaluation. This has resulted in our ability to reliably achieve excellent fertilization and pregnancy rates in couples affected by even VERY low sperm counts and / or motility. Reactive oxygen species and male infertility 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. 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 serm 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 labortory 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 encoutered from antibody levels lower than 50%. Sperm Chromatin Structure Assay (SCSA) Until several years ago the belief among most reproductive specialists was that if a man had live sperm then they were suitable for use with IVF / ICSI and if the female partner didn't get pregnant or a miscarriage ensued then it was probably an egg quality issue. Several studies had implied that the conventional sperm parameters (count, motility and morphology) 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. What has recently been shown in several studies is very interesting and in some ways unexpected. 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. By testing for sperm DNA fragmentation, many cases of formally "unexplained" infertility can now be explained. Many of those couples who have been previously unable to conceive with what would be considered extreme measures have been diagnosed with high sperm DNA fragmentation and treated. It is now very clear to see that having this information about the quality of the sperm can be tremendously helpful to couples and their physicians. There are several ways to test for sperm DNA fragmentation; the most widely used and statistically robust test is called the Sperm Chromatin Structure Assay or SCSA. The patient semen samples are frozen and shipped in a liquid nitrogen container to the SCSA reference laboratory in South Dakota. The sperm are thawed out and a stress is applied (low pH). The sperm are then labeled with a special orange colored dye that only attaches to the ends of broken DNA within the sperm cell. If the DNA is intact then no dye will attach to the sperm. A machine called a flow cytometer is used to analyze ten thousand sperm from the sample. The sperm are passed single file by a beam of light that hits the dye inside the sperm cell and reflects light at a specific wavelength causing the sperm to appear either orange (damaged) or green (normal). A computer counts the percentage of green versus orange-labeled sperm and software allows for creation of a graphic plot of the percent of damaged sperm giving an index known as the DNA fragmentation Index (DFI). The data from thousands of patients has been analyzed and correlated with the patient's clinical outcomes and references ranges were compiled. A normal sample has less then 15% of the sperm with DNA damage. Men with poor fertility potential have greater then 30% of their sperm damaged. A DFI Between 16% and 29% is considered good to fair fertility potential but becomes poorer as it approaches 27%. These numbers are thresholds meaning that above 30% the outcome for most couples was failure to have a birth even though only 30+ percent of the sperm were damaged. Under 15% most couples achieved success. The logical questions that arose were: what about the rest of the undamaged sperm in the sample? Why don't those sperm work? What causes sperm DNA fragmentation? Can the DNA fragmentation be reduced and the sperm improved? If so, How? DNA fragmentation can be thought of as a marker for other types of damage to the sperm. It is a kin to seeing the tip of the iceberg. Apparently, in semen samples with greater then 30% DNA fragmentation, other abnormalities are occurring with the non-fragmented sperm that the SCSA doesn't measure and that is why samples used with DFIs above this level do not usually result in births. The causes of high DNA fragmentation are those same causes of male factor infertility that we have known about for years such as chemical/toxin exposure, heat exposure, varicocele, infection, age, smoking, testicular cancer, radiation, and anything that increases the free radical levels in the semen among a list of many other things. It is very important to understand that sperm DNA fragmentation can change with time and it can be improved in many cases. The goal of a male factor evaluation is to seek out the causes of poor sperm quality and try to correct them so conception can occur naturally or to improve the sperm quality for IVF and maximize the chances of success. In situations where DFI can't be improved there is evidence to suggest that removing the sperm directly from the testicle via biopsy and using it with ICSI may lead to better outcomes then using poor quality ejaculated sperm. Other options include counseling patients regarding the use of donor sperm either by insemination or fertilizing a portion of the eggs harvested for ICSI with donor sperm and a portion with the patient's sperm, once again to maximize odds. The clinical utility of the SCSA is readily apparent. All men with an abnormal semen analysis are candidates for this test as well as men with normal semen analyses who have failed IVF for unexplained reasons. Those couples using egg donors or surrogates may also benefit from screening prior to going thru the procedures because the effort and costs are so great. Men with poor DFI should have a male factor evaluation including a physical examination by a male reproductive specialist. These new concepts have a significant implication on how we practice and what we recommend to couples but we must bear in mind that this test does not have a predictive values of 100% as healthy babies have been born from men with high DFI but this is fairly uncommon. Decisions concerning this advanced testing are carried out for all males presenting with sperm analysis abnormalities at the Fertility Institutes. Where SCSA is felt to be potentially helpful in resolving a problem or answering a clinical question, arrangements to obtain the test are carried out. From Resolve 2006, Dr. P. Werthman, Urologist with the Fertility Institutes
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