title: Significance of large nuclear vacuoles in human spermatozoa: implications for ICSI.
abstract: The aim of this study was to determine the extent of DNA fragmentation and the presence of denatured single-stranded or normal double-stranded DNA in spermatozoa with large nuclear vacuoles (LNV) selected by high magnification. Fresh semen samples from 30 patients were prepared by discontinuous isolate concentration gradient. Spermatozoa with normal nucleus (NN) and LNV were selected at x8400 magnification and placed on different slides. DNA fragmentation was determined by TUNEL assay. Denatured and double-stranded DNA was identified by the acridine orange fluorescence method. DNA fragmentation in spermatozoa with LNV (29.1%) was significantly higher (P < 0.001) than in spermatozoa with NN (15.9%). Therefore, cleavage of genomic DNA in low molecular weight DNA fragments (mono- and oligonucleosomes), and single-strand breaks (nicks) in high molecular weight DNA occur more frequently in spermatozoa with LNV. Similarly, the percentage of denatured-stranded DNA in spermatozoa with LNV (67.9%) was significantly higher (P < 0.0001) than in spermatozoa with NN (33.1%). The high level of denatured DNA in spermatozoa with LNV suggests precocious decondensation and disaggregation of sperm chromatin fibres. The results show an association between LNV and DNA damage in spermatozoa, and support the routine morphological selection and injection of motile spermatozoa at high magnification for ICSI.
authors.affiliation: Centre for Human Reproduction Professor Franco Jr, Ribeirão Preto, São Paulo, Brazil. franco@crh.com.br
authors.forename: J G
authors.initials: JG
authors.lastname: Franco
authors.forename: R L R
authors.initials: RL
authors.lastname: Baruffi
authors.forename: A L
authors.initials: AL
authors.lastname: Mauri
authors.forename: C G
authors.initials: CG
authors.lastname: Petersen
authors.forename: J B A
authors.initials: JB
authors.lastname: Oliveira
authors.forename: L
authors.initials: L
authors.lastname: Vagnini
mesh_terms: D000165:Acridine Orange / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004249:DNA Damage; D053938:DNA Fragmentation; D004277:DNA, Single-Stranded / Q000737:chemistry; D006801:Humans; D007248:Infertility, Male / Q000473:pathology / Q000628:therapy; D008297:Male; D020554:Sperm Injections, Intracytoplasmic / Q000379:methods*; D013094:Spermatozoa / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism
chemical_list: D002843:Chromatin; D004277:DNA, Single-Stranded; D004247:DNA; D000165:Acridine Orange