Genome-wide association study meta-analysis supports association between MUC1 and ectopic pregnancy.

STUDY QUESTION: Can we identify genetic variants associated with ectopic pregnancy by undertaking the first genome-wide association study (GWAS) leveraging two large-scale biobank initiatives? SUMMARY ANSWER: We identified two novel genome-wide significant associations with ectopic pregnancy, highlighting MUC1 (mucin 1) as the most plausible affected gene. WHAT IS KNOWN ALREADY: Ectopic pregnancy is an important cause of maternal morbidity and mortality worldwide. Despite being a common early pregnancy complication, the genetic predisposition to this condition remains understudied and no large scale genetic studies have been performed so far. STUDY DESIGN, SIZE, DURATION: A GWAS meta-analysis including 7070 women with ectopic pregnancy and 248 810 controls from Estonian Biobank and the FinnGen study. PARTICIPANTS/MATERIALS, SETTING, METHODS: We identified ectopic pregnancy cases from national registers by ICD (International Classification of Disease) codes (ICD-10 O00), and all remaining women were considered controls. We carried out standard GWAS meta-analysis and additionally annotated GWAS signals, analysed co-localization with quantitative trait loci, estimated genetic correlations and identified associated phenotypes to characterize the genetic signals, as well as to analyse the genetic and phenotypic relationships with the condition. MAIN RESULTS AND THE ROLE OF CHANCE: We identified two genome-wide significant loci on chromosomes 1 (rs4971091, P = 5.32×10-9) and 10 (rs11598956, P = 2.41×10-8) potentially associated with ectopic pregnancy. Follow-up analyses propose MUC1, which codes for an epithelial glycoprotein with an important role in barrier function, as the most likely candidate gene for the association on chromosome 1. We also characterize the phenotypic and genetic correlations with other phenotypes, identifying a genetic correlation with smoking and diseases of the (genito)urinary and gastrointestinal system, and phenotypic correlations with various reproductive health diagnoses, reflecting the previously known epidemiological associations. LARGE SCALE DATA: The GWAS meta-analysis summary statistics are available from the GWAS Catalogue (GCST90272883). LIMITATIONS, REASONS FOR CAUTION: The main limitation is that the findings are based on European-based ancestry populations, with limited data on other populations, and we only captured maternal genomes. Additionally, further larger meta-analysis or independent studies are needed to validate these findings. WIDER IMPLICATIONS OF THE FINDINGS: This study encourages the use of large-scale genetic datasets to unravel genetic factors linked to ectopic pregnancy, which is difficult to study in experimental settings. Increased sample size might bring additional genetic factors associating with ectopic pregnancy and inform its heritability. Altogether, our results provide more insight into the biology of ectopic pregnancy and, accordingly, the biological processes governing embryo implantation. STUDY FUNDING/COMPETING INTEREST(S): N.P.G. was supported by MATER Marie Sklodowska-Curie which received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No. 813707. This study was funded by European Union through the European Regional Development Fund Project No. 2014-2020.4.01.15-0012 GENTRANSMED. Computations were performed in the High-Performance Computing Center of University of Tartu. The authors declare no competing interests.

EPEK: Creation and analysis of an Ectopic Pregnancy Expression Knowledgebase.

Ectopic pregnancy (EP) is one of the leading causes of maternal mortality, where the fertilized embryo grows outside of the uterus. Recent experiments on mice have uncovered the importance of genetic factors in the transport of embryos inside the uterus. In the past, efforts have been made to identify possible gene or protein markers in EP in humans through multiple expression studies. Although there exist comprehensive gene resources for other maternal health disorders, there is no specific resource that compiles the genes associated with EP from such expression studies. Here, we address that knowledge gap by creating a computational resource, Ectopic Pregnancy Expression Knowledgebase (EPEK), that involves manual compilation and curation of expression profiles of EP in humans from published articles. In EPEK, we compiled information on 314 differentially expressed genes, 17 metabolites, and 3 SNPs associated with EP. Computational analyses on the gene set from EPEK showed the implication of cellular signaling processes in EP. We also identified possible exosome markers that could be clinically relevant in the diagnosis of EP. In a nutshell, EPEK is the first and only dedicated resource on the expression profile of EP in humans. EPEK is accessible at https://cb.imsc.res.in/epek.

Abnormal expression of SLIT3 induces intravillous vascularization dysplasia in ectopic pregnancy.

Objective: To investigate whether the morphology, capillary number, and transcriptome expression profiles of ectopic pregnancy (EP) villi differ from those of normal pregnancy (NP) villi. Methods: Hematoxylin-eosin (HE) and immunohistochemistry (IHC) staining for CD31 were conducted to compare differences in morphology and capillary number between EP and NP villi. Differentially expressed (DE) miRNAs and mRNAs were determined from transcriptome sequencing of both types of villi and used to construct a miRNA-mRNA network, from which hub genes were identified. Candidate DE-miRNAs and DE-mRNAs were validated by quantitative reverse transcription (qRT)-PCR. Correlations were identified between the number of capillaries and serum beta human chorionic gonadotropin (ß-HCG) levels and between the expression levels of hub genes associated with angiogenesis and ß-HCG levels. Results: The mean and total cross-sectional areas of placental villi were significantly increased in EP compared with NP villi. Capillary density was greatly reduced in EP villi and was positively correlated with ß-HCG levels. A total of 49 DE-miRNAs and 625 DE-mRNAs were identified from the sequencing data. An integrated analysis established a miRNA-mRNA network containing 32 DE-miRNAs and 103 DE-mRNAs. Based on the validation of hub mRNAs and miRNAs in the network, a regulatory pathway involving miR-491-5p-SLIT3 was discovered, which may have a role in the development of villous capillaries. Conclusion: Villus morphology, capillary number, and miRNA/mRNA expression profiles in villous tissues were aberrant in EP placentas. Specifically, SLIT3, which is regulated by miR-491-5p, may contribute to the regulation of villous angiogenesis and was established as a putative predictor of chorionic villus development, providing a basis for future research.

Effect and Role of miR-196b in Ectopic Pregnancy.

Ectopic pregnancy (EP) is associated with significant morbidity and mortality, but the molecular mechanism of this condition is still unclear. miR-196b, a hot research direction for the past few years, participates in the occurrence of various diseases but whether it plays a regulatory role in EP is still unclear. This research was set to investigate the expression and potential value of miR-196b in EP. qRT-PCR was utilized to determine the relative expression of miR-196b in peripheral blood of EP patients and to observe the expression changes of miR-196b before and after treatment. Correlation analysis of miR-196b with HCG and progesterone was performed. Logistic regression analysis was applied to independent risk factors affecting EP patients. TargetScan was utilized to predict the downstream target genes of miR-196b, and GO and KEGG analysis was carried out using the R language pack. qRT-PCR showed that miR-196b expression in peripheral blood of EP patients was lower than that of normal people. miR-196b expression in patients after treatment was notably higher than that before treatment. In addition, correlation analysis showed that miR-196b was positively correlated with the expression of HCG, progesterone, and estradiol. Risk factor analysis revealed that abortion history, pelvic inflammatory disease history, lower abdominal surgery history, and miR-196b were independent risk factors for EP, and the AUC of the combined ROC curve was 0.899. GO function enrichment and KEGG signal pathway enrichment found 10 potential functions and 2 potential signal pathways of miR-196b. miR-196b is expressed in EP patients, is differentially expressed according to the change in EP condition, and is expected to become a promising index for clinical diagnosis of EP.

Decorin expression in tubal ectopic and intrauterine pregnancies.

OBJECTIVE: Decorin is a leucine-rich proteoglycan, affects the proliferation, migration, and invasion of extravillous trophoblasts (EVTs). In this study, we aimed to determine the localization of decorin in the implantation site in human tubal ectopic pregnancy, to compare decorin expression levels in ectopic and intrauterine pregnancy, and to investigate the relationship between implantation depth of the tubal wall and expression levels of decorin. METHODS: 15 patients underwent salpingectomy for tubal ectopic pregnancy and 15 underwent curettage for voluntary interruption of pregnancy were included. All blocks were stained with decorin immunohistochemical staining. Trophoblastic cells of tubal Stage I-III and tubal epithelial and stromal cells were analyzed in terms of presence and intensity of decorin staining. RESULTS: Decorin was expressed in both tubal and intrauterine trophoblasts, stroma, and surface epithelium during the first trimester of pregnancy. Decorin staining intensity was significantly lower in the villous cytotrophoblasts and syncytiotrophoblasts in tubal ectopic pregnancies, compared to intrauterine pregnancies (p = 0.001 for both). Decorin staining intensity also significantly lower in the extravillous cytotrophoblasts and syncytiotrophoblasts in the tubal ectopic pregnancies (p = 0.002 and p = 0.001, respectively). There was no significant difference in the staining intensity of the trophoblasts and surface epithelial between Stage II and Stage III tubal invasion; however, the decorin expression was lower in the stroma in Stage III (p = 0.094). CONCLUSION: Decorin expression is significantly lower in trophoblastic cells of tubal ectopic pregnancies than the intrauterine pregnancies. Although it remains limited to explain the underlying cellular mechanisms, decorin seems to play a role in the development of tubal pregnancy.

Abnormally increased DNA methylation in chorionic tissue might play an important role in development of ectopic pregnancy.

BACKGROUND: Human Ectopic Pregnancy (hEP) is the second most common cause of pregnancy-related deaths in the first trimester. Without timely detection, EPs can lead to an increased rate of infertility and an elevated risk for future tubal EPs. In addition, most studies in the field focus on the effect of the fallopian tube (maternal factors) and ignore epigenetic changes in genes and proteins of the embryo, which may also cause EPs. Therefore, the present study hypothesized that embryos also play an important role in the development of EP. The study also speculated that DNA methylation is associated with ectopic pregnancy. Consequently, the effects of DNA methylation on the occurrence and development of ectopic pregnancy were investigated. Moreover, genome-wide DNA methylation of chorionic tissue from ectopic and intrauterine pregnancies was detected using Illumina HumanMethylation450 arrays. RESULTS: Forty-three hypermethylated genes involved in the regulation of adhesion as well as gene transcription and translation were identified. Furthermore, the PPI network showed that AMOTL1, SDR42E1, CAMTA1, PIP5K1C, KIAA1614, TSTD1 and DNER may play important roles in the occurrence and development of ectopic pregnancy. In addition, SDR42E1, CAMTA1 and TSTD1 displayed higher levels of methylation in ectopic pregnancy while PIP5K1C and DNER showed low degrees of methylation. CONCLUSIONS: The study reveals that abnormal increase in methylation may be an early indicator or an inducer of ectopic pregnancy. In addition, AMOTL1, SDR42E1, CAMTA1, PIP5K1C, KIAA1614, TSTD1 and DNER might play important roles in the occurrence and development of ectopic pregnancy. However, the specific molecular mechanisms are still unclear and require further studies.

An intrauterine genomic classifier reliably delineates the location of nonviable pregnancies.

OBJECTIVE: To compare the intrauterine gene expression signatures of women with surgically confirmed ectopic pregnancy (ECT) and those of women with miscarriage to inform the development of a genomic classifier for the reliable delineation of pregnancy location in women with clinically nonviable pregnancies of unknown location (NV-PULs). DESIGN: Discovery-based prospective cohort study. SETTING: Academic medical center. PATIENT(S): Women with clinically nonviable early pregnancy to include abnormal intrauterine pregnancy (AIUP), ECT, or NV-PUL. INTERVENTION(S): Endometrial (EM) pipelle sampling of the uterus was conducted at the time of scheduled surgery for clinically nonviable early pregnancy (dilation and curettage, manual vacuum aspiration, or laparoscopy). All pregnancy locations were surgically and/or histologically confirmed as intrauterine or ectopic. MAIN OUTCOME MEASURE(S): Gene expression profiles as determined by array hybridization, quantitative real-time polymerase chain reaction, and nCounter technology. RESULT(S): Intrauterine samples were obtained by EM pipelle from 27 women undergoing surgery for a clinically nonviable early pregnancy. Comparison of array-based global gene expression signatures from women with histologically confirmed ECT versus AIUP revealed 61 differentially expressed genes from which the 5 most informative were included in the pregnancy location classifier. All 5 genes (C20orf85, LRRC46, RSPH4A, WDR49, and ZBBX) were cilia-associated and showed increased expression in pipelle samples from women with ECT relative to expression in samples from women with AIUP. The 5-gene classifier demonstrated an average area under the receiver operator characteristic curve of 0.97 for the detection of ECT. In an external test set composed of publicly available EM pipelle-based gene expression data from a study with similar ECT and AIUP cohorts (n = 19), the classifier revealed an average area under the receiver operator characteristic curve of 0.84. CONCLUSION(S): Consistently increased expression of cilia-associated genes in the uterine cavity of women with ECT provides a reliable molecular signal for the delineation of pregnancy location in women with clinically assessed NV-PUL. A classifier consisting of the 5 most informative cilia-associated genes demonstrated 91% (42/46) accuracy in predicting the pregnancy location.

Cannabinoid receptor type-1 and its correlation with CB1 gene polymorphism-1359G/A in ectopic pregnancy compared to the control group.

AIM: Ectopic pregnancy (EP) is one of the most important causes of maternal mortality. This study aimed to evaluate the immunohistochemical (IHC) expression of the cannabinoid receptor type-1 (CB1) and its association with CB1-1359G/A gene polymorphism (rs1049353) in the fallopian tubes in EP compared to controls. METHODS: In this case-control study, 100 women with EP (cases) and 100 women that underwent abdominal surgery due to the hysterectomy or uterine tubal ligation (healthy controls) were included. Genotyping of CB1-1359G/A polymorphism, tissue expression of CB1 at the protein and mRNA levels were studied using restriction fragment length polymorphism, IHC method, and quantitative real-time polymerase chain reaction (qRT-PCR) analysis. RESULTS: Genotyping showed that in EP, the frequency of AA, AA+AG genotypes, and A allele was significantly higher than healthy control subjects (p = 0.001). Also, patients with EP had significantly increased IHC expression of CB-1 compared to the control samples (p = 0.016). Patients with AA and AG genotypes had a significantly higher IHC expression of CB-1 compared to the GG genotype. qRT-PCR analysis showed that patients with EP had significantly increased expression of CB-1 compared to the control samples (p < 0.001). Patients with AA and AG genotypes had higher significant mRNA expression of CB-1 compared to the GG genotype. CONCLUSIONS: CB1 is likely to be effective in creating innate immunity in humans and can affect the process of EP in the fallopian tube. CB1 is also a pathological valuable factor in identifying the pathway of inflammation during ectopic implantation.

Uterine Scarring Leads to Adverse Pregnant Consequences by Impairing the Endometrium Response to Steroids.

Uterine surgical scarring is an increasing risk factor for adverse pregnant consequences that threaten fetal-maternal health. The detailed molecular features of scar implantation remain largely unknown. We aim to study the pathologic features of uterine surgical scarring and the mechanisms of compromised pregnancy outcomes of scar implantation. We generated a mouse model of uterine surgical scarring with a uterine incision penetrating the myometrium to endometrium to examine the pathologic changes and transcriptome profiles of uterine scarring at various postsurgery (PS) time points, as well as features of the feto-maternal interface during scar implantation. We found that uterine surgical scar recovery was consistently poor at PS3 until PS90, as shown by a reduced number of endometrial glands, inhibition of myometrial smooth muscle cell growth but excessive collagen fiber deposition, and massive leukocyte infiltration. Transcriptome annotation indicated significant chronic inflammation at the scarring site. At the peri-implantation and postimplantation stages, abnormal expression of various steroid-responsive genes at the scarring site was in parallel with lumen epithelial cell hyperplasia, inappropriate luminal closure, and disorientation of the implanted embryo, restricted stromal cell proliferation, and defective decidualization. High embryonic lethality (around 70%) before E10.5 was observed, and the small amount of survival embryos at E10.5 exhibited restricted growth and aberrant placenta defects including overinvasion of trophoblast cells into the decidua and insufficient fetal blood vessel branching in the labyrinth. The findings indicate that chronic inflammation and compromised responses to steroids in uterine scar tissues are the pivotal molecular basis for adverse pregnancy consequences of scar implantation.

Contribution of genetic polymorphism of methylene tetrahydrofolate reductase on the effect of methotrexate in ectopic pregnancy patients.

BACKGROUND: Methotrexate (MTX) is the prior drug in ectopic pregnancy (EP). However, approximately 10% of patients suffer from failure by MTX therapy. Reduced folate carrier 1 (RFC1), methylene tetrahydrofolate reductase (MTHFR), and dihydrofolate reductase (DHFR) are involved in the transport and effects of MTX in vivo. In the present study, we aim to investigate the relationship between the genetic polymorphisms of RFC1, MTHFR, and DHFR and the clinical efficacy of MTX in tubal pregnancies. METHODS: 100 patients of EP were enrolled in this study. Polymorphisms of RFC1 G80A, MTHFR C677T, and DHFR A-317G were genotyped. ß-hCG level was detected in day 0, 4, and 7 after MTX injection. Association of MTX efficacy and genetic polymorphisms was analyzed. RESULTS: Methylene tetrahydrofolate reductase C677T was associated with MTX treatment (P = .017). The success rate of first MTX injection was superior in patients with harboring mutation allele of MTHFR gene than that in patients with wild-type gene (P = .001). However, there was no significant association between the polymorphisms of RFC1 G80A, DHFR A-317G, and surgical treatment (P = .709 and .476, respectively). In addition, ß-hCG level decrement was not significantly changed by MTX injection with different polymorphisms of RFC1, MTHFR, and DHFR on either day 4 (P = .214, 0.197 and 0.270, respectively) or day 7 (P = .172, .554, and .726, respectively). CONCLUSION: Our results suggested that the reliable indicator was polymorphism of MTHFR C677T in failure by MTX injection.

Estrogen receptor α is required for oviductal transport of embryos.

Newly fertilized embryos spend the first few days within the oviduct and are transported to the uterus, where they implant onto the uterine wall. An implantation of the embryo before reaching the uterus could result in ectopic pregnancy and lead to maternal death. Estrogen is necessary for embryo transport in mammals; however, the mechanism involved in estrogen-mediated cellular function within the oviduct remains unclear. In this study, we show in mouse models that ciliary length and beat frequency of the oviductal epithelial cells are regulated through estrogen receptor α (ESR1) but not estrogen receptor ß (ESR2). Gene profiling indicated that transcripts in the WNT/ß-catenin (WNT/CTNNB1) signaling pathway were regulated by estrogen in mouse oviduct, and inhibition of this pathway in a whole oviduct culture system resulted in a decreased embryo transport distance. However, selective ablation of CTNNB1 from the oviductal ciliated cells did not affect embryo transport, possibly because of a compensatory mechanism via intact CTNNB1 in the adjacent secretory cells. In summary, we demonstrated that disruption of estrogen signaling in oviductal epithelial cells alters ciliary function and impairs embryo transport. Therefore, our findings may provide a better understanding of etiology of the ectopic pregnancy that is associated with alteration of estrogen signals.-Li, S., O'Neill, S. R. S., Zhang, Y., Holtzman, M. J., Takemaru, K.-I., Korach, K. S., Winuthayanon, W. Estrogen receptor α is required for oviductal transport of embryos.

Telocytes in Inflammatory Gynaecologic Diseases and Infertility.

Women suffered with inflammatory gynecologic diseases, such as endometriosis (EMs) and acute salpingitis (AS) often complained of sub- or infertility, even in those women without obvious macroscopic anatomical pelvic abnormalities also have unexplained infertility. Generally, besides the well-known impairment of classically described oviduct cells caused by inflammatory diseases, such as the ciliated cells, fibroblasts and myofibroblasts, the involvement of the newly identified telocytes (TCs) in disease-affected oviduct tissues and potential pathophysiological roles in fertility problems remain unknown. In this chapter, TCs was investigated in rat model of EMs- and AS-affected oviduct tissues. Results showed inflammation and ischaemia-induced extensive ultrastructural damages of TCs both in cellular body and prolongations, with obvious TCs loss and interstitial fibrotic remodelling. Such in vivo pathological alterations might contribute to structural and functional abnormalities of oviduct tissue and potentially engaged in sub- or infertility. And especially, TCs connected to various activated immunocytes in both normal and diseased tissues, thus might participate in local immunoregulation (either repression or activation) and serve a possible explanation for immune-mediated pregnancy failure. Then, in vitro cell co-culture study showed that uterine TC conditioned media (TCM) can activate mouse peritoneal macrophages and subsequently trigger its cytokine secretion, thus providepreliminary evidence that, TCs are not simply innocent bystanders, but are instead potential functional players in local immunoregulatory and immunosurveillance.

[Association of vascular endothelial growth factor gene polymorphisms with ectopic pregnancy among Chinese women].

OBJECTIVE: To assess the association of vascular endothelial growth factor (VEGF) gene polymorphisms with ectopic pregnancy (EP) among Chinese women. METHODS: A case-control study was carried out, which compared 192 women with a history of EP with 210 post-menopausal controls who had two pregnancies but no history of EP for the genotypes of the VEGF gene. Polymorphisms of the VEGF gene including -460C/T, -1154G/A, -2578C/A and +936C/T were determined with a polymerase chain reaction-restriction fragment length polymorphism method. RESULTS: No significant difference was found in the genotypic and allelic distribution of the -460C/T and +936C/T polymorphisms between the two groups. Compared with the GG genotype, the VEGF -1154 AA+GA genotype could significantly decrease the risk of EP (OR=0.61, 95%CI: 0.42-0.87). Compared with the CC genotype, VEGF -2578 AA+CA genotype could significantly reduce the risk of EP (OR=0.66, 95%CI:0.44-0.99). Haplotype analysis suggested that the T-A-A (VEGF -460/-1154/-2578) and C-A-A haplotypes could significantly decrease the risk of EP compared with the T-G-C haplotype (P=0.020, OR=0.41, 95%CI:0.19-0.89, P=0.014, OR=0.29, 95%CI:0.11-0.82). CONCLUSION: The -1154A or -2578A alleles of the VEGF gene can significantly decrease the risk of EP among Chinese women. The VEGF -460C/T, -1154G/A and -2578C/A polymorphisms showed a linkage disequilibrium. Both T-A-A and C-A-A haplotypes can significantly decrease the risk of EP.

Prioritization of Susceptibility Genes for Ectopic Pregnancy by Gene Network Analysis.

Ectopic pregnancy is a very dangerous complication of pregnancy, affecting 1%-2% of all reported pregnancies. Due to ethical constraints on human biopsies and the lack of suitable animal models, there has been little success in identifying functionally important genes in the pathogenesis of ectopic pregnancy. In the present study, we developed a random walk-based computational method named TM-rank to prioritize ectopic pregnancy-related genes based on text mining data and gene network information. Using a defined threshold value, we identified five top-ranked genes: VEGFA (vascular endothelial growth factor A), IL8 (interleukin 8), IL6 (interleukin 6), ESR1 (estrogen receptor 1) and EGFR (epidermal growth factor receptor). These genes are promising candidate genes that can serve as useful diagnostic biomarkers and therapeutic targets. Our approach represents a novel strategy for prioritizing disease susceptibility genes.

Epigenetics of Notch1 regulation in pulmonary microvascular rarefaction following extrauterine growth restriction.

BACKGROUND: Extrauterine growth restriction (EUGR) plays an important role in the developmental origin of adult cardiovascular diseases. In an EUGR rat model, we reported an elevated pulmonary arterial pressure in adults and genome-wide epigenetic modifications in pulmonary vascular endothelial cells (PVECs). However, the underlying mechanism of the early nutritional insult that results in pulmonary vascular consequences later in life remains unclear. METHODS: A rat model was used to investigate the physiological and structural effect of EUGR on early pulmonary vasculature by evaluating right ventricular systolic pressure and pulmonary vascular density in male rats. Epigenetic modifications of the Notch1 gene in PVECs were evaluated. RESULTS: EUGR decreased pulmonary vascular density with no significant impact on right ventricular systolic pressure at 3 weeks. Decreased transcription of Notch1 was observed both at 3 and 9 weeks, in association with decreased downstream target gene, Hes-1. Chromatin immunoprecipitation and bisulfite sequencing were performed to analyze the epigenetic modifications of the Notch1 gene promoter in PVECs. EUGR caused a significantly increased H3K27me3 in the proximal Notch1 gene promoter, and increased methylation of single CpG sites in the distal Notch1 gene promoter, both at 3 and 9 weeks. CONCLUSIONS: We conclude that EUGR results in decreased pulmonary vascular growth in association with decreased Notch1 in PVECs. This may be mediated by increased CpG methylation and H3K27me3 in the Notch1 gene promoter region.

Pregnancy-associated microRNAs in plasma as potential molecular markers of ectopic pregnancy.

OBJECTIVE: To investigate cell-free pregnancy-associated microRNAs as molecular markers for the diagnosis of ectopic pregnancy. DESIGN: Laboratory study using human plasma samples. SETTING: Research unit in a university hospital. PATIENT(S): Plasma samples from 18 women with ectopic pregnancies (EP group), 12 women with spontaneous abortion (SA group), and 26 normal women with singleton pregnancies (NP group). INTERVENTION(S): Total RNAs containing small RNA molecules extracted from 1.2 mL of plasma. MAIN OUTCOME MEASURE(S): Plasma concentrations of cell-free microRNAs measured by quantitative real-time reverse-transcriptase polymerase chain reaction. RESULT(S): Plasma concentrations of cell-free pregnancy-associated microRNAs (miR-323-3p, miR-515-3p, miR-517a, miR-517c, and miR-518b) and serum concentration of human chorionic gonadotropin (hCG) were confirmed to have statistically significantly different plasma or serum concentrations in women with EP, SA, or NP. There was no statistically significant difference in the plasma concentrations of cell-free miR-21 between the three groups. By correlation coefficient analysis, no relationship was detected between serum hCG levels and plasma cell-free miR-517c, miR-515-3p, miR-517a, miR-518b, miR-323-3p, or miR-21 levels. Plasma concentrations of cell-free miR-517a could distinguish EP/SA from NP, yielding an area under the curve of 0.9654 (95% confidence interval, 0.9172-1.0). Plasma concentrations of cell-free miR-323-3p could distinguish EP from SA, yielding an area under the curve of 0.7454 (95% confidence interval, 0.5558-0.9349). CONCLUSION(S): Cell-free pregnancy-associated microRNAs have potential as molecular markers of ectopic pregnancy.

Embryonic miRNA profiles of normal and ectopic pregnancies.

Our objective was to investigate the miRNA profile of embryonic tissues in ectopic pregnancies (EPs) and controlled abortions (voluntary termination of pregnancy; VTOP). Twenty-three patients suffering from tubal EP and twenty-nine patients with a normal ongoing pregnancy scheduled for a VTOP were recruited. Embryonic tissue samples were analyzed by miRNA microarray and further validated by real time PCR. Microarray studies showed that four miRNAs were differentially downregulated (hsa-mir-196b, hsa-mir-30a, hsa-mir-873, and hsa-mir-337-3p) and three upregulated (hsa-mir-1288, hsa-mir-451, and hsa-mir-223) in EP compared to control tissue samples. Hsa-miR-196, hsa-miR-223, and hsa-miR-451 were further validated by real time PCR in a wider population of EP and control samples. We also performed a computational analysis to identify the gene targets and pathways which might be modulated by these three differentially expressed miRNAs. The most significant pathways found were the mucin type O-glycan biosynthesis and the ECM-receptor-interaction pathways. We also checked that the dysregulation of these three miRNAs was able to alter the expression of the gene targets in the embryonic tissues included in these pathways such as GALNT13 and ITGA2 genes. In conclusion, analysis of miRNAs in ectopic and eutopic embryonic tissues shows different expression patterns that could modify pathways which are critical for correct implantation, providing new insights into the understanding of ectopic implantation in humans.

The Lin28/Let-7 system in early human embryonic tissue and ectopic pregnancy.

Our objective was to determine the expression of the elements of the Lin28/Let-7 system, and related microRNAs (miRNAs), in early stages of human placentation and ectopic pregnancy, as a means to assess the potential role of this molecular hub in the pathogenesis of ectopic gestation. Seventeen patients suffering from tubal ectopic pregnancy (cases) and forty-three women with normal on-going gestation that desired voluntary termination of pregnancy (VTOP; controls) were recruited for the study. Embryonic tissues were subjected to RNA extraction and quantitative PCR analyses for LIN28B, Let-7a, miR-132, miR-145 and mir-323-3p were performed. Our results demonstrate that the expression of LIN28B mRNA was barely detectable in embryonic tissue from early stages of gestation and sharply increased thereafter to plateau between gestational weeks 7-9. In contrast, expression levels of Let-7, mir-132 and mir-145 were high in embryonic tissue from early gestations (≤ 6-weeks) and abruptly declined thereafter, especially for Let-7. Opposite trends were detected for mir-323-3p. Embryonic expression of LIN28B mRNA was higher in early stages (≤ 6-weeks) of ectopic pregnancy than in normal gestation. In contrast, Let-7a expression was significantly lower in early ectopic pregnancies, while miR-132 and miR-145 levels were not altered. Expression of mir-323-3p was also suppressed in ectopic embryonic tissue. We are the first to document reciprocal changes in the expression profiles of the gene encoding the RNA-binding protein, LIN28B, and the related miRNAs, Let-7a, mir-132 and mir-145, in early stages of human placentation. This finding suggests the potential involvement of LIN28B/Let-7 (de)regulated pathways in the pathophysiology of ectopic pregnancy in humans.

Association study of vascular endothelial growth factor gene polymorphisms with ectopic pregnancy in Chinese women.

OBJECTIVE: The purpose of this study is to evaluate potential associations between vascular endothelial growth factor (VEGF) gene polymorphisms and ectopic pregnancy (EP) in Chinese women. MATERIALS AND METHODS: This was a case-control study wherein 192 women with a history of EP were compared to 210 post-menopausal controls with two pregnancies and no EP for the genotyping of VEGF polymorphisms. Genotyping of the VEGF gene polymorphisms at -460C/T, -1 154G/A, -2578C/A and +936C/T were performed by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: No significant differences were found in genotype and allele distributions of the -460C/T, +936C/T polymorphisms between cases and controls. Compared with the -1154G/G genotype, the -1154(A/A+G/A) genotype could significantly reduce the risk of developing EP. For the -2578C/A polymorphism, the A/A+C/A geno- type could significantly decrease the risk of developing EP, compared with the C/C genotype. The haplotype analysis suggested that the TAA (VEGF -460/-1154/-2578) and CAA haplotypes could significantly decrease the risk of developing EP compared with the haplotype of TGC. CONCLUSION: The -1154A or -2578A alleles of VEGF gene could significantly decrease the risk of EP and might be po- tentially protective factors for EP development in Chinese women.