Impact of sperm fractionation on chromosome positioning, chromatin integrity, DNA methylation, and hydroxymethylation level.

BACKGROUND: Sperm chromosomes are nonrandomly organized in the cell nucleus, which plays an important role in the regulation of early embryo development, which is determined by the specific localization of sperm chromosomal regions carrying genes with expression crucial at the first contact with ooplasm during fertilization. Thus, the aim of this study is to determine whether the application of selective methods providing high-quality spermatozoa with good motility and/or morphology can increase the frequency of gametes with a specific positioning of chromosomes. For the first time, we used a sequential staining algorithm for consecutive analyses of the same individual spermatozoon with a fixed position, what enables one to achieve full and detailed documentation at the single cell level. METHODS: Semen samples from five normozoospermic males were collected and processed for fractionation via swim up (to select viable and motile spermatozoa) or Percoll density gradient (90%/47%; to select viable sperm with normal motility and morphology). Sperm chromatin protamination was assessed by Aniline Blue (AB) staining, and DNA fragmentation by Acridine Orange (AO) (ssDNA fragmentation) or terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (ssDNA and dsDNA fragmentation). Then, sequential staining and analyses of the same individual spermatozoon with a fixed position on a slide were performed, in the following order: (i) fluorescence in situ hybridization (FISH) for determination of positioning of chromosomal centromeres: 4, 7, 8, 9, 18, X, and Y, with so-called linear and radial estimations applied, followed by distance measurements between selected pairs of chromosomes; and (ii) immunofluorescent (IF) measurement of global sperm DNA methylation (5mC) and hydroxymethylation (5hmC) levels, which added additional data about the epigenetic layer of the sperm chromosomes' positioning. RESULTS: Our study demonstrated that high-quality sperm selection methods significantly: (i) increased the frequency of spermatozoa with good chromatin protamination (+ ~25%) and 5mC and 5hmC DNA levels (+ ~9.5%) and (ii) reduced the rate of spermatozoa with ssDNA fragmentation (- ~65%). Motile and morphologically normal spermatozoa showed distinct chromosome repositioning with sex chromosomes shifted to the nuclear periphery, a key chromosomal region of the initial interaction with the ooplasm during fertilization process. Evaluated autosomes revealed various patterns of repositioning. CONCLUSIONS: Our findings underline the validity of methods used for selection of high-quality spermatozoa in assisted reproductive technologies (ART), also in the context of the sperm chromosomal topology and chromatin integrity, crucial at the first steps during fertilization.