Figure 1. Oocyte fertilized by blue sperm.  Green sperm with fragmented DNA; blue sperm with non-fragmented DNA. (Icon made by whatwolf from flaticon)

The goal of assisted reproductive technology (ART) must be to deliver a healthy baby and, thus, it is necessary to select the best gametes. In this scenario, male factor contributes to approximately 40-50% of patients pursuing ART (1).

Semen analyses are needed to evaluate male infertility. However, about 15% of infertile men present sperm parameters regarded as normal (Table 1), and so these standard measures alone are insufficient in predicting male fertility potential. Therefore, additional tests become necessary (2).

Table 1. Normal values of sperm parameters (3)

The genetic information of the newborn is the result of the conjunction of both oocyte and sperm DNA. This should be intact, as any damage on the DNA may affect embryonic development. Consequently, sperm DNA fragmentation (SDF) has been under the scope for some time with increasing interest. This factor has been related to reduced fertilisation and pregnancy rates, abnormal embryonic development and increased risks of miscarriage and of neonatal disease in the offspring (2, 4, 5, 6, 7).

Furthermore, high SDF is associated to abnormalities in seminal parameters like concentration, morphology and motility, as well as genetic abnormalities in embryos. Nevertheless, it is estimated that around 25-40% of seminal samples with normal parameters present a high DNA fragmentation index (DFI) (2, 5).


Deoxyribonucleic acid (DNA) is the complex molecule that contains the genetic information, and it is composed of two long joined chains. Small breaks between the chains are present in some sperm cells, which could produce DNA mutations. Different effects are obtained depending on the type of fragmentation present (8) (Figure 2):

  • Single-strand DNA breaks (SSBs). Small breaks in the same chain are related to low fertilisation rate, lack of clinical pregnancy and/or an increase in time to conception. Moreover, samples with high SSB value present with low motility and morphology spermatic value (8).  

  • Double-strand DNA breaks (DSBs). Breaks in both chains are related to low implantation rate, embryos with high levels of chromosomal alterations, slower embryonic development and increased risk of miscarriage (8).

Figure 2. DNA with double-strand break and single-strand break. (Icon from flaticon with modifications)


There are mainly two ways in which DNA strand breaks could occur (9, 10):

1. Errors during spermatogenesis

Under normal circumstances, DNA nicks are generated and they are repaired during sperm maturation. If repair is impaired, sperm DNA can remain fragmented. In addition, aberrations in sperm development could be produced, which could make sperm DNA more vulnerable (7, 8, 11).

2. Oxidative Stress

Reactive Oxygen Species (ROS) are metabolites of oxygen and nitrogen, among others, and are highly reactive in that they readily oxidize other molecules. These metabolites are produced by sperm or external factors, and even though they play physiological roles in supporting sperm function, an imbalance in those levels may lead to DNA damage. Sperm contain antioxidant mechanisms to regulate this balance; however, an excess of ROS or defects in the antioxidant system can result in DNA damage. This process is associated with SSBs (8).

Even though oocytes can repair sperm DNA damage to some extent, it remains unclear whether they are able to successfully repair DSBs (7, 11). In addition, this capacity depends on the oocytes’ age, quality and external factors. Aged oocytes exhibit an inefficient and imperfect repair mechanism, thus such oocytes are unable to repair sperm DNA fragmentation.


Several studies suggest that SDF could have a variety of origins and, therefore, sperm DNA could be subjected to a multitude of damaging events (2, 12) (Figure 3). However, it is unclear whether all these factors have a direct causal relationship with DFI, and research is ongoing to understand the aetiology behind (13, 14).

Figure 3. Summary of the main cause of sperm DNA fragmentation (Icons from flaticon with modifications).

Several factors could indicate the use of a sperm DNA fragmentation test. Some of them are related to men’s lifestyle, health, environment, sperm origin (testis, male tract or ejaculate), or assisted reproduction techniques (ART) (Table 2) (2, 7, 8, 9).

Table 2. Indications to carry out sperm DNA fragmentation test. First column shows indications divided by fragmentation origin, whereas DNA fragmentation causes can be seen horizontally.

The embryologist or the medical specialist may also decide to apply SDF tests on the basis of diagnosis or previously undertaken procedures, for example:

  • Idiopathic fertility with normozoospermic parameters/abnormal semen parameters (2, 5)

  • Poor embryo quality

  • Increase of time of conception

  • Implantation failure

  • Repeated miscarriages

  • Several ART failures


Sperm DNA fragmentation can be evaluated with different tests (Table 3) (7, 8):

  • Terminal dUTP nick-end labelling (TUNEL). Fluorescence-labelled nucleotides (a variant of DNA main components) are joined in SSB or DSB. Fluorescence is observed in sperm containing DNA fragmentation (8).

  • Neutral and Alkaline COMET. Sperm membrane is broken and fluorescent substances are added. Sperm with fragmented DNA display a comet-like figure. Neutral and Alkaline COMET are two techniques used independently, but nowadays they are used simultaneously on the same sample (8).

  • Sperm Chromatin Dispersion (SCD). Acid treatments are used on sperm, and the membrane is consequently broken. Dying substances are then added and a halo is formed around sperm containing fragmented DNA (8).

  • Sperm Chromatin Structure Assay (SCSA). Sperm DNA is marked with two fluorescent substances. Green colour is observed in double-strand DNA and red colour is joined to single-strand DNA (8).

The alkaline comet assay has the highest sensitivity followed by the TUNEL, SCD and SCSA tests (5). In addition, TUNEL and Comet assays have shown the best predictability (23).Table 3. Summary of several sperm DNA fragmentation tests (7, 8).


The most commonly used techniques to eliminate sperm cells with fragmented DNA are:

  • Magnetic Activated Cell Sorting (MACS)

This technique leads to the separation of dead spermatozoa or those with fragmented DNA from those alive and without DNA fragmentation.

The semen sample is mixed with a magnetic particle containing an antibody (Ab), which can detect dead DNA fragmented-sperm. Sperm are allowed through the column; those that bind to the Ab are held back, whereas healthy sperm are drawn through the column. So, the semen sample is improved and chances of pregnancy augmented (Figure 4) [to learn more about MACS, visit our previous post here].

Figure 4. Schematic image of magnetic activated cell sorting. Blue sperm cells contain non-fragmented DNA that can pass through the column. Green sperm cells contain fragmented DNA, which are retained within the column (Magent icon from flaticon).
  • The FERTILE Chip

This is a new technique that enables selection of spermatozoa with the best motility, morphology, lowest DNA fragmentation rate and lowest quantity of ROS. Increased pregnancy rates and decreased risk of miscarriage have been suggested as the end result for this technique (24).

The Fertile chip is a microfluidic chip to whose end the semen sample is deposited. Healthy motile sperm make it to the other end of the channel, whereas poor sperm quality are retained in the inside (25).


Lifestyle modifications like stopping smoking, reducing alcohol intake and adopting a more balanced diet are the easiest way to improve SDF (Figure 5).

Several studies have demonstrated that the use of antioxidants like vitamin C, vitamin E, vitamin B, 𝛃-carotene, isoflavones, (8, 22) etc as dietary supplements can reduce oxidative DNA fragmentation or prevent oxidative stress. However, such positive effect is not observed in all patients, and so further research is needed (8). Additionally, the over-use of antioxidants might also cause various diseases and infertility (2, 5, 12).

Antioxidants added to culture media during sperm preparation have been demonstrated to reduce ROS levels produced by ART and so improve sperm parameters. Also, research on mice has shown that antioxidant inclusion during fertilisation has led to improved embryo development (26).

Men with inflammatory diseases could be treated with medications to decrease the amount of ROS and, thus, to improve fertility outcomes. Patients suffering from varicocele can undergo varicocelectomy to decrease ROS levels (2).

Recurrent ejaculation leads to the discard of older sperm cells with more SDF. Similarly, testicular sperm, directly extracted by surgery from the testis, exhibit lower SDF (2).

Figure 5. Summary of lifestyle modifications to improve DNA integrity (Icons from flaticon with modifications).


Female infertility has historically been more widely studied than male factor. Male reproductive potential is evaluated by seminal parameters, like motility, morphology, volume, total sperm count…etc. However,  these measures are insufficient as they do not assess the quality of sperm DNA. Nowadays, male factor has become a more interesting focus of attention, and so it is subjected to more and deeper research, such as SDF evaluation. Nevertheless, further studies are needed in order to determine what factors are causing SDF and to identify the optimal antioxidants regime that could reduce the DFI.

SDF must be studied in semen samples of ART patients due to its negative impact on embryo development and pregnancy success. The objective of ART is to bring home a healthy child, therefore, it is necessary to successfully carry out techniques such as SDF, when needed, in order to accomplish this goal.


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