Assessment of beta-HCG, beta-LH mRNA and ploidy in individual human blastomeres.

In human embryos, blastomeres differentiate into trophectoderm (TE) cells and inner cell mass (ICM) cells of blastocysts. Although morphologically indistinguishable, blastomeres at early cleavage stages are likely to undergo changes on a molecular level that make them destined to become ICM or TE cells. While the transcription factor Oct-4 might serve as a marker for totipotent ICM cells, human chorionic gonadotrophin might be used as the equivalent for TE cells. This study reports a reverse transcription-polymerase chain reaction procedure to assess human beta-HCG mRNA concentrations as well as ploidy in individual blastomeres from normally and abnormally fertilized human embryos. beta-HCG mRNA was detected in both euploid and aneuploid cells and in oocytes. Surprisingly, beta-LH mRNA was also detected in some euploid blastomeres. In regard to preimplantation genetic diagnosis, assessment of expression levels of beta-HCG and Oct-4 mRNA in individual biopsied cells might serve as a tool to identify embryogenic blastomeres in combination with testing for chromosome and single gene abnormalities.

Effect of incorrect gestational dating on Down's syndrome and neural tube risk assessment.

Down's syndrome risks are estimated between 15 and 20 completed weeks' gestation (cGW) using an algorithm involving maternal age and serum alpha-fetoprotein (AFP), chorionic gonadotrophin and unconjugated oestriol levels, each expressed as a multiple of the median level (MoM) at the cGW. The AFP MoM itself is the basis for screening for open neural tube defects (oNTD). Because medians change during this period, gestational dating must be accurate so that appropriate medians are used. A calculated Down's syndrome risk > 1:380 at term is generally considered to indicate a 'high-risk' pregnancy. This study focused on 378 patients with reported risk < or = 1:500 based on physician-supplied cGW (and hence considered at 'low risk' for Down's syndrome) to determine the effect of common 1-2-week dating errors on risk estimates. Using the original analytical data, each patient's risk was recalculated for each week over the 15-20 weeks, and classified into three categories: < 1:380 'low'; 1:380-1:100 'moderate'; and > 1:100 'high'. Advancing originally 'low-risk' patients by one week increased the risk by 1.09-14.1 times (median 3.18, mean 3.60); 46 (12.2%) became 'moderate' and 2 (0.5%) became 'high' risk. Advancing by two weeks increased risks 1.58-60.5 times (median 10.03, mean 12.04); 131 (36.5%) became 'moderate' and 39 (10.9%) became 'high' risk. Predictably, oNTD screening results also were affected. Although 1-2 week differences in AFP medians had little effect on most patients in this study sample, some who originally were oNTD negative became oNTD positive, whereas others who had been oNTD positive became screen negative. Thus, in many cases, a 1-2 week dating error may have only minimal effect on the estimated risks for chromosome or neural tube defects, but in other cases the effect of such an error would be significant.

Efficiency assessment of the gene trap approach.

The trapping of genes in murine embryonic stem (ES) cells offers three features in one experimental approach: 1) analysis of the expression patterns of unknown genes by using a simple staining method, 2) rapid cloning of unknown genes, and 3) generation of mutant mouse lines. We performed a gene trap screen aimed at the discovery of new genes regulating embryonic development. We have processed 209 gene trap events for expression patterns in chimeric murine embryos. Randomly tested, beta-galactosidase-positive ES cell clones resulted in vivo in 35% gene trap events showing no beta-galactosidase activity, 39% gene trap events with ubiquitous beta-galactosidase activity, and 26% gene trap events showing beta-galactosidase activity restricted to specific cell types or organs. In vitro preselection reduced gene trap events with ubiquitous beta-galactosidase activity to 10% and increased the gene trap events with restricted beta-galactosidase activity to 64%, making the screening procedure for genes expressed in a restricted manner 2.5-fold more efficient. In five of the seven gene trap insertions into genes in which the expression pattern during embryogenesis was known, the beta-galactosidase marker gene reproduced faithfully the expression pattern of the trapped gene. 5'-Rapid amplification of cDNA ends (5'-RACE) of 28 gene trap events revealed 19 novel mouse genes, 8 known mouse genes, and 1 random transsplicing event. Twelve of the 25 mouse lines that crossed to homozygosity showed overt abnormalities. The genomic structure was investigated in four of these gene trap events, which caused obvious abnormalities. In all four cases, the splice-acceptor gene trap construct was inserted into an exon. One of the 13 gene trap events that did not result in overt abnormalities was examined for the presence of wild-type mRNA. Homozygous animals were found to produce normal levels of wild-type mRNA. Evidently, gene trapping does not always provide all three of the features mentioned above. In this paper, we discuss the efficiency of gene trapping and ways in which some problems may be overcome.

Debunking the slippery slope argument against human germ-line gene therapy.

This paper attempts to debunk the slippery-slope argument against human germ-line gene therapy by showing that the downside of the slope--genetic enhancement--need not be as unethical or unjust as some people have supposed. It argues that if genetic enhancement is governed by proper regulations and is accompanied by adequate education, then it need not violate recognized principles of morality or social justice.

Assessment of polyploidy in human morulae and blastocysts using co-culture and fluorescent in-situ hybridization.

Fluorescence in-situ hybridization with DNA probes for X, Y and no. 18 chromosomes was used to analyse human morulae (n = 13) and blastocysts (n = 41), obtained after co-culture on Vero cells. On the basis of the number of hybridization signals, the proportion of embryos with more than five polyploid cells was 30.8% for morulae and 29.3% for blastocysts. These values are similar to those for mixoploidy (mosaicism of diploid and polyploid cells) observed in blastocysts of animal species. The results were confirmed by scanning electron microscopy, which showed a wide variation in the size of blastocyst nuclei, and by classical cytogenetic analysis. Mixoploidy seems to be a normal feature in preimplantation embryos and to occur very early in human embryo development. This lays open to doubt the preimplantation diagnosis of genetic errors at these stages, since results obtained from single cell analysis may not be representative of the whole embryo.