Effect of in vitro fertilization on gene expression and development of mouse preimplantation embryos.

In vitro culture (IVC) of preimplantation mouse embryos is associated with changes in gene expression. It is however, not known if the method of fertilization affects the global pattern of gene expression. We compared gene expression and development of mouse blastocysts produced by in vitro fertilization (IVF) versus blastocysts fertilized in vivo and cultured in vitro from the zygote stage (IVC) versus control blastocysts flushed out of the uterus on post coital day 3.5. The global pattern of gene expression was assessed using the Affymetrix 430 2.0 chip. It appears that each method of fertilization has a unique pattern of gene expression and development. Embryos cultured in vitro had a reduction in the number of trophoblastic cells (IVF 33.5 cells, IVC 39.9 cells, and 49.6 cells in the in vivo group) and, to a lesser degree, of inner cell mass cells (12.8, 11.7, and 13.8 respectively). The inner cell mass nuclei were larger after culture in vitro (140 microm(2), 113 microm(2), and 86 microm(2) respectively). Although a high number of genes (1912) was statistically different in the IVF cohort when compared with the in vivo control embryos, the magnitude of the changes in gene expression were low and only a minority of genes (29 genes) was changed more than fourfold. Surprisingly, IVF embryos were different from IVC embryos (3058 genes were statistically different, but only three changed more than fourfold). Proliferation, apoptosis, and morphogenetic pathways are the most common pathways altered after IVC. Overall, IVF and embryo culture have a profound effect on gene expression pattern and phenotype of mouse preimplantation embryos.

Gene expression analysis of endometrium reveals progesterone resistance and candidate susceptibility genes in women with endometriosis.

The identification of molecular differences in the endometrium of women with endometriosis is an important step toward understanding the pathogenesis of this condition and toward developing novel strategies for the treatment of associated infertility and pain. In this study, we conducted global gene expression analysis of endometrium from women with and without moderate/severe stage endometriosis and compared the gene expression signatures across various phases of the menstrual cycle. The transcriptome analysis revealed molecular dysregulation of the proliferative-to-secretory transition in endometrium of women with endometriosis. Paralleled gene expression analysis of endometrial specimens obtained during the early secretory phase demonstrated a signature of enhanced cellular survival and persistent expression of genes involved in DNA synthesis and cellular mitosis in the setting of endometriosis. Comparative gene expression analysis of progesterone-regulated genes in secretory phase endometrium confirmed the observation of attenuated progesterone response. Additionally, interesting candidate susceptibility genes were identified that may be associated with this disorder, including FOXO1A, MIG6, and CYP26A1. Collectively these findings provide a framework for further investigations on causality and mechanisms underlying attenuated progesterone response in endometrium of women with endometriosis.

Effects of oxygen tension on gene expression in preimplantation mouse embryos.

OBJECTIVE: To compare the effects of atmospheric and physiologic oxygen concentrations on the global patterns of gene expression during mouse preimplantation development. DESIGN: Comparative analysis of in vivo-produced and in vitro-produced embryos. SETTING: Research laboratory. PATIENT(S): None. INTERVENTION(S): Control embryos at the blastocyst stage that developed in vivo were collected from uteri. Experimental embryos were obtained at the zygote stage and cultured to the blastocyst stage in Whitten's medium or KSOM medium with amino acids under 20% oxygen (atmospheric) or 5% oxygen (physiologic). MAIN OUTCOME MEASURE(S): Embryo development, cell number, and gene expression assayed by microarray technology. RESULT(S): Low (physiologic) oxygen concentration is associated with faster embryo development and increased cell number. In addition, there are marked perturbations in the global pattern of gene expression, as assessed by oligonucleotide microarray, after culture in 20% oxygen as compared with 5% oxygen. CONCLUSION(S): Culture in low oxygen is associated with fewer perturbations in the global pattern of gene expression and more closely resembles that of the in vivo control embryos. These findings provide rationale for culturing human embryos in the presence of 5%, rather than 20%, oxygen.

Molecular cloning of nonsecreted endothelial cell-derived lipase isoforms.

To expand our knowledge of factors involved in lipid metabolism in the blood vessel wall, we have cloned unique molecular isoforms of endothelial cell-derived lipase (EDL) (HGMW-approved symbol/LIPG). One isoform encoded a truncated protein (EDL2a) lacking the first 80 amino acid residues of the previously characterized EDL1a isoform, including the signal peptide. A similar second clone (EDL2b) was identified that lacked not only the first 80 amino acids, but also a 74-amino-acid region that encodes a portion of the lid domain. RT-PCR analysis confirmed expression of EDL2a/2b isoforms in several human tissues and cultured cells, including endothelial cells. Western blot and immunofluorescence studies using stable transfectants revealed that EDL2a and EDL2b were localized in the cytosol, while, EDL1a was secreted into the culture medium. Cell extracts of EDL2a/2b transfectants did not have triglyceride or phospholipase activity. Thus endothelial cells express three EDL isoforms, two of which remain intracellular and do not function as lipases.

Direct treatment costs for patients with lung cancer from first recurrence to death in france.

OBJECTIVE: To determine the direct treatment cost of lung cancer management from progression to death from the viewpoint of the hospital. METHODS: A retrospective descriptive study was performed. Data from 100 patients who died of lung cancer and who had received treatment from four different types of hospital were used; the hospitals were public hospitals (teaching and non-teaching), private not-for-profit cancer centres, and private hospitals. Resource utilisation/cost data collected included the cost of diagnosis of the recurrence, the cost of hospitalisations or day care treatments and ambulatory surgery. All resources were valued in 2001 euros. RESULTS: In France, the average cost per patient was euro12 518 for the whole group (78% with non-small cell lung cancer [NSCLC], and 22% with small cell lung cancer [SCLC]), euro13 969 for patients with NSCLC and euro7369 for patients with SCLC. The higher cost of treatment in patients with NSCLC is explained by longer survival and duration of chemotherapy. In patients with NSCLC, 51% of the total cost corresponded to terminal care, with up to seven lines of chemotherapy. In patients with SCLC, the costs of diagnosis and terminal care each represented 41% of the total cost. CONCLUSIONS: The cost of treatment of recurrence of lung carcinoma is high, and is related to the number of lines of chemotherapy and the use of radiotherapy and surgery.

Identification of endothelial cell genes by combined database mining and microarray analysis.

Vascular endothelial cells maintain the interface between the systemic circulation and soft tissues and mediate critical processes such as inflammation in a vascular bed-selective fashion. To expand our understanding of the genetic pathways that underlie these specific functions, we have focused on the identification of novel genes that are differentially expressed in all endothelial cells, as well as restricted groups of this cell type. Virtual subtraction was conducted employing gene expression data deposited in public databases and 384 genes identified. These genes were spotted on custom microarrays, along with 288 genes identified through subtraction cloning from TGF-beta-stimulated endothelial cells. Arrays were evaluated with RNA samples representing endothelial cells cultured from four vascular sources and five non-endothelial cell types. These studies identified 64 pan-endothelial markers that were differentially expressed with at least a threefold difference (range 3- to 55-fold). In addition, differences in gene expression profiles among endothelial cells from different vascular beds were identified. Validation of these findings was performed by RNA blot expression studies, and a number of the novel genes were shown to be expressed under angiogenic conditions in the developing mouse embryo. The combined tools of database mining and transcriptional profiling thus provide expanded knowledge of endothelial cell gene expression and endothelial cell biology.