Changes in DNA fragmentation during sperm preparation for intracytoplasmic sperm injection over time

We assessed sperm DNA fragmentation (TUNEL) at different time periods before ICSI, and found DNA fragmentation significantly decreased after gradient and samples with TUNEL >20% were more susceptible to a significant increase over time.

Natalia Rougier, M.D., Heydy Uriondo, M.Sc., Sergio Papier, M.D., Miguel Angel Checa, M.D., Ph.D., Carlos Sueldo, M.D., Cristian Alvarez Sedó, M.Sc.

Volume 100, Issue 1, Pages 69-74, July 2013


To compare the DNA fragmentation of semen samples established by terminal deoxynucleotide transferase–mediated dUTP nick-end labeling (TUNEL) after incubation in polyvinylpyrrolidone (PVP) and hyaluronic acid (HA) for different time periods.

Comparative prospective study.

Center for reproductive medicine.

Twenty-seven semen samples from infertile patients.


Semen analysis and DNA fragmentation assays (TUNEL) were performed. Two groups were established: A) normal TUNEL (<20%); and B) Abnormal TUNEL (≥20%). TUNEL was performed in neat (T0), postgradient (TG), 1-hour postgradient (TG1), and 2-hour postgradient (TG2) samples and in TG2 samples after 0.5, 1.0, and 1.5 hours of incubation in PVP or HA.

TUNEL levels were significantly reduced after gradient separation compared with neat values. In group A, TUNEL levels were significantly higher in the TG2 + 1.5 hours in PVP and HA samples but did not reach abnormal levels. In group B, TUNEL levels were significantly higher in the TG2 + 1 hour in PVP and HA samples.

Sperm DNA fragmentation significantly decreased after centrifugation gradient, regardless of the initial levels of the sample. Samples with TUNEL ≥20% were more susceptible to a significant increase in DNA fragmentation over time, with similar increases being observed over time for samples that were incubated in HA or PVP. These data may be relevant for sperm preparation for intracytoplasmic sperm injection.

  • This study further confirmed that sperm DNA damage is a post-spermatogenesis phenomenon and the need for technique of evaluation a single sperm for IVF/ICSI. Perhaps men with elevated DFI and recurrent pregnancy loss/failed prior IVF cycles might be better served with in-cycle testicular extraction to facilitate future IVF.

  • This article highlights the need for timeliness when using freshly ejaculated sperm for ICSI, especially when there is >20% DNA fragmentation via TUNEL. It also demonstrates that good quality sperm with <20% DNA fragmentation maintained their quality over time.

    I wonder if this also holds true for sperm obtained via TESE or MicroTESE?

    I also wonder if empiric therapy for the male with antioxidant/vitamin therapy prior to and during fresh cycles could decrease ROS and thereby improve the DNA fragmentation rate?

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