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.
Highlights
Notwithstanding the significant progresses achieved in the last decade, low implantation rate per transferred embryo, likely stemming from a suboptimal uterine receptivity, still remains a major problem in assisted reproductive techniques (ART) [1]
Considering that the preanalytical variability is the major source of diagnostic uncertainty, providing SOPs for sample storage and processing is fundamental to enable proper biomarker discovery and validation studies and subsequent diagnostics development
Identification of proper markers identifying vesicles coming from the target tissue constitutes the prerogative for full leveraging of advantages of extracellular vesicles (EVs) as biomarkers in physiology and pathology conditions
Summary
Notwithstanding the significant progresses achieved in the last decade, low implantation rate per transferred embryo, likely stemming from a suboptimal uterine receptivity, still remains a major problem in assisted reproductive techniques (ART) [1]. Several factors, secreted by the endometrium into uterine fluid, control implantation by either directly affecting blastocyst development and/or by modulating the expression of key adhesion molecules. Some of these are sorted from endosomal compartments into secretory exosomes/EVs and are delivered to target tissues in a paracrine (blastocyst) and autocrine manner (endometrium itself) in a selective and specific manner. According to their size and biogenesis, EVs are broadly classified into: (i) exosomes (EXs), 30 to 100 nm in size, originated from endosomal compartment of the cell; (ii) microvesicles (MVs), ranging from 100 to 1000 nm, released from budding and fission of the plasma membrane
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