Genética Médica
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Percorrer Genética Médica por autor "Almeida-Santos, T"
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- Analysis of human spermatozoa by fluorescence in situ hybridization with preservation of the head morphology is possible by avoiding a decondensation treatmentPublication . Almeida-Santos, T; Dias, C; Brito, R; Henriques, P; Almeida-Santos, APURPOSE: Development of a hybridization technique for spermatozoa allowing the preservation of the head morphology. METHODS: FISH analysis of fixed semen samples from oligoasthenoteratozoospermia (OAT) patients with a normal somatic karyotype attending the Cytogenetics and the IVF Laboratories of a University hospital for semen analysis. In situ hybridization with centromeric probes for chromosomes X, Y, and 18 and locus specific probe for chromosome 21. RESULTS: More than 95% of the sperm heads showed clear hybridization signals and a conserved morphology including the visualization of the tail. Few cells with splitted signals were not considered. CONCLUSIONS: This is the first description of a simple and fast hybridization protocol for spermatozoa without a decondensation step, allowing preservation of the morphology of the sperm head that is particularly useful to correlate abnormal spermatozoa with specific chromosome aneuploidies. With this technique we were able to avoid troubles in interpretation of FISH spots that does not depend on the quality of nuclear decondensation, as it is the case in the previously described methods. Our goal was to demonstrate the efficiency of the method without loosing sperm head morphology. Further studies are needed to correlate the aneuploidy rates for specific chromosomes with sperm morphology.
- Cytogenetic analysis of spontaneously activated noninseminated oocytes and parthenogenetically activated failed fertilized human oocytes--implications for the use of primate parthenotes for stem cell productionPublication . Almeida-Santos, T; Dias, C; Henriques, P; Brito, R; Barbosa, A; Regateiro, FJ; Almeida-Santos, APURPOSE:Spontaneous parthenogenetically activated noninseminated oocytes and failed fertilized oocytes after ART activated by puromycin were studied to assess cleavage ability and the cytogenetic constitution of the resulting embryos. METHODS: Failed fertilized oocytes were exposed to puromycin, and whenever activation occurred, they were further cultured until arrest of development. FISH was used to assess the ploidy of spontaneous (group A) and induced parthenotes (group B). RESULTS: The mean number of oocytes exposed to puromycin and the percentage and type of activation were identical in IVF and ICSI patients. The more frequent types of activation were one or two pronuclei and one polar body suggesting that retention of the second polar body is a common event after parthenogenetic activation. CONCLUSIONS: Retention of the second polar body and chromosome malsegregation were observed after parthenogenetic activation, either spontaneous or induced by puromycin. This means that using parthenogenetic embryos for stem cell research will require great care and attention.
- Dual use of Diff-Quik-like stains for the simultaneous evaluation of human sperm morphology and chromatin statusPublication . Sousa, AP; Tavares, RS; Velez de la Calle, JF; Figueiredo, H; Almeida, V; Almeida-Santos, T; Ramalho-Santos, JBACKGROUND: Sperm chromatin status and nuclear DNA damage can be detected using well-established assays. However, most techniques are time-consuming and/or involve elaborate protocols and equipment. We have recently developed a simple and fast method to monitor sperm chromatin status in field conditions using the Diff-Quik assay which is employed in fertility clinics to assess sperm morphology with standard bright field microscopy. In the present study, we demonstrate that any Diff-Quik-like stain can easily, reproducibly and routinely monitor human sperm chromatin status as well. METHODS: Different Diff-Quik-like stains were used to assess sperm morphology and the presence of abnormal dark nuclear staining in human sperm from four ART centres. The TUNEL assay was performed in the same samples, and fertility outcomes were assessed. RESULTS: A significant correlation was found between TUNEL-positive sperm and dark sperm nuclei. Moreover, associations were also found between the percentage of dark sperm nuclei and seminal parameters, embryo development rate, embryo quality and clinical pregnancy, as well as with cryptorchidism, and there was a tendency towards an association with age. A value of 32% abnormal staining is suggested as a predictive threshold for embryo development and pregnancy. CONCLUSIONS: Our results show that any Diff-Quik-like stain, already implemented in most laboratories to assess sperm morphology, can be adapted as an indicator for chromatin status in human sperm.