Cargo small non-coding RNAs of extracellular vesicles isolated from uterine fluid associate with endometrial receptivity and implantation success


      To optimize a method of isolating extracellular vesicles (EVs) from uterine fluid and to characterize small non-coding RNAs (sncRNAs) from the EVs, with the goal of identifying novel receptivity-associated biomarkers.


      Longitudinal study comparing sncRNA expression profiles from endometrial EVs.


      University-affiliated, hospital-based fertility clinic.


      Healthy volunteers with no history of infertility (Group A) and women receiving controlled ovarian stimulation (COS)–in vitro fertilization treatment (Group B).


      In Group A, EVs were isolated from uterine fluid obtained on luteinizing hormone (LH)+2 and LH+7 in one natural menstrual cycle. In Group B, EVs were isolated from uterine fluid obtained on human chorionic gonadotropin (hCG)+2 and hCG+7 in one COS cycle. RNAs extracted from EVs were profiled using next-generation sequencing.

      Main Outcome Measure(s)

      Differential EV-sncRNAs between LH+2 and LH+7 (Group A), between hCG+2 and hCG+7 (Group B), and between pregnant and nonpregnant in vitro fertilization cycles (Group B).


      Ultracentrifugation was validated as the most efficient method to isolate EVs from uterine fluid. We identified 12 endometrial EV-sncRNAs (11 microRNAs and 1 piwi-interacting RNA) as receptivity-associated transcripts conserved in both natural and COS cycles. These sncRNAs were associated strongly with biological functions related to immune response, extracellular matrix, and cell junction. Within COS cycles, we also identified a group of EV-sncRNAs that exhibited differential expression in patients who conceived versus those who did not, with hsa-miR-362-3p most robustly overexpressed in the nonpregnant patients.


      This study is the first to profile comprehensively sncRNAs in endometrial EVs from uterine fluid and identify sncRNA biomarkers of endometrial receptivity and implantation success.
      Los pequeños ARN cargo no codificantes de vesículas extracelulares aisladas del líquido uterino se asocian con la receptividad endometrial y el éxito de la implantación.


      Optimizar un método para aislar las vesículas extracelulares (VE) del fluido uterino y caracterizar los ARN pequeños no codificantes (sncRNA) de los VE, con el objetivo de identificar nuevos biomarcadores asociados a la receptividad.


      Estudio longitudinal que compara los perfiles de expresión de sncRNA de EV endometriales.


      Clínica de fertilidad hospitalaria afiliada a una universidad.

      Paciente (s)

      Voluntarias sanas sin antecedentes de infertilidad (Grupo A) y mujeres recibiendo tratamiento de fertilización in vitro con estimulación ovárica controlada (COS) - (Grupo B).

      Intervención (es)

      En el Grupo A, los VE se aislaron del líquido uterino obtenido con la hormona luteinizante (LH) +2 y LH +7 en un ciclo menstrual natural. En el Grupo B, los EV se aislaron del líquido uterino obtenido con gonadotropina coriónica humana (hCG) +2 y hCG +7 en un ciclo COS. Los ARN extraídos de las VEs se perfilaron mediante secuenciación de próxima generación.

      Principales medidas de resultado

      EV-sncRNA diferenciales entre LH +2 y LH +7 (Grupo A), entre hCG +2 y hCG +7 (Grupo B), y entre embarazadas y no embarazadas (Grupo B) de ciclos de fertilización in vitro.

      Resultado (s)

      La ultracentrifugación se validó como el método más eficaz para aislar las EVs del líquido uterino. Identificamos 12 EV-sncRNAs de endometrio (11 microRNAs y 1 RNA que interactúa con piwi) como transcripciones asociadas a la receptividad conservadas en ciclos tanto naturales como de COS. Estos sncRNAs se asociaron fuertemente con funciones biológicas relacionadas con la respuesta inmune, la matriz extracelular y la unión celular. Dentro de los ciclos de COS, también identificamos un grupo de EV-sncRNA que exhibieron expresión diferencial en pacientes que concibieron versus aquellas que no, con hsa-miR-362-3p sobreexpresado de manera más robusta en las pacientes no embarazadas.

      Conclusión (es)

      Este estudio es el primero en perfilar de forma exhaustiva los sncARNs en los EV endometriales del líquido uterino e identificar los biomarcadores sncRNA de receptividad endometrial y éxito de la implantación.

      Palabras clave

      Líquido uterino, vesículas extracelulares, pequeños ARN no codificantes, receptividad, implantación

      Key Words

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        • Elnashar A.M.
        • Aboul-Enein G.I.
        Endometrial receptivity.
        Middle East Fertility Society J. 2004; 9: 10-24
        • Chan C.
        • Virtanen C.
        • Winegarden N.A.
        • Colgan T.J.
        • Brown T.J.
        • Greenblatt E.M.
        Discovery of biomarkers of endometrial receptivity through a minimally invasive approach: a validation study with implications for assisted reproduction.
        Fertil Steril. 2013; 100: 810-817.e8
        • Boomsma C.M.
        • Kavelaars A.
        • Eijkemans M.
        • Amarouchi K.
        • Teklenburg G.
        • Gutknecht D.
        • et al.
        Cytokine profiling in endometrial secretions: a non-invasive window on endometrial receptivity.
        Reprod Biomed Online. 2009; 18: 85-94
        • Van der Gaast M.
        • Beier-Hellwig K.
        • Fauser B.
        • Beier H.
        • Macklon N.
        Endometrial secretion aspiration prior to embryo transfer does not reduce implantation rates.
        Reprod Biomed Online. 2003; 7: 105-109
        • Matorras R.
        • Quevedo S.
        • Corral B.
        • Prieto B.
        • Exposito A.
        • Mendoza R.
        • et al.
        Proteomic pattern of implantative human endometrial fluid in in vitro fertilization cycles.
        Arch Gynecol Obstet. 2018; 297: 1577-1586
        • Azkargorta M.
        • Escobes I.
        • Iloro I.
        • Osinalde N.
        • Corral B.
        • Ibañez-Perez J.
        • et al.
        Differential proteomic analysis of endometrial fluid suggests increased inflammation and impaired glucose metabolism in non-implantative IVF cycles and pinpoints PYGB as a putative implantation marker.
        Hum Reprod. 2018; 33: 1898-1906
        • Ng Y.H.
        • Rome S.
        • Jalabert A.
        • Forterre A.
        • Singh H.
        • Hincks C.L.
        • et al.
        Endometrial exosomes/microvesicles in the uterine microenvironment: a new paradigm for embryo-endometrial cross talk at implantation.
        PLoS One. 2013; 8e58502
        • Campoy I.
        • Lanau L.
        • Altadill T.
        • Sequeiros T.
        • Cabrera S.
        • Cubo-Abert M.
        • et al.
        Exosome-like vesicles in uterine aspirates: a comparison of ultracentrifugation-based isolation protocols.
        J Transl Med. 2016; 14: 180
        • Homer H.
        • Rice G.E.
        • Salomon C.
        Embryo-and endometrium-derived exosomes and their potential role in assisted reproductive treatments–liquid biopsies for endometrial receptivity.
        Placenta. 2017; 54: 89-94
        • Fehlmann T.
        • Backes C.
        • Kahraman M.
        • Haas J.
        • Ludwig N.
        • Posch A.E.
        • et al.
        Web-based NGS data analysis using miRMaster: a large-scale meta-analysis of human miRNAs.
        Nucleic Acids Res. 2017; 45: 8731-8744
        • Robinson M.D.
        • McCarthy D.J.
        • Smyth G.K.
        edgeR: a Bioconductor package for differential expression analysis of digital gene expression data.
        Bioinformatics. 2010; 26: 139-140
        • McCarthy D.J.
        • Chen Y.
        • Smyth G.K.
        Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation.
        Nucleic Acids Res. 2012; 40: 4288-4297
        • Ritchie M.E.
        • Phipson B.
        • Wu D.
        • Hu Y.
        • Law C.W.
        • Shi W.
        • et al.
        limma powers differential expression analyses for RNA-sequencing and microarray studies.
        Nucleic Acids Res. 2015; 43 (e47-e)
        • Zuo Y.
        • Liang Y.
        • Zhang J.
        • Hao Y.
        • Li M.
        • Wen Z.
        • et al.
        Transcriptome analysis identifies Piwi-interacting RNAs as prognostic markers for recurrence of prostate cancer.
        Front Genet. 2019; 10: 1018
        • Vlachos I.S.
        • Zagganas K.
        • Paraskevopoulou M.D.
        • Georgakilas G.
        • Karagkouni D.
        • Vergoulis T.
        • et al.
        DIANA-miRPath v3. 0: deciphering microRNA function with experimental support.
        Nucleic Acids Res. 2015; 43: W460-W466
        • Jenjaroenpun P.
        • Kremenska Y.
        • Nair V.M.
        • Kremenskoy M.
        • Joseph B.
        • Kurochkin I.V.
        Characterization of RNA in exosomes secreted by human breast cancer cell lines using next-generation sequencing.
        Peer J. 2013; 1e201
        • Valadi H.
        • Ekström K.
        • Bossios A.
        • Sjöstrand M.
        • Lee J.J.
        • Lötvall J.O.
        Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.
        Nat Cell Biol. 2007; 9: 654-659
        • Kao L.
        • Tulac S.
        • Lobo Sa
        • Imani B.
        • Yang J.
        • Germeyer A.
        • et al.
        Global gene profiling in human endometrium during the window of implantation.
        Endocrinology. 2002; 143: 2119-2138
        • Borthwick J.M.
        • Charnock-Jones D.S.
        • Tom B.D.
        • Hull M.L.
        • Teirney R.
        • Phillips S.C.
        • et al.
        Determination of the transcript profile of human endometrium.
        MHR Basic Sci Reprod Med. 2003; 9: 19-33
        • Altmäe S.
        • Reimand J.
        • Hovatta O.
        • Zhang P.
        • Kere J.
        • Laisk T.
        • et al.
        Research resource: interactome of human embryo implantation: identification of gene expression pathways, regulation, and integrated regulatory networks.
        Mol Endocrinol. 2012; 26: 203-217
        • Carson D.D.
        • Lagow E.
        • Thathiah A.
        • Al-Shami R.
        • Farach-Carson M.C.
        • Vernon M.
        • et al.
        Changes in gene expression during the early to mid-luteal (receptive phase) transition in human endometrium detected by high-density microarray screening.
        Mol Hum Reprod. 2002; 8: 871-879
        • Mirkin S.
        • Arslan M.
        • Churikov D.
        • Corica A.
        • Diaz J.
        • Williams S.
        • et al.
        In search of candidate genes critically expressed in the human endometrium during the window of implantation.
        Hum Reprod. 2005; 20: 2104-2117
        • Talbi S.
        • Hamilton A.
        • Vo K.
        • Tulac S.
        • Overgaard M.T.
        • Dosiou C.
        • et al.
        Molecular phenotyping of human endometrium distinguishes menstrual cycle phases and underlying biological processes in normo-ovulatory women.
        Endocrinology. 2006; 147: 1097-1121
        • Díaz-Gimeno P.
        • Horcajadas J.A.
        • Martínez-Conejero J.A.
        • Esteban F.J.
        • Alamá P.
        • Pellicer A.
        • et al.
        A genomic diagnostic tool for human endometrial receptivity based on the transcriptomic signature.
        Fertil Steril. 2011; 95: 50-60.e15
        • Hu S.
        • Yao G.
        • Wang Y.
        • Xu H.
        • Ji X.
        • He Y.
        • et al.
        Transcriptomic changes during the pre-receptive to receptive transition in human endometrium detected by RNA-Seq.
        J Clin Endocrinol Metab. 2014; 99: E2744-E2753
        • Haouzi D.
        • Dechaud H.
        • Assou S.
        • De Vos J.
        • Hamamah S.
        Insights into human endometrial receptivity from transcriptomic and proteomic data.
        Reprod Biomed Online. 2012; 24: 23-34
        • Sha A.-G.
        • Liu J.-L.
        • Jiang X.-M.
        • Ren J.-Z.
        • Ma C.-H.
        • Lei W.
        • et al.
        Genome-wide identification of micro-ribonucleic acids associated with human endometrial receptivity in natural and stimulated cycles by deep sequencing.
        Fertil Steril. 2011; 96: 150-155.e5
        • Horcajadas J.A.
        • Riesewijk A.
        • Polman J.
        • van Os R.
        • Pellicer A.
        • Mosselman S.
        • et al.
        Effect of controlled ovarian hyperstimulation in IVF on endometrial gene expression profiles.
        Mol Hum Reprod. 2004; 11: 195-205
        • Horcajadas J.A.
        • Mínguez P.
        • Dopazo J.
        • Esteban F.J.
        • Domínguez F.
        • Giudice L.C.
        • et al.
        Controlled ovarian stimulation induces a functional genomic delay of the endometrium with potential clinical implications.
        J Clin Endocrinol Metab. 2008; 93: 4500-4510
        • Haouzi D.
        • Assou S.
        • Mahmoud K.
        • Tondeur S.
        • Rème T.
        • Hedon B.
        • et al.
        Gene expression profile of human endometrial receptivity: comparison between natural and stimulated cycles for the same patients.
        Hum Reprod. 2009; 24: 1436-1445
        • Liu Y.
        • Lee K.-F.
        • Ng E.H.
        • Yeung W.S.
        • Ho P.-C.
        Gene expression profiling of human peri-implantation endometria between natural and stimulated cycles.
        Fertil Steril. 2008; 90: 2152-2164
        • Allegra A.
        • Marino A.
        • Coffaro F.
        • Lama A.
        • Rizza G.
        • Scaglione P.
        • et al.
        Is there a uniform basal endometrial gene expression profile during the implantation window in women who became pregnant in a subsequent ICSI cycle?.
        Hum Reprod. 2009; 24: 2549-2557
        • Díaz-Gimeno P.
        • Ruiz-Alonso M.
        • Sebastian-Leon P.
        • Pellicer A.
        • Valbuena D.
        • Simón C.
        Window of implantation transcriptomic stratification reveals different endometrial subsignatures associated with live birth and biochemical pregnancy.
        Fertil Steril. 2017; 108: 703-710.e3
        • Brown J.J.
        • Papaioannou V.E.
        Extracellular matrix remodeling at implantation: role of hyaluronan.
        Molecular and cellular aspects of periimplantation processes. New York, NY: Springer New York, 1995: 125-152
        • Grund S.
        • Grümmer R.
        Direct cell–cell interactions in the endometrium and in endometrial pathophysiology.
        Int J Mol Sci. 2018; 19: 2227
        • Kattman S.J.
        • Witty A.D.
        • Gagliardi M.
        • Dubois N.C.
        • Niapour M.
        • Hotta A.
        • et al.
        Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines.
        Cell Stem cell. 2011; 8: 228-240
        • Sasaki H.
        Position-and polarity-dependent Hippo signaling regulates cell fates in preimplantation mouse embryos.
        Semin Cell Dev Biol. 2015; 47-48: 80-87
        • Yue C.
        • Chen A.C.H.
        • Tian S.
        • Fong S.W.
        • Lee K.C.
        • Zhang J.
        • et al.
        Human embryonic stem cell–derived blastocyst-like spheroids resemble human trophectoderm during early implantation process.
        Fertil Steril. 2020;
        • O'Brien J.
        • Hayder H.
        • Zayed Y.
        • Peng C.
        Overview of microRNA biogenesis, mechanisms of actions, and circulation.
        Front Endocrinol (Lausanne). 2018; 9: 402
        • Hanna J.
        • Hossain G.S.
        • Kocerha J.
        The potential for microRNA therapeutics and clinical research.
        Front Genet. 2019; 10: 478