Sperm DNA fragmentation

Deoxyribonucleic acid (DNA) is a molecule that contains the genetic information used in the development and functioning of organisms and is present in all human cells except red blood cells.

In the case of human sperm cell, the genetic information is stored in the head, and to fit into such a small space it requires sophisticated mechanisms that require the replacement of some molecules by others (in particular, histones are replaced by protamine). This results in highly condensed DNA easy to protect and transport.

In recent years, there has been growing interest in the integrity of sperm DNA and its role in cases of infertility which frequently meet the quality values established as normal. In fact, attempts have been made to postulate this parameter as a new tool for predicting male reproductive capacity and, together with the conventional sperm analysis, to further the study of the male factor.

How is fragmentation generated in the sperm DNA?

On the sperm formation — spermatogenesis — DNA strand breaks can be induced. Spermatozoa can also accumulate DNA damage during maturation and storage in the epididymis prior to ejaculation. It has been described that in advanced age in men, some organic pathologies of the reproductive system (varicocele, testicular cancer, leucospermia, etc.), as well as toxic habits, could lead to an increase in DNA damage due to excessive production of reactive oxygen species (ROS) or a decrease in seminal antioxidants.

Which techniques diagnose sperm fragmentation?

There are different techniques capable of assessing DNA fragmentation in spermatozoa, although they differ fundamentally in terms of sensitivity and the type of damage they can detect. The main techniques in clinical practice are:

• Comet assay test (COMET): in case of small portions of fragmented DNA, the sample shows an image similar to the tail of a comet. It is a highly sensitive test capable of detecting small levels of damaged DNA and does not require large numbers of cells, although it not able to discern between single and double strand breaks.

• Sperm SCSA: damaged DNA is more vulnerable to heat and acid denaturation compared to intact spermatozoa. Its major advantage is that it can assess a large number of cells by flow cytometry. It only detects single-strand damage and is an indirect method of analysis, as it requires prior exposure to acidic conditions.

• Sperm TUNEL: The TUNEL technique is based on the actual detection of single or double stranded DNA fragments, where the signal increases with the degree of fragmentation. The analysis can be performed subjectively under fluorescence microscopy or objectively by flow cytometry.

• SCD: The Sperm Chromatin Dispersion (SCD) test estimates the level of DNA fragmentation indirectly by quantifying the nuclear dispersion present in the sample. It’s a fast commercial kit, but with a high degree of subjectivity.

In which cases is it advisable to perform a sperm fragmentation study?

The integrity of the sperm’s genetic material is considered vital for normal fertilisation, proper embryo development and successful implantation and pregnancy, both in natural and assisted reproduction. In fact, single-strand damage involves multiple breakpoints in all regions of the genome, being related to oxidative stress, where in the reproductive setting it would hinder clinical pregnancy and, consequently, an increase in time to conception. In contrast, double-strand DNA breaks would be related to the lack of DNA repair in cell meiosis, favouring an increased risk of miscarriage, poor embryo quality and implantation failure in treatments [1].

Therefore, the degree and nature of DNA damage could negatively affect reproductive outcomes based on studies showing that samples from infertile males have substantially more DNA damage than those from fertile males [2].

What are the normal values for sperm DNA fragmentation?

Currently, no global consensus has been reached on the reference threshold value for the level of sperm DNA fragmentation as an indication of a male’s fertility potential. In fact, several discriminant values have been proposed according to the different assays that study the integrity of this genetic material. For example, TUNEL requires values of less than 20% in fresh semen and less than 15% in capacitated semen to consider a normal sperm sample, while the SCSA technique requires a value of less than 30%.

Can sperm DNA fragmentation be improved?

Therapeutic treatment to reduce DNA fragmentation in cases of male infertility would require the intake of oral antioxidants, elimination of toxic habits (tobacco, alcohol, drugs, etc.) as well as environmental factors (exposure to pesticides, pollution, etc.) that increase ROS.

In an IVF treatment, it would be essential to carry out the most precise and efficient sperm selection possible in order to use only those spermatozoa whose genetic material is not damaged by means of: annexin V columns (MACS), microfluidic chips, etc. Furthermore, there are studies supporting the possibility of obtaining spermatozoa directly from the testicle (TESA) with less DNA damage than those ejaculated.

Instituto Bernabeu R+D+I: studies on DNA fragmentation

Due to the fact that this parameter is a subject of interest whose controversy has been highlighted on several occasions, a research project has been developed at our centre with the aim of studying the impact of sperm DNA fragmentation in greater depth.

This study seeks to evaluate whether the fragmentation index, measured by TUNEL, of the same semen sample used in the in vitro fertilisation cycle could have a detrimental effect on fertilisation rate, embryo quality and clinical outcomes, depending on the quality of the female oocyte in 3 groups of patients: 67 with normal ovarian response, 150 with low ovarian response and 113 who underwent egg donation treatment. Finally, it was concluded that the results of assisted reproduction treatment were not affected by sperm DNA fragmentation, regardless of the fragmentation index and the origin of the eggs.

This work has been published in the journal of the British Fertility Society, Human Fertility, under the title «Sperm DNA fragmentation on the day of fertilisation is not associated with assisted reproductive technique outcome independently of gamete quality» and is available on the Instituto Bernabeu website.

Bibliographic references:

1. Ribas-Maynou J, Benet J. Single and Double Strand Sperm DNA Damage: Different Reproductive Effects on Male Fertility. Genes. 2019;10(2):105.

2. Saleh RA, Agarwal A, Nelso DR, Nada EA, El-Tonsy MH, Alvarez JG et al. Increased sperm nuclear ADN damage in normozoospermic infertile men: a prospective study. Fertil Steril. 2002;78(2):313–318.

Angel Linares, embryologist at Instituto Bernabeu