Sasoiaren menpeko erreferentzia geneen transkripzio-profilen ikerketa Chelon labrosus lazunean

Chelon labrosus lazuna oso erabilia da kutsaduraren eragina aztertzeko organismo zentinela bezala, oso kutsatuta dauden itsaso zabaleko zein itsasadarretako uretan bizirauteko gai baita. Kutsatzaileek zelulen, ehunen eta organismoen transkripzio-profilen aldaketa eragin dezaketela ondo deskribatuta dago. Aldaketa hauek neurtzeko, gene-espresioaren normalizazioa burutu behar da. Eskuarki, normalizazio prozesua burutzeko, metabolismo orokorreko geneak erabiltzen dira, hau da, erreferentzia geneak. Gene hauek, zelularen oinarrizko funtzioak garatzeko beharrezkoak diren geneak dira eta zelula guztietan konstitutiboki adierazi ohi dira. Aurretik egin diren zenbait ikerketetan ordea, gene hauen transkripzio maila egoera esperimentalen, ehun motaren, garapen fasearen edota zelularen zikloaren arabera aldatu egiten dela behatu da. Hortaz, erreferentzia geneen transkripzio mailen aldaketak, aztergai den itu genearen interpretazio okerra eman dezake. Hori dela eta, azken urte hauetan, beste metodo batzuk garatzen ari dira geneen transkripzio maila normalizatzeko, esaterako, QuanT-it OliGreen metodoa. Testuinguru honetan, Chelon labrosus lazunean toxikologia-analisiak egiteko gehien erabiltzen diren erreferentzia geneen transkripzio-profilak aztertu dira indibiduo ar zein emeetan ugalketa zikloaren zeharreko fase gametogeniko desberdinetan (bost guztira). Horretarako, erreferentzia gene bat, itu gene kontsideratuz, normalizazioa beste erreferentzia geneekiko zein QuanT-it OliGreen metodotik lortutako emaitzekiko burutu da. Azterturiko geneak β-aktina (BA), 18S RNA erribosomikoa (18S rRNA), 1-α elongazio faktorea (EF-1-α) eta glizeraldehido-3-fosfato deshidrogenasa (GAPDH) izan dira. Aztergai zeuden erreferentzia geneen transkripzio maila qRT-PCR bidez kuantifikatu ondoren, aurretik aipatutako bi metodoekiko normalizatu ziren. Normalizazioa erreferentzia geneekiko egin zenean, itu gene berak transkripzio-profil desberdinak aurkeztu zituen normalizaziorako erabili zen erreferentzia genearen arabera. QuanT-it OliGreen metodoaren bidezko normalizazioa egin zenean berriz, aztertutako erreferentzia gene guztien transkripzio-profila, fase gametogenikoen arabera aldatzen zela ondorioztatu zen. Beraz, itu gene baten transkripzio-profilaren azterketa egin nahi denean QuanT-it OliGreen metodoa erreferentzia geneak erabiltzea baino fidagarriagoa da

microRNA-based signatures obtained from endometrial fluid identify implantative endometrium

STUDY QUESTION Is it possible to use free and extracellular vesicle-associated microRNAs (miRNAs) from human endometrial fluid (EF) samples as non-invasive biomarkers for implantative endometrium? SUMMARY ANSWER The free and extracellular vesicle-associated miRNAs can be used to detect implantative endometrium in a non-invasive manner. WHAT IS KNOWN ALREADY miRNAs and extracellular vesicles (EVs) from EF have been described as mediators of the embryo-endometrium crosstalk. Therefore, the analysis of miRNA from this fluid could become a non-invasive technique for recognizing implantative endometrium. This analysis could potentially help improve the implantation rates in ART. STUDY DESIGN, SIZE, DURATION In this prospective study, we first optimized different protocols for EVs and miRNA analyses using the EF of a setup cohort (n = 72). Then, we examined differentially expressed miRNAs in the EF of women with successful embryo implantation (discovery cohort n = 15/validation cohort n = 30) in comparison with those for whom the implantation had failed (discovery cohort n = 15/validation cohort n = 30). Successful embryo implantation was considered when pregnancy was confirmed by vaginal ultrasound showing a gestational sac 4 weeks after embryo transfer (ET). PARTICIPANTS/MATERIALS, SETTING, METHODS The EF of the setup cohort was obtained before starting fertility treatment during the natural cycle, 16-21 days after the beginning of menstruation. For the discovery and validation cohorts, the EF was collected from women undergoing frozen ET on Day 5, and the samples were collected immediately before ET. In this study, we compared five different methods; two of them based on direct extraction of RNA and the other three with an EV enrichment step before the RNA extraction. Small RNA sequencing was performed to determine the most efficient method and find a predictive model differentiating between implantative and non-implantative endometrium. The models were confirmed using quantitative PCR in two sets of samples (discovery and validation cohorts) with different implantation outcomes. MAIN RESULTS AND THE ROLE OF CHANCE The protocols using EV enrichment detected more miRNAs than the methods based on direct RNA extraction. The two most efficient protocols (using polymer-based precipitation (PBP): PBP-M and PBP-N) were used to obtain two predictive models (based on three miRNAs) allowing us to distinguish between an implantative and non-implantative endometrium. The first Model 1 (PBP-M) (discovery: AUC = 0.93; P-value = 0.003; validation: AUC = 0.69; P-value = 0.019) used hsa-miR-200b-3p, hsa-miR-24-3p and hsa-miR-148b-3p. Model 2 (PBP-N) (discovery: AUC = 0.92; P-value = 0.0002; validation: AUC = 0.78; P-value = 0.0002) used hsa-miR-200b-3p, hsa-miR-24-3p and hsa-miR-99b-5p. Functional analysis of these miRNAs showed strong association with key implantation processes such as in utero embryonic development or transforming growth factor-beta signaling. LARGE SCALE DATA The FASTQ data are available in the GEO database (access number GSE178917). LIMITATIONS, REASONS FOR CAUTION One important factor to consider is the inherent variability among the women involved in the trial and among the transferred embryos. The embryos were pre-selected based on morphology, but neither genetic nor molecular studies were conducted, which would have improved the accuracy of our tests. In addition, a limitation in miRNA library construction is the low amount of input RNA. WIDER IMPLICATIONS OF THE FINDINGS We describe new non-invasive protocols to analyze miRNAs from small volumes of EF. These protocols could be implemented in clinical practice to assess the status of the endometrium before attempting ET. Such evaluation could help to avoid the loss of embryos transferred to a non-implantative endometrium. STUDY FUNDING/COMPETING INTEREST(S) J.I.-P. was supported by a predoctoral grant from the Basque Government (PRE_2017_0204). This study was partially funded by the Grant for Fertility Innovation (GFI, 2011) from Merck (Darmstadt, Germany). It was also supported by the Spanish Ministry of Economy and Competitiveness MINECO within the National Plan RTI2018-094969-B-I00, the European Union's Horizon 2020 research and innovation program (860303), the Severo Ochoa Centre of Excellence Innovative Research Grant (SEV-2016-0644) and the Instituto de Salud Carlos III (PI20/01131). The funding entities did not play any role in the study design, collection, analysis and interpretation of data, writing of the report or the decision to submit the article for publication. The authors declare no competing interests.J.I.-P. was supported by a predoctoral grant from the Basque Government (PRE_2017_0204). This study was partially funded by the Grant for Fertility Innovation (GFI, 2011) from Merck (Darmstadt, Germany). The project was also supported by the Spanish Ministry of Economy and Competitiveness MINECO within the national plan RTI2018-094969-B-I00, the European Union's Horizon 2020 research and innovation program (860303), the Severo Ochoa Centre of Excellence Innovative Research Grant (SEV-2016-0644) and the Instituto de Salud Carlos III (PI20/01131). The funding entities did not have any role in study design, sample collection, analysis and interpretation of data, report writing or decision to submit the article for publication

Mendelian randomization analysis rules out disylipidaemia as colorectal cancer cause

[EN] Colorectal cancer (CRC) has been associated with both genetic and environmental risk factors such as diet1,2, alcohol3, smoking4, physical activity5 and metabolic syndrome6,7. Metabolic syndrome is a cluster of important cardiovascular risk factors: high fasting glucose, abdominal obesity, high triglycerides (TG), reduced high density lipoprotein cholesterol (HDL) and high blood pressure. As one of the components, dyslipidemia has been thought to have an important role in inflammatory pathways, oxidative stress and insulin resistance, which could contribute to the pathogenesis of cancer. However, findings from prospective studies that have examined the association between serum dyslipidemia (TG, HDL, low density lipoprotein cholesterol (LDL) or total cholesterol (TC)) and colorectal neoplasia have been inconsistent6,8,9,10,11. It is unknown whether lipids and lipoproteins cause cancer or are intermediate or correlated factors within carcinogenic pathways. The Mendelian randomization approach can be used to establish a causal relationship between dyslipidemia and CRC. Mendelian randomization studies use the distribution of alleles in the population to simulate randomized assignment to lower or higher lipids. A previous Mendelian randomization analysis assessing the causality of dyslipidemia and CRC has been published before11. It reported an association between a genetic score for TC and the risk of CRC (OR per unit SD increase = 1.46; 95% CI: 1.20–1.79) but no significant association was found for LDL, HDL or TG. While the association of TC was strong, the interpretation of the results is not straightforward, since TC is the sum of LDL and HDL cholesterol, fractions that have been reported to have opposite effects regarding CRC and may explain the controversial findings of studies that have analyzed the association between high levels of serum TC and CRC risk8,9,12. A causal effect of lipids regarding CRC risk should have a clear mechanistic interpretation. Being TC essentially the sum of HLD and LDL, genetic instruments for TC are correlated either with LDL, HDL or both, which violates the pleiotropy assumption of Mendelian randomization studies.S

MicroRNAs libres y asociados a vesículas extracelulares como biomarcadores diagnósticos no invasivos del endometrio implantativo

[ES] Aumentar las tasas de implantación embrionaria se ha convertido en uno de los mayores retos de las técnicas de reproducción asistida. La implantación es un proceso complejo que requiere una sincronía entre el desarrollo del embrión y el endometrio, pero también una adecuada comunicación entre ambos. Los microRNAs (miRNAs) libres o asociados a vesículas extracelulares (VEs) del fluido endometrial (FE) se han descrito como mediadores de la comunicación embrión-endometrio. Por eso creemos que el análisis de miRNAs libres y asociados a VEs procedentes del FE podría utilizarse para desarrollar una metodología no invasiva que permitiera reconocer el endometrio implantativo. En este estudio prospectivo, en primer lugar, se optimizaron diferentes métodos para el análisis de VEs y miRNAs utilizando el FE de una cohorte de puesta a punto (n = 72). Dos de los métodos consistían en una extracción directa de RNA mientras que en los otros tres se realizó un enriquecimiento de VEs antes de la extracción de RNA. El RNA se analizó mediante Small RNA-seq y qPCR y se determinó que los métodos más eficientes eran los protocolos que utilizaron el enriquecimiento de VEs mediante la precipitación basada en polímeros (PBP-M y PBPB-N). A continuación, los métodos seleccionados se probaron en dos grupos de mujeres con diferente resultado de implantación (ciclo implantativo n= 45 y ciclo no implantativo n=45) y se analizaron los miRNAs diferencialmente expresados. Finalmente, se logró obtener dos modelos predictivos del endometrio implantativo (p valor <0,05) basados en tres miRNAs. El modelo 1 (PBP-M) utilizó los miRNAs hsa-miR-200b-3p, hsa-miR-24-3p y hsa-miR-148b-3p, y el modelo 2 (PBP-N) utilizó hsa-miR-200b-3p, hsa-miR-24-3p y hsa-miR-99b-5p. Además, se realizó un análisis funcional de estos miRNAs que mostró una fuerte asociación con procesos clave de la implantación embrionaria. En conclusión, gracias a este estudio se han descrito dos métodos no invasivos para analizar miRNAs procedentes del FE que podrían aplicarse en la práctica clínica para evaluar el estado del endometrio antes de realizar la transferencia embrionaria y así evitar la pérdida de embriones al ser transferidos a un endometrio que no está listo para que ocurra la implantación