What is the DrP sperm test?
It is an improved test for the sperm DNA fragmentation according to which only the fertile spermatozoa are assessed. As being fertile, are considered the live spermatozoa presenting progressive movement and physiological morphology. The method used up to nowadays for the sperm DNA fragmentation check was inaccurate, since the dead spermatozoa, those presenting non progressive movement and those with abnormal morphology were included in the assessment. We all understand that we are not interested in whether the dead spermatozoa’s DNA bears damages, nor whether the DNA of the spermatozoa presenting improper movement of morphology bears fragments, since these are not going to fertilize an oocyte (egg).
What is the sperm DNA fragmentation assay?
It is simply the assessment of the structure of the DNA in sperm cells. It consists in controlling the well-known DNA chain for cracks or fragments. Such lesions appear in every cell’s DNA when it gets old (‘apoptotic’ cell) or when an exogenous factor causes cell death. The same happens to spermatozoa. It is thus expected to find DNA fragments in dead or aged (‘apoptotic’) spermatozoa. This is not a problem since these spermatozoa will not fertilize the egg. However, in some cases apparently healthy spermatozoa may have fragments in their DNA. These cells may have normal morphology and motility and though be able to fertilize an egg. The consequences appear after fertilization, as the fragmented DNA of the spermatozoa cannot be divided properly. As a consequence, fragmentations may impede embryo development in the very first stages and lead to abortion.
What does fsDFI stands for and which is the difference from the older DFI?
The fsDFI is an improved index of DNA fragmentation which describes the percentage of the fertile spermatozoa with a high grade of fragments of their DNA over the total fertile spermatozoa. Therefore, the fsDFI stands for ‘fertile sperm DNA Fragmentation Index’. This index is much more accurate from the older index, DFI, since it describes in a more objective and standardized way the percentage of spermatozoa with fragmented DNA. In order to understand the importance of having this accuracy, let us consider a blood glucose test and compare the results of the test without having taken breakfast before, with those of the same test after having taken breakfast. In the last case, your doctor cannot make a safe diagnosis whether you are diabetic or not. He will just ask you to repeat the test without having taken breakfast before.
How does the fragmented DNA affect fertility?
The spermatozoa able to fertilize an oocyte (egg) shall not bear a fragmented DNA. This is practically impossible in Nature. Therefore, we are interested in the fertile spermatozoa that bear a DNA as less fragmented as possible. In fact, the DNA of any cell, spermatozoa included, is opening and closing in many sites repeatedly in order to transcribe the information for a cellular function. In order to open the DNA chain, an enzyme temporarily cuts the chain and rejoins it right after the transcription. If for any reason the chain remains open, cut, fragmented in many sites and the spermatozoon fertilizes n oocyte (egg), some strings of the DNA may be lost increasing the risk of early abortion.
Is it sufficient to measure only the percentage of spermatozoa with fragmented DNA?
It is not sufficient to report only the percentage of spermatozoa bearing fragmented DNA. The DrP sperm test apart from the percentage of fertile spermatozoa bearing fragmented DNA, it also describes how much these spermatozoa’s DNA is fragmented, i.e. the grade of DNA fragmentation.
Why is the grade of sperm DNA fragmentation important?
Because, after the fertilization, the oocyte (egg) is able to correct up to a certain grade the DNA fragments of the spermatozoon that fertilized it. However, if the spermatozoon that fertilized the oocyte bears too many fragments of its DNA, the oocyte may not be able to correct them all. As a result, the probability of having a bad quality embryo is increased. This is related to increased risk of abortion.
How much does the DrP sperm test cost?
The DrP sperm test costs 200euro including the semen analysis andro-test and processing for the isolation of the fertile spermatozoa.
How to interpret the results of a sperm DNA fragmentation assay
Subtantialy, the spermatozoa that are able to fertilize an oocyte shall not bear any fragmentations in their DNA. These are the spermatozoa presenting progressive motility and adequate morphology. The analysis of DNA fragmentation on sperm is expressed as a percentage of those spermatozoa with fragmentations with respect to the total. Since, in the ejaculate, there are always dead or senescent (in Biology called apoptotic) spermatozoa, it is expected that there will always be some spermatozoa with fragmentations. Generally the percentage should not exceed 30-40%, since this is usually the percentage of dead or senescent spermatozoa in a healthy semen.
Since apoptotic spermatozoa do have fragmentations in their DNA and such may be the non progressive or immotile spermatozoa (motility c and d), it could be argued that the percentage of spermatozoa with fragmentations should not exceed that of spermatozoa with motility c and d. That is, the clinician should be interested in a fragmentation percentage higher than the percentage of spermatozoa with motility c+d. This interpretation, however, does not give an accurate estimation. It is like counting blood glucose after having eaten a cake. Your doctor could not estimate which would have been the blood glucose if you wouldn’t have eaten the cake. You shall simply measure the fasting blood glucose. Therefore.. (read next paragraph)
Which is the right way to perform this analysis?
The analysis shall be performed only on the spermatozoa that are able to fertilize the oocyte, i.e. on the spermatozoa selected after washing, a procedure performed only in specialized laboratories (Spermatology laboratories). SpermLab suggested this way of performing the analysis already by the first years the DNA fragmentation test is performed on spermatozoa. However, the non-specialized laboratories keep performing the analysis in the wrong way providing the examinee with unreliable results. The reason is that they are unable to perform the selection of the ‘fertile’ spermatozoa by the washing procedure.
When the analysis of DNA fragmentation is performed on the ‘fertile’ spermatozoa, i.e. on those which the gynaecologist will use in an intrauterine insemination or an IVF, the clinical relevance of the result is immediate: none of the spermatozoa shall have fragmentations. Thus the percentage should ideally equal zero. For example, if the analysis reveals that 5% of the ‘fertile’ spermatozoa bear fragments, the result is interpreted as follows: the chance of having a spermatozoon bearing fragments to fertilize the oocyte is 5%.
Our laboratory performs DNA fragmentation analysis only on the ‘fertile’ spermatozoa, i.e. on the post wash sample.
Interpreting the results of the DrP sperm test
The fsDFI index shall ideally equal zero per cent or at least be close to the zero value. Since this index is referred only to the spermatozoa able to fertilize an oocyte (egg), if the fsDFI equals e.g. 3%, this means that the probability for the oocyte to be fertilized by a spermatozoon bearing a highly fragmented DNA is 3%. The results report of the DrP sperm test include a table describing how much fragmented is the DNA of the spermatozoa. The spermatozoa are distinguished in 5 classes according to the grade of fragmentation of their DNA:
- with the least fragmented DNA,
- with slightly fragmented DNA,
- with significantly fragmented DNA,
- with fully fragmented DNA,
- with entirely degraded DNA.
The first two classes are considered physiological, while the last three classes are pathological.
What causes the fragments?
The aging, the heat and the oxidative stress.
DNA fragmentation is the first sign of aging of every cell. Therefore, it is expected that the dead or aged spermatozoa do have fragments in their DNA.
The heat deactivates the proteins that hold the DNA chain. It is thus obvious that the exposure of the testicles to heat increases the fragments in the DNA chain of the spermatozoa. For example, in an overweight man, the testicles are not ventilated and are in close contact with his body, which has a higher temperature than the ideal for their proper function. Sedentary work and professional exposure to heat may also cause to some extent oxidative stress to spermatozoa.
Occupational exposure to petrochemicals, such as fuels, paints, organic solvents may cause oxidative stress to the spermatozoa. Moreover, oxidative stress may also be caused by toxic substances produced inside our own body:
A third degree varicocele may increase the DNA fragmentation in sperm in two ways, since it may increase the temperature of the testicles and impede the removal of the venous blood and therefore of the ROS (reactive oxygen species) from the testicles. The ROS are substances which are residuals of cell metabolism and are removed through the venous blood. A third grade varicocele shall always be removed regardless the patient’s age, since if the used blood is not removed, there is no supply of new nutritional blood. As a result, the testicle is not feed efficiently and may regress. If the function of the testicles decreases, it is not only the spermatozoa to be lost, since the testicles produce also testosterone, which is necessary even in older age. There is also a misunderstanding in regard to varicocele. It is thought that there is no need to operate it after the age of 35yo. As already argued previously, the patient’s age is not as important as the age of the varicocele at the 3rd grade, i.e. for how long the testicle is not supplied efficiently.
Oxidative stress most commonly is due to an inflammation of the testicle (often caused by an infection), or fever. An infection causes hyperaemia and therefore an increase of the testicles’ temperature. Consequently, it causes an increased DNA fragmentation in the spermatozoa. Furthermore, the immune system tries to kill the bacteria also by producing reactive oxygen species and other cytotoxic substances that affect the spermatozoa, too.
Can I have a DrP sperm test without an andro-test semen analysis?
It is indispensable to analyse and process the semen sample before performing the DrP sperm test, because we need to isolate the fertile spermatozoa first. Moreover, the andro-test provides us with information whether the genital tract is healthy. In other words, the andro-test tells whether there is a problem in the genital tract and the DrP sperm test whether that problem does affect the DNA of the spermatozoa.
Advantages of the DrP sperm test over the oxidative stress test
- The DrP sperm test assesses in a direct way what we are interested in, i.e. whether the spermatozoa have indeed been affected by oxidative factors. The oxidative stress test assesses whether there is any oxidative factor able to affect the spermatozoa, without being able to find out whether the spermatozoa are affected.
- The DrP sperm test is supplemented by the andro-test, which provides us with information whether there is a pathological condition in the genital tract which could be the cause of the DNA damage. This way, the DrP sperm test permits the urologist to advise for the appropriate treatment and restore the DNA damage. On the contrary, the oxidative stress test is not able to determine the cause of the presence of oxidative substances. It is not combined to andro-test or any semen analysis and processing of the semen sample nor is performed to the isolated fertile spermatozoa.
- The DrP sperm test is performed on the fertile spermatozoa after they are isolated from their environment, i.e. the semen liquid and the dead spermatozoa. The oxidative stress test is performed on the semen liquid. Therefore, the test is influenced by the presence of the dead spermatozoa, especially when their percentage is significantly high. Similarly, the assessment of a swimmer being alone in a pool differs from that of the same swimmer in a pool full of corpses in decomposition. Keep in mind that during the natural process of fertilization, the dead spermatozoa stay in the vagina while the fertile ones pass inside the uterus and swim alone in the fallopian tubes until they meet the oocyte to fertilize it.
- The cost for the DrP sperm test includes an analysis and processing of semen, while the cost for the oxidative stress test does not include the semen analysis and processing.
Is there a treatment?
The treatment focuses on the cause. When the agent that causes the oxidative stress is treated, the DNA quality shall be improved. However, antioxidants intake after treatment shall be beneficial.
DNA fragmentations in spermatozoa may also be due to advanced age or other endogenous factors. In those cases treatment may be more difficult.
When is this analysis needed?
This analysis is useful when regardless the semen is apparently healthy, a pregnancy is hard to achieve or there are bad quality embryos after IVF or the pregnancy arrives up to the 6th week. To avoid superfluous charging of the examinee, we recommended performing this analysis after the treatment of any agent suspected to cause oxidative stress and consequently DNA fragmentations on spermatozoa (read previous paragraph).
For example, it is pointless to charge someone for DNA fragmentation analysis, when there is an infection, if not after treatment.
Are there any similar tests?
The sperm apoptosis test is less specific, since instead of assessing the DNA fragmentation percentage on the ‘fertile’ spermatozoa, it estimates the percentage of the apoptotic (=aged) spermatozoa, which by definition bear fragments in their DNA. However, we are interested in the ‘fertile’ spermatozoa, not in the useless apoptotic ones.
An indirect way to estimate the DNA fragmentation is the measurement of ROS, which stands for Reactive Oxygen Species. ROS are related to the DNA fragmentation, since, as explained previously, it is through the increase of ROS that oxidative agents cause DNA fragmentations. By treating the causes, the ROS levels are decreased, so does the oxidative stress and consequently the DNA fragmentations, too.