Introduction: Acrosin and tubulin are structural components of the spermatozoon, involved in zona pellucida digestion and in tail movement respectively. Their cellular localization can be achieved easily by indirect immunofluorescence (IIF) and in a previous study, they were shown to be involved in the fertilization process using polyclonal antibodies. The commercial availability of monoclonal antibodies against proacrosin and tubulin has renewed the interest for a rapid diagnostic test to identify the sperm samples capable of fertilizing in vitro. The purpose of this study was 1) to correlate the IIF and morphology (strict criteria) results with the fertilization rates obtained after regular IVF or ICSI, 2) to provide biologists with a quick and efficient tool to select the most appropriate fertilization technique (IVF or ICSI). Material and Methods: A five µl aliquot from the washed sperm suspension used for regular IVF or ICSI was smeared on a microscopic slide, fixed and kept at -20 °C until use. Monoclonal antibodies against proacrosin (Biomérieux, France) or tubulin (Sigma, USA) were used for IIF assessment of the sperm acrosome and tail in a double labelling assay. The percentages of spermatozoa exhibiting normal acrosomes or normal tails were recorded separately by a single observer unaware of the fertilization outcome, using an epifluorescence microscope (400X, MTII, Olympus). The whole IIF procedure could be performed in less than 1 hour. A Papanicolaou stained smear was prepared in parallel for morphology assessment using Kruger's strict criteria. Results: Double immunofluorescent labelling of proacrosin and tubulin allowed easy identification of both acrosomal and tail defects in a single reading. In regular IVF, fertilization rates (zygotes/oocytes, %) were strongly correlated (p<0.001) with the percentages of normal fluorescence patterns, allowing identification of thresholds values (60% normal acrosomes; 80% normal tails) below which the fertilization rates were 0% to 30%. In contrast, a much weaker correlation was found with the morphology determinations, for which 30% fertilization rates were still obtained even with only 0-10% normal forms. In ICSI cycles, the fertilization rates were above 50% and were correlated neither with the IIF results nor the percentage of morphologically normal forms. Conclusions: The described IIF procedure can be easily implemented in a busy IVF unit. It provides clinicians and patients with more detailed descriptions of the sperm abnormalities. In case of abnormal sperm, it helps biologists to choose rapidly which fertilization technique should be used (IVF or ICSI).
Double fluorescenze labelling of acrosin and tubulin in human spermatozoa:a rapid diagnostic procedure to identify sperm samples with poor fertilizing ability.
MARCI, Roberto;
1997
Abstract
Introduction: Acrosin and tubulin are structural components of the spermatozoon, involved in zona pellucida digestion and in tail movement respectively. Their cellular localization can be achieved easily by indirect immunofluorescence (IIF) and in a previous study, they were shown to be involved in the fertilization process using polyclonal antibodies. The commercial availability of monoclonal antibodies against proacrosin and tubulin has renewed the interest for a rapid diagnostic test to identify the sperm samples capable of fertilizing in vitro. The purpose of this study was 1) to correlate the IIF and morphology (strict criteria) results with the fertilization rates obtained after regular IVF or ICSI, 2) to provide biologists with a quick and efficient tool to select the most appropriate fertilization technique (IVF or ICSI). Material and Methods: A five µl aliquot from the washed sperm suspension used for regular IVF or ICSI was smeared on a microscopic slide, fixed and kept at -20 °C until use. Monoclonal antibodies against proacrosin (Biomérieux, France) or tubulin (Sigma, USA) were used for IIF assessment of the sperm acrosome and tail in a double labelling assay. The percentages of spermatozoa exhibiting normal acrosomes or normal tails were recorded separately by a single observer unaware of the fertilization outcome, using an epifluorescence microscope (400X, MTII, Olympus). The whole IIF procedure could be performed in less than 1 hour. A Papanicolaou stained smear was prepared in parallel for morphology assessment using Kruger's strict criteria. Results: Double immunofluorescent labelling of proacrosin and tubulin allowed easy identification of both acrosomal and tail defects in a single reading. In regular IVF, fertilization rates (zygotes/oocytes, %) were strongly correlated (p<0.001) with the percentages of normal fluorescence patterns, allowing identification of thresholds values (60% normal acrosomes; 80% normal tails) below which the fertilization rates were 0% to 30%. In contrast, a much weaker correlation was found with the morphology determinations, for which 30% fertilization rates were still obtained even with only 0-10% normal forms. In ICSI cycles, the fertilization rates were above 50% and were correlated neither with the IIF results nor the percentage of morphologically normal forms. Conclusions: The described IIF procedure can be easily implemented in a busy IVF unit. It provides clinicians and patients with more detailed descriptions of the sperm abnormalities. In case of abnormal sperm, it helps biologists to choose rapidly which fertilization technique should be used (IVF or ICSI).I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
