Sarah E. McDonald

CB1 Expression Is Attenuated in Fallopian Tube and Decidua of Women with Ectopic Pregnancy

Background Embryo retention in the Fallopian tube (FT) is thought to lead to ectopic pregnancy (EP), a considerable cause of morbidity. In mice, genetic/pharmacological silencing of cannabinoid receptor Cnr1, encoding CB1, causes retention of embryos in the oviduct. The role of the endocannabinoids in tubal implantation in humans is not known. Methods and Findings Timed FT biopsies (n = 18) were collected from women undergoing gynecological procedures for benign conditions. Endometrial biopsies and whole blood were collected from women undergoing surgery for EP (n = 11); management of miscarriage (n = 6), and termination of pregnancy (n = 8). Using RT-PCR and immunohistochemistry, CB1 mRNA and protein expression levels/patterns were examined in FT and endometrial biopsies. The distribution of two polymorphisms of CNR1 was examined by TaqMan analysis of genomic DNA from the whole blood samples. In normal FT, CB1 mRNA was higher in luteal compared to follicular-phase (p<0.05). CB1 protein was located in smooth muscle of the wall and of endothelial vessels, and luminal epithelium of FT. In FT from women with EP, CB1 mRNA expression was low. CB1 mRNA expression was also significantly lower (p<0.05) in endometrium of women with EP compared to intrauterine pregnancies (IUP). Although of 1359G/A (rs1049353) polymorphisms of CNR1 gene suggests differential distribution of genotypes between the small, available cohorts of women with EP and those with IUP, results were not statistically significant. Conclusions CB1 mRNA shows temporal variation in expression in human FT, likely regulated by progesterone. CB1 mRNA is expressed in low levels in both the FT and endometrium of women with EP. We propose that aberrant endocannabinoid-signaling in human FT leads to EP. Furthermore, our finding of reduced mRNA expression along with a possible association between polymorphism genotypes of the CNR1 gene and EP, suggests a possible genetic predisposition to EP that warrants replication in a larger sample pool.

Attenuated sex steroid receptor expression in fallopian tube of women with ectopic pregnancy.

CONTEXT Sex steroid hormone receptor (SHR) dynamics are well-documented in human endometrium but have not been comprehensively studied in Fallopian tube (FT). OBJECTIVE The aim of the study was to compare expression patterns and hormonal regulation of SHR in FT with that described in endometrium and to determine whether SHR expression is altered in FT of women with ectopic pregnancy (EP). DESIGN Tissue was analyzed and cultured. PATIENTS OR OTHER PARTICIPANTS Women undergoing surgery for benign gynecological conditions (n = 14) and EP (n = 6) participated in the study. INTERVENTIONS Quantitative RT-PCR and immunohistochemistry were used to determine SHR mRNA expression and protein localization, respectively. SHR levels were measured in tubal explant cultures stimulated with estrogen and progestogen. RESULTS ERalpha and ERbeta mRNAs were constitutively expressed in FT during the menstrual cycle. PR-AB and PR-B mRNAs were decreased in midluteal phase compared to follicular phase. ERalpha, PR-AB, and PR-B mRNAs were down-regulated in human FT in vitro by treatment with progestogen. ERalpha, ERbeta1, ERbeta2, PR, and AR proteins localized to cell nuclei of epithelium, stroma, and smooth muscle of nonpregnant FT. In FT from women with EP, PR-B mRNA was decreased when compared to midluteal FT, and ERalpha protein was not detected. CONCLUSIONS SHR expression in FT is different from that observed in endometrium recovered at similar stages of the menstrual cycle, and expression in FT from women with EP is also altered compared with normal FT. These data are an important benchmark for furthering the understanding of normal human FT physiology, changes in expression of SHR in FT in response to progesterone, and disorders of FT function, such as EP.

11β-Hydroxysteroid dehydrogenases in human endometrium

Abstract Key reproductive events, such as menstruation and implantation, are considered to be inflammatory processes and glucocorticoids act as anti-inflammatory agents. The balance of expression of types 1 and 2 11β-hydroxysteroid dehydrogenases (11βHSD) controls the availability of cortisol to bind to the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). Expression profiles of glucocorticoid-metabolising enzymes and their cognate receptors have been characterized in the reproductive tract. We propose that factors that peripherally promote glucocorticoid action are part of an anti-inflammatory response to tissue remodelling in human endometrium. Protein and mRNA expression in endometrium were investigated using immunohistochemistry and quantitative real-time PCR. There was up-regulated expression of 11βHSD-1 at menstruation and in first trimester decidua. 11βHSD-2 and GR were expressed across the cycle. The MR expression pattern across the cycle and in decidua implies progesterone may also play a regulatory role. The precise roles and interactions of these proteins require further investigation.

Placental growth factor: a promising diagnostic biomarker for tubal ectopic pregnancy

CONTEXT Tubal ectopic pregnancy is common, but accurate diagnosis is difficult and costly. There is currently no serum test to differentiate tubal from intrauterine implantation, and an effective biomarker of ectopic pregnancy would be a major clinical advance. OBJECTIVE A key feature of successful intrauterine implantation is the establishment of a supportive vascular network, and this has been associated with the activity of placental growth factor (PIGF). We hypothesized that the local decidual environment facilitates PIGF-dependent angiogenesis and that this pathway is not active in tubal implantation. We aimed to determine whether tubal implantation is manifest by an attenuation of the normal trophoblast PIGF response and whether serum PIGF levels are different in ectopic compared with intrauterine pregnancy. DESIGN AND SETTING Tissue and serum analysis was done at a large United Kingdom teaching hospital. PATIENTS Tissue and sera were collected from gestation-matched pregnant women undergoing surgical termination of pregnancy (viable intrauterine) (n = 15), evacuation of uterus for embryonic missed miscarriage (nonviable intrauterine) (n = 10) and surgery for tubal ectopic pregnancy (n = 15). INTERVENTIONS Trophoblast was examined by immunohistochemistry and quantitative RT-PCR, and serum was analyzed by ELISA. RESULTS PIGF was localized to the cytotrophoblast cells. Expression of PIGF mRNA was reduced in trophoblast isolated from women with ectopic compared with intrauterine pregnancies (P < 0.05). Serum PIGF was undetectable in women with tubal ectopic pregnancies and reduced, or undetectable, in miscarriage compared with viable intrauterine pregnancies (P < 0.01). CONCLUSIONS Serum PIGF is a promising novel diagnostic biomarker for early pregnancy location and outcome, and large-scale studies are now required to determine its clinical utility.

Expression of secretory leukocyte protease inhibitor and elafin in human fallopian tube and in an in-vitro model of Chlamydia trachomatis infection

BACKGROUND Secretory leukocyte protease inhibitor (SLPI) and elafin are anti-protease and anti-microbial molecules with a role in innate immune defence. They have been demonstrated at multiple mucosal surfaces including those of the female reproductive tract. METHODS AND RESULTS This study details their expression in human Fallopian tubes (ampullary region) throughout the menstrual cycle (n = 18) and from women with ectopic pregnancy (n = 6), and examined their regulation by infection with Chlamydia trachomatis in an in-vitro model. Quantitative real-time PCR analysis showed that SLPI and elafin were constitutively expressed in the Fallopian tube during the menstrual cycle but were increased in ectopic pregnancy (P < 0.05 versus early-mid luteal phase, P < 0.01 versus all phases, respectively). SLPI and elafin were immunolocalised to the Fallopian tube epithelium in biopsies from non-pregnant women and those with ectopic pregnancy. An in-vitro culture model of C. trachomatis infection of the OE-E6/E7 oviductal epithelial cell line showed that elafin mRNA expression was upregulated in response to chlamydial infection. CONCLUSION These data suggest that SLPI and elafin have a role in the innate immune defence of the Fallopian tube in infection and ectopic pregnancy. Their role is likely to include regulation of protease activity, wound healing and tissue remodelling.

Ectopic pregnancy as a model to identify endometrial genes and signaling pathways important in decidualization and regulated by local trophoblast.

The endometrium in early pregnancy undergoes decidualization and functional changes induced by local trophoblast, which are not fully understood. We hypothesized that endometrium from tubal ectopic pregnancy (EP) could be interrogated to identify novel genes and pathways involved in these processes. Gestation-matched endometrium was collected from women with EP (n = 11) and intrauterine pregnancies (IUP) (n = 13). RNA was extracted from the tissue. In addition, tissues were prepared for histological analysis for degree of decidualization. We compared a) the samples from EP that were decidualized (n = 6) with non-decidualized samples (n = 5), and b) the decidualized EP (n = 6) with decidualization-matched IUP (n = 6) samples using an Affymetrix gene array platform, with Ingenuity Pathway Analysis, combined with quantitative RT-PCR. Expression of PRL and IGFBP1 was used to confirm the degree of decidualization in each group. There were no differences in PRL or IGFBP1 expression in the decidualization-matched samples but a marked reduction (P<0.001) in the non-decidualized samples. Decidualization was associated with increased expression of 428 genes including SCARA5 (181-fold), DKK1 (71-fold) and PROK1 (32-fold), and decreased expression of 230 genes including MMP-7 (35-fold) and SFRP4 (21-fold). The top canonical pathways associated with these differentially expressed genes were Natural Killer Cell and Wnt/b-Catenin signaling. Local trophoblast was associated with much less alteration of endometrial gene expression with an increase in 56 genes, including CSH1 (8-fold), and a reduction in 29 genes including CRISP3 (8-fold). The top associated canonical pathway was Antigen Presentation. The study of endometrium from tubal EP may promote novel insights into genes involved in decidualization and those influenced by factors from neighboring trophoblast. This has afforded unique information not highlighted by previous studies and adds to our understanding of the endometrium in early pregnancy.

Attenuated tubal and endometrial urocortin 1 and corticotropin-releasing hormone receptor expression in ectopic pregnancy.

Fallopian tube (FT) and endometrial urocortin 1 (Ucn1) and corticotropin-releasing hormone (CRH)-receptor (CRH-R1/CRH-R2) expression were examined using quantitative real-time polymerase chain reaction (RT-PCR) and immunohistochemistry in nonpregnant and pregnant women (intrauterine, IUP; ectopic pregnancy, EP). Tubal Ucn1 messenger RNA (mRNA) expression was higher in luteal compared to follicular phase (P < .01) and equivalent to follicular phase in FT from EP. Tubal CRH-R1/CRH-R2 mRNA was lower in luteal phase (P < .05) and in FT from EP compared to follicular phase (P < .01). Ucn1 mRNA was lower in endometrium from EP compared to IUP (P < .05). Corticotropin-releasing hormone-R1 mRNA was higher in endometrium from EP compared to viable IUP (P < .05). No differences were observed in CRH-R2 expression. Corticotropin-releasing hormone-R1 protein was primarily localized to epithelium of FT and endometrium. Quantitative analysis of tubal CRH-R1 protein expression reflected that seen at the mRNA level but endometrial expression was equivocal. The demonstration of attenuated tubal/endometrial Ucn1/CRH-R expression in EP further supports a role of the CRH-family in embryo implantation.

Characterization of the Temporal and Spatial Expression of a Disintegrin and Metalloprotease 17 in the Human Endometrium and Fallopian Tube

Metalloproteinases are thought to mediate shedding of mucins from the endometrium surface, exposing oligosaccharide ligands involved in implantation. We hypothesized that a disintegrin and metalloprotease 17 (ADAM17) is upregulated during the “window of implantation” in human endometrium but not in fallopian tube (FT) where implantation is pathological. Endometrial and FT expression of ADAM17 throughout the menstrual cycle was determined using quantitative reverse-transcription polymerase chain reaction and immunohistochemistry. The ADAM17 transcription was significantly downregulated (P < .01) during the early–midsecretory phase in the endometrium but not in the FT. The ADAM17 was localized to the surface of epithelial cells and was also detected in the endometrial stroma during the late luteal and proliferative phase of the cycle. Physiological levels of estradiol significantly (P < .05) upregulated ADAM17 transcription in vitro. Our observations do not support the role of ADAM17 in shedding of mucins during the window of implantation. The precise role of ADAM17 in the female reproductive tract requires further investigation.

Expression of the Natural Anti-Microbial Peptides, Secretory Leukocyte Protease Inhibitor and Elafin, in Human Fallopian Tube and in an In-Vitro Model of Chlamydia trachomatis Infection

Plenary Session: Trainee Plenary (Thursday, 3/19/2009, 9:00 AM 10:00 AM) Scientifi c Abstracts Reproductive Sciences Vol. 16, No. 3 (Supplement), March 2009 69ABackground: Small for Gestational Age (SGA) is defi ned as <10th centile on individualised birthweight centiles. SGA babies are at increased risk of complications in pregnancy and later life such as stillbirth, preterm delivery, cerebral palsy and heart disease and diabetes in adulthood. Metabolomics is the study of global metabolite profi les in a biological system. This technology has previously identifi ed differences in maternal plasma between normal and preeclamptic pregnancies. Aim: To examine the differences in the plasma metabolome in early pregnancy between women who develop a SGA baby and normal matched controls. Methods: 60 primigravid women who delivered a SGA baby were identifi ed within the SCOPE study (www.scopestudy.net) and matched to 60 controls. Venepuncture was performed at 15�1 weeks gestation. Samples were analysed using Ultra Performance Liquid Chromatography(UPLC)/LTQ-Orbitrap Mass Spectrometry (MS). Univariate statistical analysis was performed. Subsequently all the peaks in each class were grouped and analysed in combination by employing Multivariate Discriminant Analysis. Results: Univariate analysis of the UPLC/LTQ-Orbitrap MS data at 15 weeks gestation identifi ed 111 ?information rich? peaks (p < 0.05). The multivariate discriminant model (all 111 peaks), provided an area under the ROC curve of 0.96. Downloaded from rsx.sagepub.com at The John Rylands University Library, The University of Manchester on January 28, 2011Plenary Session: Trainee Plenary (Thursday, 3/19/2009, 9:00 AM 10:00 AM) Scienti c Abstracts Reproductive Sciences Vol. 16, No. 3 (Supplement), March 2009 69A