The chromosomal analysis of human oocytes. An overview of established procedures

The cytogenetic survey of mature human oocytes has been and remains a subject of great interest because of the prevalence of aneuploidy of maternal origin in abnormal human conceptuses, and the lack of understanding about the non-disjunction processes in human meiosis. The first attempts to analyse the chromosomal content of human female gametes were made in the early 1970s, and led to limited data because of the paucity of materials and the inadequacy of the procedure used. The years to follow brought a resurgence of interest in this field, because of the development of human IVF techniques which made oocytes unfertilized in vitro available for cytogenetic analysis. Numerous studies have since been performed. However, the difficulties in obtaining good chromosome preparations and of performing accurate chromosome identification have reduced the viability of these studies, resulting in large variations in the reported incidences of chromosomal abnormalities. The further introduction of new procedures for oocyte fixation and the screening of large oocyte samples have allowed more reliable data to be obtained and to identify premature chromatid separation as a major mechanism in aneuploidy occurrence. The last decade has been privileged to witness the adaptation of molecular cytogenetic techniques to human oocytes, and thus various powerful procedures have been tried not only on female gametes, but also on polar bodies, involving sequential and multicolour fluorescent in situ hybridization (FISH) labelling, comparative genomic hybridization (CGH), spectral karyotyping and alternative methods such as primed in situ labelling (PRINS) and peptide nucleic acid (PNA) techniques. A large body of data has been obtained, but these studies also display a great variability in the frequency of abnormalities, which may be essentially attributable to the technical limitations of these in situ methods when applied to human oocytes. However, molecular cytogenetic approaches have also evidenced the co-existence of both whole chromosome non-disjunction and chromatid separation in maternal aneuploidy. In addition, the extension of these techniques to oocyte polar body materials has provided additional data on the mechanism of meiotic malsegregation. Improvements of some of these techniques have already been reported. The further development of new approaches for the in situ analysis of human meiosis will increase the impact of cytogenetic investigation of human oocytes in the understanding of aneuploidy processes in humans.     

Multiple mutation analysis of the cystic fibrosis gene in single cells

PGD is becoming an alternative to prenatal diagnosis. The combination of IVF techniques with the PCR technology allows for the detection of genetic abnormalities in first polar bodies from oocytes and blastomeres from cleavage-stage embryos. Dealing with a genetic disease with a heterogeneous spectrum of mutations like cystic fibrosis, one of the objectives of centres offering PGD is the application of simple and efficient protocols that allow for the detection of a wide range of mutations with a single procedure. In the present work, 29 normal loci and the 31 most frequent cystic fibrosis transmembrane conductance regulator (CFTR) mutations in Southern Europe could be detected at the same time in single cells applying a modified and improved primer extension preamplification-PCR. Two different Taq polymerases were tested in isolated buccal cells heterozygous for several mutations. The protocol that gave statistically significant better results was also successful in oocytes and their first polar bodies.     

心肌双核素显像三种图像处理方法诊断冠心病的临床价值

目的 比较心肌双核素断层图、原始图和定量靶心图3种图像处理方法诊断冠心病,鉴别缺血心肌和缺血活性心肌。方法 设正常对照组20例,冠脉造影证实冠状动脉有病变的患者54例,其中冠状动脉狭窄＞50％的冠心病患者32例。在静息状态下静脉注射^99mTc-MIB I20mCi(740MBq),15min后作常规多巴酚丁胺负荷试验,在达到负荷要求时静脉注射201Tl3mCi(111MBq)。分别在注射后10min和4h作心肌双核素断层图像采集（SIEMENS SPECT仪器）。结果 （1）．心肌双核素断层图、原始图和定量靶心图诊断冠心病（冠状动脉狭窄＞50％）的灵敏度和特异性分别为100％、100％、85％和98．5％、73．7％、78．9％。（2）．断层图、原始图和定量靶心图诊断72支冠状动脉病变（冠脉造影异常）的灵敏度和特异性分别为54％、57．9％、47．2％和96．5％、87．7％、92．2％。（3）．断层图、原始图和定量靶心图诊断32支冠状动脉狭窄（狭窄＞50％）的灵敏度和特异性分别为75％、78％、72％和96．5％、87％、92．2％。结论 比较3种图像处理方法,诊断冠心病和冠脉病变差异无显著性（P＞0．05）。但结合3种图像处理方法可提高冠心病的诊断和鉴别诊断的可靠性。     

Conformation of cyclosporin A in polar solvents

A major conformation of cyclosporin A in methanol and in aqueous methanol was revealed by some simple NMR experiments. Thus, a stepwise transition of cyclosporin A conformation from 100% CDCl₃ to 100% CD₃OD was followed by ¹H NMR, which showed that the chloroform conformation of cyclosporin A was still the major one in methanol. Employing the same technique, it was also shown that the chloroform conformation of cyclosporin A was one of the major conformations in 50% aqueous methanol. This may be the first experimental determination of a major conformation of cyclosporin A in polar solvents.     

Polar sampling in k-space: Reconstruction effects

Magnetic resonance images are most commonly computed by taking the inverse Fourier transform of the k-space data. This transformation can potentially create artifacts in the im age, depending on the reconstruction algorithm used. For equally spaced radial and azimuthal k-space polar sampling, both gridding and convolution backprojection are applicable. However, these algorithms potentially can yield different res olution, signal-to-noise ratio, and aliasing characteristics in the reconstructed image. Here, these effects are analyzed and their tradeoffs are discussed. It is shown that, provided the modulation transfer function and the signal-to-noise ratio are considered together, these algorithms perform similarly. In contrast, their aliasing behavior is different, since their re spective point spread functions (PSF) differ. In gridding, the PSF is composed of the mainlobe and ringlobes that lead to aliasing. Conversely, there are no ringlobes in the convolution backprojection PSF, thus radial aliasing effects are mini mized. Also, a hybrid gridding and convolution backprojection reconstruction is presented for radially nonequidistant k-space polar sampling.     

Effects of dietary selenium supplementation on serum and liver selenium,serum malondialdehyde and liver glutathione peroxidase activity in rats consuming thermally oxidized sunflower oil

The present study compared the effects of four isocaloric diets containing (1) fresh sunflower oil not supplemented with selenium (Fresh), (2) oxidized sunflower oil not supplemented with selenium (Oxidized), (3) fresh sunflower oil supplemented with 1 ppm selenium as sodium selenite (Fresh + Se), (4) oxidized sunflower oil supplemented with 1 ppm selenium as sodium selenite (Oxidized + Se) on serum MDA concentrations, liver GPx activity and serum and liver selenium contents in growing male Sprague Dawley rats during a period of 43 days. The oxidized oil used was prepared by heating fresh sunflower oil at 180 °C for 48 h. Serum and liver selenium contents and liver GPx activity were significantly higher in the selenium supplemented groups compared to the non-selenium supplemented groups, but these parameters did not differ significantly between the oxidized oil fed groups and the fresh oil fed groups. Serum MDA concentrations increased significantly in the Oxidized group compared to the Fresh group. This suggests that the ingestion of oxidized oil resulted in, in vivo lipid peroxidation. Serum MDA concentrations remained significantly higher even in comparison of the Oxidized + Se group with the Oxidized group. Our results emphasize that the consumption of oxidized oil increases in vivo lipid peroxidation and thus can be deleterious to health. However, we did not observe a significant beneficial effect of selenium supplementation upon the ingestion of thermally oxidized oil on lipid peroxidation.     

The peptide nucleic acids as probes for chromosomal analysis: application to human oocytes, polar bodies and preimplantation embryos

Peptide nucleic acids (PNA) are synthetic DNA mimics based on an uncharged polyamide backbone, which hybridize with complementary DNA with high affinity and specificity. PNA have recently become recognized as efficient tools for in situ chromosomal identification. In the present study, this new approach has been tried on isolated human oocytes, polar bodies and blastomeres. Using centromeric PNA probes specific for chromosomes 1, 4, 9, 16, X and Y, we tested multicolour labelling PNA reaction on 27 oocytes and 23 blastomeres. Sequential PNA hybridization was performed on five oocytes and combined PNA and fluorescence in situ hybridization (FISH) reactions on two oocytes. Both the rates and the types of abnormalities observed are in agreement with results from previous FISH studies. This preliminary study indicates that PNA probes allow a reliable chromosomal analysis in isolated human oocytes and blastomeres and consequently might provide an interesting adjunct to FISH for diagnostic analysis.     

First polar body morphology before ICSI is not related to embryo quality or pregnancy rate

BACKGROUND: The aim of this study was to analyse the relationship between the first polar body (1st PB) morphology and the fertilization rate, cleavage rate, embryo quality, pregnancy and implantation rate. METHODS: This was a retrospective study on 167 consecutive cycles undergoing assisted reproduction with ICSI. The 1st PB morphology was evaluated at the moment of ICSI in the 596 injected oocytes and it was coded as intact or fragmented. The fertilization rate, cleavage rate, embryo quality (three grades), pregnancy rate, implantation rate and the time elapsed between oocyte retrieval and ICSI were evaluated. The 1st PB morphology was checked twice (denudation and ICSI) in a random sample of 180 oocytes in order to verify the effect of the in vitro culture. RESULTS: No significant relationship was found between the 1st PB morphology and the fertilization rate (P=0.703), cleavage rate (P=0.055), embryo quality (P=0.673), pregnancy rate (P=0.201) and implantation rate (P=0.511). A significant positive relationship (P=0.006) was found between the frequency of the 1st PB fragmentation and the time elapsed between denudation and ICSI. The pregnancy rate was significantly higher (P=0.008) when oocytes were injected between 5 and 7 h after retrieval rather than earlier or later. CONCLUSIONS: Our data suggest that the embryo quality, pregnancy rate and implantation rate are not related to the 1st PB fragmentation. The time which elapses between the oocyte retrieval and ICSI should be maintained at approximately 6 h in order to obtain optimal results.     

Effects of argon laser irradiation on polar excitations in frog sciatic nerve

BACKGROUND AND OBJECTIVES: Since the mechanisms underlying the effects of low-power laser irradiation on the nervous system remain unclear, we examined whether such irradiation can influence ionic channels of the nerve membrane using the law of polar excitation in isolated frog sciatic nerve. STUDY DESIGN/MATERIALS AND METHODS: Using 43 frogs (Xenopus laevis), nerve preparations were stimulated at 0.5/second using a 10-millisecond pulse at supramaximal intensity. Ar+ laser irradiation (457, 488, 514 nm; 50, 75, 100 mW) was applied for 30 minutes to the portion between the anode and cathode stimulating electrodes. RESULTS AND CONCLUSIONS: Ar+ laser irradiations (457, 488 nm; 50 mW) blocked the generation of anode-break-excitation, rather than cathode-make-excitation. Such a selective effect occurred when applying a blocker of hyperpolarization-activated cation current (Ih) channel, ZD7288. Ar+ laser irradiation may influence Na+ channels in addition to Ih channels.