Hum. Reprod. Advance Access originally published online on May 9, 2006
Human Reproduction 2006 21(8):2061-2064; doi:10.1093/humrep/del134
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Intra-individual variation in sperm chromatin structure assay parameters in men from infertile couples: clinical implications
1 Fertility Centre, Malmö University Hospital, Lund University, Malmö, Sweden 2 Andrology Laboratory, Riga Stradins University, Riga, Latvia 3 Fertility Clinic, Viborg Hospital (Skive), Skive, Denmark and 4 Section of Toxicology and Biomedical Sciences, BIOTEC-MED, ENEA CR Casaccia, Rome, Italy
5 To whom correspondence should be addressed at: Fertility Centre, Malmö University Hospital, Lund University, SE 205 02 Malmö, Sweden. E-mail: juris.erenpreiss{at}med.lu.se
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Abstract |
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BACKGROUND: Sperm DNA integrity is an important factor in the prognosis of male fertility. In this study, we investigated intra-individual variation of sperm chromatin structure assay (SCSA) parameters in infertility patients undergoing assisted reproductive techniques (ARTs). METHODS: Retrospective study of 282 consecutive patients referred for ART [intrauterine insemination (IUI), IVF or ICSI] with repeated (between 2 and 5) SCSA measurements. RESULTS: Mean coefficient of variation (CV) of DNA Fragmentation Index (DFI) for repeated SCSA measurements was 29%. A high proportion [37%; 95% confidence interval (CI): 27%, 49%] of patients with DFI >30% in the first test had DFI <30% in the second test. Also, a considerable proportion (27%; 95% CI : 16%, 40%) of patients with 21–30% DFI values in the first test had DFI >30% in the second test. CONCLUSIONS: Intra-individual variability in DFI is significant, therefore repeated SCSA measurements are recommended. The biological mechanisms behind these variations remain to be elucidated.
Key words: flow cytometry/infertility/SCSA/sperm chromatin/sperm DNA integrity
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Sperm chromatin structure and DNA integrity are known to have a crucial influence on the fertilizing process (Twigg et al., 1998
; Agarwal and Said, 2003; Sakkas et al., 2003) and on individual fertility capabilities (Evenson et al., 1999; Spano et al., 2000). Infertile men are reported to have a higher fraction of sperm with chromatin defects and DNA breaks than fertile controls (Saleh et al., 2002; Zini et al., 2002; Virro et al., 2004).
The accumulated data have indicated operative male infertility clinical threshold levels for sperm DNA damage not compatible with fertilization in vivo by either natural conception or intrauterine insemination (IUI) (Evenson et al., 1999; Spano et al., 2000; Duran et al., 2002; Saleh et al., 2003; Bungum et al., 2004). Using the sperm chromatin structure assay (SCSA), probably the most commonly applied test to assess the sperm chromatin integrity so far, these figures are set around 30% of abnormal sperm (expressed as DNA Fragmentation Index – DFI) in the ejaculate (Evenson et al., 1999; Saleh et al., 2003; Bungum et al., 2004). However, the odds ratios for fertility seem already to decrease at the DFI level of 20% (Evenson et al., 1999; Spano et al., 2000; Bungum et al., 2004). The issue of the occurrence of an analogue threshold in IVF/ICSI is still debatable (Payne et al., 2005; Erenpreiss et al., 2006; Evenson and Wixon, 2006). Nevertheless, the existing data seem to justify the option of introducing a sperm DNA damage assessment into the routine infertility workup (Agarwal and Allamaneni, 2005), at least for in vivo infertility diagnosis. However, the variability of DFI over time should be elucidated before such a recommendation can be made.
It is well known that classically measured semen variables such as sperm concentration, motility and morphology are highly variable over time (Amann, 1989; Mallidis et al., 1991; Amann and Hammerstedt, 1993; WHO, 1999; Alvarez et al., 2003). SCSA patterns and DFI values, in turn, were suggested to be homogeneous within a male over time. An average within-donor coefficient of variation (CV) of DFI <10% has frequently been reported (Evenson et al., 1991, 1999, 2000, 2002; De Jonge et al., 2004), this figure being significantly lower than those derived from common semen measurements.
However, it has been noted that DFI can fluctuate over time within certain individuals. Known factors for these variations are some drugs and high fever episodes (Evenson et al., 1991, 2000). In these cases, DFI declined to the background values after one spermatogenic cycle.
In this study, DFI has been measured in repeated samples obtained from men undergoing assisted reproductive techniques (ARTs, 2–5 cycles for each couple). These values have been monitored to check whether DFI remains stable over time.
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Materials and methods |
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Patients
A retrospective study of 282 consecutive patients referred for ART (IUI, IVF or ICSI) at the Fertility Clinic, Viborg Hospital, Skive, Denmark, during the period 2002–2003, was carried out. Between 2 and 5 SCSA measurements were performed for each man corresponding to the number of 2–5 ART treatments given to the couple.
Sperm quality measurements
Standard semen parameters
Semen samples were collected by masturbation on the day of oocyte retrieval or IUI. Samples were allowed to liquefy for 10 min. Semen volume and sperm motility were measured according to the World Health Organization (WHO) guidelines (WHO, 1999). Sperm concentration was assessed using a Makler chamber. Sperm morphology was not assessed.
SCSA
Two hundred microlitre aliquots of semen were immediately frozen at –80°C. At the end of the sample collection, samples were shipped on dry ice to the Fertility Centre of Malmö University Hospital for SCSA analysis. The SCSA was applied following the procedure described earlier, using staining with acridine orange (Bungum et al., 2004; Erenpreiss et al., 2004). Adopting guidelines published by Evenson et al. (2002), we expressed the extent of DNA denaturation by DFI, using the List View software (Phoenix Flow Systems, San Diego, CA, USA). For the flow cytometer set-up and calibration, aliquots were used from a normal human ejaculate sample retrieved from the laboratory repository.
Each time, whenever SCSA was performed, a measurement of the same reference sample was used (and always repeated after 10 measurements) for the control of the SCSA measurement stability and instrumental drifts. In addition, we have previously demonstrated high intra-assay correlation [coefficient 0.96 for DFI, measuring the same semen samples during two independent flow cytometry (FCM) sessions within one laboratory] and inter-assay stability (correlation coefficient for DFI 0.90, P < 0.0005 measuring the same semen samples in two different laboratories; mean ratio between the values in two laboratories 1.01, SD = 1.05) of SCSA (Giwercman et al., 2003; Rignell-Hydbom et al., 2005).
Data analysis
Results were expressed as means (±SD).
Coefficient of variation (CV) for DFI was calculated using the formula (SD/mean) x 100%. CV for DFI were also separately calculated for patients with 2 (n = 180), 3 (n = 66), 4 (n = 26) and 5 (n = 10) repeated SCSA measurements. In addition, CVs for DFI were assessed in the separate groups of men divided according to normal/abnormal semen standard parameters (sperm concentration >20 x 106/ml and progressive motility >50% were regarded as normal).
Furthermore, all patients were divided into four groups, according to the first DFI value: (i) DFI £ 10%, (ii) DFI 11–20%, (iii) DFI 21–30%, (iv) DFI > 30%. Univariate linear regression analysis was used to compare CV for DFI between these (excellent, good, average and bad samples of their DFI values) groups.
Thereafter, we attempted to assess the risk of high DFI in repeated SCSA measurements, depending on the DFI in the first test. Therefore, all subjects were categorized as four groups, as mentioned above, according to their DFI value in the first sample, and the proportions of men with DFI exceeding the level of 30% [binomial 95% confidence interval (CI)] in the subsequent sample were calculated.
Association between DFI and abstinence time was calculated as Spearman’s rho coefficient.
All testing were two-sided with a probability value of less than 0.05 regarded as significant. Analyses were conducted with SPSS 11.0 for Windows (SPSS, Chicago, IL, USA).
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Results |
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Mean abstinence time for the study population was 4.6 ± 3.3 days, sperm concentration: 62.9 ± 45.8 x 106/ml, total sperm count: 207.4 ± 171.2 x 106/ml and progressive motility: 62.3 ± 16.4%. There was a weak, but significant, correlation between DFI and abstinence time (
= 0.19, P < 0.001). CV of DFI for the reference sample measurements was 5–7%, either within each measurement cycle or as compared between different measurements. CV for DFI of all repeated SCSA measurements in the study population was 29%. CV for DFI for patients with 2, 3, 4 and 5 repeated SCSA measurements were 26, 35, 32 and 37%, respectively.
CV for DFI were similar in groups with both normal (n = 167) and abnormal (n = 115) sperm concentration and motility—28% and 31%, respectively.
CV for four groups of semen samples according to their first DFI value is summarized in Table I. These values were similar. A statistically significant mean difference of 6.8% (95% CI: 0.17; 13%) was found between the group with the first DFI value <10% and the group with the first DFI between 10 and 20%.
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Proportions of semen samples with DFI >30% in the second sample in these four groups of patients are summarized in Table II. Six percentage of samples with DFI £ 10% had DFI > 30% during the second measurement, 14% of samples with DFI 11–20%, 27% of samples with DFI 21–30% and 63% of samples with DFI > 30% had also DFI > 30% during the second measurement.
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Discussion |
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It has been shown earlier that for some individuals the DFI patterns can differ for some months because of drug usage and/or fever (Evenson et al., 1991
, 2000) and then return to the original pattern. However, it was suggested that DFIs, with some exceptions (Alvarez et al., 2004), are generally homogeneous within a male over time. A recent report using another method for sperm DNA integrity assessment–terminal nick-end labelling assay (TUNEL) has also shown good stability of DFI parameter over time in a small group of men (Sergerie et al., 2005).
However, previous studies were based on rather limited numbers (1–16) of both fertile and infertile men (Evenson et al., 1991, 2000; De Jonge et al., 2004; Sergerie et al., 2005). The largest study so far by Apedaile et al. (2004) reported the considerable variations of DFI in 3 of 35 infertile patients analysed. The overall CV for DFI for two repeated SCSA measurements in this study was 13%.
Data from the present study, based on 282 men, clearly demonstrate that sperm DNA damage parameters are not as homogeneous within a male over time as previously assumed. DFI variability seems to have a tendency to increase in parallel with higher numbers of repeated SCSA measurements over longer periods of time. We could not demonstrate that DFI is less variable over time in patients with excellent or good DFI levels during the first measurement as summarized in Table I. However, the clinical interest presents the risk of having the high DFI (above 30%) in the second measurement according to the level of the DFI during the first measurement. As expected, patients with DFI of 21–30% had a higher risk (27%) of having the second DFI >30%, although 11.5% of patients (21 of 182) with DFI £ 20% had DFI >30% during the second measurement. Therefore, if a patient has DFI of £10%, repetition of the SCSA measurement is not necessary because there is only a 6% risk of having DFI >30% during the second measurement. On the contrary, if a patient has DFI between 20 and 30%, there is a higher risk of DFI >30% in a subsequent sample (27%). Furthermore, if a patient has DFI >30%, there is also a high (37%) chance of DFI below 30% in the next sample.
It is well established that SCSA is a good predictor of in vivo infertility by either natural conception or IUI if the DFI exceeds 30% (Evenson et al., 1999; Spano et al., 2000; Bungum et al., 2004; Evenson and Wixon, 2006). It has been shown that DNA defects may also have a possible negative impact on the outcome of ART (Duran et al., 2002; Morris et al., 2002; Larson-Cook et al., 2003; Saleh et al., 2003; Bungum et al., 2004; Tesarik et al., 2004; Virro et al., 2004; Agarwal and Allamaneni, 2005; Greco et al., 2005; Lewis and Aitken, 2005; Spano et al., 2005; Evenson and Wixon, 2006), although data are still controversial (for review, see Erenpreiss et al., 2006), and a recent study found no association between DFI levels and IVF/ICSI outcome (Payne et al., 2005). More studies involving large numbers of subjects, together with multivariate logistic regression analysis including all the other semen variables and female factors, are needed to determine the impact of sperm DNA damage on the outcome of IVF/ICSI procedures. Meanwhile, repeated SCSA measurements over time (or before each IUI attempt) could be considered for a better prognosis of in vivo fertility potential of men and for the prognosis of the particular IUI procedure.
Oxidative stress is suggested as the main factor responsible for DNA damage in ejaculated sperm (Sakkas et al., 1999; Greco et al., 2005; Evenson and Wixon, 2006). Morphologically, abnormal spermatozoa and leukocytes are the main source of excess reactive oxygen species (ROS) generation in semen (Aitken et al., 1992). Well-known variation of semen variables (including sperm morphology) over time can theoretically lead to variable levels of ROS in semen, and, subsequently, to the variation of sperm DNA damage levels over time in some men. However, this hypothesis remains to be proved.
In conclusion, this study describes the considerable variability of sperm DNA damage within a large group of infertile men over time. It has clinically relevant implications because DFI has been shown to be a good predictor for in vivo infertility. Therefore, we suggest that SCSA should be carried out repeatedly, particularly when DFI in the first measurement is >20%. Further studies are necessary to elucidate the reasons for reported variations in sperm DNA damage levels.
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Special thanks are extended to Dr Roger Alston for his help in English editing of the manuscript.
The study was supported by grants from Swedish Governmental Founding for Clinical Research, Swedish research Council (Grant no: K2005/72X-14545-03A), The Swedish Childhood Cancer Society (Grant no: 03/016), Swedish Cancer Society (Grant no: 4423), the Gunnar Nilssons Cancer Foundation and Malmö University Hospital Foundation for Cancer Research.
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Submitted on November 8, 2005; resubmitted on February 27, 2006; resubmitted on April 4, 2006; accepted on April 7, 2006.
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