Updated Views and Data on Sperm DNA Fragmentation and Pregnancy Outcomes

 
Donald Evenson, Ph.D.

 
Lisa and Jim were happily celebrating their two years of marriage.  This was the second marriage for Jim, age 48, and the first for Lisa, age 32.  Jim has two children, ages 22 and 24, by his previous marriage. Shortly after Jim and Lisa were married they looked forward to having children together and had non-protected sex in hopes of a successful pregnancy.  Six months later their dream appeared to become true with the good news that Lisa was pregnant.  Two weeks later however, that dream chilled with a spontaneous miscarriage. Lisa’s OB/GYN physician calmed Lisa and Jim’s disappointment saying that miscarriages are a part of the human fertility experience and that they should simply continue their quest for a successful pregnancy by natural means.

A year later after their miscarriage, Lisa kept having her timely menstrual periods but no known pregnancy.  Becoming concerned, Lisa and Jim decided to go to a comprehensive infertility center staffed with doctors of varied specialties for a comprehensive evaluation of their fertility health.  Jim’s exam by a urologist found no abnormalities of his urogenital system, except for one small varicocele (enlarged testicular region where blood vessels may cause detrimental heat to the testis) that his doctor did not consider to be a problem.  Jim had a routine semen analysis where trained personnel exam the sperm under a light microscope for sperm concentration, motility and morphology. 
 

Jim’s semen analysis test came back with all parameters normal according to the standard World Health Organization guidelines.  Jim’s doctor emphasized the point that since Jim had fathered two normal pregnancies with his first wife that he was considered a normal fertile man.  Since Lisa’s examinations also came back as totally normal, this left the couple with the uncomforting diagnosis as having “idiopathic infertility” or, more simply stated, there was no known explanation for their lack of a timely, normal pregnancy.
 

Emotions began building with Jim and Lisa to search out possible answers to their dream of having children. A recommendation was made to begin with assisted reproductive technologies (ART).  Since Jim and Lisa had only limited financial resources, they opted for the least expensive method, namely, intrauterine insemination (IUI).  Further disappointment followed the lack of pregnancy with their first IUI attempt.  Two months later their clinic performed a second IUI procedure again only to be met with the same disappointment.
 

Jim and Lisa felt compelled to seek additional counsel and information.  They turned to the Internet and typed into various search engines the logical words of infertility, male infertility, female infertility, idiopathic infertility and others.  They ordered half dozen books written by medical personnel on the general topic of “how to achieve a normal pregnancy”. 
 

Armed with new knowledge and insights, they visited a well known infertility specialist who had recently attended an international infertility conference.  The specialist noted two presentations on the role of sperm DNA damage as a cause for male factor infertility; the presenters:  Donald Evenson, Ph.D., HCLD and the other Mona Bungum, M.D.  Dr. Evenson outlined his laboratory’s 25 year effort in pioneering a high tech method of detecting DNA damage in sperm. Dr. Evenson’s project began while he was on faculty at the Memorial Sloan Kettering Cancer Center in New York researching potential damage done to sperm DNA by chemotherapy medications.   Obviously, the integrity of the DNA comprising the male genome is of critical importance for the man’s contribution of his half of genetic input for a child. 
 

The discovery of this new protocol, now known as the Sperm Chromatin Structure Assay, (SCSA), was due to the invention of a scientific instrument called a flow cytometer.  Sperm cells are stained with a fluorescent dye and then forced though a glass channel of the flow cytometer that is illuminated with a laser beam.  The sperm that stain green have a zero to low level of DNA fragmentation and those that stain red have various levels of fragmented DNA.  Dr. Evenson and his Sloan Kettering colleagues announced to the scientific world the SCSA invention on the cover issue of the prestigious journal Science in December, 1980.  That 1980 publication had the summarizing statement: “We expect this assay to have application in animal husbandry, human infertility and environmental and public health”.
 

Now, fast forward a quarter century to Lisa and Jim in 2005.  Could it be that this promising speculation could be of help to Jim and Lisa 25 years later? The answer is “yes” as envisioned.  Then the obvious question: why has it taken so many years to arrive at the conclusion that the SCSA should be available for male factor infertility diagnosis and prognosis including the problem faced by Jim and Lisa? 
 

That answer lies in the testing plan developed by Dr. Evenson who was very concerned that the SCSA test would provide meaningful information to the sperm analysis laboratory.  First, extensive testing and validation was needed to determine just what the SCSA was measuring and what it meant biologically for the human infertility clinic.  Thus, Dr. Evenson, together with many national and international colleagues, spent almost two decades of research, generously funded by federal health agencies, to determine the use of the SCSA for human and livestock animal fertility as well as determining the negative effects of environmental chemicals and other reproductive toxicants on sperm DNA integrity.
 

Over two million semen analyses are done annually in the United States. he classical measurements are sperm count, morphology, viability and motility. Consensus of world wide experts has suggested levels for each of these parameters that are considered to be in the normal range or abnormal range.  While these may be the best consensus by experts, the real problem is lack of accuracy and recognizing that men who have an abnormal value may be fertile and those with normal values may be infertile.
 

The important point for Jim and Lisa was that Jim’s sperm had normal morphology values. However, in sharp contrast to looking at the “outside” of the sperm with a light microscope, the SCSA looks inside the sperm at the most important part of the sperm, namely, the integrity of the sperm DNA. Especially in this era of ICSI where sperm of various abnormalities from outside appearances can be injected into eggs and cause fertilization, nothing is as important as the quality of the DNA which is the object of measurement by the SCSA.   
 

Since the SCSA measurements of DNA integrity have generally shown very little relationship to the classical light microscope measurements of sperm count, motility and morphology, the SCSA can be considered an independent measurement that provides valuable new information to both the physician and patient.
 

All sperm quality tests, including the SCSA, provide information on the probability of infertility and not fertility.  Thus, a man’s sperm count, motility and viability value of zero would suggest that the man is infertile. But even if all the tests had good scores, this is not a guarantee for pregnancy to occur.
 

What is the consistency of semen analysis tests and DNA fragmentation tests?  The reason that most infertility clinics tell male patients to obtain at least two semen analyses spaced 1-3 months apart is that the various values of sperm count, motility and morphology can change quite dramatically over a short period of time.  The time to produce a sperm from the testis to ejaculated semen takes about three months.  Since various factors that potentially affect the numbers of sperm produced or the quality of the sperm during this three months can change, it is obviously important to check the status two or three times. 
 

It is of interest that a comprehensive study comparing routine semen quality tests with the SCSA DNA fragmentation test indicated that the SCSA results were much more consistent over time.  Even so, there are various factors that will cause a dramatic shift in sperm DNA integrity also.  An example is seen on the Internet (www.SCSAdiagnostics) where the male partner of a couple had very poor SCSA measures for two successive months with no pregnancy followed by an excellent measurement and a successful pregnancy that month.
 

Jim and Lisa’s physician who heard Drs. Evenson and Bungum speak saw the possible reason why pregnancy was not happening after a year and a half of trying for a natural pregnancy, a spontaneous abortion and two failed attempts with IUI.  Dr. Evenson showed results from two large studies, one done at the Georgetown Medical Center, Washington, DC and the second at the Danish National Hospital in Copenhagen. 
 

In the first study, 170 couples who sought to become pregnant were counseled by fertility personnel on the best methods to obtain pregnancy by natural intercourse.  Each man had a semen analysis, including a SCSA DNA fragmentation test, several days after the prime time for conception.  The women had hCG testing to determine if a biochemical pregnancy had occurred that month.  A manuscript published by Dr. Evenson and colleagues showed that the level of sperm DNA damage was low (less than 30% of the  sperm showed fragmented DNA)  in the couples that became pregnant in any of the first three months of that study.  Thus, the DNA Fragmentation Index (DFI) was less than 30% (DFI < 30%). 
 

While the SCSA was measured only during the first three months, some couples with greater than 30% sperm with fragmented DNA (>30% DFI) in the first three months became pregnant in months 4-12 of the study while others did not achieve pregnancy within the one year study.
 

The second study was done on 215 couples trying to start a family in Denmark by natural intercourse.  Likewise in this study, the statistical odds showed that these couples were ten times less probable to become pregnant when the DFI was less than 40% (considered about the same as the Evenson threshold of 30% since very few pregnancies occurred above 30%).
 

Now enter the important study of Dr. Mona Bungum in Norway. Dr. Bungum and colleagues enrolled one thousand patients in a comparative ART study.  One third of the patients had IUI, one third routine IVF and one third used ICSI fertilization of the retrieved eggs.   What captured Jim and Lisa’s physician attention was the fact that Lisa had two failed IUIs and Dr. Bungum’s large study showed that if the DFI was greater than 27%, there was 8.7 fold decreased probability of a pregnancy by IUI than when the DFI was less than 27%.   
 

Jim and Lisa’s physician decided to order a SCSA test for Jim.  Upon calling SCSA Diagnostics, it was learned that a semen sample could be collected and shipped to this diagnostic lab in two different ways.  First, Jim could go to the clinic, produce a semen sample and the clinic would freeze the sample and send it by overnight courier to SCSA Diagnostics lab.  Alternatively, since Jim and Lisa lived an hour’s drive from the clinic and Jim did not want to take off a day’s work to go to the clinic, they had SCSA Diagnostics send (Federal Express) a custom shipper to their home.  Jim was instructed to have two ejaculations prior to an abstinence period of two days when he then produced a semen sample into a collection jar that was part of a collection kit sent with the shipping container. Following the simple instructions provided, a small amount of semen was placed into a special tube and that tube was simply placed into the shipping container that was prechilled to a freezing temperature. Federal Express picked up the pre-paid shipping container for delivery the next morning at SCSA Diagnostics in Brookings, SD. This sample was then thawed and subjected to the SCSA protocol. The results were then faxed to the doctor’s office. 
 

The results showed that 41% of Jim’s sperm had DNA fragmentation.  These results placed Jim into the group of men that, statistically speaking, took a longer time to initiate a successful pregnancy, had an increased risk of spontaneous miscarriage or did not have a successful pregnancy.

Thus, it appeared to Jim and Lisa’s physician that no further attempts with IUI should be done, but go into routine IVF or for even better probabilities go to ICSI. This approach to the management of Lisa and Jim’s pregnancy quest was made clearer by Dr. Evenson’s results of a statistical analysis of SCSA DNA fragmentation studies on ~2000 clinical patients which indicated that ICSI fertilization appears to provide the best odds for a pregnancy when the man’s DFI is above 30%.  However, it is also the most costly and this was a major factor for Jim and Lisa with their limited finances. Even so, they went ahead with ICSI and were told the day after the procedure that 8 of 10 eggs fertilized, and five of the embryos looked “beautiful”.  The physician explained however, that although the majority of the embryos looked beautiful, that Jim’s sperm DNA would not contribute genetic information until day 3 and that development up to that time was maternal contribution to the embryo.  So the important question was:  Did ICSI process select sperm with mostly normal or damaged DNA?
 

Two of the best looking embryos were transferred to Lisa and then the waiting started again.  They accepted the possibility of twins, but as desperate as their feelings were for wanting a child, they decided not to run the risk of triplets with three embryos implanted.  And finally, good news!  Lisa received a phone call to say that her hCG was elevated and that pregnancy had occurred.  Furthermore, after some anxious wondering whether, once again, that only a biochemical pregnancy had occurred, more good news arrived at six weeks to confirm a clinical pregnancy.
 

Knowing that Jim had a level of sperm DNA fragmentation that put him into a statistical group of men that took  a longer time to conception and increased odds of a spontaneous abortion, they discussed with their doctor what the odds were of a normal baby with their situation.  Despite the possibility of some increased odds for spontaneous abortion, their doctor told them to relax and fully enjoy the pregnancy since the probability was very high of giving birth to a completely normal child. Jim and Lisa are waiting for their expected delivery date, and to this point, all is well.
 

Jim asked their doctor what might have caused his sperm DNA to be fragmented to the point that his DFI was above the statistical threshold of 30%.  He pointed out that a number of factors may cause a temporary increase of sperm DNA fragmentation.  These included use of very warm hot tubs or saunas, a fever in the range of 103-104oF, exposure to toxic chemicals including pesticides and herbicides, some medications like cortisone, urogenital infections, and others (examples of how these factors affect sperm DNA fragmentation can be seen on the Internet at www.SCSAdiagnostics.com). 
 

Since Jim was not aware that he was exposed to these factors, the physician thought that the most likely cause for a long term elevated DFI was Jim’s age of 48.  A study on sperm quality and age was conducted at the Lawrence Livermore National Laboratory (LLNL)  and the University of California, San Francisco, where about 100 men ranging  in age from 21 to 80 who were working or had worked at the LLNL provided a semen sample to help answer the question whether sperm quality decreased with age.  Previous thought in the medical world was that age made little or no difference and that once the sperm fertilized an egg that its work was done. 
 

Not so from current research!  Sperm quality does decrease with age, but it varies much more with men than women where there is a sharp decline after about age 36.  Dr. Evenson showed that men in their early 20’s had a DFI of approximately 5%.  In their thirties this level increased into a DFI in the range of 13-19% and in their forties into a DFI greater than 20%.  The data suggested that Jim, at age 48, had approximately a one out of three chance that his DFI would be above 30% which is the current suggested level to put Jim at risk for a reduced pregnancy outcome.  Therefore, the picture became clearer why he had easily fathered children when in his early 20’s and now that he was age 48 with a DFI of 41%, his age was likely the major contributor to the diagnosis of unexplained infertility.  The SCSA had detected a problem that was not detectable by light microscopy.  The idiopathic infertility was now explained.
 

Great progress has been made in the past quarter century for overcoming couple infertility.  The great breakthrough of in vitro fertilization is now a routine procedure in many clinics around the world.  The second major breakthrough celebrated its 10th anniversary last year, namely, ICSI, where sperm of lower quality can be injected into an egg.     With this accomplishment, many thought that any man’s sperm would be sufficient for fertilization and normal embryo development; this has not proven to be true.  While fertilization can occur even for sperm with high levels of DNA damage, ongoing genetic support of the growing embryo is dependent on a high level of DNA integrity.
 

As can be seen from the story of Lisa and Jim, there are many couples with idiopathic infertility that could be spared unnecessary tests and treatments by a simple DNA fragmentation test.  The information gained could be of significant help to both physicians and patients alike to better manage cases of couple infertility.
 
 
Contact information:

Donald Evenson Ph.D.
SCSA Diagnostics Corporation
Multiplex Research & Technology Center
807 32nd Aveue
Brookings, SD 57006
Phone:  605-692-5938
Fax:  605-692-9730
Email:  scsadon@brookings.net
 
Website:  http://www.scsadiagnostics.com
 
INCIID member profile:  http://www.inciid.org/members/member.php?cust_id=10286