Spermatozoal sensitive biomarkers to defective protaminosis and fragmented DNA

Abstract

Human sperm DNA damage may have adverse effects on reproductive outcome. Infertile men possess substantially more spermatozoa with damaged DNA compared to fertile donors. Although the extent of this abnormality is closely related to sperm function, the underlying etiology of ensuing male infertility is still largely controversial. Both intra-testicular and post-testicular events have been postulated and different mechanisms have been proposed to explain the presence of damaged DNA in human spermatozoa. Three among them, i.e. abnormal chromatin packaging, oxidative stress and apoptosis, are the most studied and discussed in the present review. Furthermore, results from numerous investigations are presented, including our own findings on these pathological conditions, as well as the techniques applied for their evaluation. The crucial points of each methodology on the successful detection of DNA damage and their validity on the appraisal of infertile patients are also discussed. Along with the conventional parameters examined in the standard semen analysis, evaluation of damaged sperm DNA seems to complement the investigation of factors affecting male fertility and may prove an efficient diagnostic tool in the prediction of pregnancy outcome.

Figure 1

Frequency of DNA fragmentation in human ejaculated spermatozoa. The percentage of DNA fragmentation in the above groups was determined using the terminal deoxynucleotidyl transferase mediated dUTP-nick end labeling (TUNEL) assay with or without Hoechst 33258 DNA staining. The arithmetic means of each group were obtained by adding the group's means, when reported by the cited studies, and dividing the sum by the number of the cited studies that evaluated that group. The Standard Error of each group shown in the graph demonstrates the variability between the results of the different studies. All five studies evaluated normal donors [13, 81, 175, 181, 182]. Three of the studies also assessed a group of infertile patients [13, 175, 181], two evaluated varicocele patients [81, 182], one evaluated cryptorchidism patients and two assessed testicular cancer patients [13, 81].

Figure 2

TUNEL assay. TUNEL-positive nuclei (with double-strand nuclear DNA fragmentation) of spermatozoa as represented by the intense (A) and dull (B) Texas red fluorescence in the nuclear region. The healthy nuclei (without DNA fragmentation) are stained blue with DAPI (C) used as counterstain.

Figure 3

CMA₃ staining. Two types of staining patterns were identified, bright and dull yellow fluorescence of the sperm nuclei (abnormal chromatin packaging) (A, B) and blue staining with DAPI in the healthy nucleus (normal chromatin packaging) seen in C.

Figure 4

The effect of age on DNA fragmentation and chromatin packaging. DNA fragmentation was assessed using the TUNEL assay, while chromatin packaging was evaluated using the chromomycin A₃ (CMA₃) staining. Sixty one oligoasthenoteratozoospermic patients were divided into two age subgroups (20–34 years old, n = 30; 35–50 years old, n = 31). Forty nine healthy fertile controls were also divided according to their age (20–34 years old, n = 26; 35–50 years old, n = 23). In the control group, the differences observed between the two age subgroups were not statistically significant (P > 0.05; Figure 4A, 4B). On the other hand, the older patient subgroup demonstrated a significantly higher percentage of TUNEL positive (P < 0.001; Figure 4A) and CMA₃ stained (P < 0.001; Figure 4B) spermatozoa compared to the younger patient subgroup.