Normal sperm morphology and chromatin packaging: comparison between aniline blue and chromomycin A3 staining

Abstract

The successful implementation of ICSI has provided a unique means of allowing couples suffering from severe male infertility to achieve their reproductive goals. However, despite the great therapeutic advantages of the technique, ICSI often provides solutions to clinicians in the absence of an aetiological or pathophysiological diagnosis. The development of a sequential diagnostic schedule for patients consulting for fertility disturbances would be an ideal method of approach. Since sperm morphology recorded by strict criteria has often been correlated with fertilization failure, the present study aimed to evaluate the relationship between normal morphology and chromatin staining among fertile and subfertile men. Both chromomycin A3 (CMA3) and acidic aniline blue (AAB) were employed to record chromatin packaging quality among 58 men visiting the andrology laboratory. Intra- and interassay variations were initially recorded for fertile sperm donors. The coefficients of variation (CV) for all intra- and inter-assay assessments were < 12%. Chromatin packaging was significantly and negatively correlated with normal sperm morphology, namely r = 0.40 (P = 0.001) and r = 0.33 (P = 0.001) for CMA3 and AAB, respectively. Receiver operator characteristics illustrated sensitivity and specificity values of 75% and 82% for CMA3 and 60% and 91% for AAB, respectively. Significantly different CMA3 and AAB staining was recorded among men with severe teratozoospermia (< 4% normal forms) when compared with normozoospermic men (> 14% normal forms), namely 49% vs. 29% for CMA3 and 51% vs. 26% for AAB staining, respectively. Chromatin packaging assessments should be a valuable addition to the sequential diagnostic programme in an assisted reproduction arena.

Evaluation of sperm’s chromatin quality with acridine orange test, chromomycin A3 and aniline blue staining in couples with unexplained recurrent abortion

“…Some studies have reported a correlation between staining of spermatozoa with AB and decreased percentage of normal spermatozoa [28]. Franken et al [16] showed that there was significantly higher percentage of AB positive spermatozoa among men with teratozoospermia when compared with normozoospermic (51% vs. 26%). Despite normal semen analysis in this study, the mean percentage of positive AB staining was 31.6% in the case group and 14.1% in the control group (p<0.001).…”

“…Three classes of head staining intensities were noted, namely unstained (gray/ white), partially stained, and entire sperm head stained dark blue (Fig. 2) [16].…”

Sperm Chromatin Structure Assay: Its Clinical Use for Detecting Sperm DNA Fragmentation in Male Infertility and Comparisons With Other Techniques

“…Aniline blue selectively stains lysine-rich histones (Terquem and Dadoune, 1983) and has been used as a clinical assessment of sperm chromatin condensation and associated infertility (Auger et al, 1990). High percentages of aniline blue-stained spermatozoa were found in patients affected by severe teratozoospermia (Franken et al, 1999), asthenozoospermia (Colleu et al, 1988), varicocele, idiopathic infertility, and with a history of unilateral cryptorchidism (Foresta et al, 1992). Furthermore, higher levels of aniline blue staining were found in subfertile men following cryopreservation than in fertile controls (Hammadeh et al, 1999b).…”

Investigation on the Origin of Sperm DNA Fragmentation: Role of Apoptosis, Immaturity and Oxidative Stress

“…Two hundred sperm were analyzed on each slide under a light microscope (Leica DM LS, Leica, Wetzlar, Germany). Sperm showing dark-blue staining were considered as AB positive, whereas those stained only weakly or not at all were considered as AB negative (43). After determining the percentage of fragmented sperm in presorted samples, the overall percentage of AB staining in such samples was calculated by (% AB staining in fragmented sperm × % fragmented sperm)/100 + (% AB staining in nonfragmented sperm × % nonfragmented sperm)/100.…”