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. 2020 Feb;113(2):364-373.e2.
doi: 10.1016/j.fertnstert.2019.09.032.

Characterization of exosomes in peritoneal fluid of endometriosis patients

Hannah M Nazri  1 , Maria Imran  1 , Roman Fischer  2 , Raphael Heilig  2 , Sanjiv Manek  3 , Rebecca A Dragovic  1 , Benedikt M Kessler  2 , Krina T Zondervan  4 , Thomas T Tapmeier  5 , Christian M Becker  1
Affiliations

Characterization of exosomes in peritoneal fluid of endometriosis patients

Hannah M Nazri et al. Fertil Steril. 2020 Feb.

Abstract
in English, Spanish

Objective: To demonstrate the feasibility of studying exosomes directly from peritoneal fluid, we isolated exosomes from endometriosis patient samples and from controls, and characterized their cargo.

Design: Case-control experimental study.

Setting: Academic clinical center.

Patient (s): Women with and without endometriosis who underwent laparoscopic surgery (n = 28 in total).

Intervention (s): None.

Main outcome measure (s): Concentration of exosomes within peritoneal fluid and protein content of the isolated exosomes.

Result (s): Peritoneal fluid samples were pooled according to the cycle phase and disease stage to form six experimental groups, from which the exosomes were isolated. Exosomes were successfully isolated from peritoneal fluid in all the study groups. The concentration varied with cycle phase and disease stage. Proteomic analysis showed specific proteins in the exosomes derived from endometriosis patients that were absent in the controls. Five proteins were found exclusively in the endometriosis groups: PRDX1, H2A type 2-C, ANXA2, ITIH4, and the tubulin α-chain.

Conclusion (s): Exosomes are present in peritoneal fluid. The characterization of endometriosis-specific exosomes opens up new avenues for the diagnosis and investigation of endometriosis.

Caracterizacion de exosomas en el fluido peritoneal de pacientes con endometriosis

Objectivo: Para demostrar la viabilidad de estudiar de exosomas directamente del líquido peritoneal, aislamos exosomas de muestras de pacientes con endometriosis y de controles y caracterizamos su carga.

Diseño: Estudio experimental caso-control.

Lugar: Centro clínico académico.

Pacientes: Mujeres con y sin endometriosis que se sometían a una laparoscopia quirúrgica (n=28 en total).

Intervención: Ninguna.

Resultados principales: Concentración de exosomas dentro del fluido peritoneal y contenido proteico de los exosomas aislados.

Resultados: Las muestras de líquido peritoneal se agruparon de acuerdo con la fase del ciclo y el estado de la enfermedad para formar seis grupos experimentales, de los cuales se aislaron los exosomas. Los exosomas fueron exitosamente aislados del líquido peritoneal en todos los grupos de estudio. La concentración varió según la fase del ciclo y el estado de la enfermedad. El análisis proteómico mostró proteínas específicas en los exosomas derivados de pacientes con endometriosis que estaban ausentes en los controles. Se encontraron cinco proteínas exclusivas del grupo con endometriosis: PRDX1, H2A tipo 2-C, ANXA2, ITIH4 y la cadena a de tubulina.

Conclusiones: Los exosomas están presentes en el fluido peritoneal. La caracterización de exosomas específicos de endometriosis abre nuevas vías para el diagnostico y la investigación de la endometriosis.

Keywords: Biomarker; endometriosis; exosomes; pathogenesis; peritoneal fluid.

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Figures

Figure 1
Figure 1
Isolation and characterization of exosomes from peritoneal fluid (PF). (A) Exosome isolation protocol. Peritoneal fluid (PF) was centrifuged twice to remove cells, and the supernatant was frozen for batch analysis. Upon thawing, the samples were spun to remove cell debris and larger, nonexosomal particles. The supernatants were pooled according to patient group to have sufficient material for downstream analysis. Exosomes were precipitated, and each pooled sample was fractionated by size exclusion chromatography. Fractions were analyzed for exosome and protein content, and the exosome-rich and protein-poor fractions were reunited as the experimental sample. The volume was adjusted to 700 μL. (B) Sample characteristics as per nanoparticle tracking analysis (NTA) analysis. The mode is the prevalent particle size, with exosome size ranging from 100–200 nm. (C) The size and concentration of exosomes within the groups in B measured by NTA. The analysis was done separately for proliferative and secretory cycle phases. The peaks indicate the presence of exosomes. (D) The comparison of exosome concentrations within samples shows statistically significant differences between cycle phases and disease stages. ****P<.0001, two-way analysis of variance with Tukey’s post-test.
Figure 2
Figure 2
Identification of exosomes within the samples. (A) Transmission electron microscopy images showing the presence of exosomes in all six sample groups. Exosomes typically show an invaginated, cup-shaped morphology as a result of sample preparation. Scale bar: 500 nm. (B) Immunoblotting of the six experimental groups. Protein was extracted from the exosome preparations and blotted with antibodies against syntenin and CD9 as markers of exosomes. Bands demonstrate the presence of exosomes in all groups (C = control; I/II = stage I/II endometriosis; III/IV = stage III/IV endometriosis). HeLa whole cell lysate was used as positive control.
Figure 3
Figure 3
Proteomic analysis of peritoneal fluid (PF) exosome cargo revealing the distinct presence of endometriosis-associated exosomes. Exosomes isolated from the PF of patients and controls were analyzed by liquid chromatography with tandem mass spectrometry according to disease stage. This allowed for the detection of five proteins found in both disease groups but absent in controls. The diagram depicts the number of proteins found in all experimental groups.
Supplemental Figure 1
Supplemental Figure 1
Protein versus particle content in fractionated sample. Plot showing the particle concentration per fraction as measured by nanoparticle tracking analysis (NTA) (black bars) compared with the free protein content (grey bars). The fractions with the highest number of particles (F8–F12) were selected, thus excluding the major portion of free (i.e. not exosome-bound) protein from downstream analysis.
Supplemental Figure 2
Supplemental Figure 2
Human Proteome Map analysis of endometriosis-specific exosome protein expression. The exosome proteins found in endometriosis stage I/II and stage III/IV groups but not in controls were searched within the Human Proteome Map database (43) to indicate their origin.
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