1 Fertility Clinic, Viborg Hospital (Skive), Resenvej 25, DK 7800 Skive, Denmark, 2 Section of Toxicology and Biomedical Sciences, BIOTEC-MED, ENEA Casaccia Research Center, Rome, Italy and 3 Fertility Centre, Scanian Andrology Centre, Malmö University Hospital, Malmö, Sweden
4 To whom correspondence should be addressed. e-mail: mona.bungum{at}sygehusviborg.dk
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: ART/DNA damage/pregnancy rate/sperm chromatin
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The sperm chromatin structure assay (SCSA), first described by Evenson et al. (1980), is a flow cytometric (FCM) technique which identifies the spermatozoa with abnormal chromatin packaging defined as susceptibility to acid-induced DNA denaturation in situ. Acridine orange (AO) staining is used, after a low pH challenge, to distinguish between denaturated (red fluorescence = single stranded) and native (green fluorescence = double stranded) DNA regions in sperm chromatin, the former being a result of DNA breaks and/or of derailments in protamine quantity and composition and/or of an insufficient level of disulfide groups (Sakkas et al., 1999
; Irvine et al., 2000
; Aitken and Krausz, 2001
). The level of DNA breaks is conveniently expressed by the DNA fragmentation index (DFI) (Larson et al., 2000
; Evenson et al., 2002
; Larson-Cook et al., 2003
), which is the ratio of red to total (red plus green) fluorescence intensities in the flow cytometric analysis. In two independent studies, DFI levels >3040% were incompatible with fertility in vivo, whatever sperm concentration, morphology and motility (Evenson et al., 1999
; Spano et al., 2000
). Furthermore, although based on limited numbers of patients, no pregnancy has been reported after in vitro ART procedures, both standard IVF and ICSI, when the DFI in raw semen was >27% (Larson et al., 2000
; Larson-Cook et al., 2003
; Saleh et al., 2003
). As a consequence, the SCSA method has been recommended for routine use in ART programmes in order to predict a couples probability of obtaining a pregnancy (Evenson and Jost, 2000
). Before recommending SCSA as routine prior to ART, however, the predictive value of this method needs to be tested in larger scale studies where other parameters of importance for the success of the treatment (e.g. sperm concentration and motility, age of the female and number of previous unsuccessful ART treatments) are taken into consideration.
The present study was set up to examine the relationship between sperm chromatin defects evaluated by the SCSA and the outcome of IVF, ICSI and intrauterine insemination (IUI), respectively.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A total of 306 patients were included in the study: 131 couples underwent IUI, 109 IVF and 66 ICSI. All demographic data including female BMI, pre-treatment levels of FSH and number of previous ART treatments for all the three categories of treatments are given in Table I.
|
Sperm chromatin structure assay (SCSA)
Sperm DNA damage was measured by SCSA following the procedure as described elsewhere (Spano et al., 2000; Evenson et al., 2002
). On the day of analysis, the samples were quickly thawed and analysed immediately. A cell suspension (12 x 106) was treated with a low-pH (pH 1.2) detergent solution containing 0.1% Triton X-100, 0.15 mol/l NaCl and 0.08 mol/l HCl for 30 s. followed by staining with 6 mg/l purified AO (Molecular Probes, Eugene, OR) in a phosphate-citrate buffer, pH 6.0. Cells were analysed using a FACSort flow cytometer (Becton Dickinson, San Jose, CA), equipped with an air-cooled argon ion laser. A total of 5000 events were accumulated for each measurement. Under these experimental conditions, when excited with a 488 nm light source, AO intercalated with double-stranded DNA emits green fluorescence and AO associated with single-stranded DNA emits red fluorescence. Thus, sperm chromatin damage can be quantified by the FCM measurements of the metachromatic shift from green (native, double-stranded DNA) to red (denatured, single-stranded DNA) fluorescence and displayed as red (fragmented DNA) versus green (DNA stainability) fluorescence intensity cytogram patterns. For the flow cytometer set-up and calibration, reference samples were used from a normal donor ejaculate sample retrieved from the laboratory repository. Off-line analysis of the flow cytometric data was carried out using dedicated software (List View, Phoenix Flow Systems, San Diego, CA). Computer gates are used to determine the proportion of spermatozoa with increased levels of red fluorescence (denatured single-stranded DNA) and green fluorescence (native double-stranded DNA). We have expressed the extent of DNA denaturation in terms of the DFI, which is the ratio of red to total (red plus green) fluorescence intensity, i.e. the level of denatured DNA over the total DNA (Evenson et al., 2002
). The DFI value was calculated for each sperm cell in a sample, and the resulting DFI frequency profile was obtained (Figure 1, upper panel). Most sperm form a unimodal distribution representing the normal population of sperm with no detectable DNA damage. Sperm with higher red fluorescence, falling in the histogram area beyond the curve of normal sperm, represent the population of abnormal sperm with detectable DFI. Additionally, we have also considered the fraction of high DNA stainable (HDS) cells. HDS represents another distinct population in semen that characterizes immature spermatozoa with incomplete chromatin condensation. The percentage of HDS cells was calculated by setting an appropriate gate on the bivariate cytogram (Figure 1, lower panel) and considering as immature spermatozoa those events which exhibit a green fluorescence intensity higher than the upper border of the main cluster of the sperm population with a non-detectable DFI.
|
Intrauterine insemination (IUI), hormonal treatment and sperm preparation
All IUI patients had a diagnosis of unexplained infertility. Normal tubal patency was confirmed by either a hysterosalpingography (HSG) or by laparascopy. IUI was only performed in cases with at least 2.5 x 106 spermatozoa per ml after gradient centrifugation. The IUI patients were stimulated with clomiphene (Pergotime; Serono Nordic, Copenhagen, Denmark) on cycle days 37 and 75 IU of recombinant FSH (rec-FSH) (Gonal F; Serono Nordic) on cycle days 79. When 12 follicles had reached a diameter of 17 mm, 10 000 IU of HCG (Profasi; Serono Nordic) was administered to induce final follicular maturation. After 36 h, a fresh semen sample was prepared by a standard density gradient centrifugation method. PureSperm, 40 and 80% (Nidacon Ltd, Gothenburg, Sweden) diluted in SpermRinseTM (Vitrolife, Gothenburg, Sweden) was used for sperm preparation. The washing procedure and dilution was performed in IVF-100TM (Vitrolife, Gothenburg, Sweden). A 1 ml aliquot of the prepared semen sample was used for insemination with a Cashmed catheter (catalogue no. IUI18; Cashmed, Surrey, UK). No luteal phase support was given.
IVF and ICSI
Hormonal treatment. IVF/ICSI patients underwent pituitary downregulation and hormonal treatment as previously described (Bungum et al., 2003). In brief, patients were downregulated with a GnRH agonist (Suprefact, Hoechst, Hørsholm, Denmark), using a long or a short protocol. Ovarian stimulation was performed with rec-FSH (Gonal F; Serono Nordic) (using an individualized dose of between 100 and 375 IU s.c. per day according to age, ovarian volume, baseline FSH and BMI). The ovarian response was monitored by ultrasound examination starting on day 8 of stimulation, and the dose of rec-FSH administered was adjusted if necessary.
Oocyte retrieval and sperm preparation. When at least one follicle had reached a diameter of 17 mm, 10 000 IU of HCG (Profasi; Serono Nordic) was administered to induce final follicular maturation. Oocyte retrieval was performed 35 h later by vaginal ultrasound-guided follicle aspiration. GameteTM (Vitrolife, Gothenburg, Sweden), a HEPES-buffered medium, was used to rinse the oocytes.
A standard density gradient centrifugation method, PureSperm, 45 and 90% (Nidacon Ltd) diluted in SpermRinseTM (Vitrolife), was used for sperm preparation. The washing procedure and dilution was performed in IVF-100TM (Vitrolife).
IVF. In cases of conventional IVF, spermatozoa with a final concentration of 150 000/ml were added to the oocytes. After incubation in a humidified incubator with a gas phase of 6% CO2, 5% O2 and 89% N2 for 90 min, the oocytes were washed three times before further culture in IVF-100TM until time of assessment for fertilization.
ICSI. In cases of ICSI, denudation of cumulus cells was performed by exposure of the oocytes to HYASETM (Vitrolife) for a maximum of 30 s. Denudation of cumulus cells was performed by the use of glass denuding pipettes (SweMed Lab, Billdal, Sweden) immediately before injection. The oocytes were washed four times in GameteTM after denudation. ICSI was performed in GameteTM covered by OVOILTM (Vitrolife) using commercially available ICSI pipettes (Cook, Brisbane, Australia). A 5 µl ultra-microdroplet of polyvinylpyrrolidone (PVP), ICSI-100 TM (Vitrolife) was spread out in a thin layer on the Petri dish. A 1 µl aliquot of the sperm preparation was introduced to the centre of the PVP droplet. After injection, the oocytes were washed twice in G.1.2TM (Vitrolife) medium before further culture in G.1.2TM
Culture media and procedure for culturing
IVF-100TM was used in the first step of culture, from oocyte retrieval to fertilization, after which the oocytes were rinsed twice in G1.2TM and cultured in G1.2TM until the morning of day 3. In cases of blastocyst culture, G2.2TM (Vitrolife) was used from the 8-cell stage on day 3 until embryo transfer.
A maximum of six fertilized oocytes were cultured in 20 µl media droplets under oil (OVOILTM). In cases with fewer than six oocytes, 10 µl droplets were used. A gas phase of 6% CO2, 5% O2 and 89% N2 was used in a humidified incubator.
Assessment of fertilization, embryo morphology classification, cryopreservation and embryo transfer
Fertilization was determined 1820 h after the IVF/ICSI procedure. The oocytes were fertilized when two distinct pronuclei were visible. Cleavage and classification of morphology was assessed 24 h later by criteria set up by Ziebe S, Groendal ML, Agerholm I and Erb K (personal communication). Strict criteria for cryopreservation were used. Only embryos with at least seven blastomeres and <20% intracellular fragments were cryopreserved on day 3. On day 5, embryos were assessed according to scoring criteria for blastocysts (Gardner and Schoolcraft, 1999). Only expanded blastocysts were cryopreserved.
A maximum of two embryos were transferred on day 2, 3 or 5 after retrieval. Embryo transfer was performed in G2.2TM. All embryo transfers were performed with a Cook Soft 5000 catheter (Cook, Brisbane, Australia).
Luteal phase support and pregnancy test
IVF/ICSI patients received luteal phase support in the form of daily vaginal administration of micronized progesterone, either 400 mg twice a day (Cyklogest; Hoechst, Copenhagen, Denmark) or 90 mg once a day (Crinone 8%; Serono Nordic) starting on the day following oocyte retrieval and continuing until the day of the pregnancy test (i.e. day 12 after embryo transfer). A positive pregnancy test was defined by a plasma HCG concentration >10 IU/l. A clinical pregnancy was defined as an intrauterine gestational sac with a heart beat 3 weeks after a positive HCG test. The implantation rate was calculated as the ratio of gestational sacs determined by ultrasound after 7 weeks in relation to the total number of embryos transferred. Birth or abortion was recorded for all patients.
Statistical methods
Statistical analysis was performed using the SPSS 11.0 software (SPSS Inc., Chicago, IL). To test inter-group differences in sperm concentration, percentage progressive motile sperm, DFI and HDS, the MannWhitney test was applied. Binary correlations were assessed by calculating Spearmans rho. The reproductive outcome measures were treated as discrete variables (±fertilization; ± biochemical pregnancy; ±clinical pregnancy; ±delivery). Since a DFI of 27% previously was reported to be the cut-off level for achieving a pregnancy by in vitro ART (Larson-Cook et al., 2003), the couples were grouped according to whether the DFI in raw semen sample was
27% or >27%. Furthermore, having assessed the results of HDS analysis in relation to the reproductive outcomes, a cut-off level of 10% was defined for this parameter. By the use of Fischers exact test, the chance of a positive reproductive outcome was calculated for the different treatment groups (IUI, IVF and ICSI) divided according to the DFI and HDS as described above (DFI:
27% and >27%; HDS:
10% and >10%). To compare the results of IVF and ICSI, in the group with DFI >27%, odds ratios (ORs) and the 95% confidence intervals (95% CIs) were calculated by the use of binary logistic regression analysis. Fischers exact test and OR calculations were also applied to compare the reproductive outcomes, i.e. biochemical and clinical pregnancy in two groups defined as follows: group 1: DFI
27% and HDS
10%; and group 2: DFI >27% or HDS >10%. A P-value
0.05 was considered as statistically significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
There was a statistically significant negative correlation between DFI and the percentage of progressively motile spermatozoa ( = 0.25; P = 0.004), but not between DFI and sperm concentration. HDS correlated in a negative manner with both sperm concentration (
= 0.36; P < 0.0005) and percentage progressively motile sperms (
= 0.25; P = 0.004).
Predictive value of DFI>27%. One biochemical pregnancy was observed in the 23 cycles with males having DFI >27% (4.3%), whereas in the 27% group, 24 of the 108 cycles resulted in a positive pregnancy test (22.2%) (P = 0.07; Table II). The clinical pregnancy and delivery rates were 4.5 versus 20.2% (P = 0.12) and 4.5 versus 17.6% (P = 0.20), respectively. In the above-mentioned case where the DFI was >27%, the actual value being 33.7%, there was a positive biochemical and subsequently even clinical pregnancy and delivery. When the cut-off level was increased to a DFI of 34%, there were no statistically significant differences with regard to any of the reproductive outcomes.
|
Predictive value of combined use of DFI and HDS. When the patients were divided into two groups, group 1, with semen samples with DFI 27% and HDS
10%, and group 2, with either DFI >27% or HDS >10%, 24 biochemical pregnancies, 21 clinical pregnancies and 19 deliveries were observed among the 95 couples in group 1, whereas only one of the 36 couples in group 2 experienced a biochemical as well as a clinical pregnancy and, finally, delivery. These differences in reproductive outcomes between group 1 and group 2 were statistically significant, the P-values being 0.002 for biochemical pregnancy, 0.008 for clinical pregnancy and 0.01 for delivery rate. The OR for biochemical pregnancy in group 1, with group 2 as reference, was 12 (95% CI 1.596). This value increased to 20 (95% CI 2.3117) when sperm concentration, percentage progressive motile, female age and treatment number were included in the model. For clinical pregnancies, the ORs in group 1 were 9.9 (95% CI 1.377) and 16 (95% CI 1.9137), without and including the above-mentioned covariates, respectively. For delivery as an outcome variable, the ORs were 8.7 (95% CI 1.168) and 14 (95% CI 1.6110), respectively, with and without inclusion of sperm concentration, percentage progressively motile spermatozoa, female age and treatment number in the model.
IVF/ICSI
There was no difference in sperm concentration, percentage of progressively motile sperm, DFI or HDS when couples with positive and negative reproductive outcome were compared, regardless of whether biochemical or clinical pregnancy was used as a criterion. This lack of a statistically significant difference was true for the whole group of IVF/ICSI-treated couples and also when the two treatment types were analysed separately (data not shown).
Similarly to the findings in the IUI group, there was a statistically significant negative correlation between both DFI and HDS and the percentage of progressively motile sperm, and additionally between HDS and sperm concentration (data not shown).
There was a considerable overlap between DFI and HDS values when couples who achieved a pregnancy were compared with those who did not (Figure 2).
|
|
Predictive value of DFI >27%. In the DFI >27% group, the results of ICSI were significantly better than those of standard IVF regarding biochemical (70.6 versus 22.2%; P = 0.007) and clinical pregnancies (52.9 versus 22.2%; P = 0.09). For delivery rates, the percentages were 47.1% in the ICSI and 22.2% in the standard IVF group (P = 0.16) (Table III), with an OR of 7.8 (95% CI 1.736) for a positive HCG test, 3.9 (95% CI 0.917.0) for clinical pregnancy and 3.1 (95% CI 0.7213) for delivery rate. After inclusion of sperm concentration, percentage progressively motile sperm, female age and treatment number in the model, the ORs for successful reproductive outcome when ICSI was compared with standard IVF increased to 26 (95% CI 1.9350), 7.7 (95% CI 0.8470) and 7.0 (95% CI 0.7565), with biochemical pregnancy, clinical pregnancy and delivery as dependent variables, respectively. Significantly higher DFI (P = 0.014) (medians 18.0 versus 16.61) and HDS (medians 7.3 versus 6.81) (P = 0.033) were found in the ICSI group compared with IVF.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In couples treated with IUI, clinical pregnancy was seen in only one out of 23 cases when the DFI exceeded 27% and/or a HDS >10% was found in the raw ejaculate prepared and used for insemination. The OR was 16 for clinical pregnancy and 14 for delivery when both these parameters were below the cut-off levels given above. This finding is in accordance with previously published in vivo studies in which the time to pregnancy (TTP) for couples with no previous infertility background has been studied (Evenson et al., 1999; Spano et al., 2000
). These studies indicated a DFI level of 3040% as a cut-off for no pregnancy. Two other recently published studies (Duran et al., 2002
; Saleh et al., 2003
) reached a similar conclusion. Saleh et al. (2003
), using the SCSA, found significantly higher DFI levels in the couples who failed to obtain a pregnancy after IUI. This study was, however, based on 11 IUI couples only. Using the TUNEL assay indicative for DNA fragmentation, Duran et al. (2002
) found that men with a DNA fragmentation level >12% had a decreased chance of fathering a pregnancy following IUI.
Apart from slightly lower (close to the level of statistical significance) HDS in men of those IUI-treated couples who achieved a biochemical pregnancy, there was no statistically significant difference in SCSA parameters between the groups with a negative and a positive reproductive outcome. The SCSA parameter, HDS, is believed to correlate to the level of immature spermatozoa and the extent of chromatin packaging (Evenson and Jost, 2000). Although previous studies indicated that high HDS could be a pejorative factor for in vivo fertility impairment (Evenson et al., 1999
, 2002), in the available materials, the associations did not reach a level of statistical significance. In our study, when a relatively large number of couples were included, an HDS of
10% was associated with a higher proportion of clinical pregnancies and births, although the statistical significance was borderline. It is therefore to be considered, as a novel finding, that the predictive value of SCSA increases when both DFI and HDS are used to define groups with high and low chances of obtaining a pregnancy after IUI. Recently, we have shown that both DFI and HDS are independently and negatively associated with sperm motility (Giwercman et al., 2003
). Motility is essential for fertilization in vivo. Impairment of the ability of spermatozoa to reach the ovum might at least partly explain the association between SCSA parameters and the probability of obtaining a pregnancy after IUI. An additional explanation could be the fact that spermatozoa with damaged DNA could be more likely to undergo apoptosis during the transport through the genital tract than spermatozoa with normal DNA integrity (Edwards, 2003
).
The present study demonstrates that even men with high levels of DNA damage can become biological fathers, when DFI exceeded 27%; however, significantly higher clinical pregnancy rates (52.9 versus 22.2%) and delivery rates (47.1 versus 22.2%) were seen after ICSI compared with IVF. When DFI exceeded 27%, the OR for a positive reproductive outcome after ICSI compared with standard IVF was 8 for biochemical pregnancy, 4 for clinical pregnancy and 3 for delivery, respectively. These ORs increased after inclusion of sperm concentration, the percentage of progressively motile sperm, number of treatments and the age of the female as co-variates. The increase in OR was statistically significant (95% CIs not including the value of 1) for biochemical pregnancy, but not for the two other reproductive outcomes, probably due to the limited number of couples included in this study. None of the previous studies dealing with this issue (Larson et al., 2000; Larson-Cook et al., 2003
; Saleh et al., 2003
) have taken into consideration the age of the female and the number of previously failed ART treatments, parameters known to be associated with the success rate of the treatment. Therefore, our findings show that DFI as assessed by means of SCSA is an independent predictor of male fertility potential in vitro.
This is the first study showing that semen samples with high rates of DNA breaks as assessed by the SCSA method are more likely to result in pregnancy in ICSI procedures, compared with traditional IVF. The finding is novel in relation to the SCSA parameters, but confirms the results of a recent study by Host et al. (2000) using the TUNEL assay.
The safety of the ICSI procedure has been questioned (Schultz and Williams, 2002), and the findings from the present study may give further reason for concern. ICSI enables fertilization to take place in spite of severely compromised semen characteristics and damaged DNA. Aitken and Krausz (2001
) proposed that sperm DNA damage is promutagenic and can give rise to mutations after fertilization, as the oocyte attempts to repair DNA damage prior to the initiation of the first cleavage. Mutations occurring at this point will be fixed in the germline and may be responsible for the induction of pathology, such as infertility (Aitken and Krausz, 2001
), childhood cancer in the offspring (Fraga et al., 1996
; Ji et al., 1997
; Aitken and Krausz, 2001
) and imprinting diseases (Cox et al., 2002
; DeBaun et al., 2003
). So far, however, follow-up studies of children born after ICSI compared with children born after conventional IVF have not been conclusive regarding the risks of congenital malformations and health problems in general (Bonduelle et al., 1996
, 1998, 2003; Kurinczuk and Bower, 1997
; Sutcliffe et al., 1999
; Wennerholm and Berg., 2000
; Hansen et al., 2002
). It has been reported previously that the highest level of sperm DNA damage usually is found in men with the poorest semen quality, i.e. ICSI candidates (Aitken and Krausz, 2001
). In the present study, the men in the ICSI group had significantly higher DFI levels compared with the men in the IVF group (median 18.0 versus 15.0%, respectively). It gives reason for concern that the most efficient ART techniques are used to treat males with the highest level of sperm DNA damage. SCSA parameters, however, refer to the DNA status of the whole cell population, not excluding the presence of a subpopulation with no significant DNA damage. Host et al. (2000
), suggested that the technician who performs ICSI, attempts to select spermatozoa with normal morphology and thereby reduces the risk of introducing spermatozoa with strand breaks. This statement may, however, be questioned, since the traditional sperm parameters such as sperm count, motility and morphology have been proven to be poorly correlated to the SCSA parameters (Evenson et al., 1991
, 1999; Spano et al., 1998
, 2000). A future challenge will be to develop methods to identify and select spermatozoa with intact DNA during the ICSI procedure.
We selected a DFI level of 27% as a cut-off for calculation of ORs in relation to the reproductive outcome. This level was defined previously in reports where the SCSA measurement was performed in other laboratories. One could ask whether each laboratory performing the SCSA analysis should have its own reference levels or not. Apart from being subject to a very limited intra-laboratory variation (Giwercman et al., 1999), however, the SCSA analysis was shown to be very robust to variation between laboratories. In an external quality control based on >180 samples, a high (
= 0.8) correlation was found between the values obtained by our laboratory and those from a control laboratory. Furthermore, not only was there a high level of correlation between the results reported by two independent laboratories that strictly followed the SCSA protocol, but the absolute DFI values obtained at two different places, using different equipment, did not on average differ by >1% (Giwercman et al., 2003
). Furthermore, in the present study, viewing the distribution of DFI values between those who became pregnant and those who did not, 27% was confirmed to be an appropriate cut-off level.
The results from this study confirm previous reports indicating a low chance of obtaining in vivo pregnancy when the DFI exceeds the level of 27%. Contrary to previous reports, the present study has shown that a DFI level >27% is not incompatible with pregnancy and delivery following IVF and ICSI. ICSI seems to be the most efficient method in cases with high levels of sperm DNA breaks. Additional large-scale trials are needed to confirm the results of the present study and to investigate further the value of SCSA in relation to other available pregnancy predictors in vivo or in vitro. Methods for selection of spermatozoa with intact DNA should be developed in order to avoid the potential risk of using gametes with chromatin breaks for ICSI treatment.
![]() |
Acknowledgements |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aitken RJ and Krausz C (2001) Oxidative stress, DNA damage and the Y chromosome. Reproduction 122,497506.
Benchaib M, Braun V, Lornage J, Hadj S, Salle B, Lejeune H and Guerin JF (2003) Sperm DNA fragmentation decreases the pregnancy rate in an assisted reproductive technique. Hum Reprod 18,10231028.
Bonduelle M, Legein J, Buysse A, Van Assche E, Wisanto A, Devroey P, Van Steirteghem, AC and Liebaers I (1996) Prospective follow-up study of 423 children born after intracytoplasmic sperm injection. Hum Reprod 11,15581564.
Bonduelle M, Joris H, Hofmans K, Liabears I and Van Steirteghem AC (1998) Mental development of 201 ICSI children at 2 years of age. Lancet 351,1553.[CrossRef][ISI][Medline]
Bonduelle M, Ponjaert I, Steirteghem AV, Derde MP, Devroey P and Liebaers I (2003) Developmental outcome at 2 years of age for children born after ICSI compared with children born after IVF. Hum Reprod 18,342350.
Braun ER (2001) Packaging paternal chromosomes with protamine. Nature Genet 28,1012.[CrossRef][ISI][Medline]
Bungum M, Bungum L, Humaidan P and Yding-Andersen C (2003) Day 3 versus day 5 embryo transfer; a prospective randomized study. Reprod Biomed Online 7,98104.[Medline]
Carrell DT and Liu L (2001) Altered protamine 2 expression is uncommon in donors of known fertility, but common among men with poor fertilizing capacity, and may reflect other abnormalities of spermiogenesis. J Androl 22,604610.
Cox GF, Burger J, Lip V, Mau UA, Sperling K, Wu BL and Horsthemke B (2002) Intracytoplasmic sperm injection may increase the risk of imprinting defects. Am J Hum Genet 71,162164.[CrossRef][ISI][Medline]
DeBaun MR, Niemitz EL and Feinber AP (2003) Association of in vitro fertilization with BeckwithWiedemann syndrome and epigenetic alterations of LIT1 and H19. Am J Hum Genet 72,156160.[CrossRef][ISI][Medline]
Duran EH, Morshedi M, Taylor S and Oehninger S (2002) Sperm DNA quality predicts intrauterine insemination outcomea prospective cohort study. Hum Reprod 17, 31223128.
Edwards RG (2003) New outlooks on IVF spermatozoa. Reprod Biomed Online 5,8991.
Erenpreisa J, Erenpreiss J, Freivalds T, Slaidina M, Krampe R, Butikova J, Ivanov A and Pjanova D (2003) Toluidine blue test for sperm DNA integrity and elaboration of image cytometry algorithm. Cytometry 52A,1927.[CrossRef][Medline]
Evenson DP and Jost LK (2000) Sperm chromatin structure assay is useful for fertility assessment. Methods Cell Sci 22,169189.[CrossRef][Medline]
Evenson DP, Darzynkiewicz Z and Melamed MR (1980) Relation of mammalian sperm chromatin heterogeneity to fertility. Science 210,1131.[ISI][Medline]
Evenson DP, Jost LK, Baer RK, Turner TW and Schrader SM (1991) Individuality of DNA denaturation patterns in human sperm measured by the sperm chromatin structure assay. Reprod Toxicol 5,115125.[CrossRef][ISI][Medline]
Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K, de Angelis P and Clausen OP (1999) Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Hum Reprod 14,10391049.
Evenson DP, Larson KL and Jost LK (2002) Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques. J Androl 23,2543.
Fraga CG, Motchnik PA, Wyrobek AJ, Rempel DM and Ames BN (1996) Smoking and low antioxidant levels increase oxidative damage to sperm DNA. Mutat Res 351,199203.[CrossRef][ISI][Medline]
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 15,830839.
Gardner DK and Schoolcraft W (1999) In vitro culture of human blastocysts. In Jansen R and Mortimer D (eds), Towards Reproductive Certainty: Infertility and Genetics Beyond 1999. Parthenon Press, Carnforth, pp. 378388.
Giwercman A, Spanò M, Lahdetie J and Bonde JP (1999) Quality assurance of semen analysis in multicenter studies. Asclepios. Scand J Work Environ Health 25, Suppl 1,2325.[ISI][Medline]
Giwercman A, Richthoff J, Hjøllund H, Bonde JP, Jepson K, Frohm B and Spanò M. (2003) Correlation between sperm motility and sperm chromatin structure assay parameters. Fertil Steril 80,14041412.[CrossRef][Medline]
Hammadeh ME, Zeginiadov T, Rosenbaum P, Georg T, Schmidt W and Strehler E (2001) Predictive value of sperm chromatin condensation (aniline blue staining) in the assessment of male fertility. Arch Androl 46,99104.[Medline]
Hansen M, Kurinczuk JJ, Bower, C and Webb S (2002) The risk of major birth defects after intracytoplasmic sperm injection and in vitro fertilization. N Engl J Med 346,725730.
Host E, Lindenberg S, Kah JA and Christensen F (1999) DNA strand breaks in human sperm cells: a comparison between men with normal and oligozoospermic sperm samples. Acta Obstet Gynecol Scand 78,336339.[CrossRef][ISI][Medline]
Host E, Lindenberg S and Smidt-Jensen S (2000) The role of DNA strand breaks in human spermatozoa used for IVF and ICSI. Acta Obstet Gynecol Scand 79,599563.
Irvine S, Twigg JP, Gordon EL, Fulton N, Milne PA and Aitken RJ (2000) DNA integrity in human spermatozoa: relationships with semen quality. J Androl 31,3344.
Ji,BT, Shu XO, Linet MS, Zheng W, Wacholder S, Ying DM and Jin F (1997) Paternal cigarette smoking and the risk of childhood cancer among offspring of non-smoking mothers. J Natl Cancer Inst 89,238244.
Kramer JA and Krawetz SA (1997) RNA in spermatozoa: implications for the alternative haploid genome. Mol Hum Reprod 3,473478.[Abstract]
Kurinczuk JJ and Bower C (1997) Birth defects in infants conceived by intracytoplasmic sperm injection: an alternative interpretation. Br Med J 315,12601265.
Larson KL, De Jonge CJ, Barnes AM, Jost LK and Evenson DP (2000) Sperm chromatin structure assay parameters as predictors of failed pregnancy following assisted reproductive techniques. Hum Reprod 15,17171722.
Larson-Cook KL, Brannian JD, Hansen KA, Kasperson KM, Aarnold ET and Evenson DP (2003) Relationship between the outcomes of assisted reproductive techniques and sperm DNA fragmentation as measured by the sperm chromatin structure assay. Fertil Steril 80,895902.[CrossRef][ISI][Medline]
Lopes S, Sun JG, Juriscova A, Meriano J and Casper RF (1998) Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection. Fertil Steril 69,528532.[CrossRef][ISI][Medline]
Morris ID, Ilott S, Dixon L and Brison DR (2002) The spectrum of DNA damage in human sperm assessed by single cell gel electrophoresis (Comet assay) and its relationship to fertilization and embryo development. Hum Reprod 17,990998.
Muratori M, Maggi M, Spinelli S, Filimberti E, Forti G and Baldi E (2003) Spontaneous DNA fragmentation in swim-up selected human spermatozoa during long term incubation. J Androl 24,253262.
Perreault SD, Aitken RJ, Baker HW, Evenson DP, Huszar G, Irvine DS, Morris ID, Morris RA, Robbins, WA, Sakkas D et al. (2003) Integrating new tests of sperm genetic integrity into semen analysis: breakout group discussion. Adv Exp Med Biol 518,253268.[ISI][Medline]
Sakkas D, Manethoz E, Manicardi G, Bizzaro D, Bianchi PG and Bianchi U (1999) Origin of DNA damage in ejaculated human spermatozoa. Rev Reprod 4,3137.
Sakkas D, Moffatt O, Manicardi GC, Mariethoz E, Tarozzi N and Bizzaro D (2002) Nature of DNA damage in ejaculated human spermatozoa and the possible involvement of apoptosis. Biol Reprod 66,10611067.
Sakkas D, Manicardi GC and Bizzaro D (2003) Sperm nuclear DNA damage in the human. Adv Exp Med Biol 518,7384.[Medline]
Saleh RA, Agarwal A, Nelson DR, Nada EA, El-Tonsy MH, Alvarez JG, Thomas AJ and Sharma RK (2002) Increased sperm nuclear DNA damage in normozoospermic infertile men: a prospective study. Fertil Steril 78,313318.[CrossRef][ISI][Medline]
Saleh RA, Agarwal A, Nada EA, El-Tonsy MH, Sharma RK, Meyer A, Nelson DR and Thomas AJ (2003) Negative effects of increased sperm DNA damage in relation to seminal oxidative stress in men with idiopathic and male factor infertility. Fertil Steril 79, Suppl 3,1597605.[CrossRef][ISI][Medline]
Schultz RM and Williams CJ (2002) The science of ART. Science 296,21882190.
Spano M, Bonde JP, Hjollund HI, Kolstad HA, Cordelli E and Leter G (2000) Sperm chromatin damage impairs human fertility. The Danish First Pregnancy Planner Study Team. Fertil Steril 73,4350.[CrossRef][ISI][Medline]
Spano M, Kolstad AH, Larsen SB, Cordelli E, Leter G, Giwereman A, Bonde JP (1998) The applicability of the flow cytometric sperm chromatin structure assay in epidemiological studies. Asclepios. Hum Reprod 13,24952505.[Abstract]
Tomsu M, Sharma V and Miller D (2002) Embryo quality and IVF treatment outcomes may correlate with different sperm comet assay parameters. Hum Reprod 17,18561862.
Sutcliffe AG, Saunders K, Thornton S, Grudzinskas JG and Lieberman, BA (1999) Children born after intracytoplasmic sperm injection: population control study. Br Med J 318,704705.
Twigg J, Fulton N, Gomez E, Irvine DS and Aitken RJ (1998a) Analysis of the impact of intracellular reactive oxygen species generation on the structural and functional integrity of human spermatozoa: lipid peroxidation, DNA fragmentation and effectiveness of antioxidants. Hum Reprod 13,14291436[Abstract]
Twigg JP, Irvine DS and Aitken RJ (1998b) Oxidative damage to DNA in human spermatozoa does not preclude pronucleus formation in intracytoplasmic sperm injection. Hum Reprod 13,18641871.[Abstract]
Ward DC (1994) The structure of the sleeping genome: implications of sperm DNA organization for somatic cells. J Cell Biochem 55,7782.[Medline]
Wennerholm UB and Bergh C (2000) Obstetric outcome and follow-up of children born after in vitro fertilization (IVF). Hum Fertil 3,5264.
World Health Organization. (1999) WHO Laboratory Manual for the Examination of Human Sperm and SpermCervical Mucus Interaction. Cambridge University Press.
Zini A, Bielecki R, Phang D and Zenzes MT (2001) Correlations between two markers of sperm DNA denaturation and DNA fragmentation, in fertile and infertile men. Fertil Steril 75,674677.[CrossRef][ISI][Medline]
Zini A, Fischer MA, Sharir S, Shayegan B, Phang D and Jarvi K (2002) Prevalence of abnormal sperm DNA denaturation in fertile and infertile men. Urology 60,10691072.[CrossRef][ISI][Medline]
Submitted on January 9, 2004; accepted on March 31, 2004.