Pregnancy with frozen-thawed and fresh testicular biopsy after motile and immotile sperm microinjection, using the mechanical touch technique to assess viability

BACKGROUND: There are few reports of pregnancy using immotile sperm, and none using a purely mechanical assessment of viability. METHODS: In this pilot study, we retrospectively analysed 66 cycles in 61 patients with determinant male factor, recording rates of fertilization, implantation, normal pregnancy and take-home babies achieved with ICSI. Sperm selection was based on morphologically normal appearance under the inverted microscope. Viability of immotile spermatozoa was assessed by the mechanical touch technique to observe tail flexibility and tail shape recovery. RESULTS: Of 17 ICSI cycles using frozen-thawed testicular sperm, six microinjected with immotile and 11 with motile sperm, we achieved fertilization rates of 65.7 and 74.3%, respectively, and five pregnancies (two and three, respectively). Of 49 ICSI cycles using fresh testicular sperm, 10 microinjected with immotile and 39 with motile sperm, we achieved fertilization rates of 73.4 and 64.4%, respectively, and 12 pregnancies (three and nine, respectively). CONCLUSIONS: Immotile (fresh and frozen-thawed) testicular sperm of normal morphological appearance can be used to achieve clinical pregnancy with ICSI. Our results strongly suggest that immotile sperm viability can be assessed by the mechanical touch technique.     

Predominance of null mutations in ataxia-telangiectasia

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder involving cerebellar degeneration, immunodeficiency, chromosomal instability, radiosensitivity and cancer predisposition. The responsible gene, ATM, was recently identified by positional cloning and found to encode a putative 350 kDa protein with a Pl 3-kinase-like domain, presumably involved in mediating cell cycle arrest in response to radiation-induced DNA damage. The nature and location of A-T mutations should provide insight into the function of the ATM protein and the molecular basis of this pleiotropic disease. Of 44 A-T mutations identified by us to date, 39 (89%) are expected to inactivate the ATM protein by truncating it, by abolishing correct initiation or termination of translation, or by deleting large segments. Additional mutations are four smaller in-frame deletions and insertions, and one substitution of a highly conserved amino acid at the Pl 3-kinase domain. The emerging profile of mutations causing A-T is thus dominated by those expected to completely inactivate the ATM protein. ATM mutations with milder effects may result in phenotypes related, but not identical, to A-T.      

High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.      

Clonal Distribution of Methicillin-Resistant Staphylococcus aureus in Poland

We report on a study of 158 methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates obtained from 1990 to 1996 in 18 different hospitals in Poland. All isolates were recovered from infection and carriage sites of patients, carriage sites of health care personnel, and hospital environment samples. Fifty-seven MRSA strains described here were studied previously and these were divided into two different clusters according to the degree of heterogeneity of methicillin resistance expression. The aim of this study was to extend the correlation between the two clusters and identify the clonal identities among all isolates by a combination of different methodologies: (i) analysis of mecA polymorphs and Tn554 insertion patterns and (ii) determination of pulsed-field gel electrophoresis patterns of chromosomal SmaI digests. Ninety-seven of 158 strains showed a heterogeneous expression of resistance to methicillin. Among these, 75 (77.3%) were ClaI-mecA type I, ClaI-Tn554 type NH (NH, no homology with transposon Tn554), and pulsed-field gel electrophoresis (PFGE) pattern A (I::NH::A); 10 isolates were III::B::M (10.3%); and the remaining clones included a few or single isolates. The isolates with homogeneous expression of resistance to methicillin (n = 61) were predominantly ClaI-mecA type III (49 of 61 [80.3%]) but had great variability in their ClaI-Tn554 and PFGE patterns. This study confirmed the existence of two main clusters of MRSA in Poland.     

Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography

In this study, we have used time-lapse video cinematography to study fertilization in 50 human oocytes that had undergone intracytoplasmic sperm injection (ICSI). Time-lapse recording commenced shortly after ICSI and proceeded for 17-20 h. Oocytes were cultured in an environmental chamber which was maintained under standard culture conditions. Overall, 38 oocytes (76%) were fertilized normally, and the fertilization rate and embryo quality were not significantly different from 487 sibling oocytes cultured in a conventional incubator. Normal fertilization followed a defined course of events, although the timing of these events varied markedly between oocytes. In 35 of the 38 fertilized oocytes (92%), there were circular waves of granulation within the ooplasm which had a periodicity of 20-53 min. The sperm head decondensed during this granulation phase. The second polar body was then extruded, and this was followed by the central formation of the male pronucleus. The female pronucleus formed in the cytoplasm adjacent to the second polar body at the same time as, or slightly after, the male pronucleus, and was subsequently drawn towards the male pronucleus until the two abutted. Both pronuclei then increased in size, the nucleoli moved around within the pronuclei and some nucleoli coalesced. During pronuclear growth, the organelles contracted from the cortex towards the centre of the oocyte, leaving a clear cortical zone. The oocyte decreased in diameter from 112 to 106 microm (P < 0.0001) during the course of the observation period. The female pronucleus was significantly smaller in diameter than the male pronucleus (24.1 and 22.4 microm respectively, P = 0.008) and contained fewer nucleoli (4.2 and 7.0 respectively, P < 0.0001). After time-lapse recording, oocytes were cultured for 48 h prior to embryo transfer or cryopreservation. Embryo quality was related to fertilization events and periodicity of the cytoplasmic wave, and it was found that good quality embryos arose from oocytes that had more uniform timing from injection to pronuclear abuttal and tended to have a longer cytoplasmic wave. In conclusion, we have shown that time-lapse video cinematography is an excellent tool for studying fertilization and early embryo development, and have demonstrated that human fertilization comprises numerous complex dynamic events.      

Induction of an adaptive response to quercetin, mitomycin C and hydrogen peroxide by low doses of quercetin in V79 Chinese hamster cells

The adaptive response is a phenomenon by which cells exposedto low, non-cytotoxic doses of a genotoxicant become significantlyresistant to a subsequent higher dose of the same or anothergenotoxic agent. Induction of the adaptive response has beenmainly studied using ionizing radiation and alkylating agentsas genotoxic agents. However, other mutagenic agents may warrantfurther study, since the adaptive response as a whole may bean important general biological mechanism to maintain geneticintegrity and thus could prevent carcinogenic initiation ofcells. The exposure to mutagenic agents present, or formed,in the diet is considered an important factor in the etiologyof human tumors and a considerable number of these agents havenot yet been identified or characterized. Flavonoids are a largegroup of polyphenolic quinoids found in a wide variety of ediblefruits and vegetables and a few, such as quercetin, presentgenotoxic activity in vitro. The mechanisms of mutagenicityof quercetin involve the production of oxygen radicals throughan autoxidation process dependent on pH value and the presenceof oxygen. Although there are few doubts regarding the mutagenicityof quercetin invitro, carcinogenicity of this flavonoid is stillcontroversial. In view of these conflicting results and theradiomimetic nature of the mutagenicity of flavonoids, we addressedthe question of cell exposure to quercetin at the low levelspresent in the diet leading to adaptation to further exposureto mutagens or carcinogens. The work reported here concernsinduction of an adaptive response by low doses of quercetinto challenging doses of quercetin and other compounds, namelyhydrogen peroxide and mitomycin C, using induction of chromosomalaberrations in V79 cells as the end point. ⁴To whom correspondence should be addressed: Tel: +351 1 3610290; Fax: +351 1 3622018; Email: jose.rueff{at}gene.unl.mailpac.pt     

Prevention of acute immunological lung lesion in rats by decomplementing treatment

The intravenous administration of nephrotoxic antibody serum to rats produced a rapid and pronounced reduction in the serum complement level; this was observed before lung lesions became apparent.

A total suppression of the acute immune lung change was observed in animals depleted of complement by treatment with heat-aggregated human γ-globulin or zymosan.

Albeit the experimental evidence presented is of indirect nature, it suggests that the complement system is involved in the mediation of the acute pulmonary injury following injection of nephrotoxic antibody serum.

     

<Emphasis Type="Italic">Trypanosoma evansi</Emphasis> in inbred and Swiss-Webster mice: distinct aspects of pathogenesis

Trypanosoma evansi (Trypanosomatidae, Kinetoplastida) is a salivarian trypanosomatid that infects eight mammal orders spread over America, Europe and Asia. In Brazil, T. evansi is the etiological agent of Mal de Cadeiras, a horse disease very often described in the region known as Pantanal do Mato Grosso. Few data concerning the genetic diversity and biology of subpopulations of T. evansi that circulate in Brazil are available. The factors that modulate the interaction of this parasite with its hosts also remain to be elucidated. Here we evaluated the course of experimental infection of six T. evansi isolates derived from domestic and wild animals in Swiss-Webster mice and three Mus musculus lineages. The follow-up included biological, immunological as well as biochemical and hematological parameters. The same isolates as well as three others were characterized by pulsed-field electrophoresis. Our results showed that T. evansi isolates displayed significant differences regarding behavior and morbidity patterns in the distinct mouse lineages. Nevertheless, these differences could not be correlated with pulsed-field electrophoresis profiles. Indeed, concerning this molecular marker, only microheterogeneity was observed. Moreover, we observed that the outcome of the infection is defined by both host genetic background and peculiarities (virulence factors) of the distinct T. evansi isolates. Anemia and hypoglycemia were the only features that could be observed in all mouse lineages, independently of the inoculated T. evansi subpopulation. In addition, our data also show that Mus musculus is a suitable model host for the study of the different pathogenetic features of T. evansi infection.