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Objective: To ascertain whether embryos classified by preimplantation genetic diagnosis (PGD) for infertility as abnormal and then plated to obtain stem cells would self-correct partially or totally in culture, producing disomic stem cells., Design: Prospective study to determine the chromosome status of embryos on day 3 and 6, as well as cultured cells derived from inner cell masses from the same embryos when cultured up to day 12., Setting: Research laboratory., Patient(s): Patients undergoing PGD of aneuploidy., Intervention(s): Of 142 embryos classified by PGD for aneuploidy as abnormal, 50 were cultured to the blastocyst stage. At that stage a fraction of the embryos underwent trophectoderm biopsy to reconfirm the PGD diagnosis. After further co-culture with feeders up to day 12, 34 embryos attached to the feeder cells. Of those, 24 were analyzed by fluorescence in situ hybridization (FISH) and the rest for the expression of Oct-4, SSEA-3, SSEA-4, TRA1-60, and TRA1-80., Main Outcome Measure(s): Disomic cells obtained from trisomic embryos., Result(s): Analysis by FISH of day-12 cultures showed that 7 were totally normal, 6 were mostly abnormal, and 11 had experienced some chromosome normalization, having between 21% and 88% normal cells. Day-12 culture was positive for Oct-4 expression by reverse transcriptase polymerase chain reaction analysis and for SSEA-3, SSEA-4, TRA1-60, and TRA1-80 by immunocytochemistry., Conclusion(s): Chromosome self-normalization occurs in a significant proportion of chromosomally abnormal embryos, possibly because of the loss of a chromosome in trisomic cells after blastocyst stage. Thus chromosomally abnormal embryos are a potential source of disomic stem cells. Not all chromosomally abnormal embryos self-corrected. Abnormal stem cells that might be derived could be used as models to study the effect of chromosomal abnormalities on human development.

Objective: We set out to characterize the expression of nine genes in human preimplantation embryos and determine whether abnormal morphology is associated with altered gene activity., Design: Reverse transcription and real-time polymerase chain reaction were used to quantify the expression of multiple genes in each embryo. The genes studied have various important cellular roles (e.g., cell cycle regulation, DNA repair, and apoptosis)., Setting: Research laboratory working closely with a clinical IVF practice., Patient(s): Over 50 embryos were donated by infertile patients (various etiologies). Among these, all major stages of preimplantation development and a variety of common morphologic abnormalities were represented., Intervention(s): None., Main Outcome Measure(s): Quantification of mRNA transcripts., Result(s): We detected an association between certain forms of abnormal morphology and disturbances of gene activity. Cellular fragmentation was associated with altered expression of several genes, including TP53, suggesting that fragmenting blastomeres are suffering stress of a type monitored by p53, possibly as a consequence of suboptimal culture conditions., Conclusion(s): Appropriate gene expression is vital for the regulation of metabolic pathways and key developmental events. Our data indicates a possible causal relationship between changes in gene expression and the formation of clinically relevant abnormal embryo morphologies. We hypothesize that embryos with expression profiles characteristic of good morphology and appropriate for their developmental stage have the greatest potential for implantation. If confirmed, this could lead to a new generation of preimplantation genetic diagnosis (PGD) tests for assessing embryo viability and predicting implantation potential.

Nitric oxide (NO) production plays an important role in regulating preimplantation embryo development. NO is produced from l-arginine by the enzyme nitric oxide synthase (NOS), which has three isoforms: endothelial (eNOS), neuronal (nNOS), and inducible (iNOS). It has been previously shown that inhibition of NO production by NG-nitro-l-arginine (l-NA) inhibits the development of two-cell embryos to the four-cell stage. However, excess NO also halts embryo development, possibly through the production of free radicals. We hypothesize that multiple NOS isoforms are expressed in order to ensure normal preimplantation embryo development and that, in this process, NO acts through the cGMP pathway. Using reverse transcription-polymerase chain reaction, mRNA for all three NOS isoforms was amplified from two-cell, four-cell, morula, and blastocyst embryos. However, blastocyst-stage embryos isolated midmorning on Day 4 of pregnancy expressed only nNOS and eNOS, whereas those isolated midafternoon again expressed all three NOS isoforms. Culture of one-cell embryos in various concentrations of Whitten (positive control), S-nitroso-N-acetylpenicillamine (SNP, a NO donor), l-NA, and/or 8-Br-cGMP demonstrated that NO is acting, at least in part, through cGMP in preimplantation embryo development. In addition, we determined that a critical concentration of NO and cGMP is required for normal embryo development and deviations from this concentration lead to developmental arrest and/or apoptosis of the embryo. This data provides support for a requirement of NO in preimplantation embryo development and one mechanism through which it regulates mitotic division in these embryos.

Oocytes, in general, are greatly enriched in mitochondria to support higher rates of macromolecular synthesis and critical physiological processes characteristic of early development. An inability of these organelles to amplify and/or to accumulate ATP has been linked to developmental abnormality or arrest. The number of mitochondrial genomes present in mature mouse and human metaphase II oocytes was estimated by fluorescent rapid cycle DNA amplification, which is a highly sensitive technique ideally suited to quantitative mitochondrial DNA (mtDNA) analysis in individual cells. A considerable degree of variability was observed between individual samples. An overall average of 1.59 x 10(5) and 3.14 x 10(5) mtDNA molecules were detected per mouse and human oocyte, respectively. Furthermore, the mtDNA copy number was examined in polar bodies and contrasted with the concentration in their corresponding oocytes. In addition, the density of mtDNA in a cytoplasmic sample was estimated in an attempt to determine the approximate number of mitochondria transferred during clinical cytoplasmic donation procedures as well as to develop a clinical tool for the assessment and selection of oocytes during in vitro fertilisation procedures. However, no correlation was identified between the mtDNA concentration in either polar bodies or cytoplasmic samples and their corresponding oocyte.

Deciphering the complex series of regulatory events that occur during early development depends partly on the ability to accurately quantify stage-specific mRNA species. However, the paucity of biological material coupled with the lack of sensitivity and/or reproducibility of the currently available quantitative methods had been severe limitations on single cell analysis. Rapid cycle DNA amplification is a highly sensitive technique for amplification of specific DNA sequences. With the addition of fluorescence probes, it is possible to monitor the log-linear phase of amplification during which the most useful quantitative data is obtained. Unknown concentrations are extrapolated from standards co-amplified producing a standard curve. Furthermore, micro volume capabilities allow for the analysis of minute samples. Consequently, this approach is ideally suited to the needs of the clinical IVF laboratory. Rapid fluorescence monitored cycling was used to examine expression levels of the housekeeping genes beta-actin and hypoxanthine guanine phosphorlbosyltransferase in individual murine/human oocytes and/or embryos. Results obtained compared favourably with those attained by others and followed the predicted temporal patterns of expression. Once informative reproductive molecular markers are identified by micro-array analysis, minimally invasive techniques can be developed to biopsy cytoplasm and/or polar bodies for clinical evaluation using rapid fluorescence monitored reverse transcription-polymerase chain reaction methods.

Objective: To determine by reverse transcription-polymerase chain reaction (PCR) whether human leukocyte antigen (HLA) class I messenger RNA (mRNA) is present in mature human spermatozoa., Design: Mature human spermatozoa was isolated from donor semen using a swim-up technique. Total RNA was extracted via guanidinium isothiocyanate-cesium chloride ultracentrifugation. By the method previously validated in our laboratory, reverse transcription-PCR was performed using primers specific for HLA class I transcripts. Positive control cells included a choriocarcinoma cell line (JEG) and human fetal tissue. Transformed peripheral blood lymphocytes (PBL) were used as a negative control for somatic cellular contamination., Results: Human spermatozoa were positive for HLA class I (-G and -B) mRNA by reverse transcription-PCR, consistent with the positive controls. We did not detect any mRNA for beta-actin, retinoblastoma (RB), CD4, or kappa light chain genes in the sperm complementary DNA samples, verifying that the class I mRNA detected was not due to somatic cellular contamination of the purified sperm samples., Conclusion: These experiments provide the first evidence that mRNA for HLA class I molecules are present in mature human spermatozoa. The physiological role of these transcripts is unknown at present. Further experiments characterizing the expression of HLA class I (-G and -B) mRNA in oocytes and preimplantation embryos are in progress.