Advanced Sperm Testing (Including Sperm DNA Fragmentation Testing)

Introduction to Advanced Sperm Testing

Basic semen analysis testing is currently the best single test that we have for predicting the fertility potential of men. However, plenty of men with low sperm counts are successful at achieving a healthy pregnancy naturally, and it is known that 15% of infertile men actually have semen parameters that fall within the normal range of the WHO 5th edition criteria. It is quite clear that there are a multitude of factors which can impact fertility that go beyond the basic parameters of density, motility, and morphology.

Many of these factors have not been discovered by science, but not for a lack of trying. The last few decades have seen many attempts to identify new ways of evaluating sperm to see if better predictive aspects can be identified and used to guide the management of couples struggling to conceive. Many avenues of exploration have led to dead ends, but some of the tests which have been developed show promise in predicting and potentially increasing the chances of establishing and maintaining a healthy pregnancy.

It must be noted that at this point in time, advanced sperm testing is not indicated in most couples who are having trouble conceiving. In fact, the American Society of Reproductive Medicine does not currently recommend any advanced testing for males in their official guidelines. However, the past decade has seen increasing evidence that a more in-depth look at sperm function may be helpful in certain clinical situations, and these will be reviewed in this website section.

There are a number of advanced sperm tests which have only limited data available at this time. These include:

1) Birefringence testing of sperm heads using polarized light

2) Raman spectroscopy

3) Annexin V Magnetic Activated Cell Sorting (AV-MACS)

4) Zeta sorting (sorting sperm by electrical charge)

5 other tests have undergone more extensive evaluation and more data is available, and we will review these in more detail:

1) DNA fragmentation index (DFI)

2) Microfluidic sperm sorting (MSS)

3) Intracytoplasmic morphologically selected sperm injection (IMSI)

4) Hyaluronan binding assay (HBA)

5) Fluorescence in situ hybridization (FISH)

Sperm DNA Fragmentation Testing

As mentioned earlier, standard semen analysis testing evaluates only the basic parameters including how many sperm are present, how well they swim, and what percent have perfectly normal shapes. However, it does not provide deeper information on the functional integrity of the sperm’s DNA, which can potentially influence that sperm’s ability to initiate and sustain a healthy pregnancy. Sperm DNA fragmentation testing involves looking at the ability of a sperm’s DNA to withstand fragmentation when placed under stress. Through several decades of research, elevated levels of sperm DNA fragmentation have been associated with lower rates of fertility success (including with IVF) as well as increased chances of pregnancy loss.

Why Is Healthy Sperm DNA Important?

Having healthy DNA is important for sperm because when a sperm finds and penetrates an egg its twenty-three chromosomes must fuse with the egg’s twenty-three chromosomes in order to form the full forty-six chromosomes of the resulting embryo. Unfortunately, the DNA of sperm is especially susceptible to damage during its long journey in search of an egg. Most of the body’s cells have robust mechanisms to defend against damage to their 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 of its unique membrane structure along with a very limited supply of antioxidants, sperm are especially susceptible to damage by reactive oxygen species. In addition, sperm have only a limited ability to repair any DNA damage that they might sustain.

TESTING FOR SPERM DNA FRAGMENTATION

There are 3 primary tests for measuring the levels of sperm DNA fragmentation:

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 studies have shown that their findings correlate fairly well with each other. DFI testing is by far the most commonly used clinically as it is available nationwide through several large commercial labs. In contrast, at this time the TUNEL and Comet assays are generally used only used at a few large research institutions which perform the testing in their own labs. The TUNEL assay does have the advantage of being able to test the levels of DNA fragmentation of very small numbers of sperm, including those extracted from the testicle. In contrast, standard commercial DFI testing uses flow cytometry technology for its testing which requires a sperm density of at least 1 million sperm/cc. Therefore, men with very low sperm counts (<1 million/cc) are generally not able to get sperm DFI testing performed.

For the rest of this section, we are going to be referring to the sperm DFI testing and its normal cut-off values. Just know that TUNEL and Comet testing are perfectly valid forms of testing for DNA fragmentation but may have different normal values depending on the laboratory at which it was performed.

DNA FRAGMENTATION INDEX (DFI)

As mentioned previously, 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 defined as 30 percent or less (that is, 30 percent or fewer 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 males with proven fertility typically have a DFI of around 8%.

Elevated levels of DFI are found in 20–30% of infertile men, including about 11% of men with completely normal semen parameters. DFI levels are not correlated with sperm density and only weakly with morphology, but a low sperm motility does increase the chances of elevated levels of sperm DFI.

IMPACT OF DFI ON FERTILITY OUTCOMES

Sperm DNA testing offers a potentially 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. The impact of elevated levels of sperm DFI on fertility outcomes can vary among couples. One reason for this is that a woman’s egg has the ability to repair some degree of sperm DNA damage once they have fused together. The eggs of younger women are hypothesized to have a greater potential for sperm DNA damage repair than those from older women.

Of note, there is no set cut-off for sperm DFI above which establishing of a successful pregnancy is not possible. Also, elevated levels of sperm DFI have not been shown to be definitively linked to elevated levels of birth defects in the subsequent children who are conceived.

Several decades of research have shown that increased levels of sperm DFI (≥30%) have been associated with poorer outcomes for natural intercourse, IUI, standard IVF, and IVF/ICSI. An early meta-analysis [Evenson D. Reprod BioMed Online 2006] of existing studies found the following relationships between a man’s DFI and fertility outcomes:

1) Natural intercourse: a DFI over 30 percent was associated with a 7-fold decrease in the rate of natural conception

2) Intrauterine insemination: a DFI over 30 percent was associated with a 7.3-fold decrease in pregnancy rate

3) Standard IVF: a DFI over 30 percent was associated with a 2-fold decrease in pregnancy rate

4) IVF/ICSI: a DFI over 30 percent was associated with a 1.5-fold decrease in pregnancy rate

This data indicates that the impact of elevated levels of sperm DFI tends to decrease as more advanced treatments are utilized from the female side. Since this 2006 study, much more detailed information has been discovered from studies regarding the impact of sperm DFI levels on fertility outcomes. This section will now review the relationship of elevated sperm DFI with timed intercourse, IUI, IVF (with or without ICSI), and recurrent pregnancy loss.

Timed Intercourse

Early studies on sperm DFI testing showed decreased rates of success with establishing a pregnancy naturally when the man’s DFI was >30%. However, there is no definitive data showing that couples with elevated sperm DFI levels will not be able to conceive naturally, especially if sperm counts and motility are good. Due to this lack of data, the American Society of Reproductive Medicine (ASRM) does not recommend sperm DFI testing as part of a first-line evaluation for couples who are having difficulty conceiving.

Conclusion: For couples who are trying to conceive naturally (with or without medications from the female side), it is currently not indicated to check sperm DFI in the man.

Intrauterine Insemination (IUI)

A relatively small number of studies have looked at outcomes for IUI when sperm DFI was elevated. Most of these studies have had very small numbers of IUI cycle data (131 cycles or less) and were also flawed by the fact that no steps were taken to try and improve the man’s sperm DFI prior to proceeding with IUI. The largest study on the topic [Bungum M. Hum Reprod 2007] looked at 387 IUI cycles in men with normal vs. elevated levels of sperm DFI. The data for this study is included below:

To summarize the study, men with elevated levels of DFI (>30%) had only a 3% pregnancy rate with IUI compared with 23.7% of men whose DFI was 30% or less. The line graph shows the odds of pregnancy depending on DFI, with “OR” meaning odds ratio. A man with an OR of 0.5 has half the chance (50%) of establishing a pregnancy compared to someone with an OR of 1.0. It can be seen that the odds of establishing a pregnancy with IUI were still fairly good up to around a DFI of 20-25%, after which they dropped considerably. Flaws with this study include that fact that the patient population was again quite small (by the standards of clinical testing) and again, no major steps were taken to try and improve each man’s DFI prior to IUI treatment.

Conclusion: There is evidence that high sperm DFI has the potential to significantly impact IUI outcomes in a negative manner. However, most clinicians at this point in time do not rely on one relatively small study from roughly a decade and a half ago to recommend that patients skip straight to IVF/ICSI if their sperm DFI is elevated. What is clearly needed are larger trials evaluating the impact of sperm DFI (after optimization) on IUI outcomes. However, men with good total motile sperm counts but persistently elevated DFI levels and who have failed 1-2 cycles of IUI may want to consider moving on to IVF/ICSI earlier rather than trying the typically recommended 3-4 cycles of IUI.

IVF/ICSI

Whenever and IVF cycles is not successful, there is always uncertainty as to why that particular cycle did not lead to a successful pregnancy. Was it a sperm problem? An egg problem? Both a sperm and egg problem? Good sperm and egg quality, but just plain bad luck?

When a clinician is trying to figure out if a sperm quality problem is playing a role in a situation of IVF failure, it is important to keep in mind that most studies shown that after an egg and sperm connect, the sperm’s DNA usually does not activate and play a role in the development process until well after fertilization occurs and the embryo reaches about the day 3 (6 to 10 cell) stage of development. [Fragouli E. MolHumReprod 2014] Therefore, the early stages of an IVF cycle (fertilization and very early embryo development) are generally not related to sperm quality. A 2019 study looking at 419 non-male factor IVF cycles confirmed that increased levels of sperm DFI had no impact on fertilization rates, but there was a lower quality of day 3 embryos as well as decreased numbers and quality of day 5 blastocysts. [Borgus E. FertSteril 2019] Of note, though later embryo progression failure can certainly be related to poor sperm quality, it must be remembered that egg quality and elements of chance can also play a role as well.

A multitude of studies have shown that standard IVF and IVF/ICSI success rates can both be negatively impacted by elevated sperm DFI levels, which in most studies is defined as >30%. Poor embryo development, decreased implantation rates, and an increased risk of miscarriage have all been shown to be linked to sperm DFI levels of 30% and above. It seems fairly clear from the data that the impact of elevated sperm DFI levels seem to be less when ICSI is used during an IVF cycle. Therefore, couples with elevated sperm DFI who are planning on doing IVF should consider utilizing ICSI during their fertility treatment. In the past, some clinicians felt that the use of ICSI completely eliminated the negative impact of elevated sperm DFI levels on IVF outcomes, but more recent data does not show this to be the case. A systematic review in 2017 of the 41 available studies on the topic concluded that: “There is sufficient evidence in the existing literature suggesting that sperm DNA damage has a negative effect on clinical pregnancy following IVF and/or ICSI treatment.” [Simon L. Asian J of Androl 2017]

Conclusion: Increased sperm DFI levels can decrease the success rates in couples undergoing both standard IVF and IVF/ICSI. ICSI seems to have better success rates in these circumstances and should be considered to be used with IVF in men with elevated sperm DFI.

Recurrent Pregnancy Loss

Recurrent pregnancy loss (RPL) is defined by the American Society of Reproductive Medicine as 2 or more clinical pregnancy losses. There are many potential risk factors for RPL from the female side, including advanced female age, blood clotting abnormalities, and as well as anatomic issues (e.g. fibroids, uterine abnormalities). However, around 40-50% of couples experiencing RPL are not found to have any apparent risk factors after an evaluation of the female. If the female evaluation is normal, standard male evaluation includes checking a karyotype to look for chromosomal abnormalities, though this testing is usually normal. There is some evidence that increased levels of sperm DNA aneuploidy by FISH testing (discussed later in this section) is a risk factor for RPL, but sperm FISH testing is rarely utilized in clinical practice. There has accumulated significant evidence that elevated levels of sperm DFI have been associated with an increased risk of RPL. This association was confirmed in a 2019 meta-analysis of the 13 available studies which showed that couples with men who had higher levels of sperm DNA (>30%) fragmentation were at significantly greater risk of suffering from issues with RPL. [McQueen DB. FertSteril 2019]

Conclusion: Elevated levels of sperm DFI have been correlated with an increased risk of RPL, though it remains unclear whether the elevated levels of fragmentation actively contributes to pregnancy loss or if it is just an associated factor. With the existing data, it makes sense to consider checking sperm DFI levels in couples with RPL if no significant risk factors are found from the female side.

PRIMARY DRAWBACK OF SPERM DFI TESTING

Sperm DFI testing has clearly shown that it can impact fertility outcomes in couples, and this information can be used to develop management strategies that address these risk factors. The primary problem of sperm DFI testing at this point in time, however, is that the testing that we have can only tell us what percentage of sperm have normal DNA versus abnormal DNA. What it cannot do is identify specifically which sperm have normal DNA and then select those exact sperm with intact DNA to be used with IVF/ICSI. This is the “holy grail” of male infertility testing: a method of identifying the individual sperm that have intact DNA and the best fertility capacity. Some new methods such as microfluidic sorting and IMSI (which are discussed later in this section) have shown the ability to isolate sperm with lower DFI levels. However, actual improvements in clinical outcomes (such as pregnancy and live birth rates) have so far not been proven when specific sperm are chosen for use with IVF/ICSI using these methods.

Current indications for sperm DFI testing

Although sperm DFI testing has several decades of accumulated data, the practical use of this data in the clinical setting is still felt to be relatively new and needs 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, most male fertility experts feel that there is enough accumulated compelling data for sperm DFI testing to play a useful role in two specific clinical situations:

1) Recurrent IVF failure

2) Recurrent unexplained pregnancy loss (if the female evaluation does not show any significant risk factors)

Another potential application is for situations in which that man has good sperm counts and motility, but the couples has failed 1 or 2 IUI cycles. When trying to decide whether to try a few more IUI cycles as opposed to moving on to IVF, it may be worthwhile to check a sperm DFI using the same collection protocol used for the previous IUI cycle(s). If sperm DFI levels are found to be elevated, then steps can be taken to improve them (and these steps will be reviewed later in the section). The man can then recheck DFI testing after ejaculating 3 days in a row (see “4 Day Rapid Ejaculation Protocol” below). If sperm DFI levels are still elevated after these interventions, then moving straight to IVF/ICSI may be the couple’s best chance for success. However, if the DFI has normalized, then repeat IUI with the same 4-day protocol could be attempted. It must be remembered that 4 days of daily ejaculation can potentially lower the man’s total motile sperm count which could translate into decreased chances of success with IUI, and therefore these numbers should be closely monitored at the time of IUI.

Some published guidelines suggest checking sperm DFI levels in cases of “unexplained infertility” in which the man has normal semen parameters and the female testing did not show any significant problems. However, with the data still fairly unclear on the significance of the impact of elevated sperm DFI on low-tech female treatment options (such as IUI), the question arises as to what to do with this information if it comes back as abnormal? ASRM guidelines for unexplained infertility recommend IUI as an effective first step in most couples [Practice Committee of ASRM Fert/Steril 2020], and therefore sperm DFI testing should generally be reserved for couples who have failed at least 1 to 2 cycles of IUI. Possible exceptions to checking sperm DFI early in the fertility process may include some particularly at-risk populations, such as post-chemotherapy patients, older men (e.g. >50 years), and those taking selective serotonin reuptake inhibitors (SSRIs), although no official guidelines have yet been established.

Risk Factors for Elevated Sperm DFI Levels

Most of the risk factors for elevated levels of sperm DFI fall into 2 main categories. The first are factors with the potential to directly damage sperm DNA integrity. The second are causes which slow the transport of sperm through the genital duct system, which is where most of sperm DNA fragmentation occurs. Of note, men do not need to have any of these risk factors to have increased sperm DFI, but many do have potentially reversible causes which can be addressed.

Risk Factors for Direct Sperm DNA Damage

1) Oxidative stress (the most common cause)

2) Tobacco use

3) Excessive heat exposure (for example, using a hot tub or laptop computer directly on the lap)

4) Obesity

5) Chemotherapy or radiation therapy

6) Air pollution exposure

7) Varicoceles

8) Aging (especially age 50 years and above) [Pino V. JBRA AssistReprod 2020]

9) Pyospermia (elevated numbers of white blood cells in the semen)

Risk Factors Which Can Delay Sperm transport

1) Prolonged sexual abstinence

2) Diabetes mellitus

3) Other neurologic problems, such as spinal cord injury

4) Selective serotonin re-uptake inhibitors (SSRIs)

MANAGEMENT OF ELEVATED SPERM DFI

Studies have shown that interventions can be to take to effectively reduce elevated DFI levels in the majority of men, and this has the potential to improve fertility outcomes in these couples. The primary approaches to reducing DFI fragmentation levels in men with abnormal testing include antioxidant therapy, treatment of reversible risk factors, and maximizing the “freshness” of the sperm specimen. There are 5 basic steps that men can take to address elevated sperm DFI levels.

1) Antioxidant therapy can decrease DFI levels by protecting the DNA of the sperm from damaging oxygen free radicals. It should be understood that there is contradictory evidence regarding the impact of antioxidants on improving sperm DFI levels. For example, a 2019 study of 1,645 infertile men found that antioxidants were able to reduce sperm DFI levels by 38.9%, and this closely matched with improvements in sperm motility as well. [Salehi P. IntJ ReprodBiomed 2019] In contrast, the 2020 randomized MOXI (Males, Antioxidants, and Fertility) Trial looking at 174 patients in 2020 did not find any improvement in sperm DFI levels with the use of antioxidants. [Steiner AZ FertSteril 2020] Until more definitive answers with larger trials can shed further light on the topic, the numerous studies showing reductions in DFI with antioxidant therapy suggests that this relatively low cost/low risk intervention is a reasonable approach that may provide some benefit for men who have increased sperm DFI levels. See the Antioxidants section of this website for more detailed information on antioxidant regimens.

2) Reversible lifestyle changes known to increase sperm DFI can be managed, including:

a) Stopping tobacco use

b) Gradual healthy weight loss in obese patients

c) Avoidance of excessive heat (hot tubs, saunas, etc.)

d) Decreasing exposure to air pollution and workplace-related chemical exposure when possible

3) Treatment of clinically significant varicoceles. The treatment of varicoceles of clinically significant size has been shown by multiple studies to have the potential to decrease sperm DFI. Some studies have shown a fairly significant decrease in sperm DFI levels after varicocele repair, such as a 2018 study of 40 patients whose average sperm DFI dropped from 34.6% to 28.3% after treatment. [ZaaZaa A. Andrology 2018] However, a meta-analysis of 6 studies on the topic found an average improvement in DFI of only 3.7% following varicocele repair [Wang YJ ReprodBiomedOnline 2012] The bottom line is that varicocele repair can potentially improve sperm DFI levels, but the impact is usually modest in most men.

4) Consider changing to a different medication if taking a Selective serotonin re-uptake inhibitors (SSRIs) if other interventions are not successful. Several studies have noted increases in sperm DFI in men taking SSRIs, with the prevailing theory being that these medications can delay the transport of sperm within the male genital duct system. A 2010 study looked at 35 healthy men who were started on paroxetine. After 5 weeks of therapy, their sperm DFI levels increased to 30.3% from a baseline (off medication) of 13.8%. [Tanrikut C. FertSteril 2010] Another study evaluated 74 men who were taking different SSRI medications and compared them with 46 men not on the medication. DFI levels in the men on SSRIs was 43.2% in comparison to 21.4% of the normal controls, and the results did not seem to differ between men taking different SSRIs (citalopram, escitalopram, fluoxetine, paroxetine, and sertraline). [Safarinejad MR. JUrol 2008] Changes of anti-depressant medications should always be done carefully and under the care of the man’s primary care physician who is managing these medications. There are not many studies looking at the impact of different non-SSRI anti-depressant drugs on sperm DFI levels so no specific recommendations can be made on best alternative medications to switch to, but it does seem clear from the available data that at this time the SSRI-class medications seem to have the most negative effect on DNA fragmentation of sperm. If felt to be safe from the standpoint of the man’s primary care provider or psychiatrist, consideration of a switch to another medication class could potentially decrease sperm DFI in men on SSRIs who have elevated levels of sperm DNA fragmentation and in whom other interventions have not been successful.

5) Getting Fresher Sperm

Most sperm DNA fragmentation does not occur during sperm formation within the testicle. Instead, sperm generally acquire most of their DNA fragmentation as they travel through the epididymis and genital duct system. Because of this, any factor which delays the transit of sperm through the ductal system can potentially increase sperm DFI levels. These include prolonged abstinence, neurologic problems (such as diabetes and spinal cord injury), and medications (such as SSRIs) which are felt to slow sperm transit times.

In contrast, getting “fresher” sperm in the ejaculate has the potential to decrease sperm DFI levels. A study in 2013 looked at 46 men who had elevated sperm DFI levels (>30%) when the semen specimen was collected with between 3 to 7 days of abstinence. By having these men ejaculate once daily for 3 days and then rechecking the DFI on the 4th day, 90% of men normalized their sperm DFI levels to 30% or less. [Pons I. JAssistReprodGenet 2013]

In my practice, I have therefore translated this data into the “4 Day Rapid Ejaculation Protocol”. For men who have an elevated sperm DFI, I start them on antioxidants and work on eliminating the risk factors as described above. I then have them repeat their sperm DFI testing using the 4 Day Rapid Ejaculation Protocol and see if the DFI has improved. If sperm DFI has improved, then this collection protocol can then potentially be used in conjunction with treatments for the female side, for example IUI if the total motile sperm count (TMC) is still good after 4 days of daily ejaculation.

Persistently Elevated Sperm DFI and Failed IVF

In men planning IVF whose sperm DFI is still elevated despite taking the above described steps, there are several options. One is to use laboratory techniques, such as microfluidic sorting or IMSI, to try and select for better quality sperm. These options will be described in more detail later in this section, but to-date have not definitively shown improvements in pregnancy and live birth rates when used in conjunction with IVF/ICSI.

Another is to try and surgically extract sperm to use with IVF/ICSI. The theory is that by removing sperm straight from the testicle, you are getting the freshest sperm possible. Multiple TUNEL assay studies have shown that in men with elevated DFIs in their ejaculated sperm, when sperm are extracted straight from the testicles these have normal DFI levels in the majority of men. A 2010 study looked at men who had an average DFI of 39.7% in their ejaculated sperm, and when testicular sperm were extracted and evaluated by TUNEL, these “fresher” extracted sperm had an average DFI of 13.3%. [Moskovtsev SI. Fert/Steril 2010] A more recent study nicely showed that sperm DFI increases as the sperm move through the male genital duct tract. In this study, the mean DFI in the ejaculated sperm of the men in the study was 23.6%, which dropped to 20.4% from sperm extracted from the vas deferens. Epididymal sperm had a mean DFI of 15.8% while extracted testicular sperm had DFI levels of only 11.4%. [Xie P. J Urol 2020] Multiple small studies have also shown higher pregnancy and lower miscarriage rates from extracted testicular sperm (as opposed to ejaculated sperm) in men with elevated DFI levels. [Esteves SC. AsianJAndrol 2015][Pabucca EG. Andrologia 2016][Arafa M. Andrologia 2017] A prospective study in 2017 looked at 228 men with elevated sperm DFI levels (30% or above) who were planning on proceeding with IVF/ICSI. The men were given the choice of using ejaculated sperm or extracting testicular sperm to use with their upcoming IVF cycle. The 148 men who chose TESA had a clinical pregnancy rate of 49.5% as opposed to 27.5% for the 80 men who used ejaculated sperm. Live birth rates were also higher (43.7%) for the testicular sperm cycles as opposed to those which used ejaculated sperm (24.9%). [Bradley CK. Andrology 2016] Although larger studies to confirm these findings would be welcome, the accumulating evidence suggests that the extraction of testicular sperm has the potential to significantly benefit the outcomes of IVF/ICSI in couples with persistently elevated levels of sperm DFI and who do not wish to use donor sperm.

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. As mentioned above, some large research institutions have their own in-house TUNEL or Comet assays which they use, but these cannot generally be ordered by clinicians not working at that particular university.

See the "Costs" section of this website for more information on the price of sperm DNA testing.

High DNA Stainability (HDS)

When sperm DFI results are run, some labs (e.g. ReproSource) also include an evaluation of the percentage of sperm with high DNA stainability (HDS). Sperm HDS measures the percentage of sperm cells with immature chromatin and therefore 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 (usually identified as “round cells” in the semen analysis report). Immature sperm cells produce oxygen free radicals which can 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.

Frankly, most male fertility experts are confused as to what to do with HDS results when they come back as abnormal. The early studies of HDS which set the “normal” range of 15% looked at chances of conceiving through natural timed intercourse. A 1999 study found that 73 couples who were successful at achieving a pregnancy within 3 months had a mean HDS of 8.95%, while 31 couples who were not able to conceive after 12 months of trying had a mean HDS of 15.03%. [Evenson DP. HumReprod 1999] Robust data confirming a negative impact of elevated HDS levels on timed intercourse and IUI have not followed. In terms of IVF outcomes, a 2015 study of 275 couples undergoing both standard IVF and IVF/ICSI showed no significant relationship between HDS levels and fertilization rates, blastocyst formation, implantations, as well as life birth rates. [Speyer BE. SystBiolReprodMed 2015] Interestingly, a study of 832 standard IVF and 770 ICSI cycles showed a small but significant increase risk of early miscarriage when HDS was 15% or above, but only in the ICSI group. [Jerre E. FertSteril 2019] So maybe if a couple is having recurrent miscarriage problems with IVF/ICSI, they could consider an attempt using standard IVF if sperm HDS levels are significantly elevated (and no other risk factors for recurrent miscarriage have been found), but the data is not entirely convincing. There is currently not good data on methods to improve sperm HDS levels when they are elevated, though antioxidant therapy could potentially decrease the impact of elevated oxidative stress associated with increased numbers of immature sperm in the ejaculate.

Microfluidic sperm sorting

Microfluidics is a field in which tiny microchannels are used to manipulate relatively small amounts of fluid. A review of the current research has shown that specialized microchannels can be used for microfluidic sperm sorting (MSS) to try and select good quality sperm for use with fertility procedures. [Samuel R. TranslAndrolUrol 2018] Depending on the design of the microchannels, selection of sperm based on shape, size, or motility can be accomplished. Channel design can also select for higher numbers of good quality sperm (to be used with IUI) or lower numbers (around 100,000 more highly selected sperm) that can be used for IVF. The theory behind the better selection of quality sperm with MSS is twofold: a gentler process of sperm sorting and the concentration of sperm with better quality.

Gentler Sperm Processing

When ejaculated sperm is used for female fertility interventions (such as IUI or IVF), it needs to be processed. This step is necessary since during normal intercourse the cervix serves as a barrier to screen out dead sperm and other elements present within the ejaculate that you do not want to pass into the uterus (such as bacteria, epithelial cells, and white blood cells). A simple wash (in which the sperm is just mixed with sterile medium, centrifuged, and then the pellet is then re-suspended with sterile media) is generally not used as this does nothing to remove the bacteria, dead sperm, and white blood cells that should not be introduced into the uterus or used in the fertility lab. Historically, two techniques have been used to prepare sperm for use with IUI and IVF:

1) Swim Up. In this approach a semen specimen is collected and placed in a container. A layer of sterile media is then carefully placed over the specimen taking care not to mix the 2 layers together. Over time, a good percentage of the motile sperm swim up into the sterile media layer while the “undesirable” elements within the ejaculate (such as poorly swimming and dead sperm as well as white blood cells and bacteria) are left in the lower ejaculate layer due to the effects of gravity. The upper layer with the highly motile sperm is then carefully siphoned off and used with IUI or IVF.

2) Density Gradient Centrifugation (DGC). With DGC a special commercially available gradient media is used along with centrifugation of the ejaculated specimen. The cells within the ejaculate separate out during centrifugation and then stop within the pre-formed media gradient at the level of material of the same density. The cells from each layer can then be selectively removed, thereby effectively removing unwanted bacteria and white blood cells.

There is no consensus within the literature as to which technique (swim up vs. DGC) produces better quality sperm for IUI and IVF. Both have been shown to decrease DFI levels of sperm, with some evidence that DGC may be somewhat more effective at this. An example is a study of 118 semen specimens with a baseline DFI of 15.3%. Sperm processing with swim up was able to reduce the DFI to 9.4%, while DGC processing lowered the DFI of the same specimens to 6.7%. [Xue X. JAssistReprodGenet 2014] There are concerns, however, that the centrifugation process associated with DGC can cause problems with sperm quality itself, as elevated levels of oxidative stress (which can damage sperm) have been noted in these samples.

Enter microfluidic sperm sorting (MSS). The potential advantages of MSS is that it has been shown to decrease sperm DFI using a very “gentle” approach which does not involve the stresses to the cells that is associated with centrifugation. MSS also processes sperm faster than the standard swim up technique, which is generally more time-consuming, but MSS usually does take a little more time than DGS. The chips used for sorting come in different sizes for processing either IUI or IVF specimens, and the average extra cost to the lab for the chips is generally around $175.

Impact on Sperm Quality

To date, the majority of research studies have looked at utilizing MSS for selecting the best quality sperm for use with IVF. A 2018 study of 70 infertile men (with an average DFI of 21%) evaluated processing their semen specimens by both DGC and MSS. Sperm DFI levels dropped to 6% with DGC and 0% with MSS. In addition, progressive motility rates of the sperm (which were 54% at baseline) improved to 91% with DGC and 100% with MSS. [Quinn MM HumReprod 2018]

Recent technology innovations have miniaturized the process for MSS culminating in relatively inexpensive “lab on a chip” (LOC) devices which can be used clinically, a commercial example of which is the Zymot Chip. A study using the Zymot Chip looked at 23 men with an average baseline DFI of 28.8% who were planning on undergoing ICSI. When the sperm was processed by DGS the DFI dropped to 21.0%, while specimens processed by MSS had DFI levels of 1.3%. [Parrella A. JAssistReprodGenet 2019]

Impact on Fertility Outcomes

Although the ability of MSS to select better quality sperm in terms of DNA fragmentation seems clear, it is still controversial as to whether the use of MSS-selected sperm actually improves fertility outcomes. The impact of MSS in IUI outcomes is quite limited. A retrospective study in 2019 looked at 295 patients planning on doing IUI who had their sperm processed using either MSS or DGC. Post wash total motile counts were similar and quite good for both groups (MSS 22.85 million vs. DGC 18.85 million) as were pregnancy rates (MSS 18.04% vs. DGC 15.15%). However, the miscarriage rates were significantly higher in the DGC group, leading to improved ongoing pregnancy rates for the MSS group (15.03%) as opposed to DGC (9.09%). [Gode F. Fert/Steril 2019]

Only a few studies so far have evaluated clinical outcomes with using MSS for IVF. The 2019 Parrella study described 4 couples who tried 11 cycles of IVF/ICSI using sperm prepared by DGC and only 1 pregnancy (which ended in miscarriage) resulted. When these same couples then underwent another 4 cycles of IVF with sperm prepared by MSS there was an ongoing pregnancy rate of 50%. [Parrella A. JAssistReprodGenet 2019] In contrast, the only prospective study to date on MSS clinical IVF outcomes evaluated 122 couples who had sperm selected by either swim up or MSS. Despite the MSS group having more high-grade embryos, the study found no difference in fertilization rates, clinical pregnancy rates, and live birth rates between the groups. [Yetkinel S. JAssistReprodGenet 2019] This study calls into question whether sperm selection using MSS has the potential to improve pregnancy outcomes in couples undergoing IVF, and more research is clearly needed. What would be interesting is to see if IVF/ICSI outcomes would be improved for select couples with persistently high sperm DFI’s, since the data clearly shows lower sperm fragmentation levels in the sperm selected using MSS.

Summary of Microfluidic Sperm Sorting

The field of microfluidics is a relatively new area of research and holds out the potential to select better quality sperm for use with female fertility treatments. A single study shows that MSS in conjunction with IUI may not improve pregnancy rates but can potentially lower the chances of pregnancy loss. For IVF outcomes, the very limited data has not yet shown a benefit in pregnancy and live birth rates with the use of this technology. What is clearly needed are more studies and data to help decipher if MSS can help to improve IVF fertility outcomes, especially select groups such as men with persistently elevated DFI levels.

Intracytoplasmic Morphologically Selected Sperm Injection (IMSI)

IMSI is an intervention that can be performed at the time of IVF/ICSI and is used to try and select the best-quality sperm to be injected into each egg. This technique (also called 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 perfectly normal shape under IMSI evaluation had lower levels of genetic aneuploidy (abnormal number of chromosomes) and lower levels of DNA fragmentation. Some early 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 early studies, others have subsequently 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. A more recent review of the available data concluded that IMSI may be beneficial in cases of “severe sperm pathology” but did not recommend its routine use with IVF/ICSI. [Osequera-Lopez I. FrontCellDevelBiol 2019]

Currently, IMSI is not currently widely available in the United States. In general, IMSI is a very labor-intensive process with the time needed to complete 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). The cost is going to vary between institutions, but generally falls between the $2,500 and $4,000 range.

Summary of IMSI

IMSI has the potential to be of help to couples with severe sperm quality problems and who have failed multiple cycles of IVF/ICSI due to poor embryo quality or progression. Couples with elevated levels of sperm DFI that are not able to be effectively decreased through interventions from the male side may also benefit, but future clinical data will be needed to support the effectiveness of IMSI in terms of clinical outcomes.

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 about $50.

2) HA-ICSI (HYALURONIC ACID-ICSI) / PICSI (PHYSIOLOGIC ICSI)

The selection of individual sperm for ICSI using hyaluronic acid binding technology is referred to either as HA-ICSI or PICSI (for simplicity, we will just refer to it as HA-ICSI in this section). HA-ICSI 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 theory is that these bound sperm are potentially healthier, and studies have shown that bound sperm have lower levels of DNA fragmentation. [Nasr-Esfahan MH. JAssistReprodGenet 2008] However, one of the concerns is the potential for 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. Early studies of HA-ICSI showed improvements in fertilization rates [Worrilow KC. HumRep 2013] and live birth rates [Mokanszki A. SystBiolReprodMed 2014]. However, a more recent randomized trial of 2772 patients showed a similar live birth rate when HA-ICSI was used (27.4%) as opposed to the control group which did not use HA-ICSI (25.2%). [Miller D. Lancet 2019] This same study, though, did show a significant decrease in miscarriage rate between the HA-ICSI group (4.3%) as opposed to the controls (7.0%).

The cost of HA-ICSI varies between labs. The HA-ICSI 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.

Summary for Hyaluronan Binding Assays

HBA testing is a relatively cheap and easy to perform test, but data on improved clinical outcomes is sparse and this test is not widely used clinically. HA-ICSI has the potential to select for sperm with lower levels of DNA fragmentation, but the most recent clinical data shows that despite lower rates of pregnancy loss, live birth rates were not significantly improved with the use of this technology. More clinical outcomes data would be helpful to assess the potential benefit of HA-ICSI and see if it may play a role in certain situations such as couples with recurrent pregnancy loss.

Fluorescence In Situ Hybridization (FISH)

Fluorescence in situ hybridization (FISH) is specialized test used to evaluate the DNA of cells. Fluorescent probes which are designed to bind to only specific areas on chromosomes are used to identify if those particular areas are present, and if so, how many copies there are. In this way, cells can be evaluated to see if they have the correct number of chromosomes (euploidy). Aneuploidy is when a cell has an abnormal number of chromosomes, and this can be due to either a cell missing a chromosome (monosomy) or having an extra copy (trisomy). FISH technology has been used extensively in scientific research since the 1970’s and can now be utilized in evaluating the genetic integrity of sperm. Normally, cells of the body have exactly 2 of each of the 24 sets of chromosomes in the body, for a total of 48 chromosomes. Since sperm are providing half of the embryo’s genetic material, they normally contain one of each of the 24 chromosomes. Sperm aneuploidy is therefore defined as when sperm are either is either missing a chromosome or has 2 sets of a particular chromosome. All populations of sperm in every man have a certain small percentage of sperm that have aneuploidy issues, but these levels have been found to be higher in men with fertility problems.

When FISH testing is performed on sperm, theoretically, all 24 chromosomes can be evaluated for aneuploidy, but this would be very expensive and time consuming to perform. In humans, the majority of abnormal chromosome combinations that result from an egg being fertilized by a sperm which is either missing or having an extra chromosome are lethal and a pregnancy does not result that cycle. However, a few chromosomal aneuploidies in embryos are compatible with life- these include chromosomes 13 (trisomy is called Patau Syndrome), 18 (trisomy is called Edwards Syndrome), and 21 (trisomy is Downs Syndrome) as well as the sex chromosomes (X and Y). These 5 chromosomes have a greater chance of leading to a clinical pregnancy that either ends in spontaneous miscarriage or significant developmental and health problems in the subsequent offspring, and therefore usually are the ones that are evaluated in standard sperm FISH testing.

Currently, sperm FISH testing is not widely used for several reason. First, the test is relatively expensive and often not covered by insurance. In addition, the test is not widely available, with only a few labs across the country performing FISH testing on sperm. [Ramasamy R. Fert/Steril 2014] In addition, many clinicians are not familiar with the test, and are reluctant to order it when they do not really know what to do if the results come back as abnormal.

Normal Values for Sperm FISH Testing

In modern FISH testing, the probes are able to evaluate about 100,000 sperm which are then analyzed by specialized imaging software. The normal ranges for different aneuploidies are based off of studies of normal fertile controls who have normal semen analysis testing. Abnormal FISH test results are generally defined as results that are greater than 2 standard deviations above the normal range.

Risk Factors for Sperm Aneuploidy

The relationship between semen parameters and increased levels of sperm aneuploidy is complex. Men with normal semen parameters can certainly have increased levels of sperm aneuploidy. In a study of 145 men with recurrent pregnancy loss, 45% of the men who had normal semen parameters were found to have elevated levels of sperm aneuploidy on FISH testing. [Ramasamy R. Fert/Steril 2015] However, men with abnormal semen analysis testing have a significantly higher chance of having abnormal sperm FISH results as well. The strongest correlation with elevated sperm aneuploidy seems to be with decreased sperm density, but abnormal morphology has been associated with an increased risk for abnormal sperm FISH tests as well. [Sarrate Z. JAssistReprodGenet 2019] The relationship between motility and sperm aneuploidy is less clear, with some studies showing a correlation and others not. [Templado C. MolHumReprod 2013]

Certain risk factors have been associated with increased levels of sperm aneuploidy including tobacco use as well as exposure to pesticides and some other occupational-related chemicals such as benzenes. [Templado C. MolHumReprod 2013] The association with increased alcohol intake remains unclear. Sperm aneuploidy rates are often noted to be significantly elevated in men who have undergone cancer treatments using chemotherapy and/or radiation therapy, and this impact can last for 2 years or longer in some circumstances. [Rives N. FertSteril 2017][Martinez G. Fert/Steril 2017]

Sperm Morphology

Sperm morphology has a complex relationship with sperm aneuploidy. As mentioned previously, abnormally high percentages of sperm with abnormal shapes by morphology increases the chances of abnormal sperm FISH testing. [Templado C. MolHumReprod 2013]. However, it has been noted that the morphology of an individual sperm cannot be used to predict if the DNA contained with that sperm is genetically abnormal. [Hwang K. TherAdvUrol 2010] Some exceptions to this are very abnormally shaped sperm, such as ones with double heads, multiple tails, round (pin) heads, and sperm lacking acrosome caps (globozoospermia). These types of extremely abnormal sperm have been noted to have significantly higher levels of sperm aneuploidy and should not be used for IVF/ICSI.

Clinical Implications of Elevated Sperm FISH Testing

The clinical implications of increased levels of sperm aneuploidy have not been fully determined at this time, but generally fall into 3 categories:

1) Decreased IVF success rates

Elevated levels of sperm aneuploidy as measured by FISH have been correlated with lower success rates with IVF/ICSI. [Hwang K. TherAdvUrol 2010]. Decreased embryo quality, increased embryo aneuploidy, decreased implantation, and lower pregnancy rates have all been associated with elevated sperm aneuploidy. [Sarrate Z. JAssistReprodGenet 2019]. Consistent with fact that sperm DNA usually does not activate until about day 3 after sperm penetration of an egg, studies have found no changes in IVF fertilization rates when sperm aneuploidy rates are elevated. [Fragouli E. MolHumReprd 2014] A 2019 study evaluated 439 men undergoing IVF who had FISH testing beforehand, pregnancy rates were significantly lower (46.6%) in men with increased sperm aneuploidy as opposed to men with normal FISH (25.0%). [Sarrate Z. JAssistReprodGenet 2019] Interestingly, the average age of the female partners in this study was 35 years. When donor oocytes were used, the difference in pregnancy rates were less pronounced: 53.3% with abnormal FISH testing vs. 61.2% with normal sperm FISH testing. This finding is consistent with the fact that eggs from younger females have the potential to repair some degree of DNA damage that may be present within sperm.

2) Increased rates of pregnancy loss

There is evidence that spontaneous abortion rates may be higher in men with increased sperm aneuploidy. [Martinez G. Fert/Steril 2017] Sperm FISH testing has therefore been suggested as a possible step in the evaluation of couples with recurrent pregnancy loss if no obvious significant risk factors have been found from the female side.

3) Future health of offspring

In IVF cycles, increased levels of embryo aneuploidy have been noted in men with abnormal sperm FISH testing. {Sarrate Z. JAssistReprodGenet 2019] These embryos may not progress if transferred but could also potentially produce viable offspring with genetic abnormalities. Studies have indeed found elevated levels of chromosomal abnormalities in the children of men with increased sperm aneuploidy, though clearly most of the children born to men with abnormal sperm FISH testing are themselves genetically normal. [Martinez G. Fert/Steril 2017] The degree of risk of transmitting genetic abnormalities in this patient population seems relatively low but certainly deserves further study.

Who Should Get FISH Testing

FISH testing can potentially be used for couples in the setting of recurrent IVF failure and/or pregnancy loss. Men with lower sperm counts (and possibly morphology) have an increased chance of sperm aneuploidy, as do men with certain risk factors (such as tobacco use). Men who want to conceive a child within a few years following treatment with chemotherapy and/or radiation (near the pelvic region) can also potentially use sperm FISH testing to evaluate their current levels of aneuploidy rates.

Where to Get Sperm FISH Testing

A few large research institutions offer sperm FISH testing from their own in-house labs. Commercially, Igenomix (www.igenomix.com) offers the Sperm Aneuploidy Test (SAT) and will mail a collection kit to your doctor’s office. The cost for the SAT test is $795.00 and is not covered by insurance for most couples.

Management of Sperm Aneuploidy

The management of abnormal FISH sperm testing does not have well-developed guidelines at this point in time. Avoidance of specific risk factors such as tobacco use and pesticide exposure can potentially help. There is some evidence that folate supplementation may reduce sperm aneuploidy in some men. [Hwang K. TherAdvUrol 2010]. At this point in time, it is unclear whether sperm processing techniques (such as microfluidic sperm sorting) can help to choose sperm with lower levels of aneuploidy. For men with documented abnormal FISH testing, interventions such as preimplantation genetic diagnosis (PGD) during an IVF cycle can help to identify which embryos have chromosomal abnormalities and make sure that these are not transferred. The use of donor oocytes could also be utilized when the female partner is older due to some evidence that eggs from younger women may be able to repair some degree of sperm DNA damage. [Sarrate Z. JAssistReprodGenet 2019]. Of course, the use of donor sperm can be utilized as well if necessary.

Summary of Sperm FISH Testing

Although elevated levels of sperm aneuploidy have clearly been linked to lower success rates with IVF/ICSI, the exact role of this test in the clinical management of infertility has yet to be established. Recurrent IVF failure and/or pregnancy loss in men with low sperm counts (and possibly low morphology) seem to be possible indications for sperm FISH testing. However, the proposed treatments (such as the addition of preimplantation genetic diagnosis/PGD) are often utilized anyway as next steps without having to spend an additional $800 to check for increased levels of sperm aneuploidy. The use of donor eggs from young fertile women tend to increase IVF success rates for most couples regardless of their situation, but it is not a particularly attractive option for most couples. Very high levels of sperm aneuploidy could prompt men to use donor sperm, but the cut-off test values for making these recommendations have not been determined.

The possible link between increased sperm aneuploidy and possible health problems of offspring is concerning. However, these findings would need to be confirmed by larger studies (as well as looking for evidence showing decreased rates of health problems with interventions such as PGD) before recommending more routine testing of sperm FISH assays on men with risk factors such as decreased sperm density. The strongest current case for use of sperm FISH testing may be for men with very strong risk factors (like relatively recent completion of chemotherapy/radiation) who want to know when it is “safe” to start trying to conceive. Unfortunately, the lack of clinical data on relative risks for poor fertility outcomes and risk to offspring are still a barrier to providing clear guidance to men whose test results come back in the abnormal range.

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, labs will continue to utilize some of the above listed tests to help to try and improve fertility outcomes.