1 School of Biological Sciences, University of Manchester, Manchester and 2 Department of Reproductive Medicine, St Mary's Hospital, Manchester, UK
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Abstract |
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Key words: Comet analysis/DNA damage/fertility/human/sperm
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Introduction |
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To assist in the risk assessment of ICSI, it would be appropriate to develop methods to measure DNA damage in the sperm and to correlate this with biological outcomes. DNA abnormalities in sperm are well documented. Cytogenetic analysis of sperm chromosomes has demonstrated sperm aneuploidy, which, although low in frequency, is associated with infertility and adverse pregnancy outcome (Egozcue et al., 2000; Shi and Martin, 2000
). Genetic information in the sperm genome may be mutated or deleted altogether which may be the cause of some cases of male infertility (Hargreave, 2000
; Foresta et al., 2001
). However, it has also become clear that other subtle genetic changes may be occurring. The nature of these is not well documented but could give rise to a spectrum of responses including: failure of fertilization, failure of preimplantation embryo development, early pregnancy loss or fetal abnormalities (Hales and Robaire, 1997
; Sakkas et al., 2000
; Shen and Ong, 2000
).
Several techniques are available to examine the integrity of sperm DNA. The sperm chromatin structure assay (SCSA) measures the susceptibility of the DNA to acid denaturization (Evenson et al., 1999). Abnormal chromatin structure is measured by flow cytometry that records the ratio of denatured to native DNA. High ratios correlate with sperm concentration and sperm head abnormalities. Additionally it has been shown that if the percentage of cells with abnormal ratios exceeds 3040% then fertility is unlikely (Evenson et al., 1999
; Larson et al., 2000
; Spano et al., 2000
). DNA strand breaks in sperm have also been directly detected by a variety of techniques. The terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) assay identifies double and single DNA strand breaks by enyzmatically labelling the free 3' OH end of the DNA with a fluorescent substrate. TUNEL-positive cells are identified either microscopically or by fluorescence-activated cell sorting (FACS). In the majority of samples only ~5% of sperm have TUNEL-detectable DNA damage (Manicardi et al., 1995
; Aravindan et al., 1997
; Sun et al., 1997
; Donnelly et al., 2000
; Irvine et al., 2000
; Oosterhuis et al., 2000
; Ramos and Wetzels, 2001
). Sperm with DNA strand breaks may persist into the ejaculate because of a failure in the mechanism of apoptosis, which normally eliminates them during spermatogenesis (Sakkas et al., 1999
) or alternatively may arise in the reproductive tract as a result of oxidative damage (Aitken and Krausz, 2001
). Fertilization after ICSI or IVF is reduced if sperm are retrieved from ejaculates containing high numbers of TUNEL-positive cells. Additionally, it has been shown that paternal smoking increases sperm DNA damage measured by the TUNEL technique and this has been suggested to account for the increase in childhood cancer (Sun et al., 1997
; Potts et al., 1999
).
The Comet assay is extensively used in somatic cells to measure genotoxic damage, especially single and double strand breaks, and was originally applied to sperm by Singh (Singh et al., 1989). This study demonstrated that sperm DNA was extremely labile in the presence of alkali and it was difficult to distinguish the level of damage between individual sperm. However, the Comet assay has been modified and used in other laboratories to investigate DNA damage in sperm and its relationship to infertility. Irvine and colleagues showed that men attending infertility clinics had a higher level of DNA damage in their sperm, which was also negatively related to semen concentration (Irvine et al., 2000
). Similar DNA changes could be generated by in-vitro treatments producing oxidative damage (Aitken et al., 1998
; Twigg et al., 1998a
; Donnelly et al., 1999
; Shen and Ong, 2000
; Ramos and Wetzels, 2001
). However, another study did not detect a difference in DNA damage between infertile men and normal controls (Hughes et al., 1996
), although subsequent reports suggest that DNA damage measured by the Comet assay is selectively increased in the sperm from infertile men after cryopreservation or in-vitro X-ray radiation (Hughes et al., 1996
; Donnelly et al., 2001). Results from the Comet assay are also correlated with DNA damage measured by the TUNEL and SCSA methods (Aravindan et al., 1997
; Donnelly et al., 2000
). Close inspection of these publications reveals significant differences in the protocols used to treat the cells prior to electrophoresis in an alkaline buffer. These methodological differences have arisen from the difficulties encountered in releasing the DNA from the sperm head due to the unique DNA compaction (Ward and Coffey, 1991
). Combined with the alkaline lability of sperm DNA, the difficulties encountered in releasing the DNA for electrophoresis have hindered the development of the Comet assays for comparative studies of male reproduction. Our laboratory has recently developed a neutral Comet assay to provide quantitative measures of DNA damage in human and murine sperm (Haines et al., 1998
). This method will produce a linear increase in DNA damage of sperm irradiated in vitro at doses that would be expected to produce DNA strand breakage. We have demonstrated that, in the mouse, spermatogonial radiation with external X-rays or internal isotopic contamination results in the appearance of substantial damage in DNA of the sperm (Haines et al., 2001
). These in-vivo treatments are known to be genotoxic and adversely effect fertility and reproduction. Additionally we have also shown that during chemotherapy using fludarabine of a patient with chronic lymphocytic leukaemia there was a substantial increase in the sperm DNA damage when measured by the Comet assay (Chatterjee et al., 2000
). DNA damage may be a good biomarker to relate to fertility problems; moreover, as the Comet assay is technically straightforward and inexpensive it may be suitable for routine measurement of DNA damage once validated by independent laboratories. We have therefore examined DNA damage in men attending for IVF/ICSI treatment using the Comet assay and correlated the DNA damage profiles in their semen with treatment cycle outcome.
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Materials and methods |
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IVF/ICSI treatment cycles
Ovarian stimulation was achieved using a conventional long protocol down-regulation involving pituitary desensitization with buserelin. Exogenous FSH was administered by a step-down protocol with an initial starting dose between 75 and 450 IU and adjusted throughout the cycle following monitoring of serum estradiol levels. HCG was administered when three or more follicles reached 17 mm, and oocytes were recovered 36 h later by ultrasound-guided retrieval. Semen samples were produced by masturbation and analysed for sperm concentration (x106/ml), percentage of sperm which were progressively motile, and percentage abnormal forms, then prepared by density gradient centrifugation using standard protocols. Briefly, sperm were washed free of seminal plasma using sperm culture medium (Medi-Cult UK Ltd, Redhill, Surrey, UK) layered upon a 40: 80% PureSperm (Hunter Scientific, Saffron Walden, Essex, UK) gradient, centrifuged at 600 g for 20 min and resuspended in the sperm culture medium (Horne et al., 1997
). For IVF treatment, oocytes were inseminated 46 h post recovery with ~1x105 sperm/ml. For ICSI treatment, single motile sperm of normal morphology were microinjected into each oocyte using standard protocols (Van Steirteghem et al., 1993
). An aliquot of each sample used for IVF or ICSI was immediately snap-frozen at 20°C until analysed for DNA damage by the Comet assay. Oocytes were cultured overnight at 37°C in 200 µl of Universal IVF medium (Medi-Cult UK).
Fertilization and embryo development
Oocytes were assessed for the presence of two pronuclei 1618 h after insemination, indicative of normal fertilization (day 1). Up to six normally fertilized zygotes were maintained in culture to cleave to the 2- or 4-cell stage (day 2), whilst excess zygotes were immediately cryopreserved. Two embryos, or occasionally one or three, were replaced in the uterus on day 2 following selection based on developmental stage and morphological criteria (Steer et al., 1992). Embryos received a stage score, with one point awarded for each intact blastomere, and a morphological grade, with 4 points awarded for even blastomeres with no fragmentation, 3 points for embryos with up to 10% fragmentation and so on. This scoring system is thought to reflect anucleate fragmentation resulting from cytokinesis and/or loss of blastomeres by apoptosis. The percentage of embryos which failed to cleave (remain at 1-cell) was also scored. Cryopreserved embryos were thawed and replaced in subsequent cycles at patient request. Implantation of fresh or frozen embryos was assessed by measuring serum ß-HCG levels 14 days after replacement, clinical pregnancy was confirmed by presence of a fetal heart on scan at 6 weeks, and live birth data were obtained by patient follow-up.
Comet DNA damage assay
Single cell gel electrophoresis of sperm DNA (Comet assay) was performed as previously described (Haines et al., 1998). Briefly, sperm cells were thawed rapidly at room temperature, cast into miniature agarose gels on microscope slides and lysed in situ to remove DNA associated proteins and allow the compacted DNA in the sperm to relax. Lysis buffer (Tris 10 mmol/l, 0.5 mol/l EDTA and 2.5 mol/l NaCl, pH 10) contained 1% Triton X-100, 40 mmol/l dithiothreitol and proteinase K, 100 µg/ml). Microgels were then electrophoresed (20 min at 25V/0.01A) in neutral buffer (Tris 10 mmol/l containing 0.08 mol/l boric acid and 0.5 mol/l EDTA, pH 8.2), during which the damaged DNA migrated from the nucleus towards the anode. DNA was visualized by staining of the slides with the fluorescent DNA binding dye SYBR Green I (Molecular Probes, Oregon, USA) and sperm identified by size and the presence of a tail. Comet measurements performed were tail length, tail moment and percentage tail DNA using a Nikon Optiphot II epifluorescence microscope and Comet Assay II software (Perceptive Instruments, Haverhill, UK); 100120 cells were analysed per semen sample (two duplicate sample slides, 50100 randomly selected cells scored per slide, up to 200 sperm in total). Measurements between the two slides were highly reproducible. For example, when the mean moments were compared for 30 randomly selected samples with moments ranging from 0.7 to 30, the values between the two slides were highly correlated (P < 0.0001, r2 = 0.975). Since intra-sample variation in this assay is so low as to be negligible, data are presented throughout as the mean of the sperm analysed on the two slides.
Statistical analysis
Data were analysed using SPSS software (SPSS Inc., Chicago, IL, USA). Comet parameters were subjected to further analysis using mean (if normally distributed) or median (if not normal) values. Principle component analysis was used to separate sperm into two populations, which were compared using the non-parametric MannWhitney U-test. Multivariate stepwise regression analysis was used to establish relationships between Comet parameters and semen analysis or treatment cycle parameters.
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Results |
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The summary results from the statistical analysis for all the samples are given in Table III. For many parameters there was no correlation with the measurements of DNA damage. However, both Comet tail length and moment were significantly correlated with age (P = 0.023 and P = 0.032 respectively) indicating that between the ages of 2947 years the levels of DNA damage in sperm increased significantly. Additionally, tail length increased with sperm motility (P = 0.011) and tail moment increased as the proportion of abnormal forms increased (P = 0.013). Table IV
presents the data analysed according to whether the sperm were used for either IVF or ICSI. IVF and ICSI men were not significantly different in age or semen characteristics such as volume or sperm concentration. However, ICSI samples did have a lower motility (P = 0.02) and percentage morphologically normal forms (P = 0.04), as expected. IVF and ICSI samples did not differ significantly in their median tail moment or length. In the ICSI group, the associations between DNA damage and age and motility were preserved, whereas the correlation with abnormal forms was lost (although this was not strong in the overall analysis). Interestingly there was a strong inverse correlation between sperm concentration and DNA damage. Furthermore, analysis of embryo cleavage showed that as DNA damage measured by the tail moment increased, embryo cleavage was impaired. Only one significant correlation was obtained for the IVF patientsbetween tail length and motility (%)which confirms the association seen in the other analyses. However, as the IVF group size was small it is unlikely that the analysis possessed the power to reveal or confirm the associations documented in Tables III and IV
(ICSI).
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Discussion |
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It is also important to consider the type of DNA damage the Comet technique is measuring, especially as the methods used for analysis of sperm vary between laboratories. Comet analysis of somatic cells electrophoresed under alkaline conditions measures single DNA strand breaks, whereas neutral conditions reportedly measure double strand breaks. However, many forms of structural damage can be converted to strand breaks during cell preparation and electrophoresis (Collins et al., 1997; Olive, 1999
). Early attempts to apply the Comet assay to sperm under alkaline electrophoresis conditions to measure single strand DNA breaks resulted in the majority of DNA migrating into the Comet tail (Singh et al., 1989
). This also occurs using the protocol we have used here and has been ascribed to the abundant alkaline-sensitive sites in the sperm DNA and reflects the fragility of the structure in vitro. More recently, several methods have been developed which rely upon harsh biochemical treatments, with or without proteinase K and reducing agents. These lyse the sperm cell, allowing the DNA to decondense by removing nuclear proteins and their crosslinks. The differences in protocols are increased by the electrophoresis conditions that range from pH 8.2 to 13 (Haines et al., 1998
; Singh and Stephens, 1998
; Hughes et al., 1999
; Irvine et al., 2000
; Donnelly et al., 2001). In spite of this, some common results have been reported, especially the observation that radiation of sperm, which is known to induce double and single strand breaks, will also increase the Comet measurements (Haines et al., 1998
; Singh and Stephens, 1998
; Hughes et al., 1999
). It is therefore likely that although the final measurement reflects DNA strand breaks in vitro, it is likely that these have arisen from a variety of in-situ DNA abnormalities ranging from strand breaks per se but also including structural abnormalities. Thus Comet analysis of sperm is most appropriately described generically as DNA damage. Which of the different protocols is the most useful for describing clinically relevant sperm DNA damage remains to be determined.
Whilst this and other studies have established that DNA damage is present in sperm, the biological significance of reproduction with these sperm is not clear. It may be predicted that high loads of DNA damage would be reflected in abnormal fertilization and development, ultimately leading to death of the embryo. In an attempt to address this question, we compared the semen analysis parameters, fertilization, embryo development and pregnancy outcome, from clinical treatment cycles for men grouped according to whether the sperm carried low or high DNA damage loads. Surprisingly, there were no significant differences in any of our measurements. It may be noteworthy that there was a trend for men in the higher DNA damage group to show poorer results in all of the development parameters and that early pregnancy loss occurred only in this group. These associations are potentially interesting and need to be confirmed by studying a larger, more clearly defined group of men.
However, multivariate analysis of the entire population of samples revealed some interesting correlations. DNA damage increased as a function of age, motility and abnormal forms. When only the men selected for ICSI treatment were analysed, the association with age and motility remained, but additionally semen sperm concentration and embryo cleavage were significantly negatively associated with DNA damage. The association of increased sperm DNA damage with abnormal forms and decreasing sperm count confirms other studies (Sun et al., 1997; Irvine et al., 2000
) and is perhaps predictable as inefficiency in spermatogenesis may lead to cells appearing in the ejaculate which either have not completed development or have escaped mechanisms to delete them within the testicular parenchyma (Sakkas et al., 2000
). There have been several studies investigating changes in male reproduction with age (Plas et al., 2000
; Kidd et al., 2001
; Paulson et al., 2001
; Rolf and Nieschlag, 2001
). Whilst it is clear that endocrine activity in ageing men is less efficient it appears that in many studies sperm production is maintained, although there may be increases in abnormal forms and a decline in motility. We did not detect an age-related decline in sperm count motility or abnormal forms, possibly because the setting of this study resulted in a heterogeneous selection of men with a limited age range of 2944 years. However, the increase in DNA damage was strongly significant. DNA fragmentation measured by TUNEL is not correlated with age (Sun et al., 1997
) although another study has suggested that DNA chromatin condensation is abnormal in sperm from ageing men (Haidl et al., 1996
). However, it is fairly clear that conception with sperm from ageing men does not adversely effect fertilization or live birth rate.
Association between sperm DNA damage and impaired fertility has previously been reported. Sperm with high levels of TUNEL labelling are more often found in infertile men, fail to decondense after ICSI and fertilization is often unsuccessful (Sakkas et al., 1996; Lopes et al., 1998b
; Host et al., 2000
). The SCSA has shown that those men with high levels of chromatin abnormalities in their sperm are likely to have poor fertility, which may be related to impaired fetal development and subsequent miscarriages (Evenson et al., 1999
; Larson et al., 2000
; Spano et al., 2000
). Although it has been shown that there is some correlation between the DNA damage assays, as it is not clear what structural changes these are measuring, it may not be helpful to compare outcomes until further work is done. However, the Comet assay has been used to compare DNA damage in sperm from fertile and infertile men. No differences in sperm Comet parameters between fertile and infertile men could be found in one report (Hughes et al., 1996
), however, the study did reveal differences after in-vitro treatment with either X-rays or hydrogen peroxide. This work suggested that sperm DNA from the infertile men was more susceptible to damage arising from oxidative free radical generation (Hughes et al., 1996
). This apparent increase in DNA fragility has also been shown by freezethawing of samples for fertile and infertile men (Donnelly et al., 2001
). The Comet assay has also detected significant correlation between DNA damage and semen parameters in an unselected population of men attending an infertility clinic (Irvine et al., 2000
). The latter study also highlighted significant differences in DNA damage between infertility patients and normospermic donors. In the combined analysis of the donors and patients there was considerable overlap of the DNA damage profiles of the two groups. However, there was a highly significant increase in DNA damage as the sperm count and the proportion of morphologically normal forms decreased. The present study confirms the associations between DNA damage and sperm concentration and abnormal forms.
Sperm motility is often used as a predictive measure in semen analyses, high motility being a prerequisite of normal sperm parameters. It was somewhat surprising therefore to find that the higher the motility of sperm in semen, the higher the DNA damage load carried by the sperm populations. In contrast, others (Barroso et al., 2000; Irvine et al., 2000
; Zini et al., 2001
) report that sperm samples with low motility carried higher loads of DNA damage (TUNEL or Comet) (Irvine et al., 2000
). ROS generated in vitro decreases motility as well as inducing sperm DNA damage (Aitken et al., 1998
; Twigg et al., 1998a
; Donnelly et al., 1999
; Shen and Ong, 2000
;Ramos and Wetzels, 2001
). DNA damage has, however, been both negatively and positively associated with sperm ROS production (Barroso et al., 2000
; Irvine et al., 2000
). These studies suggest that while nuclear DNA damage may be induced by ROS, at the same time low levels may promote sperm motility (explaining our correlation) whereas higher pathological/pharmacological concentrations impair motility (de Lamirande et al., 1997
), explaining the inverse correlation.
The most important observation from this study was that sperm containing high loads of DNA damage detected by the Comet assay gave rise to pronuclei at a normal incidence but were associated with an increase in the percentage of embryos that failed to develop after ICSI. This result is consistent with experiments in which DNA damage in human sperm was created artificially in vitro and after injection into hamster oocytes pronuclear formation was unchanged (Twigg et al., 1998b). DNA damage measured by the TUNEL assay is also negatively related to both fertilization and embryo cleavage rate after IVF, suggesting that DNA damaged sperm will fertilize less efficiently and confirming that early embryonic development is impaired (Sun et al., 1997
). The results of our study suggest that selection of the sperm for ICSI in terms of DNA damage was random and that a mechanism for the screening of sperm carrying damaged DNA operates after ICSI to ensure that only those zygotes with a relatively intact genome go on to develop. It would be reassuring to conclude therefore that implantation and pregnancy outcome would not be so adversely affected by using sperm samples carrying high loads of DNA damage. However, the size of this study does not allow us to make this conclusion, and moreover, it remains possible that low, sublethal levels of sperm DNA damage are transmitted through to embryo development. These may be insufficient to trigger a gross response such as cell cycle arrest or apoptosis prior to implantation, or early pregnancy failure, but may nonetheless be expressed in fetal or post-natal development (Hales and Robaire, 1997
; Sakkas et al., 2000
). It would be useful if screening semen samples for sperm DNA damage could contribute towards the selection of patients for ICSI. However, we cannot recommend this measurement at this time because significant numbers of sperm in a sample carrying high DNA damage loads may be genetically normal. Further research will be necessary to see if techniques can be devised to identify and select sperm with undamaged DNA for ICSI, or to remove sperm with damaged DNA from sperm samples, to enable the pregnancy outcome after ICSI to be improved. This work may also have implications for the genetic integrity and normal development of children conceived by IVF, especially ICSI.
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Acknowledgements |
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Notes |
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References |
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Submitted on August 17, 2001; resubmitted on November 22, 2001; accepted on November 29, 2001.