Sperm DNA Fragmentation Test

Infertility affects 15% of couples of reproductive age. 30-40% of men- and 40-50% of women-related causes are held responsible as effective factors. Due to such problems, many couples apply to clinicians for in vitro fertilization (IVF) therapy. For this purpose, a series of tests are performed to reveal infertility or its causes in men and women before starting the therapy.

The first of the tests to look for male-related causes is the sperm test, also called a spermiogram. In this test, in term of movement ability without problem through the female genital tract, sperm count, shape, and sperm motility are examined.

Besides, the morphological features of the sperm are evaluated by the Kruger method by staining with a special dye. The aim is to get an idea about sperm fertility capacity.

Sometimes, despite many tests being normal, fertilization does not occur and abortions occur even if fertilization occurs. One of the first things to do in such unexplained problems is to evaluate the male partner. Because sperm DNA damage is one of the most important factors of male infertility. In these couples diagnosed with unexplained infertility, DNA fragmentation rate is frequently observed in sperm samples taken from men. However, we cannot determine by the above-mentioned tests whether the sperm quality, which is of great importance for the formation and continuation of pregnancy, is dependent on DNA fragmentation.

Acceptable Sperm DNA Fragmentation Rates

There is no clear answer to reveal the limit of DNA damage rate in terms of fertility. The fact is that as sperm DNA damage rate increases, fertilization success decreases and the development of embryos that can develop from them may be impaired even if fertilization occurs. Again, unfortunately, there is no test that can distinguish the damage as important/unimportant. Generally acceptable DNA damage rates are;

  • <%15       : less fragmentation
  • %15-25   : good to fair
  • %25-50   : fair to poor
  • >50          : extremely poor sperm DNA integrity

Reasons For Sperm DNA Damage Test Request

Some of them are:

  • Unexplained infertility,
  • Recurrent fertilization failure despite interventions,
  • Embryonic development problems,
  • Recurrent abortion

Sperm DNA Fragmentation Test Methods

Many methods are used in studies to evaluate sperm DNA integrity. Commonly used are:

1. TdT-dUTP nick-end-labelling (TUNEL) method,

  • Fluorescent Microscope
  • Flow cytometri

2. Single-cell gel electrophoresis (COMET assay),

3. Sperm Chromatin Dispersion Test (Halosperm Test),

4. Fluorescent in situ hybridization (FISH)

Terminal Uridine Nick-End Labeling (TUNEL) Method

The TUNEL method, which we also do as Denge Laboratory, is the accepted method and is more frequently requested by clinicians. In this method, terminal deoxynucleotidyltransferase (TdT) DNA polymerase enzyme is used. This enzyme adds randomly deoxyribonucleotides (dUTP) to the 3’hydroxyl end of damaged DNA in single or double stranded DNA. Fluorescence added to the medium binds to these dUTPs. The amount of damaged cells is determined by measuring the bound fluorescence intensity with a fluorescent microscope or flow cytometry (Figure-1).

Single-cell Gel Electrophoresis (COMET assay)

It is a sensitive method used to determine male infertility. This method can be done in a neutral or alkaline environment. While only double chain breaks can be detected in neutral buffer, both double chain and single chain breaks can be detected in alkaline buffers. By gel electrophoresis, it is based on the principle that DNA molecules with different molecular weights and different electrical charges migrate differently in the electrical field at alkaline pH. The undamaged DNAs migrate on the gel without losing the integrity and does not form comets (tails). However, because the fragments of damaged DNA have different molecular weights and different electrical charges due to the damage, they migrate at different speeds in the electrical field and form a tail-shaped image. An idea is obtained by evaluating the resulting DNA migration images (Figure-2).

Sperm Chromatin Dispersion Test (Halosperm Test)

Sperm are placed in an agarose matrix gel and an acid solution is added to denature them. The sperm cell membrane and proteins are removed from the environment. Sperm with normal DNA release their DNA to form large halos. Sperm with little or no halo are those that contain fragmented DNA. 

Fluorescent in Situ Hybridization (FISH) Method

Sperm embedded in an agarose matrix or fixed on a slide are exposed to alkaline solution for denaturation. The ratio of single-stranded DNA increases. Elevated DNA fragmentation ratio increases the amount of single-stranded DNA. DNA damage rate is determined by in situ hybridization to DNA with fluorescent labeled probes.

References:

  1. Lewis SEM. The place of sperm DNA fragmentation testing in current day fertility management. Middle East Fertility Society J. 18(2) 2013, 78-82
  2. Bayram H, Cıncık M. Açıklanamayan İnfertil Çiftlerde Erkek Faktörü: Sperm DNA Fragmantasyonu Değerlendirmesi. Tıp Fakültesi Klinikleri. 3(3), 2020. 109-113
  3. Fidan AF. DNA hasar tespitinde tek hücre jel elektroforezi. AKÜ-Fen Bilimleri Dergisi 8(1)
  4. https://www.google.com.tr/search?q=sperm+dna+fragmentation+tunel+assay&tbm=isch&ved=2ahUKEwi93ZvVtP7zAhXBPOwKHT-PBHMQ2-cCegQIABAA&oq=sperm+dna+fragmentation+tunel+assay&gs_lcp=CgNpbWcQAzoECAAQE1DbCVjVF2CYGWgAcAB4AIABxgGIAZYHkgEDMC43mAEAoAEBqgELZ3dzLXdpei1pbWfAAQE&sclient=img&ei=IKqDYb1NwfmwB7-ekpgH&bih=657&biw=1366&hl=tr#imgrc=IubDhlRaM3-HRM (Erişim: 04.11.2021)
  5. Rahiminia T, Yazd EF et al. Sperm chromatin and DNA integrity, methyltransferase mRNA levels, and global DNA methylation in oligoasthenoteratozoospermia. Clin Exper Repr Med 45(1): 2018, 1