published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

The novel emergency contraceptive Ulipristal acetate (UPA) belongs to the progesterone receptor modulator family. A single oral dosage of 30mg UPA within 120 hours of unprotected intercourse could delay ovulation and differentiation of endometrium. Yet, whether UPA affect embryo implantation remains largely unknown. This study aims to investigate whether UPA affect endometrial gene expressions and embryo attachment onto endometrial epithelial cells. The PR-expressing human endometrial carcinoma cell line Ishikawa was used and treated with 10nM estrogen, 1μM progesterone or 4μM UPA for 24 hours. Changes in transcriptome profiles were analyzed by Affymetrix Human Gene 1.0 ST array GeneChip. Gene clustering showed the gene expression pattern after UPA treatment was similar to control (0.1% ethanol); while estrogen treated group was different from all the other groups. Totally, 8 genes were significantly increased and 1 was decreased (≥2-fold, p<0.05) after UPA treatment. All except one of the 8 up-regulated genes were also up-regulated by estrogen; while only one of them increased after progesterone treatment. Most genes that were altered by UPA were involved in angiogenesis and vascular remodeling. The effect of UPA on human embryo-endometrium attachment was carried out using an in vitro multi-cellular spheroids-endometrial epithelial cell co-culture model. Human choriocarcinoma cell line JAR and Ishikawa were used. UPA (0.04-4μM) treatment for up to 48 hours did not affect the proliferation of JAR or Ishikawa cells. Similarly, the attachment of JAR spheroids onto Ishikawa cells after 1 hour co-culture was not affected by UPA treatment. The molecules of Wnt/β-catenin signaling pathway, a pathway that is actively involved in embryo implantation, such as the β-catenin and GSK-3β, and endometrial receptive marker E-cadherin were not changed after UPA treatment. In Ishikawa cells, the expression of PR-A was induced after UPA (0.04-4μM) treatment; while PR-B increased when 0.04 or 4μM UPA used. However, the PR-A/PR-B ratio remained unchanged after all concentration of UPA treatment. The effect of UPA on spheroids attachment was further investigated with cultured human primary endometrial epithelial cells. Endometrial glandular epithelial cells were digested and isolated from endometrial biopsy taken from IVF patients on day 7 after luteinizing hormone surge (LH+7). A co-culture assay was optimized with JAR spheroids and endometrial epithelial cells that were growing on Matrigel. The attachment rate of JAR spheroids is approximately 60% after 3 hours incubation. However, after 24 hours of exposure to 4μM UPA, the attachment remained comparable to that of the control group. In conclusion, UPA could alter the expression of genes in Ishikawa cells mainly related to angiogenesis. It is likely that UPA may affect stromal decidualization and blastocyst invasion after attachment. However, UPA did not affect the expression of Wnt-signaling molecules and attachment of JAR spheroids onto either Ishikawa or human primary endometrial epithelial cell. / published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

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Our laboratory has previously isolated a 12-O-tetradecanoylphorbol-13-acetate (TPA)-nonresponsive variant line (VT-1) from mouse 3T3-L1 cells. Here the expression of type I collagen pro-α2 (pro-α2(I)) mRNA and production of type I collagen in these two cell lines is examined. In quiescent cells, the pro-α2(I) steady state mRNA levels were 4 times greater in 3T3-L1 cells than in VT-1 cells. TPA addition caused the steady-state levels of pro-α2(I) mRNA to be 6 times greater in 3T3-L1 cells than in VT-1 cells. The pro-α2(I) protein levels in the extracellular matrix or culture media of 3T3-L1 cells were substantially elevated by TPA treatment but no significant increase was detected in VT-1 cells. The correlation of collagen expression with a TPA-mediated mitogenic response suggests a new role for collagen as an early component of mitogenic signal transduction. Expression of protooncogenes and other mitogenically related genes were also examined in 3T3-L1 and VT-1 cells. c-fos, c-myc, c-jun and ornithine decarboxylase were expressed in both cell lines. These findings suggest that expression of these genes may be necessary but not sufficient for a TPA induced mitogenic response. Two of the TPA Inducible Early (TIE) genes selected had no homology with any other known sequence. Here, mRNA expression levels of these novel genes (TIE-10B and TIE-44) are examined in 3T3-L1 and VT-1 cells and in several mouse tissues. Within 30 minutes of TPA treatment, TIE gene mRNA is increased in 3T3-L1 cells; whereas, with the exception of TIE-10B, low levels or delayed mRNA expression is seen in VT-1 cells. The two genes are expressed in most mouse tissues tested. mRNA levels of both TIE genes also increased in response to serum supplemented medium. Typical mRNA expression patterns for protooncogenes c-fos, c-myc, c-jun are seen when cells are treated with TPA or serum-supplemented media. The differential mRNA expression of the TIE genes relative to other growth potentiating genes and in response to mitogens other than TPA is indicative of the complexity and wide range of genes involved in several possible pathways leading to the cells' mitogenic response. (Abstract shortened with permission of author.)

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