DNA Fragmentation

What does “ DNA fragmentation “ mean?

In each spermatozoon the genetic material (DNA) is located in his head and contains the information that is needed for the cell to perform all its functions. For the case of the spermatozoon, the cell needs information to reach the oocyte, fertilize it and subsequently contribute to the development of the fetus.

The spermatozoon is a small cell that has a small nucleus. As a result, in order for the DNA to fit into the nucleus, it should “condense”. As this process is completed, “breaks” can occur in the genetic material. So the word “fragmentation” refers to these breaks in the genetic material.

This DNA condensation does not occur in other cells, it only occurs in spermaozoa because of their small size and it is completed by the replacement of histones by protamines (the defective maturation theory). (Mechanisms of DNA Fragmentation, 2012)

DNA fragmentation of spermatozoa can also occur in the testis as a result of the apoptotic process (which is known as the abortive apoptosis theory). Following release from the testis, oxidative stress is thought to be the main mechanism responsible for the occurrence of DNA fragmentation and DNA base oxidation.  Oxidative stress is the result of toxic insult from the environment, drugs or advanced age.

DNA fragmentation prevents the proper functioning of the sperm and the fertilization of the oocyte. (DNA damage, 2012).

DNA “breaks”, detected by “DNA fragmentation”, lead to difficulty in conceiving and are considered responsible for certain cases of unexplained infertility or miscarriages.

Even in IVF attempts (IVF or ICSI), if the DNA fragmentation is high, there is a low rate of fertilization of the oocyte or even embryos of poor quality causing problems in the implantation.

DNA fragmentation is useful in cases such as:

• Unexplained infertility
• Miscarriage
• Repeated pregnancy failures in IVF cycles
• Exposure to toxic factors
• Chemotherapy or Radiation therapy
• Varicocele
• Age of the man > 45 years

There are many references in the literature for the usefulness of the sperm DNA fragmentation in various clinical conditions

• DNA fragmentation is a diagnostic tool for Male Infertility (Bungum et al, 2011)
• The rate of miscarriage is related to the levels of DNA fragmentation
• The clinical significance of DNA fragmentation in assisted reproduction
• The presence of varicocele has been associated with high rates of DNA fragmentation in sperm (Tiseo-et-al, 2016)

What are the reliable laboratory methods for the evaluation of DNA fragmentation?

Semen analysis cannot detect single or double DNA strand breaks. The most commonly used methods for the detection of DNA fragmentation are the sperm chromatin structure assay (SCSA) and the terminal
deoxynucleotidyl transferase-mediated fluorescein-dUTP nick-end labelling (TUNEL). These two
methods reveal different types  of DNA damage and the results from TUNEL and SCSA, although they show correlated results they are not equivalent. In
particular, the TUNEL assay quantifies the amount of cellular DNA
breakage by incorporating fluorescent dNTPs at single- and double stranded DNA ends in the presence of the enzyme terminal deoxynucleotidyl transferase. The SCSA method determines the extent of cellular DNA denaturation (induced by acids or heat treatment) by
measuring the metachromatic shift of acridine orange from green(indicative of intercalation into double-stranded DNA) to red fluorescence (indicative of association with single-stranded DNA).

There are other methods, such as the Hallo test, that use a commercial Kit and is based on the evaluation of ~ 500 spermatozoa  by optical microscopy. This method is very subjective and is based on the evaluation of a relatively small number of spermatozoa, which lowers its reliability.

WHAT METHOD IS USED IN ANDROLOGY-ZEGINIADOU LAB?

In Andrology-Zeginiadou DNA fragmentation is evaluated by the method that uses fluorescent dyes and is proposed by Evenson (SCSA). This method is superior to all other methods of detecting DNA fragmentation due to

•  the large number of spermatozoa  – in contrast to manual methods that evaluate maximum 500 spermatozoa, the flow cytometer makes 2 measurements of 20,000 spermatozoa each
• the objectivity of the measurement. The flow cytometer can accurately assess the fluorescent spermatozoa without showing signs of fatigue or misreading, as can occur with the human eye
• specialized software. The results of the flow cytometer are fed into a special program, which results in an “objective” measurement.

The vast majority of research studies in the literature use the method proposed by Evenson because it shows the strongest correlation with the clinical situation. Thus, the laboratory result becomes a useful tool for the choice of the most appropriate treatment for the couple.