Full-term pregnancies achieved with ICSI despite high levels of sperm chromatin damage

L. Gandini1,5, F. Lombardo1, D. Paoli1, F. Caruso1, P. Eleuteri2, G. Leter2, R. Ciriminna3, F. Culasso4, F. Dondero1, A. Lenzi1 and M. Spanò2

1 Laboratory of Seminology and Reproductive Immunology, Department of Medical Pathophysiology and 4 Chair of Health Statistics, Department of Experimental Medicine, University of Rome ‘La Sapienza’, 2 Section of Toxicology and Biomedical Sciences, ENEA CR Casaccia and 3 ART Unit, CID, Rome, Italy

5 To whom correspondence should be addressed. e-mail: loredana.gandini{at}uniroma1.it


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Sperm DNA integrity is essential for the accurate transmission of genetic information. The clinical significance of this assessment lies in its association with not only natural conception rates, but also the success of assisted reproduction technology (ART). It has been reported that sperm chromatin structure assay (SCSA) identified thresholds for negative pregnancy outcome after ART when the DNA fragmentation index (DFI), previously known as COMP{alpha}t, was >30%. METHODS: In a prospective clinical study, we examined 34 male infertile patients, the husbands of women undergoing conventional IVF or ICSI. SCSA and ART were carried out on semen aliquots taken from the same ejaculate. Fertilization rate, embryo quality and pregnancy rates were correlated to SCSA parameters, DFI and highly DNA stainable (HDS) cells. RESULTS: No differences were seen in SCSA parameter values between patients initiating pregnancies and not doing so in either ICSI or conventional IVF. Pregnancies and normal delivery were obtained even with high levels of DFI. CONCLUSIONS: There is still controversy over whether analytical techniques currently in use are able to identify the level of damage to spermatozoa. Large-scale studies should be conducted in different clinical settings to determine the effects of sperm DNA damage on the outcome of ART.

Key words: chromatin damage/ICSI/IVF/sperm chromatin/structure assay


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Over the last decade, we have seen the increasing use of assisted reproductive technology (ART) to overcome the problem of couple infertility (Katz et al., 2002Go). ARTs are now routinely proposed in the cases of couples with a severely subfertile male partner who wish to have a child. However, the pregnancy rate is still relatively low and somewhat unpredictable, given the number of variables possibly involved in the multistep process of fertilization, embryo cleavage, implantation and pregnancy.

The methods of gamete manipulation used in ART are rapidly proliferating, and in some instances outpacing the underlying science (Schultz and Williams, 2002Go). The original indications of ICSI, initially suggested for cases of severe male factor infertility, have been expanded. There are controversies about the safety of the procedure, which bypasses all processes of natural selection from sperm–oocyte physical interaction, still present in conventional IVF, as it relies on the direct injection of a sperm into the oocyte cytoplasm. There are potential concerns relating to obstetric outcome, chromosomal abnormalities, congenital malformations and developmental abnormalities in ICSI-born progeny. Following recent strong warnings about the potential risks of ART (especially ICSI) for a number of reasons (Hansen et al., 2002Go; Schieve et al., 2002Go; Stromberg et al., 2002Go; DeBaun et al., 2003Go; Moll et al., 2003Go; Orstavik et al., 2003Go), scientific debate has become quite passionate (Bonduelle et al., 2003Go; Retzloff and Hornstein, 2003Go; Tournaye, 2003Go). ICSI has made the conventional criteria of concentration, motility and morphology for assessment of sperm quality irrelevant. The need for new assays for evaluation of sperm health has thus become a priority.

The identification of sound, suitable semen and sperm characteristics enabling prediction of the chances of successful outcome after ART is an area of active research. Intuitively, the more intact and functional the spermatozoon, the higher the chances of delivering a healthy progeny. Standard semen analysis is therefore a mandatory test in the investigation of the infertile couple; however, its predictive value in ART is quite limited (Host et al., 2001Go). Moreover, this analysis gives no insight into the integrity of the genome of the male gamete. In addition to chromosome aneuplodies, more subtle abnormalities at the DNA and/or chromatin level can adversely affect normal and assisted fertilization, as well as the normal development of the embryo, fetus and child (Brinkworth, 2000Go). The presence of sperm with DNA fragmentation and chromatin abnormalities in human ejaculates is well documented (Agarwal and Said, 2003Go; Sakkas et al., 2003Go), in particular in men with poor semen quality (Lopes et al., 1998Go; Gandini et al., 2000Go; Irvine et al., 2000Go; Hammadeh et al., 2001bGo; Sakkas et al., 2002Go; Saleh et al., 2002Go; Erenpreisa et al., 2003Go; Muratori et al., 2003Go). It should be highlighted that, in some cases of severe male factor infertility, a significant proportion of spermatozoa injected into oocytes may therefore contain fragmented DNA. Injection of oocytes with spermatozoa with abnormal chromatin will probably result in failure of sperm decondensation and fertilization. In addition, due to the negative association between semen analysis parameters and sperm with DNA damage, and as extremely poor semen samples are the indication for ART, there is a high probability that these altered sperm may be used for oocyte injection, resulting in poor fertilization and/or cleavage rates. There is therefore a real risk of accidentally selecting genetically abnormal sperm which may appear motile and morphologically normal.

It has been demonstrated that chromatin-damaged human spermatozoa are able to form normal pronuclei in oocytes after ICSI just like sperm with an intact genome (Twigg et al., 1998Go). As the consequences of this for embryo development are not entirely known, a general consensus is emerging that DNA/chromatin defective sperm assessment should be integrated as a complementary diagnostic tool in any ART programme (Perreault et al., 2003Go).

Sperm DNA integrity can be determined quickly and accurately using a range of techniques (reviewed in Evenson et al., 2002Go; Agarwal and Said, 2003Go; Perreault et al., 2003Go; Sakkas et al., 2003Go) which also seem to have strong prognostic power in predicting successful ART outcomes. This is demonstrated by the accumulating body of evidence suggesting that disturbances in the organization of genomic material in sperm nuclei, evaluated using a variety of assays, are negatively correlated with spermatozoa fertility potential, whether in vivo (Evenson et al., 1999Go; Hacker-Klom et al., 1999Go; Spanò et al., 2000Go; Bonde et al., 2003Go; Carrell et al., 2003Go) or in vitro (Hoshi et al., 1996Go; Sun et al., 1997Go; Hammadeh et al., 1998Go; Lopes et al., 1998Go; Sakkas et al., 1998Go; Filatov et al., 1999Go; Esterhuizen et al., 2000Go; Host et al., 2000Go; Larson et al., 2000Go; Evenson et al., 2002Go; Morris et al., 2002Go; Tomsu et al., 2002Go; Virant-Klun et al., 2002Go; Benchaib et al., 2003Go; Larson-Cook et al., 2003Go). However, even though the trend between the percentage of sperm with abnormal chromatin/DNA damage and ART outcome is quite clear, discrepancies still emerge when the fraction of defective sperm is associated with fertilization rate, embryo quality, pregnancy rate, and whether the technique has been used in the context of IVF or ICSI. When the flow cytometric (FCM) sperm chromatin structure assay (SCSA) was applied, thresholds (calculated on neat semen) were found for negative pregnancy outcome after ART which had not been identified using conventional semen parameters (Larson et al., 2000Go; Saleh et al., 2003Go; Larson-Cook et al., 2003Go).

The objective of this study was to determine possible relationships between SCSA parameters evaluated on both neat semen and the processed aliquot to be used in the ART programme, collected on the day of the oocyte pick-up, and fertilization rate, embryo quality and pregnancy rate following ICSI and IVF.


    Materials and methods
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 Materials and methods
 Results
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Patients
We examined 34 infertile male patients, the partners of women who were undergoing IVF or ICSI. The patients had suffered from primary infertility for at least 3 years, were aged 29–45, and had not been treated medically or surgically in the 3 months prior to the study.

Patients were divided into two groups according to the ART technique employed. Group A, with 12 patients, underwent IVF; and group B, consisting of 24 patients, underwent ICSI.

Group A female partners underwent IVF either because of a bilateral Fallopian tube obstruction, or following at least four failed attempts at intrauterine insemination with ovarian stimulation under ultrasonographic and hormonal control.

Group B female partners were normal, but seminal fluid of the male partners was characterized by moderate to severe oligoasthenoteratozoospermia (OAT) and so they underwent ICSI. Two male partners had a sperm concentration/ml of 80 and 110 x 106 ml, respectively. In these cases, ICSI was performed due to two previous failed attempts at IVF (fertilization rate <30%).

The median age of female partners was 34.8 years (range 26–44) in group A and 35.1 years (range 30–44) in group B.

All patients had a normal peripheral blood karyotype, and patients with severe OAT were free of Yq microdeletion.

All patients gave their informed consent prior to the study, which was approved by the Institutional Review Board of our University.

Semen examination and in vitro treatment
On the day of oocyte pick-up, semen samples were collected by masturbation into sterile plastic jars after 3–5 days of sexual abstinence. They were allowed to liquefy for 30 min at room temperature (22°C) and were then evaluated according to the WHO criteria (World Health Organization, 1999Go). The variables taken into consideration were: ejaculate volume (ml), sperm concentration (n x 106/ml), forward motility (%) and morphology (% atypical forms). After microscopic evaluation, the samples were prepared for the assisted reproduction procedure, using swim-up for IVF and PureSperm (BioCare, Europe) gradients for ICSI.

Samples for IVF were washed twice as follows: the semen sample was diluted 1:2 with Earle’s salt solution (ESS; Sigma Chemical Co, St Louis, MO) supplemented with 5% human serum albumin (HSA) (BioCare, Europe) and centrifuged for 10 min at 300 g; the supernatant was then discarded and an aliquot of 0.5 ml of ESS was layered on the pellet; after incubation for 30 min at 37°C in 5% CO2, the supernatant was recovered.

Samples for ICSI were prepared as follows: PureSperm 100% was diluted with ESS-HSA to obtain 40 and 80% dilutions. Several PureSperm gradient columns were prepared in 5 ml Falcon tubes by gently layering 1 ml of each solution, starting from the 100% fraction at the bottom; 0.5 ml of the semen sample was stratified on top of the discontinuous PureSperm gradient columns and centrifuged for 25 min at 300 g. After centrifugation, the 100% PureSperm fraction was recovered and after thorough washing was used for insemination.

One aliquot of each of the raw semen and processed spermatozoa obtained by swim-up or PureSperm gradient were treated for evaluation of sperm chromatin structure. Briefly, after washing in TNE buffer (0.15 mol/l NaCl, 0.01 mol/l Tris–HCl, 1 mmol/l EDTA pH 7.4), pellets were re-suspended in TNE buffer containing 10% glycerol at a final sperm concentration of 2 x 106/ml and transferred to Eppendorf snap-cap tubes. The tubes were kept on dry ice until transfer to an ultra-cold freezer (–80°C) for storage until FCM analysis.

Assisted reproduction procedures
Ovarian stimulation was induced using recombinant FSH therapy at a dosage personalized for each patient for the first 5 days. Further FSH doses and the timing of the administration of 10 000 IU of HCG were adjusted according to the usual criteria of follicular maturation determined by ultrasound and estradiol findings. Pre-treatment with s.c. buserelin (2 x 100 µg, Suprefact, Hoechst Marion Roussel) was started in the midluteal phase of the cycle prior to stimulation.

Oocytes were retrieved 36 h after HCG administration by transvaginal ultrasound-guided aspiration.

Embryo quality was assessed 48 h after fertilization using the following grading system: grade 1, even, regular spherical blastomeres, intact zona, very few fragments (<10%); grade 2, uneven or irregular sized blastomeres, intact zona, no more than 20% fragments; grade 3, fragmentation of no more than 50% of blastomeres, intact zona; and grade 4, >50% fragmentation, uneven or irregular shaped blastomeres (Veeck, 1991;Go Desai et al., 2000Go).

All embryo transfers were performed 2 days after oocyte retrieval using Fryedman catheters (CCD Laboratories, Paris, France). The luteal phase was supported with progesterone (Crinone® 8%; ARES Serono SA, Rome) administered daily by the vaginal route, starting on the evening of embryo transfer.

Serum {beta}HCG levels were measured 12 days after embryo transfer. Clinical pregnancy was confirmed by transvaginal ultrasound 5 weeks after embryo transfer.

FCM SCSA
SCSA was applied following the procedure described in literature (Spanò et al., 2000Go; Evenson et al., 2002Go). On the day of analysis, samples were quickly thawed in a 37°C water bath and used immediately. A total of 1–2 x 106 cells were treated with a 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 and then stained with 6 mg/l of purified acridine orange (AO; Molecular Probes, Eugene, OR) in a phosphate–citrate buffer, pH 6.0. Cells were analysed by a FACScan flow cytometer (Becton Dickinson, San Josè, CA), equipped with an air-cooled argon ion laser. A total of 5000 events were accumulated for each measurement. Under these experimental conditions, AO intercalated with double-stranded DNA emits green fluorescence and AO associated with single-stranded DNA emits red fluorescence when excited with a 488 nm light source. Sperm chromatin damage can thus be quantified by 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.

Adopting the guidelines described in a recent publication (Evenson et al., 2002Go) and using the dedicated SCSASoft software (SCSA Diagnostics, Brookings, SD) for off-line data analysis, we expressed the extent of DNA denaturation in terms of the DNA fragmentation index (DFI), the ratio of red to total (red plus green) fluorescence intensity which previously was known as COMP{alpha}t. This is the level of denatured DNA over total DNA. The DFI value was calculated for each sperm cell in a sample, and the percentage of abnormal sperm with detectable DFI values was evaluated from the resulting DFI frequency distribution histogram.

We also considered the fraction of highly DNA stainable (HDS) cells. Their percentage was calculated by setting an appropriate gate on the scattergram (abscissa: red fluorescence, fragmented DNA; ordinate: green fluorescence, native DNA stainability) and considering as immature spermatozoa those events exhibiting a green fluorescence intensity higher than the upper border of the main cluster, which represents the sperm population with non-detectable DFI. For flow cytometer set-up and calibration, aliquots were used from a normal human ejaculate sample retrieved from our laboratory repository. Samples were measured twice, blind, and the code was broken only at the end of the measurement series. The results reported refer to the mean value of the two FCM measurements.

Statistical analysis
Descriptive statistical analysis was performed separately on the two groups. Comparisons of mean values were calculated from the appropriate test (paired t-test and Wilcoxon test, t-test for independent samples and Mann–Whitney test) for the variables HDS, DFI, motility and atypical forms. A logistic regression was performed separately for the two groups, to assess the association of pregnancy and DFI and embryo grading. Finally, correlation (Pearson’s r and Spearman correlation) of the variables with fertilization rate and DFI was calculated. Statistical analyses were also performed after logarithmic transformation of percentage variables.


    Results
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 Abstract
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 Materials and methods
 Results
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 References
 
We studied 34 couples, of whom 12 underwent IVF-ET (group A) and 22 ICSI (group B).

Group A (IVF-ET)
Table I reports the mean ± SD of sperm and SCSA parameters of the samples before and after the swim-up procedure. The improvement in sperm motility and atypical forms was statistically significant (P < 0.001). Differences were also statistically significant (P < 0.001) for the SCSA parameters DFI and HDS. An improvement in sperm chromatin quality [reduction of percentage sperm with damaged DNA (DFI fraction) and percentage spermatozoa with high DNA stainability (HDS fraction)] was seen for all samples after swim-up (Table II).


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Table I. Mean (±SD) and significance of sperm and SCSA parameters of the 12 IVF patients
 

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Table II. DFI and HDS percentage before and after swim-up, number of oocytes inseminated and fertilized, fertilization rate and full-term pregnancy outcome for each IVF patient
 
The mean ± SD of oocytes inseminated and fertilized were 10.0 ± 4.8 and 7.5 ± 3.9, respectively; the fertilization rate was 68.8%, and three pregnancies (25%) were obtained in this group. All three reached full term and five healthy children were born (two single births and one set of triplets). Table II shows, for each patient, results of DFI and HDS in raw semen and after swim-up, fertilization rate and embryo grading grouped for obtained and failed pregnancies. There was no significant correlation between DFI/HDS results before and after swim-up and the fertilization rate (P > 0.05). No significant differences were seen between DFI and HDS fractions of patients achieving and not achieving pregnancy.

Group B (ICSI)
The mean ± SD of sperm and SCSA parameters of the samples before and after PureSperm selection are reported in Table III. The improvement in sperm motility and atypical forms was statistically significant (P < 0.001). In this group, the difference in DFI values between raw semen and after Pure Sperm gradient was not statistically significant. However, the difference was statistically significant for HDS values (P < 0.001).


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Table III. Mean (±SD) and significance of sperm and SCSA parameters of the 22 ICSI patients
 
The mean ± SD of inseminated and fertilized oocytes were 9.0 ± 5.2 and 6.2 ± 4.1, respectively. The fertilization rate was 70.0%. In this group, nine pregnancies were achieved (40.9%). All reached full term and 13 healthy children were born (seven single births and two sets of triplets). Five of these pregnancies were initiated by patients whose DFI value was >27%. No statistically significant differences were seen for DFI and HDS values when the group achieving pregnancy was compared with the group who failed to father a child (Table IV). No correlation was found between percentage of spermatozoa with altered DFI and HDS and the fertilization rate and embryo grading in patients achieving and not achieving a pregnancy by ICSI (Table IV). It is worth highlighting the heterogeneity of the SCSA values on the neat samples of the nine male partners of couples achieving pregnancy after ICSI, with DFI ranging from 13.5 to 66.3%.


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Table IV. DFI and HDS percentage, fertilization rate and embryo grading for each ICSI patient
 
No significant differences in the age of male and female partners of the couples and number of oocytes retrieved were seen in the two groups (data not shown).


    Discussion
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 Materials and methods
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From the literature available, no difference has been reported between levels of embryo loss after ICSI and IVF. For example, the European IVF monitoring programme (EIM) for the European Society of Human Reproduction and Embryology (ESHRE) recently reported that, for treatments initiated during 1997, the clinical pregnancy rate per transfer for IVF was 26.1% and the delivery rate per embryo transfer 20.9%, whereas for ICSI, the corresponding rates were 26.4 and 21.5% (ESHRE, 2001Go). Our results are in general agreement with these figures. However, Sanchez et al. (1996Go) reported a miscarriage rate following ICSI greater than expected in conventional IVF, possibly reflecting the use of genomically compromised spermatozoa which conferred irreparable DNA damage to the embryo, resulting in its abortion. Moreover, a higher frequency of numerical chromosome abnormalities after ICSI than with IVF has been demonstrated (Macas et al., 1996Go) and there is evidence that genetic damage in paternal gametes could predispose offspring to disease that does not manifest itself until later in life, particularly childhood cancer (Sorahan et al., 1997Go).

As sperm genetic integrity has been implicated in fertilization and embryo development failures (Ahmadi and Ng, 1999Go; Cho et al., 2003Go), several groups have also analysed sperm nuclear integrity in terms of DNA/chromatin damage and its relationship to male infertility and ART outcome, why nuclear damage originates in certain males and how it influences the mature spermatozoon. Several methods are now available to detect sperm chromatin abnormalities, and their value and robustness are under active scrutiny (Irvine et al., 2000Go; Evenson et al., 2002Go; Erenpreisa et al., 2003Go; Perreault et al., 2003Go).

Not only sperm genetic integrity (Ahmadi and Ng, 1999Go) but also protamine deficiency can have a negative impact on male fertility, resulting in the failure of fertilization and embryo development. Knock-out mice for protamine 2 expression showed an increased rate of sperm with DNA breaks (as seen by the COMET assay) and embryo failure after ICSI (Cho et al., 2003Go), confirming the pivotal role protamines play in safeguarding DNA integrity.

The impact of sperm DNA/chromatin integrity on ART outcome has been studied using a variety of techniques. Using aniline blue staining, a method allowing detection of chromatin abnormalities in terms of excessive residual histones in the sperm chromatin assembly, it has been reported that chromatin condensation is a good predictor for IVF outcome, as fertilization and pregnancy rate were higher when the fraction of sperm with abnormal chromatin was <20% (Hammadeh et al., 1998Go). However, the same group found no significant relationships for fertilization, cleavage, implantation and pregnancy rates when applying the aniline blue technique to ICSI outcome (Hammadeh et al., 1999Go).

Using the AO method (Tejada et al., 1984Go), Hoshi et al. (1996Go) reported that when ≥50% of spermatozoa exhibited green AO fluorescence, IVF was always successful, whereas if AO green fluorescent sperm were <50%, no full-term pregnancies resulted, even after successful ICSI fertilization. Virant-Klun et al. (2002Go) noted that when the level of red fluorescent spermatozoa increased, there was a significantly lower fertilization rate after ICSI and a significantly higher number of embryos were arrested. However, no correlation was found between abnormal spermatozoa pregnancy rate and live birth rate achieved by ICSI, except in patients having no spermatozoa with single-stranded DNA, in whom the pregnancy rate was significantly higher. Katayose et al. (2003Go) used a modified AO procedure involving the derivative diamide-AO, which oxidizes nuclear protamine thiol groups. They noted a positive correlation between the fertilization rate after conventional IVF and the fraction of normal green fluorescent sperm. However, Angelopoulos et al. (1998Go) found that AO staining did not predict fertilization efficiency or pregnancy outcome in IVF cycles, and no correlations were found between chromatin decondensation and fertilization rate after ICSI (Hammadeh et al., 2001aGo).

The chromomycin A3 (CMA3) method can detect sperm chromatin abnormalities, always at the level of histone-to-protamine substitution but by examining the relative protamine abundance: decondensed, protamine-depleted spermatozoa will appear fluorescent under the microscope. Sakkas et al. (1996Go), taking normal semen values as normal morphology >20%, CMA3 fluorescence <30% and endogenous nicks <10%, reported that in a series of ICSI patients, abnormal semen samples produced more than double the number of unfertilized oocytes containing condensed spermatozoa than did normal semen samples, but no relationships were found with the fertilization rate. Moreover, CMA3 fluorescence (but not aniline blue staining) correlated with post-ICSI fertilization outcome (Razavi et al., 2003Go)

Sperm DNA damage can be measured directly by terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) and single-cell gel electrophoresis (COMET) assay. Using the TUNEL assay, negative associations were found between the percentage sperm with DNA fragmentation and fertilization and embryo cleavage rates after IVF (Sun et al., 1997Go). A significant negative association was also found between the percentage sperm with DNA fragmentation and ICSI fertilization rate (Lopes et al., 1998Go). Benchaib et al. (2003Go) found that a high proportion of sperm with fragmented DNA (>10%) was a negative factor for achievement of pregnancy when ICSI was performed, but there was no relationship when conventional IVF was carried out. However, Host et al. (2000Go) found that the level of DNA strand breaks was predictive of the success rate in IVF but not in ICSI.

Using the COMET assay, Tomsu et al. (2002Go) noted that the COMET head and tail DNA parameters could be considered potentially useful predictors of embryo quality and IVF outcomes, especially in couples with unexplained subfertility. It has also been shown that high loads of DNA damage were predictive of embryo development failure after ICSI (Morris et al., 2002Go).

SCSA is a quantitative assessment of sperm chromatin integrity defined as susceptibility of DNA to acid-induced denaturation in situ. It has been shown that the fraction of abnormal sperm detected by SCSA is higher in men with infertility problems (Zini et al., 2001aGo,b; Saleh et al., 2002Go; Schmid et al., 2003Go).

Two large independent studies (Evenson et al., 1999Go; Spanò et al., 2000Go) on the relationship between SCSA results and sperm fertilization capacity have been carried out in the USA and Europe. Both demonstrated that when >30% of sperm have abnormal chromatin as evaluated by SCSA, human male fertility is hampered, independent of sperm number, morphology and motility. SCSA also seems correlated with embryo development failures (Evenson et al., 1999Go; Bonde et al., 2003Go). Recently, SCSA has also started to be applied in the context of ART.

In the three SCSA papers published to date (Larson et al., 2000Go; Larson-Cook et al., 2003Go; Saleh et al., 2003Go), no full-term pregnancies (after ICSI, IVF or IUI) were obtained when the DFI fraction was >28%. This leads to the conclusion that SCSA-derived thresholds significantly predict negative pregnancy outcomes in couples attempting pregnancy via ART. In particular, Larson et al. (2000Go) reported a pregnancy rate of 24% per oocyte transferred if COMP{alpha}t (now termed DFI) was <27%, and 0% if COMP{alpha}t was >27%; Saleh et al. (2003Go) obtained 30% of pregnancies for IVF and 25% for ICSI; Larson-Cook et al. (2003Go) reported 29 pregnancies out of 89 FIVET/ICSI (32.5%); 28 pregnancy were obtained with a DFI <27%; one ICSI patient achieved a biochemical pregnancy with >27% DFI, but subsequently lost it before ultrasound.

The results of our ART programmes and SCSA-assessed sperm integrity do not indicate a clear predictive power for DFI (and HDS) fraction for ICSI fertility rate, embryo quality and pregnancy rate. The mean DFI value for the nine men who fathered a child (pregnancy rate 40.9%) was 32.1% (23.7% after PureSperm separation). This value was no different from that of the group of men not fathering a child (25.1%; 24.6% after PureSperm).

The categories proposed by Evenson et al. (2002Go) for individual fertility potential according to DFI fraction are: excellent <15%, good 15–24%, fair 25–30%, and poor >30% DFI, and if HDS is >15%, the fertility potential is downgraded at least one category, e.g. from excellent to good. Using these categories, five of the nine ICSI successes were characterized by excellent SCSA quality after PureSperm, and three were above the DFI threshold proposed by other authors (Larson-Cook et al., 2003Go; Saleh et al., 2003Go). This is in agreement with Larson-Cook et al. (2003Go) who reported that fertilization rate, cleavage rate and blastulation rate were not significantly related to SCSA parameters. Fertilization rates in our study were similar to those of Larson-Cook et al. (2003Go). However, Saleh et al. (2003Go) found that DFI levels were negatively correlated with fertilization and embryo quality after IVF and ICSI.

The semen quality of the IVF group is notably higher in terms of concentration, morphology, motility and chromatin structure integrity than seen in the ICSI group. In the IVF group, SCSA analysis on raw semen demonstrated seven excellent and five good samples, whereas after swim-up 11 were excellent and one was good. Pregnancies were obtained for DFI values of <10%, especially in the swim-up samples used for fertilization, well under the proposed threshold of 30–40%. However, this alone does not ensure that pregnancy is achieved, as other sperm factors must be involved in the fertilization process.

DFI values of individuals who fathered a child (11.1% raw semen, 5.8% after swim-up) were not statistically different from those who did not (13.1% raw semen, 5.4% after swim-up). We are therefore unable to draw any definitive conclusion on the predictive power of SCSA DFI, as donor semen characteristics were always of a high quality even where IVF pregnancies were obtained with ejaculates in which DFI was below the proposed threshold value for in vitro and in vivo fertility.

The swim-up technique very effectively separated the fraction of highly motile cells which were characterized, in all instances, by better sperm chromatin integrity. Mean DFI was 12.0% in the neat semen: this figure dropped to 5.5% after swim-up, confirming the evidence of previous experiments (Spanò et al., 1999Go). The swim-up procedure discriminates a subpopulation of highly motile cells with improved morphology, consistently selecting spermatozoa to give a notable improvement in sperm chromatin features as evaluated by SCSA. In particular, the fraction of DFI and HDS cells markedly decreased in all samples, and the mean value was less than half that observed in neat semen samples.

The swim-up methodology is considered that closest to the natural selection occurring in the female genital tract, emphasizing that motility and migration in a medium different from seminal plasma represent an effective, selective, non-invasive procedure to select sperm with the best chance of achieving fertilization. In contrast, PureSperm gradient, although powerful in its selection of motile and good morphology spermatozoa (Sakkas et al., 2000aGo; Tomlinson et al., 2001Go), was less effective in enriching the cell population with a higher fraction of sperm with normal chromatin structure: in fact, DFI was 28.1% in neat semen and dropped to 24.2% after PureSperm. In our series, some patients showed a higher DFI value after Puresperm than in raw semen. This has also been noted by other groups, indicating that the potential detrimental effect of density gradient centrifugation on sperm DNA integrity may be related to initial semen quality (Zini et al., 2000aGo,b), which in ICSI patients is generally poor. However, the gradient separation procedure was quite effective in reducing the sample’s HDS fraction.

In our opinion, it is not surprising that the invasiveness of the ICSI procedure allows even low quality sperm the chance to initiate a successful pregnancy. Successful pregnancies have been reported in ICSI even using testicular spermatids (Mansour et al., 2003Go), whose chromatin structure, by definition, is quite different from that acquired by sperm after spermiogenesis and epididymal maturation. Other reports in the literature using different approaches to assess sperm chromatin structure and DNA integrity [e.g. aniline blue staining (Hammadeh et al., 1998Go, 1999) or TUNEL assay (Host et al., 2000Go)] have found such characteristics to be more predictive in IVF and much less so in ICSI. Apparently, sperm chromatin integrity becomes particularly relevant when the contact between the two gametes occurs in a more natural way, such as in normal circumstances or in conventional IVF. We can speculate that in these circumstances, selective pressures operate to avoid the development of an embryo derived from sperm with a high load of genetic damage (Morris et al., 2002Go). This can be different in ICSI, where sperm are forced into the oocyte and the possibility of selecting an unwanted, genetically defective sperm is much higher. As it is likely that sperm with high DNA damage levels have contributed to successful fertilization and in vitro development, potential adverse effects remain to be clarified (Sakkas et al., 2000bGo).

Clearly, the early stages of zygote formation are completely different between ICSI and IVF (Hewitson et al., 2003Go; Terada et al., 2003Go) and the consequences of this are not well understood. We know that heavily damaged sperm can fertilize hamster oocytes and form pronuclei at the same rate as an intact sperm (Twigg et al., 1998Go). ICSI may be facilitating the transfer of genetic disorders to future generations by bypassing all natural hurdles for sperm selection without imposing more pertinent selection criteria. Sperm DNA quality is vital to the future offspring, irrespective of whether the child is conceived naturally, by IVF or by ICSI. Certainly, the biological impact of an abnormal sperm chromatin structure depends on the combined effects of the extent of DNA or chromatin damage in the spermatozoa and the capacity of the oocyte to repair that damage. Therefore, if spermatozoa selected from samples with extensively damaged DNA are used for IVF, the oocyte repair capacity may be inadequate, leading to a low rate of embryonic development and high early pregnancy loss. This concept is quite intuitive; however, although we are now reasonably able to assess the damage level of a sperm population, we cannot assess the repair capability of the oocyte and do not know if there are oocytes capable of repairing even heavily DNA damaged sperm. Neither can the possibility of selection, within a sample characterized by a high DFI fraction, of sperm with limited DNA damage compensated by the oocyte repair capabilities, be excluded.

We are aware that our study size is quite limited as the total number of patients who underwent ART was small. However, we wish to stress that for the first time, ICSI term pregnancies were obtained even with semen samples characterized by a high SCSA DFI (>30%). As fertilization is a probabilistic event between two individual gametes, we expect to find a probabilistic function describing the phenomenon instead of an all-or-nothing threshold effect for unequivocal pregnancy failure prediction. The assays available to measure abnormality or change in sperm chromatin structure or DNA have been linked most commonly with reductions in fertilization potential, but we are only beginning to evaluate their prognostic power in ART programmes. To quote some remarks of the Breakout Group on the integration of new tests for sperm genetic integrity into semen analysis (Perreault et al., 2003Go): ‘Few tests have found acceptance and routine integration into clinical settings or field studies because of technical concerns, the lack of full understanding of the biological mechanisms behind the endpoints, the lack of standardized methodologies and of consensus protocols to challenge their predictive value for abnormal reproductive outcomes of clinical importance’. Before a large-scale conclusion can be reached, randomized studies in the future must determine the effects of sperm DNA damage on the outcome of ART, and of ICSI in particular, in different clinical settings.


    Acknowledgements
 
The authors wish to thank Marie-Hélène Hayles for her assistance in English translation of the manuscript. This work was supported by a grant from the Italian Ministry of Education and Research (MIUR-COFIN) and the University of Rome ‘La Sapienza’, Faculty of Medicine.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on February 5, 2004; accepted on February 27, 2004.