Infertility is constantly increasing in Canada, where 16% of Canadian couples are experiencing difficulty conceiving. It is thought that infertility can emanate from the dysregulated communication between the embryo and the maternal endometrium. In order to allow for this window of implantation to be open at the right moment, endometrial stromal cells proliferate and differentiate by a mechanism called decidualization. Intracellular and molecular mechanisms involved in the regulation of apoptosis and cell proliferation during decidualization of the endometrium are yet to be fully understood. It has been well demonstrated previously that Akt is importantly involved in cell survival and glycogen synthesis. Akt1, Akt2 and Akt3 isoforms have distinct physiological roles; this could also be the case during decidualization and pregnancy. The aim of this study is to investigate the regulation of PI3K/Akt pathway during the decidualization process of endometrial stromal cells. Expression of Akt isoforms, Akt activity (phospho-Akt), pIκB and substrates of Akt during decidualization were measured. To our knowledge, these results are the first to suggest a decrease in levels of Akt isoforms as well as a downregulation of Akt activity in the process of decidualization of human endometrial stromal cells. We also uncovered that decidualization induced nuclear localization of p65 through the phosphorylation of IκB, its inhibitory subunit; however, Par-4, a recently uncovered regulator of cell differentiation, was displaced from the nucleus upon decidualization. Our results also suggest that HIESC cells exhibit decreased motility during decidualization and that PI3K pathway inhibition could be involved in this process. Finally, we demonstrate that specific Akt isoforms present unique effects on the successful induction of decidualization. Further analyses will involve investigations to understand the precise signaling mechanisms by which this pathway is regulated.

We have developed a rapid and accurate method to enumerate the number of CD34+ cells in peripheral blood, bone marrow, and leukopheresis samples. The method consists of a two-tube assay and a dedicated software program for data acquisition and analysis. The first reagent combination consists of (a) a nucleic acid dye to identify nucleated cells, (b) a CD45 monoclonal antibody labeled with PE/CY5 to discriminate progenitor cells from mature lymphoid, neutrophil, erythroid, and monocytic cells, (c) an IgG1 control antibody labeled with PE to establish the boundary between specific and nonspecific staining, and (d) a known number of fluorescent beads to determine an absolute count of cells. In the second reagent combination the IgG1 control antibody is replaced by a CD34 antibody labeled with PE that is used to identify the CD34+ cells in the location established by the control reagent combination. The software program uses the fluorescent beads to adjust the forward light scatter, orthogonal light scatter, and three fluorescence detectors of the flow cytometer. The expected location of the CD34+ cells is then established with the control reagent combination followed by the enumeration of the CD34+ cells per microliter of sample with the reagent combination containing the CD34 antibody. This method is sensitive enough to detect CD34+ cells in peripheral blood of normal donors and can reliably determine an increase in CD34+ cells in the peripheral blood of patients treated with chemotherapy and/or growth factors. The method alleviates some of the difficulties encountered when small numbers of CD34+ cells are enumerated. The system allows for more precise evaluations of the grafts used for bone marrow transplantation.

The occurrence of apoptosis and cell survival in the receptive uterus is intimately involved in the embryo implantation process in order to facilitate embryo attachment to the maternal endometrium. The initial stimulus leading to successful implantation might be triggered by the conceptus itself. By the end of rat embryo implantation, decidualization begins, followed by the regression of the decidua basalis on Day 14. The phosphatidylinositol 3-kinase (PI3-K) survival pathway and TGF-beta have been thought to play a role in this process. The objective of the present study was to investigate the regulation of the PI3-K/PTEN/Akt pathway in rat endometrium during pregnancy. Rats were killed on different days of pregnancy (Day 1-22 and postpartum) or pseudopregnancy (Day 1-9), and uteri were removed to collect endometrial tissues. The active form of Akt (pAkt) was increased at Day 5 of pregnancy and at Day 3 of pseudopregnancy as well as at Day 12 of pregnancy and at Day 1 postpartum. Of the three Akt isoforms (Akt1, Akt2, and Akt3), Akt3 was the only isoform phosphorylated at Day 5 during the implantation process and at postpartum as demonstrated by immunoprecipitation studies. PI3-K inhibition in vivo blocked Akt phosphorylation, reduced Smad2 phosphorylation, and reduced both TGF-beta2 and XIAP expression. PI3-K inhibition in cultured decidual cells led to inhibition of pAkt and decrease XIAP expression. These results suggest that Akt and XIAP may be important surviving signaling molecules by which apoptosis is regulated in the rat endometrium during pregnancy and that TGF-beta could be linked to this process.

Numerous morphologic features have been described in bone marrow and peripheral blood in myelodys-plastic syndrome (MDS). We draw attention to a high incidence of a subtle morphologic feature, internuclear bridging (INB), not previously recognized as a morphologic feature in MDS. The occurrence of INB in MDS suggests an underlying abnormality of mitotic division that could explain the impaired production of hematopoietic cells, the addition and deletion cytogenetic changes, and the stepwise disease progression and cytogenetic progression characteristic of MDS. Lack of awareness that INB occurs in MDS may cause confusion of MDS and congenital dyserythropoietic anemia type I, a congenital process also characterized by INB. Cancer 64:2199-2202, 1989. NTERNUCLEAR BRIDGING (INB) in erythroid precursors

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