Exosome-like vesicles in uterine aspirates: a comparison of ultracentrifugation-based isolation protocols

Background: Uterine aspirates are used in the diagnostic process of endometrial disorders, yet further applications could emerge if its complex milieu was simplified. Exosome-like vesicles isolated from uterine aspirates could become an attractive source of biomarkers, but there is a need to standardize isolation protocols. The objective of the study was to determine whether exosome-like vesicles exist in the fluid fraction of uterine aspirates and to compare protocols for their isolation, characterization, and analysis. Methods: We collected uterine aspirates from 39 pre-menopausal women suffering from benign gynecological diseases. The fluid fraction of 27 of those aspirates were pooled and split into equal volumes to evaluate three differential centrifugation-based procedures: (1) a standard protocol, (2) a filtration protocol, and (3) a sucrose cushion protocol. Characterization of isolated vesicles was assessed by electron microscopy, nanoparticle tracking analysis and immunoblot. Specifically for RNA material, we evaluate the effect of sonication and RNase A treatment at different steps of the protocol. We finally confirmed the efficiency of the selected methods in non-pooled samples. Results: All protocols were useful to isolate exosome-like vesicles. However, the Standard procedure was the best performing protocol to isolate exosome-like vesicles from uterine aspirates: nanoparticle tracking analysis revealed a higher concentration of vesicles with a mode of 135 +/- 5 nm, and immunoblot showed a higher expression of exosome-related markers (CD9, CD63, and CD81) thus verifying an enrichment in this type of vesicles. RNA contained in exosome-like vesicles was successfully extracted with no sonication treatment and exogenous nucleic acids digestion with RNaseA, allowing the analysis of the specific inner cargo by Real-Time qPCR. Conclusion: We confirmed the existence of exosome-like vesicles in the fluid fraction of uterine aspirates. They were successfully isolated by differential centrifugation giving sufficient proteomic and transcriptomic material for further analyses. The Standard protocol was the best performing procedure since the other two tested protocols did not ameliorate neither yield nor purity of exosome-like vesicles. This study contributes to establishing the basis for future comparative studies to foster the field of biomarker research in gynecology

Exosome-like vesicles in uterine aspirates : a comparison of ultracentrifugation-based isolation protocols

Altres ajuts: This work was supported by the "Fondo Europeo de Desarrollo Regional" FEDER (RTC-2014-3110-1), the AECC (Grupos Estables de Investigacion 2011 - AECC- GCB 110333 REVE), the Fundació La Marató TV3 (2/C/2013).Altres ajuts: MSCBS/RD12-0036-0035Uterine aspirates are used in the diagnostic process of endometrial disorders, yet further applications could emerge if its complex milieu was simplified. Exosome-like vesicles isolated from uterine aspirates could become an attractive source of biomarkers, but there is a need to standardize isolation protocols. The objective of the study was to determine whether exosome-like vesicles exist in the fluid fraction of uterine aspirates and to compare protocols for their isolation, characterization, and analysis. We collected uterine aspirates from 39 pre-menopausal women suffering from benign gynecological diseases. The fluid fraction of 27 of those aspirates were pooled and split into equal volumes to evaluate three differential centrifugation-based procedures: (1) a standard protocol, (2) a filtration protocol, and (3) a sucrose cushion protocol. Characterization of isolated vesicles was assessed by electron microscopy, nanoparticle tracking analysis and immunoblot. Specifically for RNA material, we evaluate the effect of sonication and RNase A treatment at different steps of the protocol. We finally confirmed the efficiency of the selected methods in non-pooled samples. All protocols were useful to isolate exosome-like vesicles. However, the Standard procedure was the best performing protocol to isolate exosome-like vesicles from uterine aspirates: nanoparticle tracking analysis revealed a higher concentration of vesicles with a mode of 135 ± 5 nm, and immunoblot showed a higher expression of exosome-related markers (CD9, CD63, and CD81) thus verifying an enrichment in this type of vesicles. RNA contained in exosome-like vesicles was successfully extracted with no sonication treatment and exogenous nucleic acids digestion with RNaseA, allowing the analysis of the specific inner cargo by Real-Time qPCR. We confirmed the existence of exosome-like vesicles in the fluid fraction of uterine aspirates. They were successfully isolated by differential centrifugation giving sufficient proteomic and transcriptomic material for further analyses. The Standard protocol was the best performing procedure since the other two tested protocols did not ameliorate neither yield nor purity of exosome-like vesicles. This study contributes to establishing the basis for future comparative studies to foster the field of biomarker research in gynecology. The online version of this article (doi:10.1186/s12967-016-0935-4) contains supplementary material, which is available to authorized users

Estadificación prequirúrgica del carcinoma endometrial mediante ecografía en dos y tres dimensiones

Objectius: El nostre objectiu era comparar el rendiment diagnòstic de l'ecografia transvaginal (ETV) i la ressonància magnètica (RM) per predir la profunditat de la invasió miometrial (IMP) i la invasió estromal cervical (IEC) en pacients amb càncer d'endometri (CE). Mètodes: Estudi prospectiu i consecutiu que inclou totes les pacients amb CE diagnosticades entre l'octubre del 2013 i el juliol del 2018 a l'Hospital Vall d'Hebron de Barcelona. Es va realitzar una estadificació preoperatòria amb ETV i ressonància magnètica seguida d'una estadificació quirúrgica. La histologia final es va considerar com a estàndard de referència. Es van calcular la sensibilitat, l'especificitat, les raons de verosimilitut i la precisió diagnòstica per a les dues tècniques d'imatge per a la predicció de la IMP i la IEC. Es va calcular l'índex d'acord. Es van utilitzar les directrius STARD 2015. Resultats: Es van incloure un total de 177 pacients consecutivament. La sensibilitat va ser major per a ETV en comparació amb la ressonància magnètica, tant per a la predicció de IMP (68% (95% IC 55-79) enfront del 60% (95% IC 47-72), respectivament) com de IEC (49% (95% CI 35- 62) contra el 29% (IC del 95% 18-43), respectivament). Les especificitats van ser similars per a la predicció de IMP (ETV 84% (95% CI 77-90) i ressonància magnètica 92% (95% CI 85-96)) i iguals per a IEC (95% (95% CI 90-98). L'índex d'acord entre ETV i RN va ser de 0,74 per a IMP i 0,99 per a IEC. Conclusions: El rendiment diagnòstic de la ETV no és inferior a la ressonància magnètica per a la predicció de IMP i IEC en el carcinoma endometrial i pot tenir un paper com a tècnica d'imatge de primera línia en l'avaluació preoperatòria del càncer d'endometri.Objetivos: Nuestro objetivo fue comparar el rendimiento diagnóstico de la ecografía transvaginal (ETV) y la resonancia magnética (RM) para predecir la profundidad de la invasión del miometrio (IMP) y la invasión del estroma cervical (IEC) en pacientes con cáncer de endometrio (CE). Métodos: Estudio prospectivo y consecutivo que incluye todas las pacientes con CE diagnosticadas entre octubre de 2013 y julio de 2018 en el Hospital Vall d'Hebron de Barcelona. La estadificación preoperatoria se realizó con ETV y RM seguida de estadificación quirúrgica. La histología final se consideró como estándar de referencia. Se calcularon la sensibilidad, la especificidad, las razones de probabilidad y la precisión diagnóstica para ambas técnicas de imagen para la predicción de IMP y IEC. Se calculó el índice de concordancia. Se utilizaron las pautas STARD 2015. Resultados: Se incluyeron un total de 177 pacientes de forma consecutiva. La sensibilidad fue mayor para ETV en comparación con RM tanto para la predicción de IMP (68% (95% CI 55-79) versus 60% (95% CI 47-72), respectivamente) como para la predicción de IEC (49% (95% CI 35- 62) versus 29% (IC 95% 18-43), respectivamente). Las especificidades fueron similares para la predicción de IMP (ETV 84% (95% CI 77-90) y ETV 92% (95% CI 85-96)) e iguales para IEC (95% (95% CI 90-98). El índice de acuerdo entre ETV y RM fue 0,74 para IMP y 0,99 para IEC. Conclusiones: El rendimiento diagnóstico de ETV no es inferior a la RM para la predicción de IMP y la IEC en el carcinoma de endometrio y puede desempeñar un papel como técnica de imagen de primera línea en la evaluación preoperatoria del cáncer de endometrio.Objectives: We aimed to compare the diagnostic performance of transvaginal ultrasound (TVUS) and magnetic resonance imaging (MRI) to predict the depth of myometrial invasion (DMI) and cervical stromal invasion (CSI) in patients with endometrial cancer (EC). Methods: Prospective and consecutive study including all EC diagnosed between October 2013 and July 2018 at the Vall d'Hebron Hospital in Barcelona. Preoperative staging was performed with TVUS and MRI followed by surgical staging. Final histology was considered as the reference standard. Sensitivity, specificity, likelihood ratios and diagnostic accuracy were calculated for both imaging techniques for the prediction of DMI and CSI. Agreement index was calculated. The STARD 2015 guidelines were used. Results: A total of 177 patients were consecutively included. Sensitivity was higher for TVUS compared to MRI both for the prediction of DMI (68% (95%CI 55-79) versus 60% (95%CI 47-72), respectively) and CSI (49% (95%CI 35-62) versus 29% (95%CI 18-43), respectively). Specificities were similar for the prediction of DMI (TVUS 84% (95%CI 77-90) and MRI 92% (95%CI 85-96)) and equal for CSI (95% (95%CI 90-98). The agreement index between TVUS and MRI was 0.74 for DMI and 0.99 for CSI. Conclusions: The diagnostic performance of TVUS is not inferior to MRI for the prediction of DMI and CSI in EC and can play a role as a first line imaging technique in the preoperative evaluation of endometrial cancer.Universitat Autònoma de Barcelona. Programa de Doctorat en Pediatria, Obstetrícia i Ginecologi

Hysteroscopic myomectomy without anesthesia

Scarce literature about myoma removal without anesthesia has been published. The aim of this paper is to evaluate the feasibility of a new alternative for a hysteroscopic myomectomy in a conventional office setting, without need for anesthesia. Step-by-step description of the surgical technique has been provided, based on video images. An office hysteroscopy was performed in a Gynecological Endoscopy Department of a tertiary European hospital. A 49-year-old woman was referred for management of severe hypermenorrhea. Consent and approval were received from the patient and the institutional review board, respectively. The introduction of a Truclear ® hysteroscopic polyp morcellator of 5.5 mm with optic of 0 degrees into the uterine cavity did not require any kind of anesthesia or cervical dilatation. The use of saline flow helped distend the cavity and identify a submucosal myoma. Under direct vision, a full myomectomy was performed via mechanical energy with continuous cutting movements, without any complication. After the procedure was completed, the excised material was aspirated through the device into a collecting pouch. A successful complete morcellation of a Type-0 submucosal leiomyoma with a polyp morcellator device was performed in an outpatient setting. Good medical results, good tolerance by the patient besides lower surgical risks due to mechanical instead of electrical energy are shown. In conclusion, this video demonstrates that a hysteroscopic myomectomy can be performed successfully in office with lower risk of complications from the procedure and without use of general anesthesia besides good tolerance by the patient

Exosome-like vesicles in uterine aspirates: a comparison of ultracentrifugation-based isolation protocols

Background: Uterine aspirates are used in the diagnostic process of endometrial disorders, yet further applications could emerge if its complex milieu was simplified. Exosome-like vesicles isolated from uterine aspirates could become an attractive source of biomarkers, but there is a need to standardize isolation protocols. The objective of the study was to determine whether exosome-like vesicles exist in the fluid fraction of uterine aspirates and to compare protocols for their isolation, characterization, and analysis. Methods: We collected uterine aspirates from 39 pre-menopausal women suffering from benign gynecological diseases. The fluid fraction of 27 of those aspirates were pooled and split into equal volumes to evaluate three differential centrifugation-based procedures: (1) a standard protocol, (2) a filtration protocol, and (3) a sucrose cushion protocol. Characterization of isolated vesicles was assessed by electron microscopy, nanoparticle tracking analysis and immunoblot. Specifically for RNA material, we evaluate the effect of sonication and RNase A treatment at different steps of the protocol. We finally confirmed the efficiency of the selected methods in non-pooled samples. Results: All protocols were useful to isolate exosome-like vesicles. However, the Standard procedure was the best performing protocol to isolate exosome-like vesicles from uterine aspirates: nanoparticle tracking analysis revealed a higher concentration of vesicles with a mode of 135 +/- 5 nm, and immunoblot showed a higher expression of exosome-related markers (CD9, CD63, and CD81) thus verifying an enrichment in this type of vesicles. RNA contained in exosome-like vesicles was successfully extracted with no sonication treatment and exogenous nucleic acids digestion with RNaseA, allowing the analysis of the specific inner cargo by Real-Time qPCR. Conclusion: We confirmed the existence of exosome-like vesicles in the fluid fraction of uterine aspirates. They were successfully isolated by differential centrifugation giving sufficient proteomic and transcriptomic material for further analyses. The Standard protocol was the best performing procedure since the other two tested protocols did not ameliorate neither yield nor purity of exosome-like vesicles. This study contributes to establishing the basis for future comparative studies to foster the field of biomarker research in gynecology

Exosome-like vesicles in uterine aspirates: a comparison of ultracentrifugation-based isolation protocols

Background: Uterine aspirates are used in the diagnostic process of endometrial disorders, yet further applications could emerge if its complex milieu was simplified. Exosome-like vesicles isolated from uterine aspirates could become an attractive source of biomarkers, but there is a need to standardize isolation protocols. The objective of the study was to determine whether exosome-like vesicles exist in the fluid fraction of uterine aspirates and to compare protocols for their isolation, characterization, and analysis. Methods: We collected uterine aspirates from 39 pre-menopausal women suffering from benign gynecological diseases. The fluid fraction of 27 of those aspirates were pooled and split into equal volumes to evaluate three differential centrifugation-based procedures: (1) a standard protocol, (2) a filtration protocol, and (3) a sucrose cushion protocol. Characterization of isolated vesicles was assessed by electron microscopy, nanoparticle tracking analysis and immunoblot. Specifically for RNA material, we evaluate the effect of sonication and RNase A treatment at different steps of the protocol. We finally confirmed the efficiency of the selected methods in non-pooled samples. Results: All protocols were useful to isolate exosome-like vesicles. However, the Standard procedure was the best performing protocol to isolate exosome-like vesicles from uterine aspirates: nanoparticle tracking analysis revealed a higher concentration of vesicles with a mode of 135 +/- 5 nm, and immunoblot showed a higher expression of exosome-related markers (CD9, CD63, and CD81) thus verifying an enrichment in this type of vesicles. RNA contained in exosome-like vesicles was successfully extracted with no sonication treatment and exogenous nucleic acids digestion with RNaseA, allowing the analysis of the specific inner cargo by Real-Time qPCR. Conclusion: We confirmed the existence of exosome-like vesicles in the fluid fraction of uterine aspirates. They were successfully isolated by differential centrifugation giving sufficient proteomic and transcriptomic material for further analyses. The Standard protocol was the best performing procedure since the other two tested protocols did not ameliorate neither yield nor purity of exosome-like vesicles. This study contributes to establishing the basis for future comparative studies to foster the field of biomarker research in gynecology