Table of contents
What is sperm DNA fragmentation?
Sperm DNA fragmentation is a type of male fertility test that measures the amount of damaged DNA in a semen sample.
Deoxyribonucleic acid (DNA) is a molecule that contains genetic information and is 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, genetic information is stored in the head, and to fit into such a small space, complex mechanisms are required that require the replacement of some molecules with others (in particular, histones are replaced by protamine). This results in a high condensation of DNA that is easy to protect and transport.
In recent years, there has been increasing interest in sperm DNA integrity and its impact in cases of infertility when routine sperm analysis is normal. Attempts have been made to evaluate this parameter as a new method for predicting male reproductive capacity and, together with routine semen analysis (sperm analysis), to further study the male factor of infertility.
The integrity of sperm genetic material is considered vital for normal fertilization, proper embryo development and successful implantation (attachment of the embryo to the uterine lining) and pregnancy development in both natural fertilization and assisted reproductive technologies (ART). All men have some damage to sperm DNA. Single-stranded damage includes multiple breakpoints throughout the genome associated with oxidative stress, which in reproductive settings can prevent clinical pregnancy and, as a result, increase the time to conception. In contrast, DNA double-strand breaks may be associated with a lack of DNA repair in cellular meiosis, contributing to an increased risk of miscarriage, poor embryo quality, and implantation failure in infertility treatment [1].
Oxidative stress occurs continuously in the human body and can damage sperm DNA. Environmental factors (plastics, solvents, preservatives, heavy metals) and lifestyle factors (smoking, excessive alcohol consumption, excessive caffeine consumption, psychotropic drugs, severe prolonged stress) can significantly increase oxidative stress levels and, accordingly, increase the damage (level of fragmentation) of male germ cell DNA.
In this article, we will look at more information about DNA fragmentation, how to interpret the results, and how it differs from other fertility tests.
Checking Sperm Quality
For decades, male fertility testing has relied on a basic semen analysis that looks at the concentration, morphology, and motility of sperm, known as a spermogram. Yet, about 30% of the 5 million men in the United States who experience infertility still don’t know why they’re infertile. A semen analysis assesses basic criteria but does not provide a complete or predictive assessment of male fertility.
Sperm are very small, but they carry a huge amount of genetic information to the egg. While our understanding of sperm DNA damage has increased, the exact cause remains unknown. One important caveat is that the amount of sperm damage detected does not always correlate with the spermogram results. In other words, you can have a good spermogram but have significant damage to your sperm DNA.
Interestingly, the egg has some ability to repair damaged sperm DNA during fertilization. However, scientists believe that the threshold for DNA damage may exceed the ability of eggs to repair it, especially in women over the age of 40 [3].
What are the DNA fragmentation scores?
a.) Excellent to good (less than or equal to 15% DFI)
b.) Good to fair (greater than 15% to less than 25% DFI)
c.) Fair (greater than or equal to 25% to less than 50% DFI)
d.) Very poor (greater than or equal to 50% DFI)
Fig. 1. Single- and double-stranded DNA breaks
What are the methods for diagnosing sperm fragmentation?
Sperm Chromatin Structure Analysis (SCSA)
Many tests have been developed to determine the damage to sperm DNA. However, they all use slightly different procedures and provide different measurement standards. The SCSA test involves treating the sperm with a chemical dye. Damaged DNA turns red, normal DNA turns green.
Fig. 2 SCSA method, damaged DNA in red, undamaged DNA in green.
The sample is then passed through a computerized machine with special software that reads about 5,000 sperm cells. The machine then classifies the ratio of green to red sperm to determine the DNA fragmentation index (DFI). It is possible that the SCSA test can detect damage in 20% of couples who previously had no explanation for infertility.
Sperm chromatin dispersion (SCD) test
SCD (sperm chromatin dispersion) is a simple and rapid test that detects sperm DNA fragmentation; it can be performed without the need for sophisticated laboratory equipment. Sperm are attached to a slide using agarose, incubated with an acidic solution that denatures the DNA, and then with a customized lysis solution that removes most of the nuclear proteins. As a result, the DNA loops of healthy sperm form chromatin halos, while damaged (fragmented) sperm do not produce such halos.
Fig. 3. SCD method, spermatozoa with intact DNA form a specific halo, with damaged DNA – do not form a halo, are dense.
TUNEL
The TUNEL assay is another of the most common methods for assessing sperm DNA fragmentation. The principle of TUNEL is to label the breaks present in DNA with deoxynucleotides (usually deoxyuridine triphosphate, dUTP). dUTP can be directly conjugated to a fluorescent dye or labeled with biotin. Fluorescence is measured using flow cytometry or fluorescence microscopy.
The results are expressed as a percentage of damaged (green) sperm to normal (blue) sperm. We perform this method of analysis to determine DNA fragmentation in our clinic.
Fig. 4. TUNEL method, spermatozoa with damaged DNA are green, with undamaged DNA are blue.
A landmark study has been published (Borges 2019) demonstrating how high versus low levels of sperm DNA fragmentation affect IVF success and embryo quality. This study used the sperm chromatin dispersion (SCD) test to assess sperm DNA fragmentation. In evaluating the sperm of 200 men, the DNA fragmentation levels of sperm with low levels (<30% DNA fragmentation) were compared to those with high levels (>30% DNA fragmentation). The results of IVF treatment were then compared between the two groups.
- The fertilization rate of eggs was the same between the two groups
- The number of high-quality embryos on day 3 of development differed and was lower with high sperm DNA fragmentation (36.4% vs. 23.8%)
- The percentage of embryos at the blastocyst stage (blastulation level) was also different (56.2% vs. 39.0%), and the quality of embryos at day 5-6 of development was lower with high sperm DNA fragmentation
- The rate of embryo implantation was lower with high DNA fragmentation (46.0% vs. 33.2%)
- The abortion rate before 20 weeks of pregnancy was higher with high DNA fragmentation (17.8% vs. 39.9%).
According to the results of this study, a high sperm DNA fragmentation rate affects the quality of embryos, the implantation rate and the increase in the rate of abortion.
What is the clinical benefit of a DNA fragmentation test?
Thus, the degree and nature of sperm DNA damage can negatively affect the outcome of infertility treatment, based on studies showing that samples from men who have had difficulty conceiving have significantly more DNA damage than samples from fertile men. Since sperm defects can be hidden, the SDF (sperm DNA fragmentation) test can provide additional information to assess sperm quality in men with an unknown history of infertility.
Can sperm DNA fragmentation be improved?
To reduce DNA fragmentation rates in cases of male infertility, antioxidants are prescribed, eliminating toxic habits (smoking, alcohol, drugs, etc.), as well as harmful environmental factors (exposure to pesticides, pollution, etc.) that increase oxidative stress in cells. Taking antioxidants should be prescribed by your andrologist, requires understanding their effect in each specific case, adding cofactors, certain trace elements and amino acids that enhance cell support and reduce the level of DNA fragmentation. The most effective antioxidants include:
Ubiquinol (coenzyme Q10),
Resveratrol (trans resveratrol),
Omega 3, with a mandatory combination with natural tocopherol acetate (for example, wheat germ oil),
Addition of zinc, vitamin D, L-carnitine.
During the IVF program, it is important to conduct the most accurate and effective selection of sperm by means of microfluidic chips in order to use only those sperm whose genetic material has not been damaged.
References:
- Ribas-Maynou J, Benet J. Single and Double Strand Sperm DNA Damage: Different Reproductive Effects on Male Fertility. Genes. 2019;10(2):105.
- Borges E Jr, Zanetti BF, Setti AS, Braga DPAF, Provenza RR, Iaconelli AJr. Sperm DNA fragmentation is correlated with poor embryo development, lower implantation rate, and higher miscarriage rate in reproductive cycles of non–male factor infertility. Fertil Steril; 2019 Sep; 112(3): 483-490. doi:10.1016/j.fertnstert.2019.04.029.Epub 2019Jun 11. PMID: 31200969.
- Oocyte ability to repair sperm DNA fragmentation: the impact of maternal age on intracytoplasmic sperm injection outcomes
Amanda Souza Setti 1, Daniela Paes de Almeida Ferreira Braga 2, Rodrigo Rosa Provenza 3, Assumpto Iaconelli Jr 2, Edson Borges Jr 2
