1 Servicio de Análisis Clínicos, Hospital San Agustín, Linares, Jaén, Spain and 2 Unidad de Reproducción, Hospital Virgen de la Nieves, 18014 Granada, Spain
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Abstract |
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Key words: chromatin/flow cytometry/propidium iodide/spermatozoa
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Introduction |
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The assessment of chromatin status is very important when evaluating the ability of spermatozoa to fertilize. Many techniques have been described for the evaluation of chromatin status, such as optical microscopy (Krzanowska, 1982; Huret, 1984
; Rosenborg et al., 1990
), electron microscopy (Jamil, 1984
; Lipitz et al., 1992
) and flow cytometry (Engh et al., 1992
; Zucker et al., 1992
; Molina et al., 1995
; Samocha-Bone et al., 1998
; Evenson et al., 1999
; Spanò et al., 1999
). Sperm chromatin defects have been correlated with the reduced ability of spermatozoa to fertilize both in the context of assisted reproduction techniques (Bianchi et al., 1996
; Hoshi et al., 1996
; Sakkas et al., 1996
; Hammadeh et al., 1998
; Lopes et al., 1998
; Filatov et al., 1999
; Gopalkrishnam et al., 1999
) and in the general population (Evenson et al., 1999
; Hacker-Klom et al., 1999
; Spanò et al., 2000
). Moreover, patients with fertility problems have often been characterized by an increased frequency of spermatozoa with abnormal chromatin (Spanò et al., 1984
; Engh et al., 1992
; Foresta et al., 1992
; Kosower et al., 1992
; Liu and Baker, 1992
; Hughes et al., 1996
).
In this study, different groups of patients were examined, both fertile and infertile, in order to analyse the standard parameters of semen analysis and those that depend on the state of sperm chromatin. Thus, it was possible to observe differences between patient groups, using the technique of flow cytometry after DNA staining with the fluorochrome, propidium iodide (PI).
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Materials and methods |
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Semen samples were obtained by masturbation after 3 days of sexual abstinence, and collected in a sterile plastic container. They were allowed to liquefy for 30 min at 37°C, after which an analysis was performed to measure the following parameters: concentration, motility, percentage of normal forms, percentage of spermatozoa with head anomalies, vitality, hypoosmotic test, mixed agglutination reaction test and volume of ejaculate (WHO, 1992).
The ejaculates were used to study sperm chromatin status by analysing the uptake of PI, using flow cytometry with a FACScan IV cytometer (Becton Dickinson, Mountain View, CA, USA). In this study, only one of the three fluorescence detectors available to the FACScan IV was used, to measure the fluorescence corresponding to the red colour of PI. PI-stained cells were analysed in a flow cytometer equipped with a 488 nm argon laser as the light source and a 560 nm optical filter. For each determination 10 000 sperm nuclei were measured.
The flow cytometry parameters analysed were: condensation of sperm chromatin; resistance to decondensation after treatment with 1% sodium dodecyl sulphate (SDS) plus 6 mmol/l ethylenediaminetetraacetic acid (EDTA) decondensing solution in borate buffer for 5 min; and the tendency to achieve a state of hyperstability after 6 h of incubation at 37°C and subsequent treatment with SDSEDTA (Molina et al., 1995). Three aliquots of the semen sample were taken for analysis. The first was directly stained with 50 µg/ml PI, using the commercial Cycle test kit (Beckton Dickinson). This permitted analysis of the state of condensation of the sperm chromatin, as this is directly related to PI uptake. The second aliquot was treated with SDSEDTA and then stained with PI, while the third aliquot was incubated for 6 h in B2 medium (Bio Mérieux, Marcy l'Etoile, France) at 37°C and 5% CO2 and then treated with SDSEDTA. This method was employed in order to observe the tendency of the spermatozoa to achieve a state of hyperstability. The mean channel (MCH) of fluorescence was used to analyse the accessibility and, consequently, the degree of staining (`stainability') of sperm DNA after staining with PI.
For each sample, the percentage of variation of stainability after the decondensation step under normal and hyperstability conditions was calculated as follows:
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Results were presented as the mean ± SEM. All percentage data were transformed using the formula: log(x + 0.5)/(100.5 x).
This transformation has been suggested as a suitable method to normalize percentage data (Atkinson, 1985). Variance analysis was used to examine group differences, and comparisons were made with Bonferroni's test. Significance was defined as P < 0.05. Simple linear regression analysis was used to show the degree of linear association between chromatin status and seminal parameters. A stepwise multiple regression was used to predict sperm chromatin status; a variable was included if its partial regression coefficient was significant at the 0.05 level, and was eliminated if its partial regression coefficient failed to reach significance at the 0.10 level. Statistical analyses were performed using the BMDP statistical package (BMDP Statistical Software, Los Angeles, CA, USA).
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Results |
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Discussion |
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The significant differences observed between the male factor and varicocele groups and the pre-vasectomy group have many possible explanations, including incomplete replacement of histones by protamines, aberrant ratios of protamine 1 to protamine 2, high concentrations of non-oxidized SH groups in protamine molecules, or the occurrence of DNA breaks (Balhorn et al., 1988; Auger et al., 1990
; Belokopytova et al., 1993
; De Yebra et al., 1993
; Bench et al., 1996
; Aravindan et al., 1997
; Filatov et al., 1999
).
Others (Foresta et al., 1989) have studied the different types of pathologies that damage the structure of sperm chromatin. On evaluating the percentage of sperm heads with chromatin denaturation, using acridine orange (Tejada et al., 1984
), these authors found that varicocele, cryptorchidia and orchitis resulting from parotiditis all result in spermatozoa having a chromatin that is less resistant to chemical denaturalization, though the incidence of this effect is not the same for all individuals and pathologies. The presence of varicocele is usually associated with abnormality in seminal parameters and in the histology of the testicle, while varicocelectomy has been shown to improve semen quality and increase the rate of pregnancies achieved (Ito et al., 1986
; Takihara et al., 1990
). DNA evaluation of testicular cells by flow cytometry has shown that spermatogenesis in males with varicocele is usually lower (Takihara et al., 1990
). The factors responsible for chromatin disorders in spermatozoa from the varicocele group remain unclear, though suggestions are that: (i) apoptosis is abnormally frequent in the sperm cells of the patients with varicocele, and plays a significant role in the spermatogenetic dysfunction associated with varicocele (Simsek et al., 1998
); or (ii) there are increasing concentrations of reactive oxygen species (Koksal et al., 2000
) and a reduction in antioxidant defences (Barbieri et al., 1999
) in higher grades of varicocele.
It should also be noted that, for those parameters that are indicative of sperm chromatin, males who are sterile due to immunological factors did not present statistically significant differences from the pre-vasectomy and `no apparent cause' groups. This suggests that the effect of the antisperm antibodies occurs at the membrane or cytoplasm level but not at the nuclear level. It has been suggested (Naz, 1992; Naz and Menge, 1994
) that there is an effect at the nuclear level in antisperm antibodies, as these could inhibit development of the pronuclei in the zygote. Furthermore, anti-DNA antibodies have been found frequently to be present in males with antisperm antibodies (D'Cruz et al., 1994
).
There seems to be a certain association between semen of lower quality, according to conventional measurements (WHO, 1992), and a reduction in the condensation of sperm chromatin. However, when analyses were carried out of the different flow cytometry histograms for the state of sperm chromatin in those who were infertile due to male factor, the histograms varied from being almost identical to those of normozoospermic males to showing virtually the whole sperm population as hypocondensed.
When the different seminal parameters were related, it was found that the best correlations (values of 0.70.9) corresponded to parameters that are indicative of the state of sperm chromatin. This is logical, as these parameters are completely interrelated, and reflect the evolution of the sperm nucleus during spermatogenesis and subsequent maturation. However, when the correlation of sperm chromatin parameters with standard parameters for semen analysis was analysed, low values (~0.5) were found, suggesting that these reflect completely different physiological processes during spermatogenesis, and thus there is little correlation. The standard seminal parameters presented low correlation coefficients (0.40.6), which was in agreement with the findings of others (Wang et al., 1988).
The stepwise multiple linear regression analysis performed to determine which seminal variables present the best relationship with the degree of condensation of sperm chromatin, revealed these to be motility, percentage of normal forms in the ejaculate, and the concentration of spermatozoa. Nevertheless, these parameters are only able to predict 50% of the variability observed in the condensation of sperm chromatin, as they reflect biological processes which differ from those representing nuclear quality (Spanò et al., 1998; Evenson et al., 1999
). The origin of this weak relationship is not clear; pathological agents such as oxidative stress may affect spermatozoa at different levels at the same time, including mitochondrial function affecting motility, the acrosome and membrane functions affecting morphology and vitality (Dadoune et al., 1988
), and DNA affecting accessibility of PI and, consequently, chromatin-related parameters.
The parameters related to resistance to the decondensation of sperm chromatin include motility and ejaculate volume. The volume effect might be explained in individuals with a low level of prostatic secretion, as this would be manifested by a reduction in ejaculate volume, as well as a reduction in resistance to decondensation, due to the lower level of prostate-derived zinc in the ejaculate, as has been suggested by others (Kvist, 1980; Kvist and Eliasson, 1980
; Kvist et al., 1988
; Björndahl and Kvist, 1990
).
Finally, multiple linear regression analysis found the parameters most likely to predict the variability in the attainment of hyperstability by the sperm chromatin to be motility and sperm morphology, though these only predicted 45% of the hyperstability. Therefore, it is clear that there is relatively little correlation between the parameters related to sperm chromatin and the other conditions, as each is a reflection of the different processes occurring during spermatogenesis.
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Notes |
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References |
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Submitted on October 10, 2000; accepted on November 27, 2000.