Assessment of Chromatin Maturity in Human Spermatozoa: Useful Aniline Blue Assay for Routine Diagnosis of Male Infertility

Abstract

During spermatogenesis, sperm chromatin undergoes structural changes and results in a high condensation. This nuclear compaction would be useful as a predictor of sperm fertilization capacity and pregnancy outcome. We purpose to evaluate firstly the relationship among chromatin maturity assessed by aniline blue staining (AB) and the semen parameters in infertile men. Secondly, we analyzed whether the sperm gradient density centrifugation is effective to select mature spermatozoa. Fifty-one ejaculates were investigated by semen analysis and stained for chromatin condensation with AB to distinguish between unstained mature sperm and stained immature sperm. AB was applied also on 12 ejaculates which proceeded by density gradient centrifugation to compare the rates of immature sperm before and after selection. Neat semen were divided into two groups: G1 (n = 31): immature sperm <20% and G2 (n = 20): immature sperm ≥20%. No significant differences were detected in sperm concentration, motility, and normal morphology between G1 and G2. However, the rates of some morphology abnormalities were higher in G2: head abnormalities (P = 0.01) and microcephalic sperm (P = 0.02). We founded significant correlation between sperm immaturity and acrosome abnormalities (r = 0.292; P = 0.03). Sperm selection has significantly reduced the rates of immature sperm. A better understanding of chromatin structure and its impact on the sperm potential is needed to explore male infertility.

Scrotal heat stress causes sperm chromatin damage and cysteinyl aspartate-spicific proteinases 3 changes in fertile men

“…A total of 1 mL of the semen sample (mixed with hypoosmotic medium) from the HOS test was centrifuged at 500 g for 5 min, and then the supernatant was discarded and the sperm pellet was resuspended in 0.1 mL of a hypo-osmotic solution. To perform AB staining, sperms were stained with AB as described in a previous report [26,27]. The slides were prepared by smearing 10 μL of each HOS semen sample.…”

Nuclear sperm quality in total polymorphic teratozoospermia and its impact on intracytoplasmic sperm injection outcome

“…Sperm DNA fragmentation which is linked to chromatin decondensation (Agarwal & Said, ) may affect embryo development, blastocyst formation and pregnancy outcome (Herbemont & Sifer, ; Sellami et al, ). In fact, sperm chromatin compaction is involved in the protection of paternal genome throughout the transit of spermatozoa in male and female genital tracts and its interaction with the oocyte (Kim et al, ).…”

Sperm chromatin maturity and integrity correlated to zygote development in ICSI program

“…Our finding suggests that infertility in ICSI patients might be associated with a low level of sperm maturity in their ejaculates. Sellami et al [2013] reported the degree of sperm chromatin maturity was significantly correlated with the average number of sperm head abnormalities and the acrosome abnormalities in infertile men. Other studies demonstrated significant correlations between sperm chromatin immaturity and the decrease of sperm count and progressive motility [Kazerooni et al 2009;Aoki et al 2005].…”

“…Generally, the quality of the sperm chromatin plays an important role in the success of ovum fertilization [de Lamirande et al 2012;Hofmann and Hilscher 1991]. Different simple methods based on different staining or fluorochromes have been proposed in recent years to assess sperm chromatin quality, using many staining methods for DNA integrity such as aniline blue (AB), toluidine blue (TB), and chromomycin A3 (CMA3) for protamination and chromatin integrity assay as well as fragmentation assays [de Lamirande et al 2012;Hekmatdoost et al 2009;Hofmann and Hilscher 1991;Kazerooni et al 2009;Kim et al 2013;Pizzol et al 2014;Sellami et al 2013;Tsarev et al 2009]. The usefulness of these methods in the evaluation of male infertility and as prognostic markers for successful assisted reproductive programs is still debatable, partly because standardization has not been reached for some of these methods [Pizzol et al 2014].…”