DNA Fragmentation Dynamics in Fresh Versus Frozen Thawed Plus Gradient-Isolated Human Spermatozoa

Gosálvez, Jaime; Torre, J. de la; López‐Fernández, Carmen; Pérez-Gutiérrez, Laura; Ortega, Leonor; Caballero, Pedro; Núñez, Rocio

doi:10.3109/19396360903515430

Cited by 34 publications

(22 citation statements)

References 36 publications

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“…12 An aliquot of 50 ml of each DGC-recovered semen sample was used for the SDF assessment. To avoid the undesirable effect of iatrogenic DNA damage to the sperm after processing, which produces a variable and dynamic increase in SDF values, 13,14 all sperm samples were assessed for SDF immediately after sperm selection.…”

Section: Sample Collection and Preparationmentioning

confidence: 99%

The ability of sperm selection techniques to remove single- or double-strand DNA damage

Enciso¹,

Iglesias²,

Galán³

et al. 2011

Asian J Androl

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A wide variety of techniques for the preparation of sperm are currently available, of which the most commonly employed are densitygradient centrifugation (DGC) and swim-up (SUP). To date, these methods appear to be effective in selecting functional sperm for assisted reproduction techniques (ART), but they may have negative effects on sperm DNA. In this study, the ability of these semen processing techniques to eliminate spermatozoa containing single-and double-strand DNA damage was assessed by the two-tailed comet assay and the sperm chromatin dispersion test in 157 semen samples from patients seeking assisted reproduction treatment. Our results indicated that SUP and DGC are equally efficient in eliminating spermatozoa containing double-strand DNA damage and sperm with highly damaged (degraded) DNA, as characterized by the presence of both single-and double-strand DNA breaks. However, DGC is more efficient than SUP in selecting spermatozoa that are free from single-strand DNA damage. Future studies should characterise the importance of the various types of DNA damage and examine the sperm processing protocols used in each laboratory to determine their ability to eliminate DNA damage and hence, prevent the potential transmission of genetic mutations via ART.

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Section: Sample Collection and Preparationmentioning

confidence: 99%

The ability of sperm selection techniques to remove single- or double-strand DNA damage

Enciso¹,

Iglesias²,

Galán³

et al. 2011

Asian J Androl

Self Cite

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“…This is an important aspect of the study, since DNA damage, far from being a static parameter, increases as the samples are incubated in vitro. This effect is observed both in fresh 38 and in cryopreserved thawed samples. 39 Consequently, the observed values at the time of ICSI fertilisation can be very different to those assessed before or after the time of fertilisation.…”

Section: Oocytes and Spermatozoamentioning

confidence: 80%

“…Congruent with this idea are those reports claiming that the predictive value of SDF is low when ICSI is used. 14,15,21 It should be noted that while the incidence of SDF in the selected population may in fact decline after sperm selection, there is still a reasonable chance of accidently selecting one of the remaining underlying sub-population of DNA damaged spermatozoa that was not initially excluded in the selection procedure, i.e., a certain level of damaged spermatozoa still remains in the sample, even under the most stringent and rigorous conditions for sperm selection (see data in Zini et al 42 Gosálvez et al 38 and Enciso et al 43 ). By way of an example, if we analyse the data shown by Santiso et al 48 in Figure 1, in some cases and after a swim-up procedure, we are selecting sperm subpopulations containing shorter telomeres than those considered as normal.…”

Section: Sperm Selection and Sperm Dna Damagementioning

confidence: 99%

“…These sperm are essentially cryptic in terms of SDF detection, waiting 'under the surface', ultimately to be detected, depending on the degree of damage imposed by ex vivo manipulation or iatrogenic damage prior to use in ART. We proposed that dynamic assessment of SDF by incubation of sperm in vitro 38,39 would allow for the detection of this sub-population; 61 this cryptic damage is essentially ignored by single assessments of DNA damage. It is possible that this cryptic subpopulation may contain sufficient DNA damage to prevent pregnancy, especially if the oocyte is not capable of DNA repair.…”

Section: The Iceberg Effectmentioning

confidence: 99%

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Can DNA fragmentation of neat or swim-up spermatozoa be used to predict pregnancy following ICSI of fertile oocyte donors?

Gosálvez

Caballero²,

López‐Fernández³

et al. 2013

Asian J Androl

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This study compared the potential of assessing sperm DNA fragmentation (SDF) from neat semen and the subsequent swim-up (SU) procedure to predict pregnancy when conducting ICSI of fertile donor oocytes. Infertile females (n581) were transferred embryos resulting from intracytoplasmic sperm injection (ICSI) of their partner's spermatozoa and proven donor oocytes. This model normalized the impact of female factor in putative sperm DNA repair. Semen was blindly assessed for SDF using Halosperm immediately following ejaculation (NS) and after swim-up at the time of ICSI fertilisation. There was a decrease in SDF values of the ejaculated semen sample following the swim-up protocol (P50.000). Interestingly, pregnancy could be equally predicted from SDF values derived from either neat or swim-up semen samples. Receiver operator curves and the derived Youden's indices determined SDF cutoff values for NS and SU of 24.8% and 17.5%, respectively. Prediction of pregnancy from NS SDF had a sensitivity of 75% and a specificity of 69%, whereas for SU SDF was 78% and 73%, respectively. While increased levels of SDF negatively impact reproductive outcome, we have shown that a reduction in SDF following sperm selection using ICSI with proven donor oocytes is not mandatory for achieving pregnancy. This suggests that a certain level of DNA damage that is not detectable using current technologies could be impacting on the relative success of assisted reproductive technology (ART) procedures. Consequently, we propose a modification of the so called 'iceberg model' as a possible rationale for understanding the role of SDF in reproductive outcome.

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“…However, it has still not been clearly established whether there exists a direct relationship between cryopreservation and sperm DNA damage. Gosálvez et al (2010), suggest that the incidence of increased single-and/or double-stranded DNA breaks following cryopreservation is not a direct consequence of the freezing process, but rather, that modifications to the chromatin-SNBP interaction as a result of freeze-thawing predisposes the sperm DNA to fragmentation that manifests, not immediately, but over time following thawing. In most mammalian species, including but not limited to the koala (Johnston et al 2012a), stallion ), ram and Asian elephant (Elephas maximus; Imrat et al 2012), the basal level of sperm DNA damage observed immediately after thawing does not differ significantly from that observed in fresh spermatozoa.…”

Section: The Effect Of Cryopreservation On Dna Integritymentioning

confidence: 99%

Amphibian sperm DNA fragmentation

Pollock¹

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There are extremely limited studies that have investigated DNA fragmentation in amphibians and even less information exists pertaining to DNA fragmentation in the spermatozoa.A DNA fragmentation assay applied and validated for amphibian spermatozoa would not only provide the first evidence of this phenomenon in a new major taxon, but also prove a valuable tool for developing improvements in ART used in the captive propagation of threatened and endangered species. Consequently, the fundamental aim of this project was the development and validation of the sperm chromatin dispersion (SCD) test for amphibian spermatozoa with the purpose of investigating the dynamic behaviour of amphibian sperm DNA in response to cryopreservation.The SCD test was first applied to African clawed frog (Xenopus laevis) as an amphibian sperm model using a species-specific modified lysing solution for protein depletion. Sperm DNA fragmentation (SDF) was assessed immediately following activation of sperm motility (T0) and again after one hour and 24 hours of incubation in order to produce a range of spermatozoa with differing levels of DNA damage. The SCD procedure resulted in the production of three nuclear morphotypes; amphibian sperm morphotype 1 (ASM-1) and ASM-2 showed no evidence of DNA damage, whereas ASM-3 spermatozoa were highly fragmented with large halos of dispersed DNA fragments and a reduced nuclear core. Levels of SDF revealed by the SCD test were highly correlated with results produced using in situ nick hybridisation with DNA-specific molecular probes (ISNT) and the double comet assay (r = 0.9613). The alkaline step of the double-comet assay revealed the extensive presence of structural single-stranded DNA breaks (SSB) and provided grounds for the suggestion that alkali labile sites (ALS) are a constitutive feature of African clawed frog sperm chromatin.SDF is a dynamic process and has been shown to increase with incubation at room temperature as well as following cryopreservation. However, the mechanisms of DNA cryoinjury and the resulting effect on the potential reproductive output of ART remain open to interpretation.Consequently, dynamic changes to the basal level of DNA fragmentation were examined in fresh African clawed frog spermatozoa in comparison with the post-thaw integrity of cryopreserved (-80 °C) and frozen (-20 °C) sperm DNA. SDF was examined initially at T0 and over incubation of up to 60 minutes. The difference in SDF was only marginal for fresh, frozen (-20 °C) and cryopreserved (-80 °C) spermatozoa examined at the onset of the experiment immediately upon thawing, however, a significant increase (p = 0.004) in SDF, albeit of a low magnitude, was observed in activated ii cryopreserved (-80 °C) spermatozoa following incubation. Although African clawed frog spermatozoa appeared to be highly resistant to cryopreservation-induced DNA damage, cryopreserved-thawed spermatozoa yielded a significantly lower (p = 0.0065) fertilisation rate than fresh spermatozoa.In order to provide further insight into cryo...

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DNA Fragmentation Dynamics in Fresh Versus Frozen Thawed Plus Gradient-Isolated Human Spermatozoa

Cited by 34 publications

References 36 publications

The ability of sperm selection techniques to remove single- or double-strand DNA damage

The ability of sperm selection techniques to remove single- or double-strand DNA damage

Can DNA fragmentation of neat or swim-up spermatozoa be used to predict pregnancy following ICSI of fertile oocyte donors?

Amphibian sperm DNA fragmentation

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