Advanced Sperm Testing
This section is for couples who have suspected functional sperm problems that go beyond the standard semen parameters of density, motility, and morphology. At this point in time, advanced sperm testing is not indicated in most couples who are having trouble conceiving. The most common candidates for advanced sperm testing are couples who have failed multiple cycles of IVF for unknown reasons, especially in the presence of poor embryo progression and/or poor embryo quality (see below). Some advanced sperm tests, such as DNA fragmentation index, can also be performed for couples with unexplained recurrent pregnancy loss.
A wide array of advanced sperm tests are currently under evaluation by the scientific community. Although many potentially promising tests have been discovered, none has yet proven to be the game-changing breakthrough that everyone is looking for. When one of these new tests is developed, it tends to follow a particular pattern in which the initial research studies on a small number of patients show a significant positive correlation between the sperm test results and fertility outcomes. The clinical fertility community gets excited that a breakthrough in sperm testing has been found that will change the treatment options for couples with recurrent IVF failures. However, subsequent larger-scale studies by other institutions often find that these tests are not quite as clinically useful as first anticipated, and the search goes on.
The holy grail of advanced sperm testing is a test that will have the ability to not only determine which patients have high levels of functional sperm impairment but also determine which individual sperm have good fertility potential and isolate them for use in IVF/ICSI.
Unfortunately, no test to date can do all this. However, several tests have shown themselves to be potentially clinically useful:
1) DNA fragmentation index (DFI)
2) Intracytoplasmic morphologically selected sperm injection (IMSI)
3) Hyaluronan binding assay (HBA)
4) Birefringence testing of sperm heads using polarized light
5) Raman spectroscopy
6) Membrane-based electrophoresis
7) Microfluidic testing
8) Magnetic activated cell sorting (MACS)
Later sections in this chapter will review a few of these tests in further detail, including the sperm DNA fragmentation testing, IMSI, and HBA. This does not mean that these are necessarily the best of the above-listed tests, only that these three seem to have the most published scientific data associated with them to date.
Sperm DNA Fragmentation Testing
When a sperm successfully enters an egg, the twenty-three chromosomes of its DNA must fuse with the egg’s twenty-three chromosomes in order to form the full forty-six chromosomes of the resulting embryo. As described earlier, the male’s DNA contribution generally starts to play a role after fertilization has occurred, and therefore abnormalities in sperm DNA typically play a larger role in subsequent embryo development.
Nuclear chromatin is the material combination of DNA, RNA, and proteins in the nucleus of cells. Most of the body’s cells have robust mechanisms to defend against damage to the cell’s DNA, as well as the ability to repair some types of damage that do occur. Unfortunately, the sperm cell has a very different structure compared to most other cell types in the body, because it needs to be compact, mobile, and strong enough to swim long distances and successfully interact with an awaiting egg. Because it has a unique membrane structure and a very limited supply of antioxidants, sperm are especially susceptible to damage by reactive oxygen species. In addition, sperm have only minimal ability to repair any DNA damage that they might sustain.
Sperm DNA integrity can be evaluated by one of three methods:
1) DNA fragmentation index (DFI; also called SCSA and SDFA)
2) TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)
3) Comet assay (single cell gel electrophoresis)
All three of these testing methods are reliable, and their findings correlate fairly well with each other. The one most commonly used commercially is DFI testing. At this time, the TUNEL and Comet assays are more frequently used in research at large academic institutions, which perform the testing in their own labs. The TUNEL assay does have the advantage of being able to test the DNA fragmentation index of very small numbers of sperm, including those extracted from the testicle (as opposed to DFI testing, in which a sperm density of at least 1 million per cc from an ejaculated specimen is generally required).
While sperm DNA testing offers another useful way to examine the ability of sperm to interact successfully with an egg that goes beyond the standard semen parameters of density, motility, and morphology, at this point in time sperm genetic testing can only tell us what percentage of sperm have normal DNA versus abnormal DNA. No sperm DNA test can currently identify specifically which sperm have normal DNA and then select those exact sperm with intact DNA to be used with IVF/ICSI.
DNA Fragmentation Index (DFI)
The most widespread, commercially available assay for assessing sperm DNA integrity is DFI testing. Commercially, DFI testing can be found under such names as SDFA (sperm DNA fragmentation assay) and SCSA (sperm chromatin structure assay).
DFI testing is an indirect assay that measures the ability of sperm chromatin to resist DNA denaturation after exposure to stress, such as a mild acid. In this test, around five thousand sperm are evaluated by a process called flow cytometry, and results are given as a percentage of sperm cells with fragmented DNA.
Normal DFI is under 30 percent (that is, less than 30 percent of sperm exhibit fragmentation when exposed to stress). Often labs will define a borderline DFI result range of 20–30 percent, but most guidelines consider 30 percent to be the actual normal cutoff (of note, some labs use 25% as their normal cut-off). Studies have shown that healthy fertile males typically have a DFI of around 7 percent or less.
Elevated levels of DFI are found in about 10 percent of normal fertile men versus 20–30 percent of infertile men. Studies have found that about 8 percent of infertile men who have normal standard semen parameters have an abnormally high DFI on testing. DFI levels are not necessarily correlated with semen parameters and have only a weak correlation with strict morphology findings.
Causes of increased sperm DFI include:
1) Oxidative stress (the most common cause), such as that associated with pyospermia
2) Tobacco use
3) Excessive heat exposure (for example, using a laptop computer on the lap, or using a hot tub)
5) Chemotherapy or radiation therapy
6) Air pollution exposure
8) Aging (DFI is about 13 percent in men between twenty and twenty-nine years old, versus about 50 percent in men between sixty and eighty)
9) Prolonged sexual abstinence
10) Spinal cord injury
11) SSRI use (healthy volunteers taking paroxetine [Paxil] had an average increase in sperm DFI
from 14 percent to 30 percent after four weeks of use, with no change in standard
Impact of DFI on Fertility Outcomes
The impact of a high DFI on fertility varies significantly between couples. One reason for this is that a woman’s egg is able to repair some degree of sperm DNA damage, and this amount of repair varies among individuals—for example, eggs from younger women may have more repair capacity.
Abnormal DFI has been associated with poorer outcomes for natural intercourse, IUI, standard IVF, and IVF/ICSI.
1) Natural intercourse: a DFI over 30 percent is associated with a 7-fold decrease in the rate of
2) Intrauterine insemination: a DFI over 30 percent is associated with a 7.3-fold decrease in
3) Standard IVF: a DFI over 30 percent is associated with a 2-fold decrease in pregnancy rate
4) IVF/ICSI: a DFI over 30 percent is associated with a 1.5-fold decrease in pregnancy rate
(although some experts think that DFI has a minimal impact if ICSI is used)
Sperm DNA testing is still relatively new, and the above findings need to be verified by additional large, controlled studies. Currently the American Society of Reproductive Medicine does not recommend sperm DFI testing for routine male fertility evaluations. However, there is emerging compelling evidence that DFI testing can play a useful role in several specific clinical situations: recurrent unexplained pregnancy loss, recurrent unexplained IVF failure, and couples in which the man’s semen analysis is normal but who still have unexplained persistent infertility.
Couples with otherwise normal semen parameters who are not having success with timed intercourse or intrauterine insemination can have sperm DFI testing to help them decide if moving straight to IVF/ICSI would be beneficial (if a highly abnormal DFI is found) versus trying longer with the low-tech treatments (if the DFI is normal). Some clinicians also use sperm DNA testing to assess the integrity of sperm DNA in various at-risk populations, such as post-chemotherapy patients or older men, although no official guidelines have yet been established.
Treatment of Elevated Sperm DFI
Three main approaches are usually undertaken to reduce DFI fragmentation levels in men with abnormal testing, including antioxidant therapy, treatment of reversible risk factors, and maximizing the freshness of the sperm specimen.
Antioxidant therapy can decrease sperm DFI by protecting DNA from damaging free radicals.
Reversible risk factors known to increase sperm DFI can be managed, including:
1) Stopping tobacco use
2) Weight loss in obese patients
3) Avoidance of excessive heat (hot tubs, saunas, etc.)
4) Treatment of clinically significant varicoceles
5) Change from a SSRI antidepressant to another type of antidepressant medication (such as
Wellbutrin) that may have less impact on sperm ductal transport (this should be done under
the care of the patient’s primary care provider or psychiatrist)
Most sperm DNA fragmentation occurs within the epididymis and genital duct system; less occurs in the testicles. Therefore, getting the freshest sperm possible can decrease the sperm DFI in some men. I generally have men with sperm DFI problems who have an upcoming sperm DFI test or IVF cycle ejaculate daily for one week, and then abstain for one day prior to collecting their semen specimen. Studies have shown that in men with an elevated DFI, daily ejaculations for a week can potentially decrease DFI readings by over 40 percent.
Some men with unexplained IVF failure or pregnancy loss cannot decrease their DFI to the normal range despite all of the above interventions. Several small studies have shown that in these circumstances, sperm surgically taken directly from the testicle typically have lower DFI scores than ejaculated sperm (DFI 24–40 percent with ejaculated sperm vs. 5–13 percent with testicular sperm). These extracted sperm can then be used for IVF/ICSI. Small studies have found higher pregnancy rates using these fresh extracted sperm when compared with cycles that used the ejaculated sperm. It is controversial as to whether sperm freezing significantly increases sperm DFI in men with abnormal fragmentation levels to begin with. In men with normal fertility, any mild increase in DFI associated with freezing likely has little clinical impact. However, to optimize sperm quality, I prefer to use fresh sperm extractions for men with persistently elevated DFI levels and recurrent IVF failure.
Ordering Sperm DNA Testing
Sperm DFI testing is generally not performed at regular semen analysis testing labs. There are a few specialty companies that perform this testing, including SCSA Diagnostics and Reprosource.
See the "Costs" section of this website for more information on the price of sperm DNA testing.
High DNA Stainability (HDS)
Some sperm DNA tests include an HDS evaluation, which measures the percentage of sperm cells with immature chromatin. Elevated HDS levels indicate the presence of higher levels of immature sperm. In men with sperm production problems, increased numbers of immature sperm are often present on standard semen analysis testing. The presence of immature sperm cells can potentially increase oxidative stress on the sperm. In healthy, fertile men, average HDS is about 6 percent, though up to 15 percent is still considered normal.
Elevated HDS readings have been associated with longer times to pregnancy with IUI and IVF in some studies. However, at this point in time, I do not think the clinical data supporting HDS testing are particularly compelling.
Intracytoplasmic Morphologically Selected Sperm Injection (IMSI)
IMSI is an intervention that can be performed at the time of IVF/ICSI and is used to select the best-quality sperm to be injected into each egg. This technique (motile sperm organelle morphology examination, or MSOME) is based upon evaluating sperm at very high magnifications using a specially designed microscope. Standard ICSI evaluations of sperm generally take place at 300–400× magnification, while IMSI evaluates sperm at magnifications of up to 6,000×. This allows a more thorough assessment of sperm cell structure, including evaluation for specific structures such as vacuoles within the sperm heads.
Studies have shown that approximately 65 percent of sperm that were selected under standard 300–400× magnification as good candidates for use with ICSI in fact showed abnormalities of sperm structure when evaluated at 6,000× magnification with IMSI. Various studies have found that sperm assessed as having normal shape under IMSI evaluation had lower levels of genetic aneuploidy (abnormal number of chromosomes) and lower levels of DNA fragmentation. Some studies have shown improved embryo quality (after day 3), increased pregnancy rates, improved embryo implantation rates, and decreased miscarriage rates when IMSI-selected sperm are used. Improved pregnancy rates have also been found when IMSI is used in couples who have failed multiple cycles of standard IVF/ICSI.
Despite the promising findings of these studies, other studies have shown no improvement in fertility outcomes with IMSI. A 2013 Cochrane Review looking at all the available good-quality studies did not find compelling evidence of improvements in fertility outcomes or decreases in miscarriage rates with the use of IMSI, and concluded that further trials are necessary before IMSI can be recommended in clinical practice. Other downsides to IMSI are that it is a very time-consuming process and that it is not widely available in the United States at this time.
The cost of IMSI is going to be different for each institution, and is also likely going to vary depending upon the number of eggs that are retrieved for IVF (as more eggs means that more better-quality sperm need to be identified to inject into each egg). In general the cost of IMSI at a facility will likely range between $2,500 and $4,000.
My overall impression is that IMSI has the potential to be of help to couples who have failed multiple cycles of IVF/ICSI due to poor embryo quality or progression and are trying to optimize their chances of success for a subsequent cycle. I hope that future clinical data will support the role of IMSI for couples with recurrent unexplained IVF failures, and that this technology will become more available in the United States at a reduced cost.
Hyaluronan Binding Assay (HBA)
The human egg is surrounded by a layer called the cumulus oophorus complex, which is primarily made up of a substance called hyaluronic acid. This layer acts as an efficient sperm selection barrier, since only mature, normally functioning sperm are generally allowed to pass through and initiate fertilization. Sperm that are able to bind to hyaluronic acid have been shown in some studies to have lower levels of sperm DNA fragmentation, resulting in better embryo quality and development.
Testing of sperm binding to hyaluronic acid has been used in two ways:
1) Hyaluronan binding assay (HBA)
This simple, inexpensive test looks at the percentage of sperm that will successfully bind to hyaluronic acid. Normal levels have been reported to range from 60 percent to 80 percent or higher. Some labs have used this test in couples who need IVF, but it remains unclear whether standard IVF or IVF/ICSI should be used. If the HBA shows abnormally low binding, then that patient’s sperm may have trouble with normal egg/sperm interactions, and adding ICSI may improve the couple’s outcomes. The average cost of HBA is $50.
2) PICSI (physiologic ICSI)
This involves exposing sperm to a sheet of hyaluronic acid, and then choosing sperm for ICSI from those that have successfully been bound after five to ten minutes of incubation. The thinking is that these bound sperm are potentially healthier. Some studies have shown that using these sperm results in improvements in embryo quality and implantation rates, with a trend toward improved fertilization and pregnancy rates with PICSI (as opposed to standard ICSI). However, other studies have not shown improvements in fertility outcomes using PICSI. A potential criticism is that PICSI may be choosing sperm with lower overall motility, since the poorly motile are easily bound, while extremely motile sperm may break free and so not be identified for use with ICSI. The cost of PICSI is going to vary between labs. The PICSI dishes themselves are produced by Origio and are not particularly expensive, but there is lab time involved with using them and selecting out the bound sperm. My estimation of a general cost for PICSI based upon other lab related activities would be about $500 to $1,000.
I think HBA testing may provide some useful information about men who might benefit from IVF/ICSI as opposed to standard IVF. PICSI might also be of benefit in men with recurrent IVF/ICSI failure of unknown cause, although more studies are needed to prove definitive improvements in pregnancy and live birth rates.
Conclusion for Advanced Sperm Testing
The search continues for the perfect advanced sperm test which can not only identify which men have abnormal sperm, but then identify the exact “normal” sperm which are present so that they can be selected and used for IVF/ICSI. In the meantime, some labs will continue to utilize some of the above listed tests to help to try and improve fertility outcomes.