Hum. Reprod. Advance Access originally published online on November 6, 2007
Human Reproduction 2008 23(1):4-10; doi:10.1093/humrep/dem353
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Sperm chromatin structure assay parameters measured after density gradient centrifugation are not predictive for the outcome of ART
1 Centre of Reproductive Medicine, Malmö University Hospital, 205 02 Malmö, Sweden 2 Section of Toxicology and Biomedical Sciences, BAS-BIOTEC-MED, ENEA Casaccia Research Center, Rome, Italy 3 The Fertility Clinic, Viborg Hospital (Skive), 7800 Skive, Denmark
4 Correspondence address. E-mail: mona.bungum{at}med.lu.se
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Abstract |
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BACKGROUND: The sperm chromatin structure assay (SCSA) parameter DNA fragmentation index (DFI) has been shown to predict in vivo and in vitro fertility. So far most SCSA studies have been based on SCSA analysis performed on neat semen. The aim of this study is to assess whether SCSA analysis of sperm prepared by density gradient centrifugation (DGC) could add more information in regard to the prediction of treatment outcome.
METHODS: The study included 510 assisted reproductive technique (ART) cycles. SCSA was performed in neat semen and post DGC. SCSA results were expressed in terms of DFI and high DNA stainability (HDS) cell fractions. The outcome parameter was clinical pregnancy (CP).
RESULTS: Scatter-plot diagrams demonstrated that for DGC samples, no DFI cut-off values could be set for in vivo or in vitro fertility. In intrauterine insemination, IVF and ICSI groups the mean difference (95% CI) in DFI post DGC between those who achieved CP and those who did not was 0.2% (–1.7 to 2.0%), 0.4% (–1.9 to 2.8%) and 1.3% (–3.1 to 5.9%), respectively, none of these being statistically significant. The corresponding differences for HDS were 0.1% (–1.3 to 1.5%), 0.1% (–0.7 to 0.9%) and 0.6% (–1.6 to 2.7%), respectively (all P-values >0.6).
CONCLUSIONS: SCSA performed in semen prepared by DGC cannot predict the outcome of ART.
Key words: sperm chromatin structure assay/density gradient centrifugation/DNA fragmentation index/high DNA stainability/assisted reproduction
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Introduction |
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During recent years, there has been an increased focus on the role of sperm DNA and chromatin integrity in infertility (reviewed in Evenson et al., 2002
; Spanò et al., 2005; Erenpreiss et al., 2006; Evenson and Wixon, 2006a; Aitken and De Iuliis, 2007). A variety of tests have been described to assess sperm chromatin integrity (reviewed in Erenpreiss et al., 2006). One of these tests, the sperm chromatin structure assay (SCSA) has shown to be a good predictor of fertility, in vivo (Evenson et al., 1999; Spanò et al., 2000; Bungum et al., 2004, 2007) as well as in vitro (Evenson and Jost, 2000; Larrson et al., 2000; Larson-Cook et al., 2003; Saleh et al., 2003; Bungum et al., 2004, 2007; Gandini et al., 2004; Virro et al., 2004; Check et al., 2005; Boe-Hansen et al., 2006; Evenson and Wixon, 2006b). Recently, in the largest SCSA study focusing on outcome of assisted reproductive techniques (ARTs) published so far (998 cycles), we demonstrated that the SCSA parameter DNA fragmentation index (DFI) can be used as an independent predictor of fertility in couples undergoing intrauterine insemination (IUI). Moreover, when DFI exceeded 30%, the odd ratio of pregnancy was three times higher when comparing intracytoplasmic sperm injection (ICSI) to standard in vitro fertilization (IVF) (Bungum et al., 2007).
Spermatozoa used for ART are, in a vast majority of cases, prepared by density gradient centrifugation (DGC) or by a swim-up preparation in order to favour the isolation of motile and morphologically normal spermatozoa. Several studies have shown that, even though various levels of efficiency are reported, both sperm separation methods are quite effective in sorting out spermatozoa with nicked DNA and poorly condensed chromatin as evaluated by a variety of the sperm DNA integrity assays: SCSA, (Golan et al., 1996; Larson et al., 1999, 2000; Spanò et al., 1999; Gandini et al., 2004; Zini et al., 2000a, 2000b), terminal transferase-mediated DNA end-labelling (TUNEL) (Younglai et al., 2001; Lachaud et al., 2004; Morrell et al., 2004; Piomboni et al., 2006), Comet assay (Donnelly et al., 2000, 2001; McVicar et al., 2004) and chromomycin A3 (CMA3) (Sakkas et al., 2000; Hammadeh et al., 2001; Tomlinson et al., 2001). The need to minimize the occurrence of sperm exhibiting minimal DNA damage is an active research issue and new procedures for achieving this goal have recently been described and proposed (Said et al., 2006; Ainsworth et al., 2007).
The SCSA parameters, DFI and HDS (high DNA Stainability), reflect the fraction of defective sperm either because of the presence of DNA breaks or of poorly condensed chromatin. Most of the SCSA–ART studies published so far, where associations have emerged between the SCSA parameters and the ART success outcome, were based on the analysis carried out on neat, unprepared semen. Notable exceptions are the two small studies by Larson et al. (2000) and Gandini et al. (2004), which are based on only 24 and 34 couples, respectively. In these studies, the SCSA parameters were measured before and after the sperm separation procedure. They concluded that DFI and HDS measured after sperm preparation were not predictive in relation to the outcome of IVF and ICSI. Therefore, it was still unclear whether measuring sperm chromatin integrity on samples prepared for use in an ART procedure is advantageous in relation to assessments done on neat semen samples.
We aimed, therefore, in a larger setting, to investigate how effectively DGC sorts out spermatozoa with nicked DNA and poorly condensed chromatin and to assess whether SCSA analysis of semen samples prepared by DGC could add more information in regard to the outcome of ART. The present study includes 510 ART cycles: IUI, IVF and ICSI.
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Material and Methods |
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Patients
The study was based on a cohort of consecutive infertile couples undergoing ART at Viborg Hospital during the period April 2002–December 2003. A total of 510 cycles (197 IUI, 220 IVF and 93 ICSI) from 396 couples were included in the study. There were 78 couples who contributed with more than one treatment. The data on predictive value of SCSA performed on neat semen (998 cycles) have already been reported (Bungum et al., 2007
). Of these 998 samples used for IUI–IVF–ICSI, 510 contained enough material for performing SCSA analysis after DGC. When comparing the background characteristics for the 510 samples included in the present study and the remaining 488 there were no significant differences concerning female age, or DFI or HDS on neat samples, the mean values (SD) being 31.6 (4.1) versus 31.5 years (4.3 years), 20.3 (12.0) versus 21.8% (13.2%) and 8.8 (3.8) versus 10.1% (4.9%), respectively. In order to obtain sufficient number of spermatozoa for SCSA analysis, only those men having a sperm concentration of at least 1 x 106 ml–1 in neat semen were included. The choice of fertilization method (IUI, IVF or ICSI) was based upon infertility diagnosis. Although couples diagnosed with unexplained infertility were referred to IUI, the IVF group mainly consisted of couples with female infertility factors. The criteria for performing ICSI were a sperm concentration lower than 500 000 progressive motile spermatozoa after DGC.
The study was approved by the Ethics Committee of Viborg County, and all patients provided written informed consent.
Semen collection and analysis
Semen samples were collected by masturbation at the day of ovum-pick up or IUI. Sperm concentration was assessed by use of Makler–chamber and motility was scored according to the World Health Organization (WHO) guidelines (WHO, 1999). Sperm morphology was not assessed.
After semen analysis, within 1 h from the time of ejaculation, 100 µl of the raw semen sample was frozen at –80°C for later SCSA analysis.
Density gradient centrifugation
A standard DGC using PureSperm, 45 and 90% (Nidacon Ltd, Gothenburg, Sweden) diluted in G-SpermTM (Vitrolife, Gothenburg, Sweden), was applied for sperm preparation. After layering of semen (a maximum of 3 ml) on the top of the two layer PureSperm (45 and 90%), the sample was centrifuged at 300 g for 15 min. Thereafter the pellet was washed twice in IVF-100TM (Vitrolife, Gothenburg, Sweden) and centrifuged at 200 g between the washing steps. The final dilution of the sample was performed in IVF-100TM (Vitrolife, Gothenburg, Sweden) and the spermatozoa were incubated in 5% CO2 in air, 37°C until the insemination, IVF or ICSI fertilization procedure. Immediately after the DGC procedure 100 µl of the prepared sample was frozen at –80°C for later SCSA analysis.
Sperm chromatin structure assay
The principles and procedure to measure sperm DNA damage by flow cytometry (FCM) SCSA are described in details elsewhere (Evenson et al., 1999; Spanò et al., 2000; Bungum et al., 2004).
Briefly, on the day of analysis, the samples were quickly thawed and analysed immediately. An aliquot of unprocessed semen (
13–70 µl) was diluted to a concentration of 1–2 x 106 sperm/ml with TNE buffer (0.01 M Tris–HCl, 0.15 M NaCl and 1 mM EDTA, pH 7.4). This cell suspension was treated with an acid detergent solution (pH 1.2) containing 0.1% Triton X-100, 0.15 mol/l NaCl and 0.08 N HCl for 30 s, and then stained with 6 mg/l purified Acridine Orange (AO) (Polysciences Inc., Warrington, PA, USA) in a phosphate–citrate buffer, pH 6.0.
Cells were analysed using a FACScan flow cytometer (Becton–Dickinson, San Jose, CA, USA), equipped with an air-cooled argon ion laser. A total of 10 000 events were accumulated for each measurement at a flow rate 200–300 cells/s. AO, i.e. intercalated in double-stranded DNA emits green fluorescence, whereas AO associated with single-stranded DNA emits red fluorescence. Off-line analysis of the flow cytometric data was carried out by using dedicated software (SCSASoft; SCSA Diagnostics, Brookings, SD, USA). The percentage of abnormal sperm with detectable DFI (%DFI) was calculated from the DFI frequency histogram obtained from the ratio between the red and total (red plus green) fluorescence intensity (Evenson et al., 2002). High DNA stainability (%HDS) was calculated on the basis of the percentage of sperm with high levels of green fluorescence, which are thought to represent immature spermatozoa with incomplete chromatin condensation (Evenson et al., 2002). For the flow cytometer set-up and calibration, a reference sample was used from a normal donor ejaculate sample retrieved from the laboratory repository. The intra-laboratory coefficient of variation was found to be 4.5% for DFI and 10% for HDS, respectively.
SCSA was performed both prior to and after DGC. The SCSA parameters obtained on neat and prepared samples were designated DFI-1/HDS-1 and DFI-2/HDS-2, respectively. The data on predictive value of SCSA performed on neat semen have already been reported (Bungum et al., 2007).
Art-procedures
In IUI-patients, all hormone stimulation and insemination procedures were performed as previously described (Bungum et al., 2004). In IVF/ICSI patients, hormonal treatment, oocyte retrieval (OR), gamete handling, culture and embryo transfer (ET) were performed as previously described (Bungum et al., 2004).
Statistical analysis
Background characteristics of the couples, according to the treatment type and outcome are given in Table I.
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Primarily, for each of the three treatment modalities we evaluated, changes in the DFI and HDS following DGC by using linear regression analysis.
Thereafter, we focused on the predictive value of SCSA parameters obtained on DGC samples using clinical pregnancy (CP) as treatment outcome. A CP was defined as an intrauterine gestational sac with a heart beat 3 weeks after the serum β-HCG, taken 12 days after ET. Delivery (D) was also available as potential outcome. However, it was planned only to perform statistical analyses with regard to predictive value of DFI-2 and HDS-2 in relation to D if calculations based on CP as outcome indicated that SCSA performed on DGC sperm could predict treatment outcome.
DFI-2 and HDS-2 were compared for those who achieved CP and those who did not using linear regression models. Thereafter, in order to find a possible threshold effect, for the DGC samples, we evaluated a scatterplot diagram to compare DFI-2 and HDS-2 levels in the group, which achieved CP and the one which did not. In a recent study (Bungum et al., 2007), where all 998 cycles were included in the analysis we found a DFI threshold of 30% to be predictive of the outcome of IUI and for ICSI versus IVF. In order test whether the same trend could be found in the subset of 510 samples included in the present study we performed, using Fisher's exact test (www.graphpad.com), the following comparisons with CP as outcome: for IUI DFI-1 
30% versus DFI-1 > 30%; for DFI-1 > 30% IVF versus ICSI.
Otherwise, statistical analyses were performed using SPSS 14.0 for Windows (SPSS Inc., Chicago, USA). The term ‘statistically significant’ is used to denote a two-sided P-value <5%.
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Results |
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In the present study, a majority of the samples showed an improvement in DFI and HDS following DGC (Tables I and II). Only for 6 (1%) of the 510 samples DFI increased after preparation. DFI increased from 22.2, 12.4, 24.9, 17.4, 16.2 and 18.8% to 48.7, 20.6, 30.3, 30.9, 18.9 and 57.9%, respectively. With exception of three (0.5%) samples, all samples having a DFI above 30% in neat semen, showed a DFI decreased to below 30% after DGC (data not shown). Mean (95% CI) decrease in DFI following DGC was for all treatment groups 15.1% (14.0–16.2%) whereas the corresponding figures for HDS were 4.4% (4.0–4.9%). Changes in DFI and HDS following DGC, separately for each treatment group (IUI, IVF and ICSI), are specified in Table II. In relative terms, the mean (SD) DFI reduction following DGC was 75.9 (27.6), 65.7 (84.7) and 63.8% (61.7%) for IUI, IVF and ICSI groups, respectively. For HDS the corresponding values were 42.9 (45.6), 54.9 (42.3) and 35.5% (48.5%).
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The study population was then divided into subgroups according to treatment type (IUI, IVF and ICSI) and whether they obtained pregnancy or not. In pregnant IUI, IVF and ICSI groups mean (SD) DFI-2 values were 4.5 (5.1), 5.3 (8.6) and 8.7% (11%) compared to the non-pregnant groups whose values were 4.2 (5.3), 4.5 (8.2) and 8.0% (10%), respectively. The corresponding HDS-2 values for the pregnant groups were 4.7 (4.3), 3.4 (2.4) and 5.5% (3.9%) and for non-pregnant groups 4.5 (3.9), 3.5 (3.2) and 6.4% (5.6%) (Table III). For none of the three treatment types were statistically significant differences in DFI-2 or HDS-2 found when comparing those who achieved pregnancy and those who did not (for all comparisons: P > 0.6).
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For all three treatment groups DFI-2 and HDS-2 levels in pregnant and not pregnant groups were more or less completely overlapping. Scatter-plot diagrams demonstrated that no cut-off values could be set for in vivo or in vitro failure in achieving pregnancy, neither for DFI-2 nor HDS-2 (Fig. 1).
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Regarding neat sperm samples, when using DFI-1 = 30% as a threshold (Bungum et al., 2007
), for IUI a significantly lower chance of obtaining a CP was seen in the group with a DFI > 30% compared to the group with a DFI 30% (P < 0.005). In the DFI-1 > 30% group, comparing IVF and ICSI a trend toward higher OR for the latter was seen, however without reaching the level of statistical significance (P = 0.6) (data not shown). |
Discussion |
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In this study, we only focused on the possible predictive value of SCSA analysis performed on sperm obtained after DGC. On the basis of the same group of patients, we have previously reported SCSA data obtained by using the corresponding neat semen sample (Bungum et al., 2007
) and similar trends, regarding the predictive value of DFI, were seen in the subset of cycles tested in this study. The main conclusion that can be drawn from the current study is that, unlike SCSA analysis performed on neat semen, assessments done on sperm prepared by DGC cannot predict the outcome of IUI, IVF and ICSI.
Several previous authors have recommended the necessity to set-up studies aiming at clarifying whether processed semen also have a predictive value in ART (Tomlinson et al., 2001; Spanò et al., 2005; Tarozzi et al., 2007). Tomlinson et al. (2001) suggested assessing sperm DNA integrity in its appropriate context; sperm DNA fragmentation in neat semen with reference to in vivo conception and in post-preparation samples in relation to ART has been requested. So far, only two of the SCSA–ART studies published have used prepared semen for the SCSA analysis (Larson et al., 2000; Gandini et al., 2004). Larson et al. (2000) in 24 IVF–ICSI patients studied the effect of neat versus density gradient centrifuged semen in regard to the pregnancy outcome and found, similarly to our recently published results (Bungum et al., 2004, 2007), that DFI in neat semen can be predictive of the treatment outcome. SCSA parameters of the DGC samples were however, not predictive of pregnancy outcome. Larson et al. (2000) indicated that elevated SCSA values in neat semen reflected chromatin abnormalities within the entire sperm population that were not completely eliminated by sperm preparation techniques. Gandini et al. (2004) included 34 IVF–ICSI patients. No differences were seen in SCSA parameter values between patients initiating pregnancies and those who did not. This was the case before as well as after sperm preparation, DGC used for ICSI and swim-up for IVF. However, whereas the two previous studies were performed on relatively low numbers of treatments and type II error could, therefore, not be excluded, the current study, due to its size, can be considered as more conclusive.
Also the majority of reports using other techniques for sperm chromatin integrity assessment failed to find any association between DFI in sperm population selected by DGC or swim-up and treatment outcome (Morris et al., 2002; Benchaib et al., 2003, 2007; Seli et al., 2004; Huang et al., 2005; Hammadeh et al., 2006; Muriel et al., 2006). On the other hand, using TUNEL, Borini et al. (2006) in ICSI patients found DFI >10% in DGC prepared semen to be discriminative for CP. In a study focusing on the relationship between sperm DNA fragmentation evaluated by TUNEL and IUI outcome, Duran et al. (2002), used washed semen samples and found no pregnancy if DFI exceeded the level of 12%.
In the present study, majority of the samples showed an improvement in DFI and HDS after DGC. Only 1% of the samples showed an increased DFI after preparation. With the exception of three samples, all samples having a DFI above 30% in neat semen DFI decreased to below 30% after DGC which means that the preparation has removed a significant amount of spermatozoa with DNA breaks as measured by SCSA. There are several papers demonstrating that, although with different net performances, sperm preparation procedures can decrease the fraction of defective sperm initially present in the neat semen (Golan et al., 1997; Larson et al., 1999, 2000; Spanò et al., 1999; Donnelly et al., 2000, 2001; Sakkas et al., 2000; Zini et al., 2000a; Hammadeh et al., 2001; Tomlinson et al., 2001; Younglai et al., 2001; Gandini et al., 2004; Lachaud et al., 2004; McVicar et al., 2004; Morrell et al., 2004; Piomboni et al., 2006). On the other hand, unchanged or worse results have been reported by Zini et al. (1999, 2000a,b) and Tomsu et al. (2002).
On the basis of the results of the current and previous study (Bungum et al., 2007), we conclude that the prognostic ability of SCSA is limited to analyses performed on neat semen. This supports the theory suggested by Larson et al. (2000) that elevated DFI in neat semen may reflect chromatin or other abnormalities within the entire sperm population interfering with the fertilizing ability of the sperm but not completely eliminated by DGC or swim-up. Although SCSA is a generalized test of sperm chromatin stability, it is unclear precisely which types of sperm DNA breaks (single/double strand breaks) SCSA detects. It cannot be excluded that some unknown type of damage may also be present in the sperm although the DNA does not denaturate during the SCSA procedure. These chromatin structure defects may remain in the DGC material without being reflected by DFI. Why such abnormalities, if related to sperm DNA breaks, cannot be measured by SCSA, remains to be elucidated.
Moreover, possibly after DGC the resulting sperm cohort is characterized by very low levels of DFI. The gradient of the resulting sperm cohort, in our hands characterized by very low levels of DFI, is not contrasted enough and we thus loose the possibility of discriminating between the high and low levels of DFI in the correlation with the ART outcome.
Therefore, as clearly stated in the SCSA guidelines (Evenson et al., 2002), SCSA analysis should be performed on raw semen aliquots.
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Funding |
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The study was supported by grants from Viborg County Research Foundation, Swedish Governmental Founding for Clinical Research, Swedish Research Council (Grant No K2005–72X-14545-03A), the Swedish Cancer Foundation (Grant No CAN 2006/1142), Gunnar Nilssons cancer Foundation and Malmö University Hospital Foundation.
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Acknowledgements |
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The assistance of the staff of the Fertility Clinic, Viborg hospital (Skive) and Katarina Jepson at Reproductive Medicine Centre, Malmö University Hospital are gratefully acknowledged.
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References |
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Submitted on August 21, 2007; resubmitted on October 1, 2007; accepted on October 8, 2007.
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