Screening of single nucleotide polymorphism in matrix metalloproteinase-2 (MMP2) and tetraspanin CD63 genes in Chlamydia trachomatis-infected tubal ectopic pregnancy patients.

OBJECTIVE: Tubal ectopic pregnancy (EP) is a leading cause of maternal morbidity and mortality. Studies have suggested that infection-induced inflammatory responses are major risk factors for EP. The aim of the present study was to find an association between MMP2 and CD63 gene variants and risk of EP during Chlamydia trachomatis infection in an Indian population. METHODS: Fallopian tube samples of 120 EP and 120 tubal ligation women were collected. C. trachomatis was detected by PCR. The genotyping of MMP2 (rs17859882 G/T, rs7201A/C) and CD63(rs2231464 C/T, rs376086542 A/G) gene variants was done by qualitative real-time PCR using allelic discrimination method (VIC- and FAM-labeled). RESULTS: The frequency of GG or GT genotype of MMP2 G/T polymorphism (rs17859882) was 66.6% in infected EP and 36.7% in uninfected EP and 22% in tubal ligation controls (P < 0.0001), while the frequency of AC or CC genotype of MMP2 A/C polymorphism (rs7201) was 66.6% in infected EP and 20.6% in uninfected EP and 13.5% in tubal ligation controls (P < 0.0001). The frequency of CT or TT genotype of CD63 C/T polymorphism (rs2231464) was 74% in infected EP and 21.8% in uninfected EP and 11.8% tubal ligation controls (P < 0.0001), while the frequency of AG or GG genotype of CD63 A/G polymorphism (rs376086542) was 48.1% in infected EP and 41.3% in uninfected EP and 18.6% tubal ligation controls (P < 0.0001). CONCLUSIONS: The present study revealed a strong association between the presence of gene variants MMP2 (rs17859882 G/T, rs7201A/C) and CD63 (rs2231464 C/T, rs376086542 A/G) and risk of tubal EP during C. trachomatis infection.

[High expression of long noncoding RNA UCA1 promotes invasion, migration and epithelial-mesenchymal transition of trophoblasts in vitro].

OBJECTIVE: To investigate the role of urothelial carcinoma antigen 1 (UCA1) in regulation of invasion, migration and epithelial-mesenchymal transition (EMT) of trophoblast HTR-8/SVneo cells and its association with tubal pregnancy. METHODS: Cultured HTR- 8/SVneo cells stimulated with interleukin-6 (IL-6) were examined for changes in UCA1 expression and cell migration ability using qRT-PCR and scratch assay, respectively. A HTR-8/SVneo cell model with UCA1 silencing was constructed by transient transfection, and the migration and invasion abilities of the cells were assessed using Scratch assay and Transwell assay; qRT-PCR and Western blotting were performed to detect the mRNA and protein expression levels of EMT markers. RESULTS: HTR-8/SVneo cells stimulated with IL-6 exhibited significantly increased migration ability and up-regulated expression of UCA1 (P < 0.01). UCA1 silencing obviously suppressed migration and invasion abilities of HTR-8/SVneo cells (P < 0.01), significantly up-regulated the mRNA and protein expressions of EMT epithelial marker E-cadherin (P < 0.01), and down-regulated the expressions of the mesenchymal markers integrin ß3, vimentin and N-cadherin (P < 0.05). CONCLUSION: UCA1 may be a key gene that promotes the occurrence of tubal pregnancy and thus provides a new therapeutic target for tubal pregnancy.

Decreased Toll-like receptor-2 messenger ribonucleic acid and increased Toll-like receptor-4 in the tubal epithelium next to the infiltrated trophoblasts during tubal pregnancy.

OBJECTIVE: To explore the expression patterns of Toll-like receptor (TLR)2 and TLR4 in the tubal epithelial cells next to the infiltrated trophoblasts at the maternal-fetal interface during tubal pregnancy. DESIGN: Prospective, observational study. SETTING: University-based obstetrics and gynecology hospital. PATIENT(S): Thirty-seven women undergoing salpingectomy for tubal ampullary pregnancy and nine nonpregnant patients with benign uterine or appendix disease. INTERVENTION(S): Oviduct tissues with ectopic gestations were separated into implantation site (group 1) and nonimplantation site (group 2). Tissues from ampullary fallopian tubes during mid-secretory phase (group 3) were collected as the control group. Immunohistochemistry and quantitative real-time polymerase chain reaction were performed. MAIN OUTCOME MEASURE(S): Differences of TLR2 and TLR4 expression patterns between group 1 and group 2 and between the pregnant group (combined group 1 and group 2) and the nonpregnant group (group 3). RESULT(S): Comparing the pregnant group with group 3, TLR4 messenger RNA (mRNA) and protein were both significantly up-regulated in the pregnant group. In contrast, TLR2 mRNA was significantly down-regulated, whereas TLR2 protein showed a tendency toward reduction. Detailed analysis between group 1 and group 3 revealed statistically significantly higher TLR2 and TLR4 protein in group 1. In terms of mRNA, TLR4 expression was still shown to be significantly increased in group 1, whereas TLR2 expression was markedly decreased in group 1. CONCLUSION(S): Decreased TLR2 mRNA and increased TLR4 in the tubal epithelial cells next to the infiltrated trophoblasts may be associated with aspects of the pathophysiology of tubal ectopic pregnancy in immune defense.

L-selectin ligands expression in human fallopian tube epithelia of tubal pregnancies.

The objective of the study was to evaluate the expression of L-selectin ligands in tubal epithelia during tubal ectopic pregnancy. Sixteen fallopian tube samples from ectopic pregnancies and four normal control fallopian tubes from women undergoing sterilization were obtained for the study. Oviduct tissues from ectopic pregnancies were separated into implantation sites and matched nonimplantation sites. Expression of L-selectin ligands was evaluated by immunohistochemistry with antibodies against HECA-452 and MECA-79 and by real-time PCR. Immunoreactivity levels against HECA-452 and MECA-79 were significantly higher at the implantation site than at the paired nonimplantation site or at the normal oviducts. Moreover, compared with MECA-79 staining, stronger HECA-452 staining was observed in the implantation and nonimplantation groups. HECA-452 histologic scores at implantation sites correlated with serum human chorionic gonadotropin levels. Increased expression of L-selectin ligands may be involved in the implantation process in tubal pregnancy.

The onset of human ectopic pregnancy demonstrates a differential expression of miRNAs and their cognate targets in the Fallopian tube.

Human ectopic pregnancy (EP) is a leading cause of pregnancy-related death, but the molecular basis underlying the onset of tubal EP is largely unknown. Female Dicer1 conditional knockout mice are infertile with dysfunctional Fallopian tube and have a different miRNA expression profile compared to wild-type mice, and we speculated that Dicer-mediated regulation of miRNA expression and specific miRNA-controlled targets might contribute to the onset of tubal EP. In the present study, we used microarray analysis and quantitative RT-PCR to examine the expression of miRNAs and core miRNA regulatory components in Fallopian tube tissues from women with EP. We found that the levels of DICER1, four miRNAs (let-7i, miR-149, miR-182, and miR-424), and estrogen receptor α distinguished the tubal implantation site from the non-implantation site. Computational algorithms and screening for interactions with the estrogen and progesterone receptor signaling pathways showed that the four miRNAs were predicted to target ten genes, including NEDD4, TAF15, and SPEN. Subsequent experiments showed differences in NEDD4 mRNA and protein levels between the implantation and non-implantation sites. Finally, we revealed that increases in smooth muscle cell NEDD4 and stromal cell TAF15, in parallel with a decrease in epithelial cell SPEN, were associated with tubal implantation. Our study suggests that changes in miRNA levels by the DICER-mediated miRNA-processing machinery result in aberrant expression of cell type-specific proteins that are potentially involved in the onset of tubal EP.

DNA sequencing identifies mutation of the von Hippel-Lindau gene in tubal pregnancy.

Genetic mutations in the von Hippel-Lindau (VHL) gene are common in certain diseases. The effects on the VHL gene in the tubal pregnancy tissues are unknown but with further study, it was found that the VHL gene may be related to prognosis or therapy selection. This study was conducted to analyse the VHL gene in tissues of human fallopian tube and tubal pregnancy. A total of 35 patients undergoing salpingectomy for tubal pregnancy were recruited into the experimental group. Samples of ampullary fallopian tube during mid-secretory phase were collected from 10 patients with benign uterine disease as the control group. Fluorescent dye dideoxy termination method was performed to detect three exons sequences of the VHL gene in tissues of both the human fallopian tube and tubal pregnancy. The DNA sequences of three exons of VHL gene coding region in tubal pregnancy were not found in mutations. The present study suggested that the VHL gene mutations were not related with tubal pregnancy.

Expressions of candidate molecules in the human fallopian tube and chorionic villi of tubal pregnancy exposed to levonorgestrel emergency contraception.

BACKGROUND: Cases of ectopic pregnancy (EP) following levonorgestrel (LNG) emergency contraception (EC) failure were reported, however, the effects of LNG on tubal microenvironment or chorionic villi in EP have not yet been documented. METHODS: Fifty-five women with tubal pregnancy were divided into two groups according to whether LNG-EC was administrated during the cycle of conception. The serum concentrations of beta-hCG, E2 and P were measured. The mRNA and protein expressions of estrogen and progesterone receptors, leukemia inhibitory factor, vascular endothelial growth factor, inducible nitric oxide synthase, and endocannabinoid receptor - CB1 in the ectopic implantation site and chorionic villi were examined. RESULTS: Compared to those unexposed to LNG-EC, women with tubal pregnancy exposed to LNG-EC during the cycle of conception had no statistically significances in the serum concentrations of beta-hCG, E2 P, nor in the pathological types of tubal pregnancy or the expressions of ER-alpha, PR, LIF, VEGF, iNOS and CB1. CONCLUSIONS: The expressions of candidate molecules in the fallopian tube and chorionic villi were not altered by exposure to LNG-EC. A routine therapy with no additional intervention might thus be applied to tubal pregnancy exposed to LNG-EC.

Increased placental expression and maternal serum levels of apoptosis-inducing TRAIL in recurrent miscarriage.

INTRODUCTION: Recurrent miscarriage (RM; ≥3 consecutive pregnancy losses) occurs in 1-3% of fertile couples. No biomarkers with high predictive value of threatening miscarriage have been identified. We aimed to profile whole-genome differential gene expression in RM placental tissue, and to determine the protein levels of identified loci in maternal sera in early pregnancy. METHODS: GeneChips (Affymetrix(®)) were used for discovery and Taqman RT-qPCR assays for replication of mRNA expression in placentas from RM cases (n = 13) compared to uncomplicated pregnancies matched for gestational age (n = 23). Concentrations of soluble TRAIL (sTRAIL) and calprotectin in maternal serum in normal first trimester (n = 35) and failed pregnancies (early miscarriage, n = 18, late miscarriage, n = 4; tubal pregnancy, n = 11) were determined using ELISA. RESULTS: In RM placentas 30 differentially expressed (with nominal P-value < 0.05) transcripts were identified. Significantly increased placental mRNA expression of TNF-related apoptosis-inducing ligand (TRAIL; P = 1.4 × 10(-3); fold-change 1.68) and S100A8 (P = 7.9 × 10(-4); fold-change 2.56) encoding for inflammatory marker calprotectin (S100A8/A9) was confirmed by RT-qPCR. When compared to normal first trimester pregnancy (sTRAIL 16.1 ± 1.6 pg/ml), significantly higher maternal serum concentration of sTRAIL was detected at the RM event (33.6 ± 4.3 pg/ml, P = 0.00027), and in pregnant women, who developed an unpredicted miscarriage 2-50 days after prospective serum sampling (28.5 ± 4.4 pg/ml, P = 0.039). Women with tubal pregnancy also exhibited elevated sTRAIL (30.5 ± 3.9 pg/ml, P = 0.035). Maternal serum levels of calprotectin were neither diagnostic nor prognostic to early pregnancy failures (P > 0.05). CONCLUSIONS: The study indicated of sTRAIL as a potential predictive biomarker in maternal serum for early pregnancy complications.

Reduced expression of aquaporin 9 in tubal ectopic pregnancy.

Previous studies demonstrate significant roles for passive water channels (aquaporins, AQPs) in maintaining water homeostasis in cell membranes of endometrial cells during decidualisation and embryo implantation. However, there is little information regarding the role of AQPs in the human fallopian tube, specifically their role in human tubal ectopic pregnancy. In this study we took tissue samples from the site of implantation of tubal ectopic pregnancy (group 1, N = 30, mean age 32 years, range 23-42) and the corresponding non-implantation site in women undergoing salpingectomy for tubal pregnancy (group 2). Ampullary fallopian tubes during mid-secretory phase were collected as control group (group 3, N = 17, mean age 37 years, range 30-50). Thin sections were prepared and stained with anti-AQP9, and, for estrogen and progesterone receptors in each group. Immunohistochemical studies showed that AQP9 proteins localize in the cytoplasm of epithelial cells of Fallopian tube. Expression of AQP9 was significantly reduced during tubal pregnancy compared to controls (group 1 vs. group 3, P = 0.036; group 2 vs. group 3, P = 0.029), and, this reduced expression was not related to estrogen receptor or progesterone receptor status (group 2, ER vs. AQP9, Pearson r = 0.173, P = 0.361; PR vs. AQP9, Pearson r = 0.124, P = 0.514, respectively). Similarly, there is no correlation between AQP9 and estrogen receptor or progesterone receptor status in the normal group (group 3, ER vs. AQP9, Pearson r = -0.026, P = 0.923; PR vs. AQP9, Pearson r = -0.292, P = 0.255, respectively). Reduced expression of AQP9 in human fallopian tube may contribute to aspects of pathophysiology of tubal ectopic pregnancy.

Endometrial inhibin/activin beta-B subunit expression is related to decidualization and is reduced in tubal ectopic pregnancy.

CONTEXT: Ectopic pregnancy is common but remains difficult to diagnose accurately. There is no serum test to differentiate ectopic from intrauterine gestation. OBJECTIVE: Our objective was to investigate differential gene expression in decidualized endometrium of ectopic pregnancy. DESIGN: Tissue and serum analysis informed by microarray study was performed. SETTING: The study was performed at a large United Kingdom teaching hospital. PATIENTS OR OTHER PARTICIPANTS: Women undergoing surgical termination of pregnancy (n = 8), evacuation of uterus for miscarriage (n = 6), and surgery for tubal ectopic pregnancy (n = 11) were included in the study. Endometrium was collected from normally cycling women undergoing hysterectomy. INTERVENTIONS: Decidualized endometrium was subjected to microarray analysis, morphological assessment, and immunohistochemistry. Endometrial stromal fibroblasts were cultured in the presence of decidualizing stimuli. MAIN OUTCOME MEASURES: Differential expression of potentially secreted molecules was calculated. RESULTS: Inhibin/activin beta-B expression was lower in decidualized endometrium from ectopic pregnancies when compared with that of ongoing pregnancies (P < 0.01) or miscarriages (P < 0.01). The localization of the beta-B subunit was more marked in decidualized than nondecidualized stroma. Decidualization of stromal fibroblasts in vitro was associated with increased beta-B expression (P < 0.05). Endometrial stroma of ectopic pregnancies was less decidualized morphologically (P < 0.05), with lower prolactin (P < 0.01) and IGF binding protein-1 (P < 0.005) expression. Serum activin B was lower in ectopic pregnancies (P < 0.005) than in intrauterine pregnancies, whereas there was no difference in progesterone concentrations. CONCLUSIONS: Despite similar concentrations of progesterone, the endometrium of ectopic pregnancies is less decidualized than intrauterine pregnancies. Expression of the beta-B subunit is related to decidualization and can be detected in the circulation as activin B. Serum activin B concentrations are lower in ectopic pregnancy.

Interleukin-11 expression: its significance in eutopic and ectopic human implantation.

Embryo implantation and subsequent decidualization, trophoblast invasion and formation of a functional placenta are crucial for establishment and maintenance of pregnancy. Interleukin-11 signalling has been shown to be obligatory for adequate decidualization and trophoblast invasion in mice. Defects in IL-11 signalling in mice result in trophoblast over-invasion and fetal loss. The pathological situation of human tubal pregnancy resembles that of IL-11Ralpha(-/-) mice concerning these symptoms. As our interest is focused on the human early pregnancy, we compared IL-11 expression at the implantation site of ectopic tubal pregnancy (EP) to 1st and 2nd trimester of normal intrauterine pregnancies (IP), and to the normal cycling endometrium. The mRNA expression of IL-11 and IL-11Ralpha was analysed by semiquantitative RT-PCR. Protein expression was detected by western blotting and immunohistochemistry. IL-11Ralpha is expressed constitutively in all tissue specimens analysed. IL-11 is expressed predominantly during follicular and early luteal phase of the menstrual cycle. In IP, IL-11 expression peaks during the 1st trimester and declines from the beginning of the 2nd trimester onwards. In tubal abortions, IL-11 expression is reduced in comparison to vital EP and IP. Cultured primary endometrial and decidual epithelial cells were analysed for hormonal regulation of IL-11 by enzyme-linked immunosorbent assay and RT-PCR. IL-11 is up-regulated by estrogen and down-regulated by progesterone. Overall, our results indicate that in humans, IL-11 signalling is significantly involved in regulation of trophoblast invasion. In the case of tubal abortion, inadequate IL-11 signalling may therefore result in dysregulation of trophoblast invasion.

HOXA10 gene expression in human fallopian tube and ectopic pregnancy.

OBJECTIVE: The molecular mechanisms underlying ectopic implantation have not been well characterized. Here we investigate HOXA10 gene expression at the site of ectopic implantation as compared with the endometrium and with the normal fallopian tube. STUDY DESIGN: Northern blot analysis was used to evaluate HOXA10 gene messenger RNA level in various segments of normal pregnant and nonpregnant human fallopian tube, ectopic pregnancy, and endometrium. RESULTS: Normal human fallopian tube expressed minimal levels of HOXA10 gene messenger RNA in the nonpregnant state. A trend toward a greater expression of HOXA10 gene was observed in the normal fallopian tube during pregnancy, but the difference was not statistically significant (P =.075). HOXA10 gene messenger RNA expression was up-regulated significantly at the site of implantation in ectopic pregnancy (P <.001), and its expression approached that of the endometrium during normal pregnancy (P =.33). CONCLUSION: HOXA10 gene expression is up-regulated at the ectopic implantation site in the fallopian tube, approaching that of the endometrium in normal intrauterine gestation. Inherently increased HOXA10 gene expression in the fallopian tube or dysregulation of HOXA10 gene expression by an abnormally implanting blastocyst may play a role in ectopic implantation.

Expression of beta hCG and alpha CG mRNA and hCG hormone in human decidual tissue in patients during tubal pregnancy.

We recently showed that endometrial tissue produces hCG during the secretory phase of the menstrual cycle. Based on these findings, we hypothesized that the decidua should also be able to secrete hCG. We examined the decidualized endometrium of patients with extrauterine pregnancies. Decidual specimens were obtained for mRNA extraction and paraffin embedding from 24 patients that were between weeks 6-11 of tubal pregnancy. Tissues were evaluated and classified into one of three groups based on the endometrial differentiation that took place prior to conception: (A) high secretory transformation, (B) diminished transformation with restricted decidualization and (C) inferior endometrial proliferation. Decidual gland hCG secretion was demonstrated immunohistochemically and by Western blotting. Serum hCG levels were higher (P < 0.0001) in patients from group A than group C. mRNA expression of both the beta subunit (beta-hCG) and alpha subunit (alpha-CG) was determined by RT-PCR. Furthermore, the specificity of beta-hCG amplification was confirmed by restriction enzymes. beta-LH amplification was not found. Moreover, the degree of endometrial transformation and the level of decidualization was found to correlate with hCG hormone staining and beta-hCG mRNA expression. hCG protein in the decidua was present in the glands of the compact layer and in the spongy layer, and was more pronounced in previously transformed high secretory endometrium than in inferior endometrium. In conclusion, this study provides the first evidence that hCG is produced in the decidua of patients during extrauterine pregnancies and might play a possible paracrine role.

Local fetal signal is not required for maintaining IGFBP gene expression in the human decidua: evidence from extrauterine pregnancies.

Insulin-like growth factor-II (IGF-II) from the invading extravillous cytotrophoblasts (EVTs) and insulin-like growth factor binding proteins (IGFBPs) from the maternal decidua interact at the feto-maternal interface and regulate implantation and placentation. To determine whether a local stimulus from the fetus is important in the regulation of IGFBP gene expression in the human decidua, we compared the expression of IGFBP genes in intra- and extrauterine (tubal) pregnancies. The expression of IGF-II and IGFBP-1 to IGFBP-6 mRNAs was determined by in-situ hybridization in the Fallopian tubes of extrauterine pregnancies and concurrent decidua (n = 6), and in the placentae and Fallopian tubes of intrauterine pregnancies (n = 6). All six IGFBP mRNAs were identified in the decidualized endometrium and decidualized Fallopian tubes of intra- and extrauterine pregnancies, with IGFBP-1 mRNA being the predominant mRNA. IGFBP-4 was the second most predominant mRNA and was slightly more abundant in the decidua of extrauterine pregnancies than of intrauterine pregnancies. IGF-II mRNA was expressed mainly in cells of fetal origin. The fact that the IGFBP mRNAs were expressed similarly in both intra- and extrauterine pregnancies indicates that the local physical stimulus from an implanting fetus is not necessary to induce or maintain decidual IGFBP gene expression.

Triploid abortus presenting as an ectopic pregnancy.

In this article we present a case of an ectopic gestation having morphologic features of a partial hydatidiform mole and demonstrating triploidy by flow cytometry in a patient presenting at 9 weeks' gestation. We include brief comments on partial hydatidiform mole.