ICSI and SCSA Sperm Diagnositics 
by Donald P. Evenson, Ph.D., HCLD 
and Kjersten Larson-Cook, Ph.D.

Amy and Todd have been trying to conceive for 2 years. Initially, when they decided to start a family, Todd was certain they would become pregnant very quickly. In his previous marriage, Todd had conceived each of his two children, now 20 and 22 years old, within 3 months. However, on Amy’s 35th birthday, the couple realized they could no longer explain away their problem and decided to consult a fertility specialist.

Even with Todd’s previous fertility, their physician informed them that Todd should have a semen analysis. Todd questioned if this was necessary remembering a physician saying “Men just get better with age, like a fine wine.” But, not wanting to seem uncooperative, Todd made an appointment for his semen analysis, which measures the concentration, motility and morphology (shape) of his sperm. He was not worried but was pleased to learn that, as a 55 year old man, his results came back completely normal.

Meanwhile, Amy’s test results also were normal. It seemed that there was no identifiable cause for their struggle with infertility. This made the situation more complicated. If there was some identifiable cause like Todd’s sperm count being extremely low, or Amy’s fallopian tubes being blocked, it seemed it would have been easier to determine the appropriate treatment. Without a known cause, Todd and Amy were diagnosed as having idiopathic infertility. They felt more frustrated than ever in their failure to conceive.

Due to the length of their infertility and Amy’s advancing age, their physician recommended that the couple begin in vitro fertilization. Their physician explained that as a general protocol Amy’s eggs would be fertilized using a technique called intracytoplasmic sperm injection (ICSI), where individual sperm from Todd would be injected into each of Amy’s eggs. Todd questioned the use of this technique since he believed that it only was used in cases where men had severe and irreversible sperm problems. However, the doctor explained they used this technique to increase fertilization rates and the odds that there would be high quality embryos to transfer to Amy.

The couple proceeded though one ICSI cycle. They were optimistic learning that all of Amy’s 20 eggs had fertilized normally, and that the embryos were developing beautifully. The embryos were allowed to mature in culture media for 3 days at which time 4 were transferred to Amy’s uterus. The couple waited anxiously to take a pregnancy test but were devastated to find that Amy had a negative BhCG. What happened? Everything seemed perfect at the time of transfer. The couple continued through 2 frozen embryo transfers – and still no pregnancies.

Amy and Todd were frustrated and saddened by their experience, but wished to try again. Their physician recommended that in this ICSI cycle they should culture the embryos for 5 days before transferring them to Amy. In this way, she said they would be more able to identify the embryos that were most healthy and would have the best chance to implant and develop into a healthy baby. Ten eggs were harvested and again all fertilized beautifully and developed nicely through day 3. Yet, on day 4, many of the embryos were beginning to show signs of abnormal development and by day 5 only 2 embryos of poor quality remained viable for transfer. Todd and Amy again faced a negative BhCG.

What was happening?

Why had everything looked so promising and without explanation seemingly crashed at Day 4 of embryo development?
ICSI bypasses all requirements of normal sperm motility and sperm/oocyte oolemma interaction. The only requirement for fertilization and early embryonic development is oocyte activation and adequate DNA integrity. Surprising to many, the male genome (sperm DNA) is silent in the first 3 days of embryo development. The oocyte drives the development and cleavage of the early embryo. On Day 4, a critical switch occurs, and the male genome is turned on. At this time, the maternal and paternal genomes begin to work in concert to orchestrate the activities of the newly developing embryo.

It is at this time – Day 4 – when abnormalities in the paternal genome may begin to have what can be profoundly devastating effects on embryo development. Therefore, using sperm with abnormal DNA for ICSI may lead to normal fertilization and early embryo development yet be followed by embryo death expressed as a failure to implant (no chemical pregnancy) or early pregnancy loss (prior to an ultrasound-confirmed heartbeat.)

Abnormalities in the paternal genome (sperm DNA) are weakly related to sperm concentration, motility, and morphology. Therefore, men like Todd with excellent sperm quality may still harbor sperm with poor DNA quality that contribute to infertility. Thus, a test of DNA integrity is very helpful in deciding which couple would most likely benefit from ICSI and which couple would be at greater risk for failed cycles. Table I lists conditions when DNA fragmentation testing could potentially benefit a couple.

An international consensus group stated that “practical and reliable tests to detect damaged DNA in sperm are needed in order to optimize adverse developmental defects in the embryos.” Furthermore, this group agreed that “the remarkable consistency of the methodology makes the SCSA® [sperm chromatin structure assay] an ideal technique to detect sperm cell damage” and “that the SCSA® is a good predictor, relative to other sperm measures, for clinical diagnosis of male infertility.”

SCSA® Diagnostics, Inc.’s team of scientists invented and patented the Sperm Chromatin Structure Assay (SCSA®) test and has over 30 years of experience studying how disease, environmental toxicants and age affect chromatin (DNA and protein) structure and function. These studies have included researchers from around the world and resulted in over 125 peer-reviewed journal articles. During that time, the ability of the SCSA® test to identify men at risk for infertility has been validated and is now offered by SCSA® Diagnostics, Inc. to physicians and patients around the world.

The SCSA® test measures the percent of sperm in a semen sample that has fragmented DNA as well as the extent of that DNA fragmentation. In the SCSA® test, sperm with very low levels of fragmentation fluoresce green, while sperm with moderate to high levels of fragmentation fluoresce red. A flow cytometer measures the ratio of red to green fluorescence in each of 5000 sperm. The percent of sperm with DNA fragmentation (red fluorescence) is expressed as the DNA Fragmentation Index (DFI).
The SCSA® test is practical for clinical evaluations of sperm DNA quality due to the speed of analysis and the consistency of results. The clinical report includes two SCSA® test measurements of the same ejaculate to ensure accuracy. Typically, these two measurements yield nearly identical results. SCSA® test results also are extremely consistent among samples collected from an individual man over time. Indeed, the SCSA® test provides much greater consistency both within a single sample and between samples collected over time than is provided by standard measures of semen quality such as concentration, motility and morphology. This highly accurate SCSA® test analysis and Clinical Report can be completed and made available to physicians and patients located across the country within a week. In addition to the speed and reliability of the SCSA® test, the large numbers of sperm that are evaluated make the test statistically robust, providing consistent evidence for the relationship between sperm DNA fragmentation and function. The first large scale human fertility study using the SCSA® test identified sperm DNA Fragmentation Index (DFI) thresholds of 0-15%, 16-29%, ?30% that related to high, moderate and very low natural fertility potential, respectively. Specifically, if ?30% of the sperm in a sample had SCSA®-detected DNA fragmentation, the risk for infertility was significantly higher.

A pilot study including 27 men showed that the percentage of sperm with DNA damage was significantly greater in men with no pregnancy (31.1 % DFI ? 3.2) than in men that initiated pregnancy following IVF or ICSI (15.4 % DFI ? 4.6). In addition, the study identified a DFI threshold (?27%) for infertility following IVF and ICSI indicating the potential value of the SCSA® test in predicting negative ART outcomes (Larson et al., 2000). Larger studies including hundreds of infertile couples have validated that ?30% DFI places men at a significantly greater risk for infertility. Although this risk is influenced by a myriad of potential sources (age of maternal partner, clinical techniques etc.), exceeding this DFI threshold appears to decrease pregnancy rates by 50% or more.

Therefore, exceeding the 30% DFI threshold is telling of problems within the entire sperm population. Otherwise, the other 70% of the sperm in the population without elevated levels of DNA fragmentation would support normal pregnancies. As a possible explanation, Dr. Evenson, innovator and developer of the SCSA® test, describes the “tip of the iceberg” phenomenon. When the physical stress of the SCSA® test is enough to draw out and detect 30% of the sperm as abnormal, the damage within the entire sperm population is sufficient to increase the risk of infertility.

Because the SCSA® test is not representative of WHO parameters, it is a predictor of infertility, not fertility potential. Fertility may be affected by many other factors (e.g., poor motility, acrosome deficiency) even when the DNA quality is excellent. In contrast, DNA quality may be very poor in men with normal semen parameters that would otherwise be diagnosed with idiopathic infertility. A number of cases have been documented where a man with normal conventional semen parameters had multiple failed IVF/ICSI attempts, even using donor eggs. Twelve couples with multiple failed ART cycles and no known male factors progressed to sperm donor following discussion of their abnormal SCSA® test results (>30% DFI). Nine of these couples conceived within 3 sperm donor cycles after years of infertility.

Estimating the risk of infertility based on DNA fragmentation also may be valuable to men diagnosed with cancer. These men must decide prior to treatment if they wish to cryopreserve their semen for future ART. The DNA quality in cancer patients ranges from excellent to very poor prior to any treatment. Those men who have excellent sperm DNA quality may wish to cryopreseve their sample, while those with very poor DNA quality may wish to begin cancer treatment immediately, avoiding the delay and expense required for semen cryopreservation and storage.

A man’s age, long periods of abstinence, high fever (>103°F) and the presence of leucocytes (indicating infection) appear to be related to a significant increase in sperm DNA damage. Data also show that exposure to environmental stresses, pollutants, as well as cigarette smoking, may have a negative effect on sperm DNA quality.

DNA fragmentation by the SCSA® test reflects the quality of sperm that have just undergone maturation in the male reproductive tract. While an excellent-quality sample is likely reflective of previous and future quality, poor DNA quality may be transient due to high fever, medications, physical or mental stress, or other unidentified conditions. Therefore, if SCSA® test results are poor, it is necessary to question the patient as to whether he has been exposed to conditions that may negatively affect sperm quality, correct these conditions if possible, and repeat the SCSA® test in 2 to 3 months.

The SCSA® test provides a rapid, unbiased, quantitative assessment of sperm DNA fragmentation that is an independent predictor of natural as well as in vitro infertility. This prognostic information provides invaluable information to couples who may consider donor insemination or discontinuing ART when sperm DNA quality is very poor putting them at a significantly greater risk for not achieving a pregnancy or early miscarriage. This prognostic information provides a significant medical, emotional, and financial benefit to couples considering ART, donor sperm or adoption.

Because Todd’s standard semen analysis was normal, their medical team felt certain the failed cycles were associated with female factors. Amy underwent a myriad of tests, but no problem was identified. After two failed ICSI cycles, Todd and Amy did not know which way to turn. Should they continue with another cycle or begin looking into egg donation? During this time, Todd and Amy’s physician learned about the SCSA® test and felt that sperm DNA fragmentation may have contributed to the poor embryo development and ultimately the couple’s failure to conceive naturally and through IVF. She suggested they check the sperm DNA fragmentation levels – just as a precaution – before undergoing more costly and invasive procedures. She explained that the test was $200 plus shipping ($150) and that this was a small price for the information it could provide.

Certain it was a female factor, Amy and Todd were hesitant, but at the urging of their physician, they decided to have the SCSA® test completed. Todd had a high level of DNA fragmentation ~ 48% DFI (well above the 30% DFI threshold that places couples at risk for infertility.) Recognizing their low odds for success with Todd’s sperm, they decided to go ahead with donor sperm. Amy became pregnant on her first cycle, and the couple is now awaiting the birth of their baby, which is expected in just 2 months.
The experience of the couple outlined above demonstrates the importance of the diagnostic and prognostic information provided by the SCSA® test. They may have made different decisions going into ART had they known of Todd’s increased risk for infertility. They could have saved time, money and emotional costs associated with failed ART and proceeded more quickly to the treatment that allowed them to successfully begin building their family.

Table I. Conditions Indicating SCSA® Testing

• abnormal Semen Analysis
• Unexplained Infertility
• Persistent Infertility after Treatment of Female
• Recurrent Miscarriage
• Prior to Assisted Reproductive Technologies
• Cancer in Male: Before and after Treatment
• Advancing Male Age

Commonly asked questions about the SCSA® test.

Important questions are raised by couples similar to the one described above and are outlined here.

Q: My physician requested that I have the SCSA test and gave me your telephone number. What do I need to do?
A: We are able to send the sperm shipping container to your physician’s office or directly to your home. We will send all of the necessary supplies to the address you request. The sample collection, packaging and shipping procedures are simple and completely explained on our Web site as well as in the instructions provided. In our experience, people have very few questions and do not make mistakes.

Q: What is the cost of the SCSA test?
A. The test costs $200, which includes pre-cooling the shipping container, packaging all of the materials and instructions, preparing the Federal Express paperwork for shipping and return, and measuring and analyzing the sample for DNA fragmentation. All of the paperwork for the return shipping is included so you need only call your Federal Express station for pick-up. When the sample arrives at SCSA Diagnostics, the sample is then thawed and treated by a very precise procedure. Of importance, two independent measurements are made for each sample to be absolutely certain that the results are correct. The results are then processed by a special computer program, and the clinical report prepared.

Q: What is the cost of the Federal Express overnight shipping?
A: For round trip shipping in the US, the cost is $150. The reason for this amount is due to the fact that the shipping container is fairly large (2 ft. tall) so that the tank liner can absorb enough liquid nitrogen to keep your sample frozen and safe on the trip back to our laboratory.

Q: How often do male-factors contribute to infertility?
A: In about 50% of infertile couples, male-factors play a significant role in infertility.

Q: How often is DNA fragmentation a significant source of infertility? 

A: In couples that seek a male infertility evaluation, up to one out of four male partners are identified by the SCSA® test as having levels of DNA fragmentation known to put them at risk for infertility..

Q: What exactly is the sperm chromatin structure assay (SCSA®)?
A: The Sperm Chromatin Structure Assay (SCSA®) is an assessment of sperm DNA fragmentation that identifies men with a highly reduced probability of initiating and supporting a successful pregnancy. The percentage of sperm in a semen sample with fragmented DNA is reported as the DNA Fragmentation Index (% DFI). More detailed information may be found on SCSA Diagnostics Web page, www.SCSAdiagnostics.com.

Q: What is sperm chromatin?
A: The 23 chromosomes in the sperm head are made up of chromatin, which consists of DNA and proteins.

Q: Semen analysis showed that my sperm concentration, motility and morphology are normal. Can I still have high levels of DNA fragmentation?
A: Yes. Sperm that appear to be completely normal by all the standard measurements may have high levels of DNA fragmentation.

Q: I fathered two children, now 16 and 18 yrs old, with my previous wife. Isn’t this proof that my sperm are fertile?
A: Although some older men are highly fertile, the natural aging process appears to be associated with increasing levels of sperm DNA fragmentation. Like many men, aging may have decreased your fertility potential.
Q: Are there medications that may influence my SCSA® test results?

A: Preliminary results indicate that some medications may influence SCSA® test results. Therefore, we recommend if you are on medication, consult with your doctor about temporarily reducing or discontinuing your medication for 2-3 months to have the SCSA® test. If a significant improvement has occurred your doctor may suggest either that you proceed with attempts for fertility at that time, or that you freeze the sperm so that you can go back on medication and use the sperm when your wife cycles.

Q: I was recently sick with a high fever. Could this influence my test results?
A: Yes. A high fever (>103°F) has been shown to impact SCSA® test results, and you should wait 2-3 months to have the test.

Q: Is there a required abstinence period required before collecting semen for the test? 
A: Yes, there is an important window of time. Abstinence time should not be less than 2 days or greater than 3 days.
Q: What SCSA® test results would indicate that we are at high risk for infertility? 

A: When ?30% of the sperm in your sample contain high levels of DNA fragmentation (?30% DFI, DNA Fragmentation Index), your sample results are similar to men that, as a group, have a significantly reduced probability of initiating a pregnancy that will go to term.

Q: Have you ever seen pregnancies in men with ?30% DFI? 
A: Yes, but in studies of hundreds of patients from very highly respected clinics, we have seen that pregnancy rates drop by 50% to 100%. Therefore, poor results do not mean that you will not initiate a pregnancy, but that your chances for success are less. 

Q: Can assisted reproductive techniques (ART) like in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) overcome high levels of DNA fragmentation? 
A: Men with high levels of fragmentation also are at high risk for failed ART cycles.

Q: If a semen sample has a high percentage of sperm with fragmented DNA, is this percentage permanent or might it improve over time?
A: SCSA® test results are significantly more stable over time than results obtained by conventional semen analysis (sperm concentration, motility and shape). However, some men bordering on infertility may have relatively unstable DNA quality as assessed by the SCSA® test. Therefore, if the SCSA® test indicates a high level of DNA fragmentation in one month, it is recommended that a second SCSA® test be done two to three months later.

Q: What causes sperm DNA fragmentation? 
A: DNA fragmentation in sperm may be the result of many factors including, but not limited to, disease, diet, drug use, high fever, elevated testicular temperature, air pollution, cigarette smoking and advanced age.

Q: How long will it take to receive our SCSA® test results? 
A: We are typically able to provide results to your physician within one week of receiving your sample.
Q: What do SCSA® test results indicate? 

A: A man’s SCSA® test results place him in one of three statistical categories of fertility potential. These categories were derived from a comprehensive study of male fertility without assisted reproductive techniques. Excellent fertility potential ~ <15% DFI, Good to fair fertility potential ~ 15-29.9% DFI, Poor fertility potential ~ ?30% DFI.

Dr. Evenson is a Distinguished Professor of Biochemistry at South Dakota State University and President and Director of SCSA® Diagnostics, Inc. a private corporation that is dedicated to using the SCSA® test to determine the relationship between infertility and DNA fragmentation in sperm. Dr. Evenson invented and developed the SCSA® test and has over 20 years experience measuring thousands of animal and human sperm samples. He has published more than 125 peer reviewed research papers and has presented many invited lectures around the world. His research on the SCSA® test has been continuously supported over the past two decades by competitive, federally funded grants.