16 What’s the signifcation of nuclear vacuoles in human sperm? Qual é o signifcado dos vacúolos nucleares no espermatozóide humano? JG Franco Jr 1,2 , RLR Baruff 1 , AL Mauri 1 , CG Petersen 1,3 , JBA Oliveira 1 , L Vagnini 4 1 Centro de Reprodução Humana Prof Franco Jr - Ribeirão Preto, SP, Brasil 2 Departamento de Ginecologia e Obstetrícia – Faculdade de Medicina de Botucatu – Universidade Estadual Paulista Julio de Mesquita - UNESP – Botucatu, SP, Brasil 3 Pós-graduando, Departamento de Ginecologia e Obstetrícia – Faculdade de Medicina de Botucatu – Universidade Estadual Paulista Julio de Mesquita - UNESP – Botucatu, SP, Brasil 4 CPDP – Centro Paulista de Diagnóstico e Pesquisa – Ribeirão Preto, SP, Brazil. ABSTRACT The aim of this study was to determine the extent of DNA fragmentation and the presence of single/denatured or double stranded of DNA in sperm with large nuclear vacuoles (LNV) selected by high-magnifcation. A total of 30 patients had fresh semen samples prepared by discontinuous concentration gradient. Sperm with normal nucleus (NN) and LNV were selected at 8400x magnifcation and placed in different slides. DNA fragmentation was determined by TUNEL assay. Denatured and double stranded DNA was identifed by acridine orange fuorescence method. The percentage of DNA fragmentation in LNV sperm (29%) was signifcantly higher (P<0.001) than NN sperm (15.8%). 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 LNV. Identically, the percentage denatured stranded DNA in sperm with LNV (67.9%) was signifcantly higher (P <0.0001) than NN sperm (33%). The high level of denatured DNA in sperm with LNV suggests precocious decondensation and disaggregation of sperm chromatin fbers. Our results support an association between LNV sperm and DNA damage, and the routine selection and injection of morphological motile sperm at high magnifcation for ICSI. The adverse effect (DNA fragmentation or denaturation) leads to concern particularly about the possibility of iatrogenic transmission of genetic abnormalities. Keywords: human sperm, nuclear vacuoles, ICSI, DNA fragmentation, denatured stranded DNA RESUMO O objetivo deste estudo foi determinar a extensão da fragmentação do DNA e a presença de DNA dupla fta ou fta simples (desnaturada) em espermatozóides com vacúolos nucleares largos (VNL) selecionados pela microscopia de alta magnifcação. Um total de 30 pacientes tiveram amostras de esperma fresco preparadas por gradiente descontínuo. Os espermatozóides com núcleo normal (NN) e com VNL foram selecionados a magnifcação de 8400x e colocados em laminas diferentes. A fragmentação do DNA foi determinada através do método do TUNEL. O DNA desnaturado (fta simples) e o dupla fta foram identifcados pelo método de acridina orange. O percentual de fragmentação do DNA nos espermatozóides VNL (29%) foi signifcativamente maior (p<0.001) do que nos espermatozóides NN (15.8%). Dessa forma, a quebra do DNA genômico em fragmentos de baixo peso molecular (mono e oligonucleossomos), e quebras em cadeia única (nicks) em DNA com alto peso molecular ocorre mais frequentemente no espermatozóide com VNL. Igualmente, a percentagem de cadeia desnaturada de DNA (fta simples) em espermatozóides com VNL (67.9%) foi signifcativamente maior (p<0.0001) que em espermatozóides NN (33%). O alto nível de DNA desnaturado em espermatozóides com VNL sugere descondensação e desagregação precoce das fbras da cromatina espermática. Nossos resultadosindicam uma associação entre o espermatozóide com VNL e dano no DNA, além de substanciar o uso da alta magnifcação na seleção e injeção dos espermatozóides para ICSI. Palavras-chave :: esperma humano, vacúolos nucleares, ICSI, DNA fragmentação, cadeia desnaturada de DNA I NTRODUCTI ON It is known that IVF/ICSI is associated with increased (although relatively low) risk of birth defects and genetic and epigenetic abnormalities in children. To date, it is unclear whether the ICSI procedure or the underlying infertility is responsible for these defects (Cox et al., 2002; Hansen et al., 2002; Ludwig et al., 2005). The risk of birth defects (major and minor) was found to be signifcantly higher (odds ratio ~ 1.5) in children conceived by IVF/ ICSI than in those naturally conceived. The prevalence of chromosomal abnormalities (de novo abnormalities) was found to signifcantly higher (1.6%/0.5%) in children conceived by ICSI than naturally (Bonduelle et al,1998). Epigenetics abnormalities, such as errors in DNA methylation, Artigo Original Recebido em:01/08/2008 Aceito em:28/08/2008 Copyright - Todos os direitos reservados a SBRA - Sociedade Brasileira de Reprodução Assistida DOI: 10.5935/1518-0557.2008.12.3.03