1 Département de Médecine de la Reproduction, Hôpital Edouard Herriot, Place dArsonval, 69373 Lyon cedex 08, 2 INSERM U418, Hôpital Debrousse, 29 Rue Soeur Bouvier, 69322 Lyon cedex 05, France
3 To whom correspondence should be addressed at: Laboratoire de Biologie de la Reproduction, 8 avenue Rockefeller, 69373 Lyon Cedex 08, France. e-mail: benchaib{at}rockefeller.univ-lyon1.fr
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Abstract |
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Key words: ART/DNA fragmentation/embryo quality/human sperm
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Introduction |
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Materials and methods |
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Ovarian stimulation
After three weeks of desensitization by GnRH analogues (Decapeptyl®; Ipsen), ovarian stimulation was achieved by recombinant FSH (Gonal-F®; Serono, or Puregon®; Organon), and monitored by endovaginal echography and plasma estradiol. When the follicles reached the desired diameter, 36 h before oocyte retrieval, 10 000 IU of hCG (Organon) was administered. The oocyte retrieval was carried out under general anaesthesia by a vaginal ultrasonographic-guided aspiration.
Sperm preparation for ART
The sperm were prepared using a discontinuous PureSperm gradient (Nicadon, Gothemberg, Sweden). The gradient consisted of 3 layers of 1 ml of PureSperm: 90, 70 and 50%. On the 50% layer was deposited 1 ml of semen. The gradient was then centrifuged at 300 g for 20 min. After centrifugation, the 90% layer was collected and washed with 5 ml of Ferticult Flushing (FertiPro N.V., Beernen, Belgium) at 600 g for 10 min. The pellet was then resuspended in IVF Medium (Scandinavian IVF, Gothenburg, Sweden) for IVF or in HEPES IVF medium for ICSI.
Embryos
At 1618 h after insemination or microinjection, the oocytes were assessed for fertilization (two pronuclei (PN) stage). Then 48 h after oocyte retrieval, the embryos were classified according to their morphology. Classification was as follows: grade A: no fragmentation and four regular cells; grade B: <25% fragmentation; grade C: between 25 and 50% fragmentation and grade D: >50% fragmentation (Ebner et al., 2001). The transfer of the embryos took place either at 48 h, or at 72 h, or at the blastocyst stage. When the transfer was carried out at 48 or 72 h, the supernumerary embryos were cryopreserved if their morphological states allowed it (grade A or B). If they were not cryopreserved, they were cultivated in sequential medium until the blastocyst stage was reached, and if one or more good quality blastocysts were obtained, those were then cryopreserved. When a transfer at the blastocyst stage was programmed, embryos were cultivated in sequential medium: P-1 Medium (Irvine Scientific, Santa Ana, CA, USA) for the two first days, Blastocyst Medium (Irvine Scientific) for the last days of culture. After the transfer, the remaining good morphology blastocysts were cryopreserved. A clinical pregnancy was assessed by the succession of 3 positive plasma
hCG and ultrasound detection of a fetal heartbeat.
Semen sample preparation for DNA fragmentation study
The detection of cells with fragmented DNA was performed on the spare sperm suspension that was used for ART procedure (the volume was between 50 and 60 µl of selected motile sperm suspension). All patients had given previously their informed consent for the study. The cells were spread out over sialinized slides. Cell fixation was carried out by a methanol/acetic acid mixture (3 volumes/1 volume) for 20 min. The cells were permeabilized with phosphate buffer saline (PBS) with 1% of Triton X100 (Sigma). Cells with fragmented DNA were revealed by TUNEL Technique by use of the Apoptag plus Kit (Oncor, Illkirch, France). The cells were all first neutralized with the balanced buffer. Then, sperm cells were incubated in a moist chamber at 37°C, for 1 h, with the Terminal d Transferase (TdT) solution in order to allow DNA elongation. The elongation was revealed by incubation of the cells with anti-digoxigenin antibody coupled to peroxidase, during 30 min in a dark moist chamber. The peroxidase was revealed with DiAminoBenzidine (DAB). Counterstaining of the sperm nucleus was performed with Harris haematoxylin. A positive control was done on positive slides supplied in the Oncor Kit. The cells were observed under a microscope (Zeiss, Oberkochen, Germany) equipped with x100 oil-objective. The sperm with fragmented DNA had their nuclei stained in brown, whereas the other cells nuclei were blue-grey. On each slide 500 cells were counted, the percentage of sperm with fragmented DNA were thus determined.
Statistical evaluation
Statistical analysis was performed with SPSS for Windows software package version 10.1 (SPSS Inc., Chicago, IL, USA). The 2-test was used to analyse the qualitative parameters. For the comparison of quantitative parameters, the Students t-test and ANOVA with mixed factors (mixed linear model) were used. Spearmans correlation coefficients were calculated. Statistical differences were considered significant at P < 0.05, for some comparisons the power analysis was calculated when no significant differences were found to ensure that the lack of significance could not be attributed to low sample number. The construction of a receiver-operating characteristic (ROC) curve allowed us to determine the thresholds values of DNA fragmentation having a prognostic role.
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Results |
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For the men, the mean age was 35.5 ± 4.9 years. In 38.5% of the cases, the sperm used for the ART procedure presented anomalies according to World Health Organization (WHO) standards (World Health Organization, 1999). An oligospermia was found in 26% of the cases, an asthenospermia in 19.2% and a teratospermia in 15% of the cases. These anomalies were not always isolated.
The mean (± SD) quantity of FSH administered to the patients was of 2823 ± 1351 IU, the average time of stimulation was of 12.1 ± 1.6 days and the estradiol rate at the day of the puncture was of 2675 ± 1556 pg/ml. The results were summarized according to the ART procedure used (Table I).
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DNA fragmentation and pregnancy
The pregnancy rate was influenced neither by the sperm characteristics, nor the type of ART procedure.
According to the ART procedure, when IVF was performed, DNA fragmentation was not statistically different whether a pregnancy was established or not. When ICSI was carried out, DNA fragmentation was significantly lower when a pregnancy was obtained (P < 0.05) irrespective of the age of the embryos at the time of the transfer (Figure 4).
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Discussion |
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Ahmadi and Ng (1999a) showed that high sperm DNA fragmentation did not impair fertilization but precluded the blastocyst formation. For example, oxidative damage to sperm DNA does not impede pronucleus formation after micro-injection in hamster eggs (Twigg et al., 1998
). This observation is rather logical since it is generally assumed that the first steps of development are under the dependence of the maternal transcripts, thus the paternal expression would normally start at the 68 cell stage; it must be reminded that in the majority of cases embryo transfers are performed at day 2 or day 3, i.e. before the paternal effect can be expressed. However, recent studies have shown that the paternal genome could play a role very early in the human oocyte development, as soon as the first cell cycle (Tesarik et al., 2002
). This could explain the controversies about the impact of sperm DNA fragmentation on fertilization rate, since this early effect is certainly less important than the alternative effect observed at the 68 cells stage; it is therefore a logical hypothesis that DNA fragmentation may indeed impair fertilization, but only for highly fragmented DNA as observed in the present study. Whereas there are still controversies about the impact of high sperm DNA fragmentation on the fertilization rate, there is a wider agreement concerning their negative effects on embryo development (Ahmadi and Ng, 1999a
; Morris et al., 2002
) and pregnancy rate (Ahmadi and Ng, 1999a
; Host et al., 2000
; Larson et al., 2000
; Tomlinson et al., 2001
).
In our study this deleterious effect of high sperm DNA fragmentation rate was significant in ICSI cycles, but not significant in IVF cycles, in disagreement with Tomsu et al. (2002) who used Comet assays for evaluating sperm DNA damage. This difference may be explained as follows: since most ICSI cycles are performed because of poor sperm characteristics (that is not the case in IVF), it is logical to find a higher rate of sperm cells with elevated DNA fragmentation in ICSI than in IVF cycles. Moreover, it is possible that IVF steps lead to a natural selection of sperm: the selected fertilizing spermatozoon will be morphologically normal and highly motile, and supposed to have an intact DNA. This hypothesis is supported by recent studies, including the one of Van Dyk et al. (2000)
who suggested that the human zona pellucida had the capacity to select against aneuploid sperm. It was shown by several authors that in a poor quality sperm population (according to classical criteria), DNA damage is found at a high level (Sun et al., 1997
; Lopes et al., 1998
; Gandini et al., 2000
; Irvine et al., 2000
; Younglai et al., 2001
); however, such sperm with DNA damage would have very few chances of fertilizing the oocyte if they were used in an IVF procedure. The problem is different in ICSI, where the choice of the spermatozoon to be injected is made according to very rough criteria, and in case of very poor sperm characteristics, it is even not possible to select one normal motile sperm so the risk of injecting one spermatozoon with impaired DNA is high.
These observations can have a number of applications in certain situations. For example, it has been shown that testicular extracted sperm have lower DNA fragmentation than sperm retrieved from the epididymis (Steele et al., 1999). So if the choice between testicular and epididymal sperm is possiblein cases of obstructive azoospermiatesticular extracted sperm should be preferred to epididymal sperm for ICSI in order to obtain embryos with a good development potential.
Considering the overall data (ICSI and IVF cycles), we showed an important drop in the relative risk of pregnancy as the proportion of sperm with DNA fragmentation reached higher than 18%. The specificity values reached 90 and 95% for fragmentation proportions of 15 and 18% respectively. With the aim of defining the best strategy for couples, it seems more important to look for high specificity rather than a high sensitivity: thus the observation of no pregnancy occurring in our study for fragmentation higher than 30%, can constitute a strong argument to counsel some couples, who have already made several attempts with systematic embryo transfers, but without pregnancy, and who wonder about the usefulness of beginning one more IVF treatment cycle. Nevertheless, the sample size does not allow the drawing of definitive conclusions, since the area under the ROC curve is 0.5, which represents a minimal acceptable value.
For those patients exhibiting a high proportion of sperm with fragmented DNA (20%), a solution would consist in the eliminationor at least the reductionof the cells with damaged DNA from the sperm population prepared for ART procedures. The cells could be sorted with flow cytometry. This is technically possible, but the problem is to be sure that the technique by itself, which necessitates DNA labelling and laser illumination, has no deleterious effects on sperm DNA. Donnelly et al. (2000
) suggested that Percoll density gradient centrifugation was useful in isolating a sub-population of sperm exhibiting a lower proportion of fragmented DNA than in the original semen, using both the Comet and the TUNEL assays. However, the values measured in our study were already obtained after density gradient selection, and one can expect that the percentages of DNA fragmented sperm in the original ejaculate were much higher. Moreover, some doubts may be raised about the DNA quality of the cells that are not labelled by the Comet or TUNEL assays, but may be in a pre-fragmentation state. For example, if the high level of DNA fragmentation is due to the presence of reactive oxygen species or to the activation of apoptotic factors, it is doubtful that the DNA in these negative cells will remain undamaged.
In conclusion, our data indicate that the proportion of sperm with DNA fragmentation influences the fertilization rate for a threshold value above 10%, and an implantation rate of ICSI-derived embryos. Since no pregnancy was obtained if >20% of selected sperm were TUNEL positive, this factor may have a good predictive value in cases of successive failures of implantation for apparently good quality embryos.
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References |
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Submitted on July 26, 2002; resubmitted on December 24, 2002; accepted on February 5, 2003.