1 Assisted Conception Unit, Birmingham Women's Hospital, UK, 2 Department of Animal Biology, University of Modena and Reggio Emilia, Italy, 3 Institute of Biology and Genetics, University of Ancona, Italy and 4 Department of Obstetrics and Gynaecology, Yale University School of Medicine, Connecticut, USA
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Abstract |
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Key words: semen parameters/sperm chromatin/sperm morphology/sperm nuclear DNA/sperm preparation
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Introduction |
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There is an increasing body of evidence to suggest that the sperm nuclear integrity should be routinely examined. Poor chromatin packaging has been shown to correlate with numerous reproductive outcomes: anomalies in fertilization related to ICSI (Sakkas et al., 1996), poor fertilization after IVF and ICSI (Esterhuizen et al., 2000
; Lopes et al., 1998
), the fertility of couples after intercourse (Evenson et al., 1999
; Spano et al., 2000
) and a higher incidence of pregnancy loss (Evenson et al., 1999
). Sperm donors have also been found to exhibit lower levels of nuclear DNA damage when specifically compared to infertility patients (Irvine et al., 2000
). The underlying mechanisms responsible for the existence of DNA-damaged spermatozoa are unclear, although irregularities in apoptosis during spermatogenesis may be involved (Sakkas et al., 1999a
, b
).
We and others have previously shown that sperm DNA/chromatin integrity improves after preparation by density gradient centrifugation (Colleu et al., 1996; Golan et al., 1997
; Angelopoulos et al., 1998
; Larson et al., 1999
; Sakkas et al., 2000
). Using a relatively large study population, our aim was to increase the understanding of the relationship between sperm quality, as measured by conventional semen parameters, and DNA/chromatin integrity. We present data on how these relationships alter following sperm preparation by discontinuous density gradient centrifugation and the possible implications for assisted reproduction.
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Materials and methods |
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Sperm preparation was carried out for assisted conception by discontinuous density gradient centrifugation, using 0.5 ml volumes of PureSperm® (Nidacon, Gothenberg, Sweden). Briefly, 0.5 ml of a 45% suspension was layered over 0.5 ml of 90% and centrifuged for 20 min (300 g). Spermatozoa in the 90% PureSperm® pellet were fixed in 3.5% paraformaldehyde. Three smears were then prepared on slides and left to air dry. Three smears were also prepared from the raw semen fraction. In patients undergoing IVF, smears were prepared from the semen and the prepared fraction of spermatozoa was only taken once insemination had been performed.
In-situ nick translation (NT) assay and Chromomycin A3 (CMA3) staining
We have previously shown that the CMA3 fluorochrome is a useful tool for assessing the packaging quality of the chromatin in spermatozoa and may allow an indirect visualization of protamine deficiency (Bianchi et al., 1993; Bizzaro et al., 1998
). In addition to the accessibility of CMA3 to mature human sperm chromatin, `endogenous' NT experiments (that is nick translation not preceded by endonuclease treatment) indicate that the presence of DNA nicks occurs in an appreciable, even if variable, number of human ejaculated spermatozoa (Manicardi et al., 1995
). Although the two techniques are closely related, the CMA3 is more indicative of a gross anomaly in the nucleus, i.e. the way the DNA is packaged, while nick translation represents damage in the actual DNA itself. NT was performed as previously described (Manicardi et al., 1995
) by omitting the endonuclease treatments, since, in the presence of pre-existing DNA endogenous nicks, the DNA polymerase I, by virtue of its 5'3' exonuclease activity, can catalyse movement of the nicks along the double helix. The only difference to the previously described method was that Digoxigenin-11-dUTP (Roche, Monza, Italy) was used.
For CMA3 staining, slides were treated for 20 min with 100 µl CMA3 solution (0.25 mg/ml McIlvaine buffer, pH 7.0, containing 10 mmol/l MgCl2) (Manicardi et al., 1995). They were then rinsed in buffer, air-dried and mounted with a 1:1 mixture of PBS and glycerol. In nearly all cases an operator, working blind, examined at least 500 spermatozoa on each coded slide. The nick translation and CMA3 staining were predominantly of all-or-nothing type and the rare cells showing ambiguous fluorescence were not considered. Fluorescence analysis was performed using a Zeiss Axioplan (Zeiss, Oberkochen, Germany).
IVF
IVF and sperm preparation were performed in IVF-500 medium (Scandinavian IVF, Gothenburg, Sweden). Once collected, oocytes were inseminated overnight in pools of up to 5 in 1 ml IVF-500 medium under oil (OvOil, Scandinavian IVF) containing 34x105 motile spermatozoa per ml overnight. Once fertilized, embryos were placed in IVF media for 23 days of culture until transfer. Stimulation and luteal support were as per our standard published procedure (Sharif et al., 1995). Pregnancy was assessed after 67 weeks under ultrasound by the observation of a fetal heartbeat.
Statistical analyses
Seminal parameters, CMA3 and NT were correlated to IVF rates using Spearman's ranked correlation. Semen parameters and DNA damage in normozoospermic individuals were compared with those of oligozoospermic and teratozoospermic men using the non-parametric MannWhitney test. Logistic regression was used to assess the impact of the various semen parameters plus CMA3 and NT on fertilization.
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Results |
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Discussion |
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In this study, sperm nuclear integrity as assessed by NT demonstrated a very clear relationship with sperm concentration, motility and morphology. Normozoospermics had a lower percentage of spermatozoa with DNA damage and CMA3 positivity compared with oligozoospermic and teratozoospermic men. Irvine and co-workers (Irvine et al., 2000) demonstrated a similar association with sperm count when they compared comet assay results from a group of sperm donors with those of infertility patients. Surprisingly, in their study, NT results did not agree with sperm count, despite a very strong correlation with comet results, and the suggestion was that the comet assay had a higher resolution than NT. It may be that the much larger study population in the present study enabled us to detect significant associations with NT. They found no associations between DNA damage and sperm morphology, whereas in the present study, an increased percentage of DNA-damaged spermatozoa was observed in teratozoospermic patients (<15% normal forms). A similar, yet much stronger, relationship has been found when comparing CMA3 with normal forms using 4% as a threshold value (Esterhuizen et al., 2000
). The lack of a clear-cut relationship between sperm count and morphology suggests that when we examine morphology and NT or CMA3 we are looking at distinct populations of spermatozoa. We had previously shown that morphologically normal spermatozoa show differences in chromatin packaging when comparing spermatozoa from normozoospermic men with those possessing abnormal sperm parameters (Bianchi et al., 1996
). In addition, when we studied both NT and CMA3 staining in the same sperm population we found the existence of different classes of spermatozoa in the human ejaculate. Subsequently, we proposed that in most ejaculates the majority of healthy spermatozoa contain compact chromatin (highly protaminated CMA3 negative spermatozoa) and unbroken DNA. The other two main classes, however, represent sub-groups of spermatozoa that have anomalies, and contain either loosely packaged chromatin (probably under-protaminated CMA3 positive spermatozoa) but unbroken DNA or contain both loosely packaged chromatin (probably under-protaminated) and nicked DNA. We concluded that the relative proportion of these classes of spermatozoa in a patient's ejaculate might have been a useful tool for assessing his fertility (Manicardi et al., 1995
).
What does the added assessment of DNA damage in the sperm nucleus show us? The above studies indicate that the assessment of the integrity of the sperm nucleus is highly indicative of the possible fertility status and therefore competence of human spermatozoa. NT will indicate anomalies that have occurred during the remodelling of the nuclear DNA in spermatozoa and in doing so is more likely to observe sperm anomalies not indicated by morphology. It may also be able to indicate if there is damage arising from factors such as heat exposure (Setchell, 1998; Setchell et al., 1998
) or the generation of reactive oxygen species following exposure to leukocytes within the male reproductive tract (Aitken et al., 1989
, 1991
).
Others are also accumulating data, indicating that nuclear integrity measurement, using tools such as NT, adds significantly to the diagnostic power of the semen analysis. Possibly the best reported test is that used for many years by Evenson's group. They and others have provided strong evidence of a relationship between sperm nuclear DNA integrity, as assessed using the sperm chromatin structure assay (SCSA), and fertility after both normal intercourse (Evenson et al., 1999; Spano et al., 2000
) and assisted reproduction techniques (Larson et al., 2000
). The SCSA measures the susceptibility of sperm nuclear DNA to heat- or acid-induced denaturation in situ followed by staining with acridine orange. Evenson et al. found cases where the classical criteria (concentration, motility and morphology) were within the normal ranges, but the SCSA values were poor, and not compatible with good fertility after intercourse (Evenson et al., 1999
).
In many of the above studies, couples attempting to achieve pregnancy without the aid of assisted reproduction techniques were examined. One of the confounding problems of relating semen parameters to overall IVF outcome is that a certain population of spermatozoa is selected during the semen preparation procedure. It is likely that regardless of the initial sample, a degree of homogenization occurs after sperm preparation. This study has shown that simple density gradient centrifugation can enrich the sample both with morphologically normal forms and spermatozoa with improved nuclear integrity. This `normalizing' effect of density gradient preparations may be the reason why sperm parameters pre-preparation had little prognostic value, in terms of fertilization and pregnancy using IVF. This indicates a need to assess spermatozoa in context, i.e. raw semen parameters in conjunction with natural conception, or intracervical insemination and prepared parameters in relation to IUI/IVF and ICSI. Concurrently, the normalizing effect led to a population of spermatozoa in the insemination drop that showed no correlation with fertilization or day 23 embryo development. However, morphological enrichment correlated with fertilization rates and, perhaps more importantly, NT values of the spermatozoa in the insemination drop were significantly lower in the pregnant group when compared to the non-pregnant group. The NT data support previous results using the SCSA test (Evenson et al., 1999; Spano et al., 2000
). It seems likely that morphological sperm parameters are important up to the fertilization step, and the importance/influence of DNA integrity is realized later, becoming the most important sperm parameter related to the establishment and continuation of a pregnancy. We know that there is no difference for example in fertilization rates, when using the hamster egg penetration test after ICSI, when comparing normal and DNA-damaged spermatozoa (Twigg et al., 1998
). In addition, UV-irradiated mouse spermatozoa that have nuclear DNA damage are able to fertilize normally but there are significant reductions in implantation rates and birth rates when compared to control spermatozoa (Ahmadi and Ng, 1999
).
We have demonstrated that along with the classical semen parameters, the assessment of nuclear integrity improves the characterization of the semen sample. However, the study needs to be expanded by performing NT or similar on a larger group of patients, eliminating as far as possible complicating influences, e.g female factors. In doing so, we will improve and possibly `fine-tune' our current semen analyses for patients attempting complex assisted reproductive treatments.
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Acknowledgements |
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Notes |
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References |
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Submitted on February 28, 2001; accepted on June 26, 2001.