Unsuitability of multinucleated human blastomeres for preimplantation genetic diagnosis

Multinucleated blastomeres (MNBs) were detected in 30.4% of230 cleaved but subsequently arrested human embryos, and in66.7% of 21 cleaved embryos rejected after preimplantation geneticdiagnosis. A total of 71 MNBs from both groups of embryos wasanalysed by fluorescence in-situ hybridization (FISH) usingsimultaneous X and Y chromosome detection. The sex chromosomeanalyses of these MNBs suggests the existence of at least threemechanisms of MNB generation. In 95% of the embryos in whichmononucleated and multinucleated blastomeres were analysed,the sex of the MNBs corresponded with the sex of the entireembryo. However, the number of sex chromosomes per MNB and theirdistribution in each nucleus varied greatly, indicating theirunsuitability for aneuploidy diagnosis at the preimplantationstage.     

The HLA genetic constitution of the Bushmen (San)

The HLA-A, -B, and -C antigens of 290 and the DR antigens of 212 !Kung San individuals were characterized. The most frequent antigens were HLA-A30 gene frequency (gf) = 0.193, Bw58 (gf = 0.303), Cw6 (gf = 0.327), DR4 (gf = 0.273), and DQw3 (gf = 0.553). An unexpected finding was the low frequency of the classic African black antigen Bw42 (gf = 0.004). Marked differences as well as similarities in HLA gene frequencies were observed between the San and the South African Negroes, supporting the view that they had a common origin and were then separated for a very long time. During this period differences developed as a result of selective advantage in the Negroes following the pastoralist-agriculturalist way of life as opposed to the hunter-gatherer way of life. The picture is further complicated by the fact that gene flow, mostly from the San to the southern African Negroes, took place when they met again a few hundred years ago. The data also illustrate HLA haplotypes, linkage disequilibria, and four-locus haplotypes not previously seen in other human populations. The most frequent four-locus haplotype in the San, HLA-Aw43, Cw7, B7, DRw6 was also different from A30, Cw2, Bw42, DR3, the most common among the South African Negroes.     

Preimplantation genetic diagnosis and chromosome analysis of blastomeres using comparative genomic hybridization

Numerical chromosome errors are known to be common in early human embryos and probably make a significant contribution to early pregnancy loss and implantation failure in IVF patients. Over recent years fluorescent in situ hybridization (FISH) has been used to document embryonic aneuploidies. Many IVF laboratories perform preimplantation genetic diagnosis (PGD) with FISH to select embryos that are free from some aneuploidies in an attempt to improve implantation, pregnancy and live birth rates in particular categories of IVF patients. The usefulness of FISH is limited because only a few chromosomes can be detected simultaneously in a single biopsied cell. Complete karyotyping at the single cell level can now be achieved by comparative genomic hybridization (CGH). CGH enables not only enumeration of all chromosomes but gives a more complete picture of the entire length of each chromosome and has demonstrated that chromosomal breakages and partial aneuploidies exist in embryos. CGH has provided invaluable information about the extent of mosaicism and aneuploidy of all chromosomes in early human conceptuses. CGH has been applied to clinical PGD and has resulted in the birth of healthy babies from embryos whose full karyotype was determined in the preimplantation phase.     

Comparisons of virulence of influenza virus recombinants in ferrets in relation to their behaviour in man and their genetic constitution.

Two parent viruses, A/Finland/4/74(H3N2) and A/Okuda/57(H2N2), virulent and attenuated respectively for man, showed similar differences of virulence in ferrets as judged by estimations of 50% minimal infectious doses (MID50), the level and persistence of nasal infection, the height and duration of pyrexia and the level of lung infection. In ferrets, two recombinant clones, WRL 94(H3N2) and WRL 105(H3N2), were almost as virulent as A/Finland and indistinguishable from one another, a result which agreed well with genetic analysis (Hay et al. 1977); the RNA pieces of these recombinants appeared identical and largely derived from the virulent parent (A/Finland). The results in ferrets did not agree with tests on clone WRL 94 in small numbers of human volunteers but they were not inconsistent with those on clone WRL 105 in larger numbers. It is possible therefore that careful tests in ferrets may yield more accurate information on the virulence of strains than limited tests in human volunteers. A rapid test for virulence in ferrets is described. It could be used to screen many additional recombinants thereby yielding information on the genetical basis of virulence and indicating possible vaccine strains for more thorough testing in ferrets and in man.     

河北省手足口病病例的病原构成及EV71病毒基因特征分析

目的了解河北省不同地区、不同严重程度手足口病病例的病原构成情况及EV71病毒的基因特征。方法采集河北省不同地区的HFMD患者粪便、疱疹液、咽拭子标本进行核酸检测和病毒分离,同时结合所收集的HFMD患病例的居住地、疾病严重程度信息加以分析。选取18株EV71阳性分离株进行VP1编码区基因扩增和核苷酸序列测定和分析,与其它38株各基因型和基因亚型的EV71代表株构建系统发生树。结果2009年河北省HFMD临床诊断病例的EV阳性率为65．13％,其中以EV71为主,占阳性病例的58．0％（752／1296）。秦皇岛、邯郸、保定、邢台地区手足口病例以EV71感染为主,而衡水、沧州等地区则以CA16为主。轻型病例中EV71阳性率为37．74％,重症病例中EV71阳性率为80．64％,死亡病例检测13例,均为EV71阳性。18株EV71分离株的VP114核苷酸同源性为94．9％～99．8％,与C4亚型代表株的VP1区核苷酸同源性最高,为91．9％～99．6％。进化树结果显示,河北省EV71分离株与c4亚型代表株处于同一分支,并在C4a进化分支的不同簇中。结论2009年引起河北省手足口病流行的病原体主要为EV71和CA16。秦皇岛、邯郸、保定、邢台地区手足口病例以EV7I感染为主,而衡水、廊坊、沧州等地区则以CA16为主。EV71是重症病例的主要致病病原体。河北省EV71分离株为c4亚型C4a进化分支。     

Characterization of multinuclear hepatocytes induced in rats by mitemcinal (GM-611), an erythromycin derivative

Mitemcinal is an erythromycin derivative with motilin agonistic action, developed as a gastrointestinal motor-activating agent. The characteristics of mitemcinal-induced multinuclear hepatocytes (MNHs, hepatocytes with three or more nuclei per cell) from detailed morphological observations together with the results of a study on the mechanisms of MNH formation by combining cytocentrifuge preparations with 5-bromo-2'-deoxyuridine cumulative labeling are reported. MNHs were observed only in rats in the high-dose groups of the subchronic study, with a higher incidence in females and reversibility after twenty-eight days of drug withdrawal, but not observed in dogs. In the chronic study, the incidence increased relative to the dose. Histopathologically, MNHs were preferentially observed in the centrilobular zone, without nuclear atypia or mitotic figures. In the cell kinetic study, the labeling pattern of MNHs included all-positive, all-negative, and mixed labeling patterns of nuclei. The all-negative pattern indicated that the cells were formed by fusion of nondividing cells. The current results indicate that the cell kinetic approach effectively demonstrated the mechanism of mitemcinal-induced MNHs as fusion of hepatocytes and that drug-induced disturbance of mitosis is not involved in the multinucleation of MNHs by mitemcinal.     

The constitution of styrene-polysulfone

2:1 styrene-polysulfone was subjected to an alkaline degradation with potassium tert-butoxide in dimethylsulfoxide at 50°C. A random and not an unzipping degradation was operating throughout the decomposition and β-phenylbutyrophenone and other materials were obtained as the products, indicating dimeric and head to tail sequences are dominating in the polymer chain. NMR spectrometry was also undertaken to get a further information on the chain constitution, where repeating (? St? St? SO₂? ) sequence is believed to be the principal part of the polymer chain as compared to the NMR spectra of polystyrene, poly-α-methyl-styrene, and the high styrene containing styrene-polysulfone.     

Characteristics of body constitution and their relations to success in learning

Somatotype, finger dermatoglyphic pattern type, emotional stability level and foreign languages learning successfulness have been analyzed in 297 male cadets (aged 17-20 years) of the Military Institute of Physical Training. The cadets studied most frequently belonged to macrosomal and mesosomal somatotypes. In the study of finger patterns, loops were found to be most common (61.5% of all the patterns), while ringlets (33.4%) and arc patterns (5.1%) were less frequent. The amount of ulnar loops increased, while that of ringlets became less in the direction from micro- to macrosomal type. Almost half (46.9%) of the cadets appeared to be ambiverts, 30.8% were intraverts and the rest were extraverts. Loop patterns on all the fingers to a greater extent were found in cadets with high level of neuroticism; the cadets having lower neuroticism level were characterized by a combinations of loops with arcs on the left hand and arcs with ringlets on the right one. The cadets differing in foreign language learning successfulness level were different in their dermatoglyphic patterns and, especially in the prevalence of pattern combinations. So, among the excellent pupils the loop-arc combinations were 2.7 times more common and combinations of all three types of patterns (arcs, loops, ringlets) were 1.4 times more common.     

植入前遗传学诊断中卵裂球固定技术改进

目的改进植入前遗传学诊断卵裂球固定技术,减少卵裂球细胞丢失,获得更好的固定效率、更高的荧光原位杂交成功率,简化操作程序.方法体外授精治疗周期废弃胚胎的96个卵裂球细胞,分别用3种不同的固定技术进行固定,固定后使用X、Y着丝粒探针进行荧光原位杂交,比较其固定效率和杂交效率.结果改进方法固定率和杂交率均为100%.而两种传统技术的固定率分别为90%,83.3%;杂交率为80%,73.3%.结论这种新的固定卵裂球技术从根本上消除了卵裂球标本的丢失,使PGD结果更为可靠;同时简化了操作程序,值得推广.     

The Schinzel syndrome in a mother and daughter

The Schinzel Syndrome was identified in a mother and daughter. This report expands the phenotype associated with this disorder.     

The frequency and developmental capability of human embryos containing multinucleated blastomeres

The frequency of multinucleated blastomeres (MNB) in 2- and 4-cell stage human embryos was recorded immediately before embryo transfer using a high-power inverted microscope. About 44% of patients (150/338) possessed embryos exhibiting MNB. The appearance of this nuclear abnormality was not correlated with maternal age. Overall, 15% of the otherwise good quality embryos (274/1885) that developed after monospermic fertilization contained several multinuclei (from two to seven) in at least one cell. Quite often MNB were found within all cells of the embryo (50% in 2-cell embryos). Blastomere multinucleation was significantly higher in 2-cell than 4-cell embryos (P <0.0001). This suggests that a considerable number of human embryos become abnormal during the first embryonic division. The embryos containing MNB were usually excluded for uterine transfers, with the exception of 19 cases when only such embryos could be replaced (6%; 19/338 patients). The results demonstrated that embryos with MNB may implant (4/19 cases; 21%) and they can lead to both spontaneous abortions and the successful birth of healthy infants (two cases). The fact that in the successful cases, 2-cell stage embryos with a mononucleated and a binucleated blastomere were transferred also suggests that due to the cell totipotency, development of a healthy baby is possible from one normal blastomere. Since multinucleation in early embryos may reflect gross chromosomal abnormalities or development of mosaic embryos, it is advisable not to replace embryos with MNB. Occasional transfers, however, can be considered because defective embryos may sometimes develop normally.      

Preimplantation diagnosis: Primer extension preamplification for detection of multiple genetic loci from single human blastomeres

A new technology called primer extension preamplification (PEP),which has been applied to single spermatozoa, increases theamount of polymerase chain reaction (PCR) templates by amplifyingDNA of the whole genome. The current investigation was aimedat applying PEP to single human blastomeres. Two blastomereswith nuclei from arrested embryos were selected for this study.Using three different PEP protocols (experiments I, II and III),DNA from single blastomeres was amplified using 15-base oligonucleotiderandom primers. The efficiency of the procedure was determinedby further amplifications of aliquots of the PEP products withtwo specific sequences. Three aliquots from each PEP productwere used as PCR templates for the human X chromosome (X) orthe exon 10 of the cystic fibrosis gene (CF). PCR amplifiedproducts were analysed by gel electrophoresis. In experimentI, when X primers were used, positive signals were detectedin all 10 embryos (100%), 90.0% (18/20) of the blastomeres,and in 80.0% (96/120) of the replicates. When CF primers wereamplified, all embryos (100%, 10/10), 90.9% (18/20) of the blastomeresand 78.3% (47/60) of the replicates were positive. In experimentII, efficiency was significantly reduced when total time forthe procedure was minimized from 8 h to 5 h and 45 min. Althoughthe time was further reduced to 4 h and 40 min in experimentIII, the efficiency remained the same as in experiment I whenthe volume of PEP was reduced from 60 µl (experimentsI and II) to 40 µl. One out of 132 control replicates(0.8%) was contaminated. The present study indicates that PEPcan be successfully applied to single blastomeres allowing forsimultaneous detection of approximately 20 DNA sequences.     

Feasibility study of repeated fluorescent in-situ hybridization in the same human blastomeres for preimplantation genetic diagnosis

In order to increase the number of chromosomes examined in each blastomere, we have developed a repeated fluorescent in-situ hybridization (FISH) procedure by which six or more chromosomes can be analysed per blastomere of a human embryo. Three consecutive FISH procedures with directly-labelled fluorescent Vysis DNA probes were carried out for examination of chromosomes X, Y, 11, 13, 18 and 21 in the same blastomeres (n = 126) and lymphocytes (n = 164). Based on the initial number of nuclei, the percentages of nuclear loss and presence of signals were 3 and 92% respectively in blastomeres; 6 and 91% respectively in lymphocytes after the first FISH; 7 and 87% respectively in blastomeres and 10 and 86% respectively in lymphocytes, after the second FISH. These percentages were 13 and 78% respectively in blastomeres and 14 and 81% respectively in lymphocytes after the third FISH. The FISH procedure was repeated successfully in a couple for preimplantation genetic diagnosis of chromosomal aneuploidies in biopsied blastomeres of their embryos in our clinic. In conclusion, it is feasible to carry out repeated FISH procedures in the same blastomeres. Six or more chromosomes of a single blastomere may be examined using this procedure.      

Diagnostic efficiency, embryonic development and clinical outcome after the biopsy of one or two blastomeres for preimplantation genetic diagnosis

BACKGROUND: Preimplantation genetic diagnosis or screening (PGD, PGS) involves embryo biopsy on Day 3. Opting for one- or two-cell biopsy is a balance between the lowest risk for misdiagnosis on the one hand and the highest chance for a pregnancy on the other hand. METHODS: A prospective controlled trial was designed and 592 ICSI cycles were randomly assigned to the one-cell (group I) or the two-cell group (group II). Primary outcomes were diagnostic efficiency and embryonic development to delivery with live birth (analysed by cycle). The false-positive rate for the PCR cycles is presented as a secondary outcome (analysed by embryo). RESULTS: A strong significant correlation was observed between embryonic developmental stage on Day 3 and post-biopsy in vitro development on Day 5 (P < 0.0001). The influence of the intervention on Day 3 was less significant (P = 0.007): the biopsy of one cell is less invasive than the biopsy of two cells. PCR diagnostic efficiency was 88.6% in group I and 96.4% in group II (P = 0.008). For the fluorescence in situ hybridization (FISH) PGD cycles no significant difference in efficiency was obtained (98.2 and 97.5% in group I and II, respectively). Similar delivery rates with live birth per started cycle were obtained [58/287 or 20.2% in group I versus 52/303 or 17.2% in group II, P = 0.358; the absolute risk reduction = 3.05%; 95% confidence interval (CI): -3.24, 9.34]. Post-PGD PCR reanalysis showed six false positives in 97 embryos (6.2%) in group II and none in group I (91 embryos reanalysed). No false negatives were found. CONCLUSIONS: While removal of two blastomeres decreases the likelihood of blastocyst formation, compared with removal of one blastomere, Day 3 in vitro developmental stage is a stronger predictor for Day 5 developmental potential than the removal of one or two cells. The biopsy of only one cell significantly lowers the efficiency of a PCR-based diagnosis, whereas the efficiency of the FISH PGD procedure remains similar whether one or two cells are removed. Delivery rates with live birth per started cycle were not significantly different.     

The impact of direct-to-consumer marketing of cancer genetic testing on women according to their genetic risk

PurposeTo assess the impact of direct-to-consumer marketing for genetic testing among women of varying genetic risk for breast and ovarian cancer.MethodsTelephone surveys were conducted with 315 women in Denver, Colorado, one target audience for the Myriad BRACAnalysis ad campaign. Genetic risk was determined from personal and family history and grouped by probability of having a BRCA1/2 mutation (low <5%, moderate 5–<10%, high ≥10%).ResultsHigh-risk women were more knowledgeable about BRACAnalysis and more likely to recall the media ads than were low-risk women (60 vs. 39%, P < 0.01). After seeing the ads, about 40% of women were more interested in testing and about 10% expressed increased worry about developing breast or ovarian cancer. Women across all risk groups overstated the benefits and appropriateness of testing. An equal percentage of high- and low-risk women (51 and 60%) felt that they would benefit from genetic testing.ConclusionThe campaign effectively reached a large audience. Concern about breast cancer was not appreciably increased. A large percentage of low-risk women (not candidates for testing) expressed interest in testing, suggesting the campaign was too broad. A campaign targeted at high-risk women, who may benefit from testing might be preferred.     

The origin of Palestinians and their genetic relatedness with other Mediterranean populations.

The genetic profile of Palestinians has, for the first time, been studied by using human leukocyte antigen (HLA) gene variability and haplotypes. The comparison with other Mediterranean populations by using neighbor-joining dendrograms and correspondence analyses reveal that Palestinians are genetically very close to Jews and other Middle East populations, including Turks (Anatolians), Lebanese, Egyptians, Armenians, and Iranians. Archaeologic and genetic data support that both Jews and Palestinians came from the ancient Canaanites, who extensively mixed with Egyptians, Mesopotamian, and Anatolian peoples in ancient times. Thus, Palestinian-Jewish rivalry is based in cultural and religious, but not in genetic, differences. The relatively close relatedness of both Jews and Palestinians to western Mediterranean populations reflects the continuous circum-Mediterranean cultural and gene flow that have occurred in prehistoric and historic times. This flow overtly contradicts the demic diffusion model of western Mediterranean populations substitution by agriculturalists coming from the Middle East in the Mesolithic-Neolithic transition.