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Review
. 2021 Mar;53(2):e13874.
doi: 10.1111/and.13874. Epub 2020 Oct 27.

Sperm DNA fragmentation testing: Summary evidence and clinical practice recommendations

Sandro C Esteves  1   2   3 , Armand Zini  4 , Robert Matthew Coward  5   6 , Donald P Evenson  7   8 , Jaime Gosálvez  9 , Sheena E M Lewis  10   11 , Rakesh Sharma  12 , Peter Humaidan  3   13
Affiliations
Review

Sperm DNA fragmentation testing: Summary evidence and clinical practice recommendations

Sandro C Esteves et al. Andrologia. 2021 Mar.

Abstract

We herein summarise the evidence concerning the impact of sperm DNA fragmentation in various clinical infertility scenarios and the advances on sperm DNA fragmentation tests. The collected evidence was used to formulate 41 recommendations. Of these, 13 recommendations concern technical aspects of sperm DNA fragmentation testing, including pre-analytical information, clinical thresholds and interpretation of results. The remaining 28 recommendations relate to indications for sperm DNA fragmentation testing and clinical management. Clinical scenarios like varicocele, unexplained infertility, idiopathic infertility, recurrent pregnancy loss, intrauterine insemination, in vitro fertilisation/intracytoplasmic sperm injection, fertility counselling for men with infertility risk factors and sperm cryopreservation have been contemplated. The bulk evidence supporting the recommendations has increased in recent years, but it is still of moderate to low quality. This guideline provides clinicians with advice on best practices in sperm DNA fragmentation testing. Also, recommendations are provided on possible management strategies to overcome infertility related to sperm DNA fragmentation, based on the best available evidence. Lastly, we identified gaps in knowledge and opportunities for research and elaborated a list of recommendations to stimulate further investigation.

Keywords: assisted reproductive technology; male infertility; practice guideline; semen analysis; sperm DNA fragmentation.

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Conflict of interest statement

SCE declares the receipt of unrestricted research grants and lecture fees from Merck outside the submitted work. PH reports receipt of unrestricted research grants from Merck, IBSA, Gedeon Richter, and MSD, and lecture fees from Merck, Gedeon Richter, MSD, and IBSA outside the submitted work. AZ declares shares in YAD‐Tech neutraceuticals. DPE is president director of SCSA Diagnostics, a company with a commercial interest in sperm DNA damage. SEML is an employee of Examenlab Ltd., a university spin‐out company with a commercial interest in sperm DNA damage. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
TUNEL assay (Flow Cytometry). TUNEL assay using BD Accuri C6 flow cytometer (top). Boxes (bottom panels) include representative plots of a positive sample. (a) Forward scatter versus side scatter or ‘Plot 1’: Gate is drawn, and small debris and larger nonsperm cells are excluded. Spermatozoa stained with propidium iodide (PI) with a flame‐shaped gate are gated in the forward scatter (FSC) versus side scatter (SSC) plot. (b) Gating strategy for PI positive cells. (c) Plot of a positive sample. SSSC‐A: Side scatter area; FSC‐A: forward scatter area; FL2‐A: fluorescence in the red or propidium iodide channel‐area; FL1‐A: fluorescence in the green or FITC‐area; Q1‐UR: Quadrant 1‐upper right; Q1‐UL: Quadrant 1‐ Upper Left; Q1‐LL: Quadrant 1‐Lower Left and Q1‐LR: Quadrant 1‐Lower Right. Asterisk indicates that virtual gain is applied to the data by aligning with the negative peak of a standard sample with known DNA fragmentation
Figure 2
Figure 2
TUNEL Assay (Fluorescence Microscopy). Visualisation of sperm DNA damage using terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Digoxigenin‐dUTP is incorporated to DNA breaks using a terminal transferase; anti‐digoxigenin‐FITC is used to label the sites where digoxigenin‐dUTP is present (green colour). TUNEL + represents spermatozoa presenting DNA damage. Slides were counterstained with propidium iodide (red colour). TUNEL‐ represents spermatozoa free of DNA breaks
Figure 3
Figure 3
Sperm chromatin structure assay (SCSA). Test data (SCSA Diagnostics, Brookings, USA). Left panel (top box): raw data from a flow cytometer showing each of 5,000 spermatozoa as a single dot on a scattergram. Y‐axis = green fluorescence with 1,024 gradations (channels) of DNA stainability (intact double‐stranded DNA). X‐axis = red fluorescence with 1,024 gradations of red fluorescence (single‐strand DNA). Axes shown are 1,024/10. Line at Y = 75 marks the upper boundary of DNA staining of normal sperm chromatin; above that line are spermatozoa (dots) with partially uncondensed chromatin allowing more DNA stainability. Bottom left corner shows gating out of seminal debris. Middle panel: Raw data from left panel are converted by SCSAsoft software (or equivalent) to red/red + green fluorescence. This transforms the angled sperm display in the left panel to a vertical pattern that is often critical for accurately delineating the percentage of spermatozoa with fragmented DNA. Y‐axis = total DNA stainability versus. X‐axis = red/red + green fluorescence (DFI). Right panel: Frequency histogram of data from middle panel showing computer gating into %DFI and Mean DFI. Bottom box: SCSAsoft software calculations of mean of two independent measures of mean and standard deviation (std dev) of median DFI, %DFI and %HDS (high DNA stainability)
Figure 4
Figure 4
Alkaline Comet assay under fluorescence microscopy. (a) Sperm sample of a patient exhibiting elevated sperm DNA fragmentation (SDF). Several comets are shown which represent spermatozoa with DNA fragmentation. The longer and brighter the ‘Comet’ tail, the more fragmentation is present. (b) Spermatozoon with DNA fragmentation (open arrow), and another one with a hardly visible ‘Comet’ tail (white arrow), representing a cell with minimal DNA fragmentation. As the Comet test measures the amount of damage in each cell, it is rare to find a perfect spermatozoon with 0% damage, even from fertile donors
Figure 5
Figure 5
Sperm Chromatin Dispersion test (SCD) under bright‐field microscopy (Halosperm, Halotech DNA, SL, Madrid, Spain). (a) Sperm sample of an individual presenting with normal level of sperm DNA fragmentation (SDF). (b) Sperm sample of a patient with varicocele presenting with elevated SDF. Open arrowheads indicate spermatozoa with halos of dispersed chromatin representing a normal DNA molecule with no fragmented DNA. Black arrowheads indicate spermatozoa with small or absent halos of dispersed chromatin, representing spermatozoa with fragmented DNA. Arrows in ‘b’ indicate spermatozoa with fragmented‐degraded DNA
Figure 6
Figure 6
Two‐dimensional (two‐tail) Comet assay for simultaneous mapping of single‐strand DNA damage (SS‐DB; Y‐axis) and double‐strand DNA damage (DS‐DB; X‐axis) in spermatozoa. (a) Normal spermatozoa showing DNA displacement in the Y‐axis due to the structural presence of alkaline labile sites. (b) Presence of DS‐DB in the X‐axis after electrophoresis under neutral conditions. (c) Presence of SS‐DB along the Y‐axis after alkaline electrophoresis. (d) Presence of both SS‐DB and DS‐DB affecting a single spermatozoon. Arrows indicate the perpendicular sense of each electrophoresis
Figure 7
Figure 7
Pictorial summary of the recommendations for sperm DNA fragmentation testing and possible management in couples with elevated sperm DNA fragmentation. IUI, intrauterine insemination; IVF, in vitro fertilisation; ICSI, intracytoplasmic sperm injection; RPL, recurrent pregnancy loss
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References

    1. Abdelbaki, S. A. , Sabry, J. H. , Al‐Adl, A. M. , & Sabry, H. H. (2017). The impact of coexisting sperm DNA fragmentation and seminal oxidative stress on the outcome of varicocelectomy in infertile patients: A prospective controlled study. Arab Journal of Urology, 15, 131–139. 10.1016/j.aju.2017.03.002 - DOI - PMC - PubMed
    1. Aboulmaouahib, S. , Madkour, A. , Kaarouch, I. , Sefrioui, O. , Saadani, B. , Copin, H. , Benkhalifa, M. , Louanjli, N. , & Cadi, R. (2018). Impact of alcohol and cigarette smoking consumption in male fertility potential: Looks at lipid peroxidation, enzymatic antioxidant activities and sperm DNA damage. Andrologia, 50(3), e12926. 10.1111/and.12926 - DOI - PubMed
    1. Agarwal, A. , Cho, C. L. , Majzoub, A. , & Esteves, S. C. (2017). The Society for Translational Medicine: Clinical practice guidelines for sperm DNA fragmentation testing in male infertility. Translational Andrology and Urology, 6(Suppl. 4), 720–733. 10.21037/tau.2017.08.06 - DOI - PMC - PubMed
    1. Agarwal, A. , Gupta, S. , Du Plessis, S. , Sharma, R. , Esteves, S. C. , Cirenza, C. , Eliwa, J. , Al‐Najjar, W. , Kumaresan, D. , Haroun, N. , Philby, S. , & Sabanegh, E. (2016). Abstinence time and its impact on basic and advanced semen parameters. Urology, 94, 102–110. 10.1016/j.urology.2016.03.059 - DOI - PubMed
    1. Agarwal, A. , Hamada, A. , & Esteves, S. C. (2012). Insight into oxidative stress in varicocele‐associated male infertility: Part 1. Nature Reviews Urology, 9, 678–690. 10.1038/nrurol.2012.197 - DOI - PubMed