Embryo quality and IVF treatment outcomes may correlate with different sperm comet assay parameters*

M. Tomsu1,4, V. Sharma2 and D. Miller3

1 Jessop Wing, Royal Hallamshire Hospital, Tree Root Walk, Sheffield S10 2SF, 2 Assisted Conception Unit, St James's University Hospital, Leeds LS7 9TF and 3 Centre For Reproduction, Growth and Development, Level D, Clarendon Wing, Leeds General Infirmary, Leeds LS2 9NS, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Standard semen parameters have proven poor at predicting the outcomes of IVF treatment cycles. As recent studies suggest that the male genome may play an important role in early embryogenesis, this study attempts to correlate the level of sperm DNA damage in fresh semen and prepared sperm with the outcomes of conventional IVF treatment cycles. METHODS: Forty patients embarking on IVF treatment were recruited into this prospective observational study. Both fresh semen and PureSperm®-prepared sperm were processed using a modified comet assay 3–6 months prior to the patients' IVF treatment cycles. Comet head DNA (mean and integrated head density) and tail DNA parameters (length and moment) were measured separately. RESULTS: Significant correlations between total sperm concentration and between comet length, moment, mean head density with embryo quality were detected in fresh semen and prepared sperm. Surprisingly, no significant correlations between head and tail parameters were detected. CONCLUSIONS: Comet head and tail DNA parameters appear to be potentially useful as predictors of embryo quality and IVF outcomes, especially in couples with unexplained subfertility. The lack of correlation between head and tail parameters may be due to a different mechanism of DNA damage within these two compartments.

Key words: comet assay/embryo quality/IVF/sperm DNA


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Traditionally, in IVF treatment cycles, poor embryo quality has been regarded as an oocyte-related problem. In contrast to embryo quality, which is a good indicator of successful pregnancy outcome, standard semen parameters have proved disappointing at predicting the outcome of IVF treatment cycles. In this respect, any improvement in our ability to predict pregnancy outcome using sperm samples would be welcomed in view of their easy accessibility and costeffectiveness. In addition, standard semen parameters appear to have an influence on embryo development that continues into the blastocyst stage after IVF/ICSI treatment cycles (Shoukir et al., 1998Go).

The poor predictive value of semen profiling may be due partly to variable quality control in the assessment of semen parameters in IVF treatment centres and in andrology laboratories. Similarly, sperm function tests, in addition to not being cost-effective, are usually complex, intensely laboratory-based and most can only assess the fertilizing ability of sperm using non-human oocytes which require animal handling facilities and where there may be moral and ethical constraints. This unsatisfactory situation, together with improvements in our understanding of the cell and molecular biology of human sperm, has led investigators to focus their attention on the male gamete and in particular its genome.

Evidence is now accumulating on the importance of sperm DNA integrity during both fertilization and embryogenesis. Even though, during micromanipulative IVF treatment, oxidative damage to sperm DNA does not preclude fertilization (Twigg et al., 1998Go), several authors have reported significant correlations between sperm DNA damage and fertilization (Sakkas et al., 1996Go; Lopes et al., 1998Go) as well as pregnancy rates (Hammadeh et al., 1996Go; Larson et al., 2000Go). Moreover, it was shown recently that in natural conception, sperm DNA status is an essential prerequisite to the achievement of a successful pregnancy (Evenson et al., 1999Go; Spano et al., 2000Go). Using the sperm chromatin structure assay (SCSA), which measures susceptibility to acid denaturation, these studies found a high susceptibility to acid denaturation in the sperm of men whose female partners were significantly delayed in their time to conception. Prior to these findings it had been thought that damage to sperm DNA can only potentially have an effect on IVF where the natural process of sperm migration in the reproductive tract is circumvented.

In this prospective observational study, we recruited subfertile men about to embark on IVF treatment and measured the levels of their sperm DNA damage in both fresh semen and PureSperm®-prepared sperm using a modified comet assay. IVF outcome data was then prospectively collected during their treatment cycles and attempts made at correlating these with the level of DNA damage detected earlier. Our goal was to test whether various comet assay parameters may be useful as predictive indicators of embryo quality and hence a useful prognostic indicator of IVF outcomes in conventional IVF treatment cycles.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Local ethics committee approval was obtained for this study. Patients attending the Assisted Conception Unit were approached and written consent obtained. A total of 40 patients who satisfied the selection criteria were recruited. The selection criteria included female age of partner <40 years; good correlation between semen analysis at the time of recruitment and IVF treatment cycle and treatment within 6 months of recruitment. All patients with sperm concentration <20x106/ml were excluded.

Sperm preparation
The sperm sample was produced by masturbation into a clean sterile container and allowed to liquefy at room temperature for 30 min. Sperm count and motility was assessed in a Makler chamber (World Health Organization, 1992Go). The presence of sperm-bound antisperm antibody was checked using the commercial SpermMar direct IgA and IgG test (Fertipro NV, Microm, Thame, UK) according to the manufacturer's instructions. Briefly, on a microslide, 10 µl of fresh semen and latex particles were placed and mixed using the edge of a coverslip. For the IgG test only, 10 µl of SpermMar antiserum were also added, mixed and incubated for 2–3 min. The coverslip was put on the mixture and the sample observed under the microscope. At least 100 cells were observed under the microscope and the percentage sperm attached to the latex beads, if any, was counted. The semen samples were then divided into two aliquots. One aliquot was kept in seminal plasma at room temperature and the other aliquot prepared as if for insemination during IVF treatment cycles. Briefly, this involved layering ~2–3 ml of the seminal fluid onto a PureSperm (Hunter Scientific, Saffron Walden, Essex, UK) discontinuous gradient (40 and 80%). Centrifugation was carried out for 14 min at 250 g. All the supernatant was removed with a sterile disposable pipette. The pellet was resuspended in Earle's medium and centrifuged again at 250 g for 5 min to remove any residual PureSperm. Samples in seminal plasma were washed twice at 500 g for 5 min using Dulbecco's phosphate-buffered saline (PBS). The sperm concentration was then diluted to a concentration of 1x106/ml.

Modified single-cell gel electrophoresis (comet assay)
The comet assay was performed using a modified method as previously described (Hughes et al., 1996Go; Donnelly et al., 2000Go). Poly-L-lysine-coated clear microscopic slides (BDH laboratory supplies, London, UK) were precoated with 100 µl of 1% normal melting point agarose in PBS (Sigma, Poole, Dorset, UK) and kept overnight to dry out or for >=2 h prior to performing the assay. Approximately 1x105 sperm in 10 µl of PBS were mixed with 75 µl of 0.5% low melting point agarose at room temperature. This was rapidly pipetted on top of the agarose layer and covered with glass cover slips. The agarose was allowed to solidify before the cover slips were gently removed. Another layer of 0.5% low melting point agarose was pipetted on top of the second layer and allowed to solidify at room temperature.

Lysis of the cells was carried out for 1 h at 4°C by removing the cover slip and immersing the slides in a Coplin jar containing freshly prepared cold lysis solution (2.5 mol/l NaCl, 100 mmol/l Na2 EDTA, 10 mmol/l Tris; pH 10, with 1% Triton X-100 (Sigma) added just before use. Following lysis, the slides were incubated with 10 mmol/l dithiothreitol (DTT; Sigma) for 30 min at 4°C followed by a further incubation at 20°C with 4 mmol/l of lithium diiodosalicylate (LIS; Sigma) for 90 min.

A horizontal gel electrophoresis tank was filled with electrophoresis solution containing 300 mmol/l NaOH, 1 mmol/l EDTA, pH >=13.0 at 12–15°C. The slides, after carefully draining the lysis solution, DTT and LIS, were placed side by side in this electrophoresis solution and kept for 20 min to allow the DNA to unwind. The electrophoresis buffer was adjusted at a level of ~0.25 cm above the slides surface.

Electrophoresis was performed for 10 min at 25 V (0.714 V/cm) adjusted to 300 mA by either raising or lowering the buffer level in the tank. After electrophoresis the slides were drained and flooded with three changes of neutralization buffer (0.4 mol/l Tris; pH 7.5) each for 5 min. When analysis of the slides could not be carried out on the same day they were stored by dipping in 100% ethanol for 5 min in a Coplin jar and kept overnight to dry out at room temperature before being stored in an airtight desiccator.

Ethidium bromide (20 µg/ml dissolved in distilled water) was used to stain the sperm DNA. Fifty cells from each slide were captured with a Sensys photometrics camera, mounted on a Zeiss fluorescent microscope (excitation filter of 515–560 nm from a 100 W mercury lamp and barrier filter of 590 nm), using the Iplab spectrum software (Scanalytics Inc., Fairfax, VA, USA) on a Powermac G3 Apple computer. The slides were marked vertically into five equal segments and 10 cells randomly selected from each segment from top to bottom. Analysis of comet parameters was performed using NIH image package (Scion Corp., Frederick, MA, USA), available by anonymous file transfer protocol (FTP) from: http://zippy.nimh.nih.gov or the Scion corporation website: http://www.scioncorp.com. The mean head density (MHD), the integrated head density (IHD), comet moment and tail length were measured and the mean of both fresh semen and prepared sperm recorded for each patient. The MHD is defined as the sum of all grey values in the selection (comet head) divided by the number of pixels. The IHD is the comet head area density minus the background fluorescence. The comet moment is the product of the fluorescence in the comet tail and the tail length measured from the edge of the comet head.

Reproducibility
Although just 50 cells were used to assess sperm DNA damage, reproducibility of the assay was checked using the fresh semen of 10 subfertile men recruited to this study. Duplicate slides were processed simultaneously from each patient. One of the slides was assessed immediately for sperm DNA damage and the second set of slides stored for analysis >=6 months later. The coefficient of variation ranged from 0 to 3% for the head parameters (MHD) and from 0 to 5% for the tail parameters (comet length and moment) in all duplicate samples.

IVF procedure
Oocytes were retrieved in the selected women undergoing conventional IVF treatment cycles as described in detail (Salha et al., 2001Go). Briefly, this involved achieving pituitary desensitization using either naferelin intranasal spray or s.c. buserelin acetate commencing on the first day of the menstrual cycle. Multiple follicular stimulation was commenced using highly purified FSH or HMG. Transvaginal ultrasound scan criteria only were used to determine the date of HCG administration. A standard dose of 5000 IU HCG was administered i.m. 35–37 h prior to oocyte retrieval. All oocytes with normal appearances were inseminated with 100000 sperm/ml at 40–42 h after HCG administration. Oocytes were examined for fertilization (presence of two pronuclei) at 16–20 h after insemination.

Embryo grading
Embryo quality was assessed morphologically, 2 days after fertilization by using a modified grading system (Veeck, 1986Go). Briefly, grade 1 and 2 embryos have no or very few fragments in the cytoplasm with equal size blastomeres and therefore are considered the best embryos. Grade 3 and 4 embryos have significant or severe fragmentation; little cytoplasmic fragmentation with blastomeres of distinctively unequal size.

Pregnancy outcome
Embryo transfer was performed on day 2 immediately following embryo grading. A urinary pregnancy test was performed 14 days after oocyte retrieval followed by ultrasound scan 2–3 weeks later if the pregnancy test was positive. Demonstration of positive fetal heartbeat by transvaginal ultrasound scan was taken as successful establishment of pregnancy. Four patients had their embryos frozen due to significant risk of OHSS (n = 2) or unexpectedly difficult transfers (n = 2).

Statistical analysis
Parametric and non-parametric tests were performed depending on the distribution of the data. All normally distributed data had parametric analysis of variance. The four patients who did not have embryo transfers were excluded from analysis of pregnancy outcome. Spearman's and Pearson's (normally distributed data) correlation coefficient values were used to determine the significance of the correlations found in all data. Both the comet moment and IHD were log-transformed to achieve a normally distributed data. P < 0.05 was deemed statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The median age of the men recruited was 35 years (range 22–45). The indications for IVF treatment for all the 40 couples were as follows: 13 unexplained subfertility, 18 tubal factor, eight anovulatory and one multiple factor (tubal and anovulatory) subfertility. Excluding the single couple suffering from multiple factor subfertility, we found no statistically significant difference between the clinical indication for IVF and sperm DNA damage as shown in Figure 1Go.



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Figure 1. The bar chart shows the means of prepared sperm comet moment (log10). Error bars are 95% confidence intervals. No statistically significant difference was noted between the three groups of patients. Note only one patient in multiple factor group. Unexp. = unexplained; Anov. = anovulation.

 
A strong positive correlation between MHD and embryo quality was observed in fresh semen (r = 0.698; P < 0.008) as well as prepared sperm (r = 0.718; P < 0.006) from the unexplained subfertility group. Similarly, an inverse relationship with IHD was observed with embryo quality in both fresh semen (r = –0.567; P < 0.044) and prepared sperm (r = –0.554; P < 0.049). However, no correlations with clinical pregnancy rates were observed. No such relationships involving the MHD or IHD were observed in patients suffering from tubal or anovulatory subfertility.

As expected, a good correlation was found between comet length and moment of fresh semen (r = 0.751; P < 0.0001) and prepared sperm (r = 0.691; P < 0.0001). There was an inverse relationship between the mean of MHD and the mean of IHD in fresh semen (r = –0.733; P < 0.0001) and prepared sperm (r = –0.677; P < 0.0001). These analyses indicated that the assay was set up correctly. There was no correlation between total sperm concentration and embryo quality (P = 0.065) at the initial semen analysis, but the sperm concentration at the time of IVF treatment appeared to correlate with embryo quality (P < 0.030). There was no correlation, at any time, between percentage motility and good embryo quality.

The semen parameters at routine semen analysis and at time of IVF treatment were not significantly different (Table IGo). Furthermore, no statistically significant difference was found in the four comet assay parameters from both fresh and prepared sperm with fertilization per se. Table IIGo also shows the IVF treatment outcomes according to female partner age and embryo quality, where good embryo quality was defined as >=75% grade 1 and 2 embryos out of the total number of embryos and two-pronuclear (2PN) stage post-fertilized oocytes.


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Table I. The relationship between standard semen parameters at recruitment and at the time of insemination during IVF treatment
 

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Table II. Outcomes of IVF procedures in the female partners of the patients recruited
 
We found the best correlation with the establishment of clinical pregnancy (r = 0.440; P < 0.007), if the number of grade 1 and 2 embryos was >75% of the total number of embryos (and 2PN stage oocytes). IHD, however, was correlated with failure to establish a pregnancy.

A significant correlation was observed between comet length, moment and MHD with embryo quality in both fresh semen and prepared sperm (Figures 2, 3 and 4GoGoGo). However, IHD of both fresh semen and prepared sperm did not show any significant correlation with embryo quality. A significant correlation was observed between IHD of both fresh semen (r = 0.381; P < 0.022) and prepared sperm (r = 0.394; P < 0.017) with failure to establish a pregnancy. These data suggest that comet IHD is a useful measurement of late rather than early embryo failure. Only two out of the 40 patients recruited had positive sperm antibody tests and were therefore not included for statistical analysis. They both had low levels of IgA and IgG antisperm antibodies (<30%). The indication for IVF treatment in one of the two patients was tubal factor (whose partner conceived with twins) whilst the other couple suffered from anovulatory subfertility (partner failed to conceive).



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Figure 2. Shows the inverse relationship between comet length and embryo quality. Significantly longer comet lengths in patients with poorer embryo quality (<75% grade 1 and 2 embryos) was observed in both fresh semen (r = –0.421; P < 0.004) and prepared sperm (r = –0.421; P < 0.007). (->) = median values.

 


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Figure 3. Shows the inverse relationship between fresh semen comet moment (log-transformed) and embryo quality. The correlation coefficient was weak, but significantly higher levelsof sperm DNA damage are shown in the men with <75% good embryo quality indicating higher levels of DNA damage in their fresh semen (r = –0.223; P < 0.05) and prepared sperm(r = –0.332; P < 0.036). (->) = median values.

 


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Figure 4. The correlation between mean head density (MHD) and embryo quality shows that couples with good embryo quality (>=75% grade 1 and 2 embryos) had significantly better MHD than couples with poorer embryo quality (<75% grade 1 and 2 embryos) in fresh semen samples (r = 0.338; P < 0.032). The same relationship was observed in prepared sperm (r = 0.359;P < 0.018). Values of MHD are arbitrary. (->) = median values.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In contrast with other reported studies, where a relationship was found between sperm DNA status and fertilization (Duran et al., 1998Go), our study found no such relationship. It has been reported previously that oxidative sperm DNA damage in ICSI cycles does not preclude fertilization (Twigg et al., 1998Go). Moreover, we found no correlation between sperm DNA damage as measured by the comet length, moment and MHD with pregnancy rates per se, even though IHD was found to correlate well with failure to conceive. This lack of correlation was found in both fresh and prepared sperm. A good correlation, however, was observed between embryo quality and successfully established pregnancy (P < 0.007) and between embryo quality and comet tail length, moment and MHD. Our evidence therefore, suggests that both tail length and MHD are predictive of successful pregnancy establishment and that IHD is a useful predictor of failed pregnancy. This finding suggests that IHD is able to measure DNA breaks that are irreparable in the long term, leading to failure in the later stages of embryonic development. A similar finding, in ICSI cycles, has shown that increased sperm susceptibility to acid denaturation (sperm chromatin structure assay) was a good predictor of negative pregnancy in both fresh and prepared sperm (Larson et al., 2000Go). Furthermore, the findings of a strong correlation between MHD and IHD with embryo quality in only the unexplained subfertility group suggest the presence of hidden sperm anomalies contributing to poor embryo quality (Host et al., 2000Go).

In terms of standard semen profiles (excluding sperm morphology) we did not find any correlations with comet assay parameters apart from total sperm concentration, which showed an inverse relationship with the comet length and moment of prepared sperm. Similarly, other reported studies have found a negative correlation between sperm DNA damage and semen quality (Irvine et al., 2000Go). However, another study (Hughes et al., 1996Go) did not find any relationship between parameters of semen quality and sperm DNA damage using the comet assay. The inverse correlation between sperm concentration and embryo quality, which is strongly predictive of pregnancies in vitro, may be indicative of a similar relationship between sperm DNA status and pregnancy rates in vivo found in couples attempting to conceive naturally (Spano et al., 2000Go).

Our main objective in this study was to look for correlations between sperm DNA status at both routine and prepared semen analysis with IVF outcomes—the first time, to our knowledge that this has been attempted. To achieve this, we used the comet assay to quantify changes in the sperm comet head and tail, and to determine the relationship (if any) with IVF outcomes. One of the principles of the comet assay is that nicked double-stranded DNA tends to remain in the comet head and short fragments of nicked double- and single-stranded DNA migrate into the tail area (Klaude et al., 1996Go). The remaining DNA in the head may loosen its supercoiled structure due to alkaline unwinding effects (McKelvey-Martin et al., 1993Go) to which sperm are particularly sensitive (Singh et al., 1989Go). Using the modified assay where the post-lysis incubation is carried out under alkaline conditions, these effects may be responsible for the differential staining of the comet head, providing our measurements of mean and IHD. Given that the potential risk associated with the transmission of defective genes to future offspring is thought to be more significantly associated with double-stranded DNA damage, as single-stranded breaks are probably repaired with time, monitoring for DNA damage in the sperm head separately from the tail would appear to be a worthwhile exercise. Furthermore, a recent report has shown a good correlation between archived sperm comet MHD with reduced hyperactivation and the zona penetration test (Chan et al., 2001Go).

In this respect, certain inferences can be made from this study regarding the information that each of these comet parameters have provided. Amongst the four measured comet assay parameters, comet length had the best correlation with embryo quality, and hence successful establishment of pregnancy, followed by the MHD. The MHD had shown significant correlation with embryo quality in both fresh semen and prepared sperm. On the other hand, the IHD appears to be a good predictor of failed pregnancies. Surprisingly, there was no significant correlation between the comet head parameters (MHD and IHD values) and tail parameters (moment and length). Our results therefore, indicate that DNA damage in the comet head may not necessarily be reflected in the comet tail, perhaps due to different mechanisms of DNA damage.

Our findings of a differential comet head staining may be explained by high pH `alkaline' decondensation occurring during the assay, leading to increased staining of DNA by ethidium bromide in the same way that Acridine Orange or Aniline Blue stains decondensed DNA. An alternative explanation is that reactive oxygen species (ROS), produced by sperm themselves and leukocytes, induce oxidative DNA fragmentation that remains nuclear-bound. This initial insult can then possibly be followed by induction of apoptosis, which leads to further de-novo double-stranded DNA damage, ultimately affecting the quality of sperm that reaches the oocyte. Such mechanism of DNA damage, which has recently been demonstrated to occur in sperm exposed to H2O2 (Ramos and Wetzels, 2001Go), could account for the correlation between IHD with failed pregnancy outcomes.

The precise aetiology of sperm DNA damage in subfertile men has so far proved elusive. Various factors, however, have been implicated including defective chromatin packaging (Bianchi et al., 1996Go; Molina et al., 2000Go) and the effect of ROS (Aitken et al., 1989Go, 1991Go; Iwasaki and Gagnon, 1992Go; Lopes et al., 1998Go). More recently, sperm cell apoptosis has been linked to poor semen quality (Sakkas et al., 1999Go; Gandini et al., 2000Go) and may ultimately prove to be a significant factor in the aetiology of sperm DNA damage. Indeed, recent studies have shown a positive correlation between comet assay parameters and apoptosis using the TUNEL assay (Donnelly et al., 2000Go) and annexin V staining (Oosterhuis et al., 2000Go).

The reproducibility of the comet assay using a similar protocol to ours and counting 50 nuclei for analysis of sperm DNA damage has been previously demonstrated (Hughes et al., 1996Go). These authors found a coefficient of variation of <=6% measured in triplicate slides of 10 individuals studied, in keeping with our findings. Thus, it appears that using 50 nuclei for sperm DNA damage analysis is a valid method of detecting significant differences between semen samples in our laboratory and others, although we would recommend the use of 100 nuclei in order potentially to increase the sensitivity of the assay, as we are currently doing in our ongoing experiments. In our limited experience so far, this may involve the use of duplicate slides for each sample, especially in patients with low sperm concentrations.

In conclusion, we have demonstrated that sperm concentration was weakly correlated with embryo quality. We also found that a significant correlation between sperm DNA damage and IVF outcomes exists, which could be potentially useful as a prognostic test in couples about to embark on IVF treatment cycles. Clearly, however, the relationship between sperm concentration, apoptosis and outcomes of IVF treatment cycles needs further investigation. One of the criticisms of previous reports regarding the predictive value of sperm DNA status has been that, the semen used for these studies could not be used for insemination in view of damage caused by the assays. Data obtained from this study supports the view that semen obtained within 6 months of the actual treatment can be used to predict pregnancy outcome, even if that sample is itself not used in the procedure. Couples about to embark on IVF treatment could therefore be tested within 6 months in advance of the treatment and counselled against the risk of poor outcomes. In addition, couples with repeated `unexplained' poor embryo quality or pregnancy failure can be tested using this assay, in order to investigate whether high sperm DNA damage could be the underlying aetiology for their poor IVF outcomes, especially if they belong to the unexplained subfertility group.

Our detailed analysis of the two components of the comet formed during this assay indicates to us that the mechanism of DNA damage in the comet head may be different from the damage reflected by the tail DNA. Furthermore, both comet head DNA damage and tail damage significantly correlate with outcomes of IVF treatment, be it failure to establish a pregnancy or a successful pregnancy outcome. Between them, IHD and tail length may be able to predict the likelihood of successful pregnancy or failure. This prediction appears to be independent of the elapsed time (within 6 months) between the comet assay procedure and time of IVF treatment.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank S.E.M.Lewis at The Queen's University Hospital, Belfast, UK for the modified comet assay protocol and all the embryologists at the Assisted Conception Unit, St James's University Hospital and Leeds General infirmary, Leeds, UK for helping out with the collection of semen samples.


    Notes
 
4 To whom correspondence should be addressed. E-mail: mustitomsu{at}aol.com Back

* Presented in part at the 17th Annual Meeting of the European Society of Human Reproduction and Embryology, Lausanne, Switzerland on July 1-4, 2001. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Aitken, R.J., Clarkson, J.S., Hargreave, T.B., Irvine, D.S. and Wu, F.C. (1989) Analysis of the relationship between defective sperm function and the generation of reactive oxygen species in cases of oligozoospermia. J. Androl., 10, 214–220.[Abstract/Free Full Text]

Aitken, R.J., Irvine, D.S. and Wu, F.C. (1991) Prospective analysis of sperm–oocyte fusion and reactive oxygen species generation as criteria for the diagnosis of infertility. Am. J. Obstet. Gynecol., 164, 542–551.[ISI][Medline]

Bianchi, P.G., Maniacardi, G.C., Urner, F., Compana, A. and Sakkas, D. (1996) Chromatin packaging and morphology in ejaculated human spermatozoa: evidence of hidden anomalies in normal spermatozoa. Mol. Hum. Reprod., 2, 139–144.[Abstract]

Chan, P.J., Corselli, J.U., Patton, W.C., Jacobson. J.D., Chan, S.R. and King, A. (2001) A simple comet assay for archived sperm correlates DNA fragmentation to reduced hyperactivation and penetration of zona-free hamster oocytes. Fertil. Steril., 75, 186–192.[ISI][Medline]

Donnelly, E.T., O'Connell, M., McClure, N. and Lewis, S.E. (2000) Differences in nuclear DNA fragmentation and mitochondrial integrity of semen and prepared spermatozoa. Hum. Reprod., 15, 1552–1561.[Abstract/Free Full Text]

Duran, E.H., Gurgan, S., Gunlap, S., Enginsu, M.E., Yarali, H. and Ayhan, A. (1998) A logistic regression model including DNA status and morphology of spermatozoa for prediction of fertilization in vitro. Hum. Reprod., 13, 1235–1239.[Abstract]

Evenson, D.P., Jost, L.K., Marshall, D., Zinaman, M.J., Clegg, E., Purvis, K., de Angelis, P. and Claussen, O.P. (1999) Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in human fertility clinic. Hum. Reprod., 14, 1039–1049.[Abstract/Free Full Text]

Gandini, L., Lombardo, F., Paoli, D., Caponecchia, L., Familiari G., Verlengia, C., Dondero, F. and Lenzi, A. (2000) Study of apoptotic DNA fragmentation in human spermatozoa. Hum. Reprod., 15, 830–839.[Abstract/Free Full Text]

Hammadeh, M.E., Al-Hasani, S., Stieber, M., Gauss, C., Rosenbaum, P., Georg, T., Diedrich, K. and Schmidt, W. (1996) The effect of chromatin condensation (Aniline Blue staining) and morphology (strict criteria) of human spermatozoa on fertilization, cleavage and pregnancy rate in an intracytoplasmic sperm injection programme. Hum. Reprod., 13, 2468–2471.

Hughes, C.M,. Lewis, S.E.M., McKelvey-Martin, A. and Thompson, W. (1996) Comparison of baseline and induced DNA damage in human spermatozoa from fertile and infertile men, using the modified comet assay. Mol. Hum. Reprod., 2, 613–619.[Abstract]

Host, E., Lindenberg, S. and Smidt-Jensen, S. (2000) DNA strand breaks in human spermatozoa: correlation with fertilization in vitro in oligozoospermic men and in men with unexplained infertility. Acta Obstet. Gynecol. Scand., 79, 189–193.[ISI][Medline]

Irvine, D.S., Twigg, J.P., Gordon, E., Fulton, N., Milne, P.A. and Aitken, R.J. (2000) DNA integrity in human spermatozoa: relationship with semen quality. J. Androl., 2, 33–44.

Iwasaki, A. and Gagnon, C. (1992) Formation of reactive oxygen species in spermatozoa of infertile patients. Fertil. Steril., 57, 409–416.[ISI][Medline]

Klaude, M. Eriksson, S., Nygren, J. and Ahnstrom, G. (1996) The comet assay: mechanism and technical considerations. Mut. Res., 363, 89–96.[ISI][Medline]

Larson, K.L., Dejonge, C.J., Barnes, A.M., Jost, L.K. and Evenson, D.P. (2000) Sperm chromatin structure assay parameters as predictors of failed pregnancy following assisted reproductive techniques. Hum. Reprod., 15, 1717–1719.[Abstract/Free Full Text]

Lopes, S., Jurisicova, A., Sun, J.G. and Casper, R.F. (1998) Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa. Hum. Reprod., 13, 896–900.[Abstract]

McKelvey-Martin, V.J., Green, M.H.L., Schmezer, P., Pool-Zobel, B.L., De Meo, M.P. and Collins, A. (1993) The single cell gel electrophoresis (comet asaay): a European review. Mut. Res., 288, 47–63.[ISI][Medline]

Molina, J., Castilla, J.A., Fontes, J., Mendoza, N. and Martinez, L. (2000) Chromatin status in human ejaculated spermatozoa from infertile patients and relationship to seminal parameters. Hum. Reprod., 16, 534–539.[Abstract/Free Full Text]

Oosterhuis, J.G., Mulder, A.B., Kalsbeek-Batenburg, E., Lambalk, C.B., Shoemaker, J. and Vermes, I. (2000) Measuring apoptosis in human spermatozoa: a biological assay for semen quality. Fertil. Steril., 74, 245–249.[ISI][Medline]

Ramos, L. and Wetzels, A.M.M. (2001) Low rates of DNA fragmentation in selected motile spermatozoa assessed by the TUNEL assay. Hum. Reprod., 16, 1707–1707.

Sakkas, D., Urner, F., Bianchi, P.G., Bizzaro, D., Wagner, I., Jaquenoud, N., Manicardi, G. and Campana, A. (1996) Sperm chromatin anomalies can influence decondensation after intracytoplasmic sperm injection. Hum. Reprod., 11, 837–843.[Abstract]

Sakkas, D., Mariethoz, E. and St John, J.C. (1999) Abnormal sperm parameters in human spermatozoa are indicative of an abortive apoptotic mechanism linked to the Fas-mediated pathway. Exp. Cell Res., 251, 350–355.[ISI][Medline]

Salha, O., Dada, T. and Sharma, V. (2001) Influence of body mass index and self-administration of hCG on the outcome of IVF cycles: a prospective cohort study. Hum. Fertil., 4, 37–42.

Shoukir, Y., Chardonnes, D., Campana, A. and Sakkas, D. (1998) Blastocyst development from supernumerary embryos after intracytoplasmic sperm injection: a paternal influence? Hum. Reprod., 13, 1632–1637.[Abstract]

Singh, N.P., Danner, D.P., Tice, R.R., McCoy, M.T., Collins, G.D. and Schneider, E.L. (1989) Abundant alkali-sensitive sites in DNA of human and mouse sperm. Exp. Cell Res., 184, 461–470.[ISI][Medline]

Spanò, M., Bonde, J.P., Hjøllund, H.I., Kolstad, H.A., Cordelli, E. and Leter, G. (2000) Sperm chromatin damage impairs human fertility. Fertil. Steril., 73, 43–50.[ISI][Medline]

Twigg, J.P., Irvine, D.S. and Aitken, R.J. (1998) Oxidative damage to DNA in human spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm injection. Hum. Reprod., 13, 1864–1871.[Abstract]

Veeck, L.L. (1986) Atlas of the Human Oocyte and Early Conceptus, Vol. 1. Williams & Wilkins, Baltimore.

World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and SemenCervical Mucus Interaction, 2nd edn. Cambridge University Press, Cambridge.

Submitted on September 4, 2001; resubmitted on December 31, 2001; accepted on March 19, 2002.