ANALYSIS OF OXIDATIVE STRESS, DNA DAMAGE AND APOPTOSIS IN HUMAN SPERMATOZOA AND THEIR CORRELATION WITH SEMEN QUALITY OF INFERTILE MEN

number: 
1411
إنجليزية
department: 
Degree: 
Imprint: 
Medicine
Author: 
Ali Fuad Hadi Al-Hashimi
Supervisor: 
Dr. Faik H. Mohammed (Supervisor
Dr. Usama S. Al-Nasiri
year: 
2006

Abstract:

Sperm DNA quality is vital to successful reproduction for the correct conveyance of genetic material from one generation to the next. Poor quality sperm DNA may be a major factor in male infertility. Different theories have been proposed to explain the origin of DNA damage in mature spermatozoa from infertile men, including defective sperm chromatin packaging, apoptosis and oxidative stress (OS). The goal of this study was to elucidate the interrelations between these factors and the involvement of apoptosis and reactive oxygen species (ROS) in inducing DNA damage in ejaculated spermatozoa. To examine these issues, a randomly selected group of nonazoospermic infertile patients were included in this study (n = 40), who were attending the andrology clinic at Al-Kadhemia Teaching Hospital with a history of infertility of at least 1 year duration. Infertile patients were classified to 5 subgroups according to the WHO criteria (1992), most of the subgroups were asthenozoospermic (4 subgroups, n = 33). Controls consisted of samples obtained from healthy donors of proven fertility (n = 20). Four main assays were studied in ejaculated spermatozoa from infertile patients and compared to conventional semen analysis. These are the (green Acridine Orange [AO] fluorescence test) that measures DNA integrity and maturity by calculating the percentage of spermatozoa that enclose native double-stranded DNA. In addition, both early and late apoptosis were investigated, by assessing mitochondrial membrane depolarization (Mitolight™ mitochondrial membrane potential [MMP] assay) and DNA fragmentation (Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labeling [TUNEL] assaybased ApopTag® technology); respectively. Finally, OS in spermatozoa was estimated through the assay of sperm malondialdehyde (MDA), a thiobarbituric acid-reactive substance (TBARS). Infertile patients had significantly higher percentage of cytoplasmic droplets (P = 0.0001), higher level of TBARS, higher percentage of depolarized mitochondria,
higher percentage of fragmented DNA and lower percentage of mature spermatozoa (P = 0.00001). All asthenozoospermic subgroups had higher levels of cytoplasmic droplets, TBARS and depolarized mitochondria. The observation that ejaculated spermatozoa from infertile patients were positive for both early and late apoptotic markers supports the "abortive apoptosis" theory and reflects a possible role for which in male infertility. However, despite a negative correlation observed between late apoptosis (TUNEL assay) and DNA integrity (AO assay) (P = 0.024), there was no correlation with early apoptosis. urthermore, comparing the levels of both assays indicated that the total amount of DNA damage cannot be explained by apoptosis alone. On the other hand, results of the TBARS assay in infertile patients, were significantly correlated with the levels of both early and late apoptotic assays (P = 0.00001; and P = 0.007; respectively),
and inversely with the native double-stranded DNA (P = 0.007). Therefore, DNA integrity was more significantly correlated with ROS than with apoptosis (P = 0.007; and P = 0.024; respectively). Levels of cytoplasmic droplets were significantly correlated with TBARS in all patients (P = 0.00001) and in most
asthenozoospermic subgroups, while they correlated only with early mitochondrial apoptosis, whether in patients or in subgroups and not with late apoptosis. From all of these observations, it can be concluded that OS contributes most likely to DNA damage and may be responsible, at least in some part, for induction
of apoptosis in spermatozoa from infertile patients.