Sperm DNA Fragmentation

Dr. Yasmin Magdi Abd-Elkreem

overview• Conventional semen analysis test remains the most

widely used predictor for male infertility diagnosis .• based on the descriptive microscopic evaluation of

ejaculate sperm, including concentration, motility and morphology.

• not discriminate effectively between the sperm of fertile and infertile men.

• DNA integrity assessment has been suggested as an independent additional marker of fertility to give most conclusive information on the sperm DNA.

overview• Spermatogenesis : Complex, diverse, 74 days• Sperms prone for disruption by potential

targets.• Most significant - free radicals.• Species with unpaired electrons, highly

reactive.• Although sperm DNA fragmentation does not

constitute a mutation in its own, it still is a promutagenic change of the male genome.

Etiology of sperm DNA damage• The term ‘chromatin’ refers to the

macromolecular complex that contains the intranuclear DNA.

• The structure of chromatin is stabilized by interactions between its main compounds, DNA and DNA-binding proteins.

• Sperm cell chromatin is characterized by highly condensed structure.

• Condensation of sperm DNA is necessary in order to accommodate the paternal genome into the relatively small sperm cell head, to repress gene expression during spermiogenesis and to protect the paternal genetic message making it inaccessible to nucleases or mutagens.

Main etiologies of sperm DNA damage

Testicular cancer AgingEnvironmental toxins ObesityHormonal disturbances

Smoking

Testicular hyperthermia

Varicocele

Drugs , chemotherapy and radiation

Inflammation

Cryopreservation in ART programs

Febrile illness

Spinal cord injury

Etiology of sperm DNA damage• The etiology of sperm DNA damage is

most probably multi-factorial but compromised chromatin remodeling, oxidative stress and abortive apoptosis are commonly described theories.

• Sperm DNA damage may arise from combinations of all three mechanisms.

1.Abnormal chromatin remodeling

2.Oxidative stress3.Abortive apoptosis

Abnormal chromatin remodeling

• Deficiencies in recombination and chromatin packaging during spermiogenesis.

• occur due to deficient protamination or abnormalities in protamine content.

1. Due to abnormal function of transcriptional or translational regulators (a general abnormality in spermatogenesis).2. induce apoptosis which could explain the link between underprotamination and poor sperm quality in infertile men (checkpoint regulators for spermatogenesis). 3. makes spermatozoa more vulnerable to attack by nucleases, free radicals that can cause oxidative stress or (environmental) mutagens.4. may also be compromised by abnormal topoisomerase II activity (responsible for break and repair DNA during protamination)

Oxidative stress

• ROS production is a physiological phenomenon that is normally balanced by anti-oxidants.

• Sperm cells have no defense mechanism, apart from the characteristic tight packaging of the DNA and anti-oxidants present in seminal plasma.

• DNA fragmentation can be caused by oxidative stress, due to free radicals [reactive oxygen species (ROS)].

Abortive apoptosis

• Normal spermatogenesis is a dynamic process in which cell production and cell death are well balanced.

• Cells to be eliminated will enter a cascade of reactions leading to apoptotic cell death.

• Cutting of sperm DNA by endonucleases is one of the earliest events in this cascade, that is completed by phagocytosis and elimination of appropriately earmarked sperm cells by Sertoli cells.

• If this system does not operate efficiently and the process is not completed, damaged cells may escape apoptosis.

DNA damage and infertility• sperm DNA fragmentation (SDF) tests can

differentiate fertile from infertile males.• The assessment of DNA integrity in sperm

could be an independent marker of fertility.• higher levels of chromatin damage in men

with severe sperm defects.• The negative impact of high levels of

sperm DNA damage negatively effect on natural pregnancy.

DNA fragmentation and semen parameters• There is a strong association between the

presence of nuclear DNA damage in the mature spermatozoa of men and poor semen parameters.

• sperm motility, concentration, and morphology had significant positive correlations with non-apoptotic and DNA non-fragmented sperm.

• The application of these findings in clinical practice can ultimately increase implantation and pregnancy rates in patients where ICSI is the treatment of choice.

Our data conclusively showed a negative correlation between degree of DNA fragmentation and morphology of sperm in

native semen samples.

DNA damage and ART• The structure of spermatozoa DNA is very

unique, highly specialized in order to control time-appropriate maturation of the zygote.

• Damage to sperm DNA may occur as a result of uni or multi-factors. This damage thereby may have negative effects on ART procedures, and could lead to failure of fertilization.

• Sperm DNA damage significantly contributes to the growing number of infertility cases, and should be a part of a modern andrology lab.

Assays to quantify sperm DNA damage• Different methods may be used to evaluate the

status of the sperm chromatin for the presence of abnormalities or simply immaturity

• staining techniques: acidic aniline blue (AAB) and toluidine blue (TB) stains,

• fluorescent staining techniques: sperm chromatin dispersion (SCD) test, chromomycin A3 (CMA3), DNA breakage detection– fluorescent in situ hybridization assay (DBD–FISH), in situ nick translation (NT), and flow cytometricbased sperm chromatin structure assay (SCSA).

• Some assays employ more than one method for the analysis of their results.: acridine orange (AO) and terminal deoxynucleotidyl transferase-mediated fluorescein- deoxyuridine triphosphate-nick end labeling (TUNEL) assays.

• Methods less frequently used: high-performance liquid chromatography (HPLC).

1. Acidic aniline blue (AAB) stain

Principle : • The AAB stain discriminates between lysine-rich

histones and arginine/cysteine-rich protamines.• This technique provides a specific positive

reaction for lysine and reveals differences in the basic nuclear protein composition of ejaculated human spermatozoa.

• Histone-rich nuclei of immature spermatozoa are rich in lysine and will consequently

• take up the blue stain. • On the other hand, protaminerich nuclei of

mature spermatozoa are rich in arginine and cysteine and contain relatively low levels of lysine, which means they will not be stained by aniline blue.

Technique:I. Slides are prepared by smearing 5 μl of either

raw or washed semen sample. The slides are air-dried and fixed for 30 minutes in 3% glutaraldehyde in phosphate- buffered saline (PBS).

II. The smear is dried and stained for 5 minutes in 5% aqueous aniline blue solution (pH 3.5).

III. Sperm heads containing immature nuclear chromatin stain blue, and those with mature nuclei do not take up the stain.

IV. The percentage of spermatozoa stained with aniline blue is determined by counting 200 spermatozoa per slide under bright field microscopy.

1. Acidic aniline blue (AAB) stain

1. Acidic aniline blue (AAB) stain

Clinical significance:• a clear association between abnormal sperm

chromatin end male infertility.• The correlation between the percentage of aniline

blue-stained spermatozoa and other sperm parameters remains controversial.

• Good predictor for IVF.• It cannot determine the fertilization potential and the

cleavage and pregnancy rates following ICSI. Advantages and limitations:• simple and inexpensive• ordinary microscope

2. Sperm chromatin dispersion test

Principle:• The SCD test is based on the principle that when

sperm are treated with an acid solution prior to lysis buffer, the DNA dispersion halos that are observed in sperm nuclei with nonfragmented DNA after the removal of nuclear proteins are either minimally present or not produced at all in sperm nuclei with fragmented DNA.

Technique: • The spermatozoa are immersed in a melted

agarose matrix at 37°C. On a slide, a drop of 8 ul of mixed sperm agarose initially treated with an acid solution (solution 1) for 7 min to denature the DNA with DNA breaks, and directly treated with lysing solution (solution 2) for 20 min to deproteinize the nucleoids.

• After removal of nuclear proteins, fixation was done using ethanol and then slide was stained using solution 3 for 6 minutes and solution 4 for 7 minutes.

• Non-fragmented sperm DNA appears with a core and with a peripheral halo of dispersion of DNA loops. Fragmented sperm DNA appears with very small or no halo of DNA dispersion.

2. Sperm chromatin dispersion test

Advantages and limitations:• It does not require the determination of color or

fluorescence intensity. • Simple, fast, and reproducible, and its results are

comparable to those of the SCSA.• little is known about its limitations and its clinical

significance.

2. Sperm chromatin dispersion test

Conclusion

• Sperm chromatin assessment is an independent measure of sperm quality that provides better diagnostic and prognostic capabilities than standard sperm parameters for male fertility potential (idiopathic infertility).

• There are multiple assays that may be used for evaluation of the sperm chromatin status.

• Most of these assays have many advantages as well as limitations. The choice of which assay to be performed depends on many factors.

• The establishment of a cutoff point between normal levels in the average fertile population and the minimal levels of sperm DNA integrity required for achieving pregnancy still emains to be investigated. Except for the SCSA.

References • Gardner DK, Weissman A, Howles CM, Shoham Z.

Textbook of Assisted Reproductive Techniques. 3rd ed. Vol. 1. In: Agarwal A, Erenpreiss J, Sharma R. Sperm chromatin assessment. United Kingdom: Informa healthcare, 2009:67-84.

• Aspinder Singh and Ashok AgarwalThe Role of Sperm Chromatin Integrity and DNA Damage on Male InfertilityThe Open Reproductive Science Journal, 2011, 3, 65-71.

• A.Agarwal and Tamer M.Said. Role of sperm chromatin abnormalities and DNA damage in male infertility. Human Reproduction Update, Vol.9, No.4 pp. 331±345, 2003

Thank You !

For contact: E-mail: Yas.magdi@hotmail.com