Clues to Non-Invasive Implantation Window Monitoring: Isolation and Characterisation of Endometrial Exosomes

Abstract

Despite the significant advances in the last decades, low implantation rate per transferred embryo still remains a major concern in assisted reproductive techniques, highlighting a need to better characterize endometrial receptivity also by mean of specific biomarkers. Based on physiology and on the intimate contact with endometrium as the tissue of interest, in this study we developed and validated an optimized protocol that uses extracellular vesicles (EVs) recovered from uterine flushings and from a cervical brush, the latter never used until now as an EVs source, as surrogates for endometrial biopsies. This method combines the safety of sampling with the ability to study the expression profile across the uterine cycle. We have compared the yield and composition of EVs recovered from different biofluids samples and fractions thereof, opting for chemical precipitation as the EV isolation procedure, assuring the highest yield without introducing any bias in specific EV recovery. Moreover, collected EVs, in particular exosome-like vesicles, express putative endometrial markers, such as glycodelin A and receptors for estrogen and progesterone, thus confirming their endometrial origin. We also identified uterine flushing EVs, in particular those recovered from its mucous fraction, as the richest source of endometrial transcripts, likely correlated to cellular (epithelial) origin of these vesicles. Finally, our pilot quantitative assessment of three endometrial gene profiles, in samples collected at different time points along the luteal phase, revealed the fluctuations apparently recapitulating gene expression variability prior reported during the menstrual cycle. Unlike tissue biopsy that is subjected to inter- and intra-sample differences, our data suggest that EVs from liquid biopsies (from uterine flushings and a cervical brush) obtained through less-invasive procedures, can be substrate to detect and track the tissue representative expression profiles, better depicting the total endometrium complexity.

Global transcriptomic changes occur in uterine fluid-derived extracellular vesicles during the endometrial window for embryo implantation

“…UF samples were obtained by lavage of the endometrial cavity with 2.5 ml of sterile saline solution using a balloon hysterosonography catheter to avoid vaginal contamination. As previously reported, recovered volume may vary (from 0.8 to 1.8 ml in our series) ( Luddi et al , 2019 ). For EVs isolation, UFs were freshly processed to avoid freezing/thawing cycles.…”

“…In this study, we sought to determine the RNA content of EVs released in the uterine microenvironment in order to understand its potential value as a proxy of endometrial receptivity status. Purified EV populations containing exosomes have been previously isolated and characterized in uterine fluid ( Ng et al , 2013 ; Vilella et al , 2015 ; Luddi et al , 2019 ). Different protocols for their isolation have been assessed ( Vilella et al , 2015 ; Campoy et al , 2016 ) and the standard procedure by differential centrifugation was found as the best approach in terms of providing sufficient materials for proteomic and transcriptomic analyses.…”

Extracellular vesicles in embryo implantation and disorders of the endometrium

“…Proteome profiling of the uterine fluid that contains EVs have identified a panel of proteins that has specificity and sensitivity of 91.7% for distinguishing proliferative and receptive phase samples; the panel can also differentiate RIF non‐invasively with 91.7% specificity and 96.6% sensitivity 34 . In another study, EVs have been isolated from uterine flushes and cervical brush and characterized using molecular markers 112 . These EVs express putative endometrial markers, such as glycodelin A and receptors for oestrogen and progesterone, thus, confirming their endometrial origin, the levels of these markers vary by stage of the cycle 112 …”

Isolation and Characterization of Equine Uterine Extracellular Vesicles: A Comparative Methodological Study

“…In the mare, little is known about the uterine EVs and their contribution to embryo-maternal interactions leading to successful pregnancy. Due to the very unique features of reproduction in equids, current results from other mammalian species such as mouse, sheep, bovine, pig or human [ 14 , 23 , 24 , 25 ], cannot be simply applied to horses. Studies in the mare have focused on EVs from follicular fluid and their miRNAs and protein cargo, as a possible new form of cell communication within the ovarian follicle [ 26 ].…”