The Association between Smoking and Ectopic Pregnancy:Why Nicotine Is BAD for Your Fallopian Tube

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Diagram comparing array data in each of the four analytical groups.

<p>In the OE-E6/7 cell line the addition of 40 ng/ml cotinine (‘Low Cell’) altered the expression of 946 genes (increasing 585 genes and reducing the expression of 361 genes). Of the 676 genes (361 increased and 315 reduced) altered by the addition of 400 ng/ml cotinine (‘High Cell’), 135 were shared with the ‘Low Cell’ treatment. In FT explants the addition of 40 ng/ml cotinine (‘Low Tissue’) altered the expression of 596 genes (137 increased and 459 reduced). Explant treatment with 400 ng/ml cotinine (‘High Tissue’) changed 2419 genes (increasing 313 and reducing 2106), 265 of which were shared with the ‘Low Tissue’ treatment. The effect of cotinine in Fallopian tube therefore is mainly to inhibit gene expression. When comparing the effect of cotinine in the OE-E6/7 cells when compared to FT explants common were genes identified. There were 16 altered genes the ‘Low Tissue’ group shared with the ‘Low Cell’ group and 27 genes shared with the ‘High Cell’ group. There were 111 genes that changed expression in common between the ‘High Tissue’ and ‘Low Cell’ group and 54 with the ‘High Cell’ group. There was only one up-regulated gene and one down regulated gene in common to all groups.</p

Assessment of cell death in the FT.

<p>A) Frequency of immunopositive cells in the FT of non-smokers (clear bars: n = 11) and smokers (filled bars: n = 10). Relative expression of of <i>CASP3</i> (B) and <i>CASP9</i> (C) transcripts within the FT of smokers and non smokers and (D) their correlation to each other in a single FT sample. Observed differences are significant at * <i>P</i><0.05. Gene expression was related to a <i>G6PDH</i> internal control.</p

TaqMan RT-PCR analysis of BAD and BCL2 transcript abundance in FT from smokers and non-smokers.

<p>Relative expression of <i>BAD</i> (A) and <i>BCL2</i> (B) in the FT of non-smokers (clear bars: n = 11) and smokers (filled bars: n = 10). Observed differences are significant at * <i>P</i><0.05, **<i>P</i><0.01. Gene expression was related to a <i>G6PDH</i> internal control.</p

Immunohistochemistry.

<p>A) BAD (brown) is expressed in the epithelium (E), most prominently towards the lumen (L), of the FT and not in the stroma (S). B) BCL2 (brown) immunolabelling showing predominant epithelial staining with occasional cell staining in the stroma. C) Higher power BCL2 immunolabelling showing no staining in the ciliated epithelial cells (black arrow) with staining in interspaced cells with no obvious cilia (red arrow). D) Scanning EM highlighting the two populations of epithelial cells with and without cilia. E) Apoptotic cells (brown) identified by immunolabelling for cleaved caspase 3. The arrow shows an apoptotic cell in the tubal epithelium. F) Higher power view showing a cell stained for cleaved caspase 3 (arrow) at the epithelial and stromal junction. G) Cells stained by the proliferation marker Ki-67 (brown). H) Higher power view showing a cell stained for Ki-67 (arrow) in the tubal epithelium. I) Representative image of a section of FT from a smoker immunostained for BAD showing the apical smooth protuberances or ‘epithelial bledding’ (arrow). J) Section of FT immunostained for BAD with the ciliated epithelial cells with no surface ‘epithelial blebbing’.</p

Assessment of changes in cell proliferation and morphology in the FT of smokers.

<p>A) Frequency of Ki67 positive cells in the FT of non-smokers (clear bars: n = 11) and smokers (filled bars: n = 10). B) Relative expression of Cyclin D1 transcripts within the FT of smokers and non smokers Expression of <i>CCND1</i> in the FT of smokers and non smokers. C) Histoscore analysis of the degree of ‘epithelial blebbing’ in non-smokers compared to smokers. Observed differences are significant at * <i>P</i><0.05. Gene expression was related to a <i>G6PDH</i> internal control.</p