80 Evaluation of extracellular vesicles from culture medium of human embryos as a possible method of pre-implantation genetic diagnosis

Abstract

Noninvasive methods are the clue to increase the efficiency of invitro-derived embryo selection without decreasing their competence. Embryos selection based on their morphology is the most used method but only 40% of selected embryos are able to implant and develop correctly. In humans, pre-implantation genetic diagnosis increases the efficiency of selection by excluding embryos with chromosomal abnormalities. However, pre-implantation genetic diagnosis needs embryonic cells, which might compromise embryo viability. On the other hand, embryos release extracellular vesicles (EVs: microvesicles and exosomes) to the culture medium that contain biological cargo-like proteins and mRNA lipids, and might contain genomic DNA (gDNA). For this study we evaluated the culture medium from embryos generated by intracytoplasmic sperm injection in a certified fertility clinic. Embryos were cultured in Global Total serum-free medium. The embryos were assessed at Day 3 of development and classified in three categories: top, fair, and poor quality. Corresponding medium was collected for isolation of EVs. The nature of EVs was confirmed by their size and concentration using nanoparticle tracking analysis (NTA), presence of surface markers (CD9, CD63, CD81, and CD40L), and morphology using transmission electron microscopy. A correlation analysis between NTA results (EV size and concentration) and embryo quality was performed. To evaluate chromosomal abnormalities of gDNA present in isolated EVs from embryo culture medium, microarray-based comparative genomic hybridization (aCGH) assay was performed. In a second experiment, aCGH analysis was performed and compared between arrested embryos and EVs isolated from corresponding culture medium. Isolated nanoparticles from embryo culture medium were positive to all markers CD9 (30.9%), CD63 (27.2%), CD81 (31.7%), CD40L (8.7%) and had a morphology accordingly to exosomes. The analysis of NTA data indicated that top-quality embryos had EVs with higher diameter (mean: 112.17nm, mode: 91.74nm) than embryos classified as fair (mean: 108.02nm, mode: 89.67nm) and poor quality (mean: 102.78nm, mode: 88.17 nm; P<0.05). The aCGH analysis showed the representation of the 23 pairs of chromosomes in EVs from culture medium and the chromosomal abnormalities were detected in chromosome 4 (C4: 6/15 (40%)) and chromosome 13 (C13: 6/15 (40%)). In the second experiment, the aCGH assay also showed abnormalities in different chromosomes from samples of EVs from culture medium (24.9%) and were more frequent than those observed in the arrested embryos (8.7%; P=0.03). However, the rate of similitude in chromosomal abnormalities between EVs and their respective embryo was 70-80%. In conclusion, the size and gDNA of EVs from culture medium might be an alternative to evaluate the competence of human embryos. This research was supported by FONDECYT-1170310 and Corfo 17Cote-72437, Chile.

Omics insights into extracellular vesicles in embryo implantation and their therapeutic utility

“…In recent years, EVs have been detected in ESM and UFs obtained from human [33, 53, 77, 78, 80–82, 124, 125], bovine [55, 73, 79, 126–129], ovine [54, 57], porcine [76, 130], and murine [74, 131] species, suggesting that EVs may be an evolutionarily‐conserved function in reproductive biology (Table 1). The functional spectrum of EVs in reproductive biology is continuously expanding [63, 132–135], implicating EVs in gamete development and function (i.e., sperm activation [136], capacitation [137], acrosome reaction [138, 139], and oocyte maturation [140–142]), fertilisation [143], embryo development [144], implantation [43, 44, 66, 145] and invasion [43, 44, 66], endometrial receptivity [84, 146] and embryo‐maternal crosstalk [147].…”

Exploiting Microfluidics for Extracellular Vesicle Isolation and Characterization: Potential Use for Standardized Embryo Quality Assessment

“…A large number of studies have characterized and investigated the function of EVs in reproduction, including characterization of EVs recovered from male and female reproductive tract fluids such as the prostate (98), epididymis (98), vagina (99), endometrium (100), follicle (101) and oviduct (102,103), and their roles in physiologic and pathologic reproductive processes (103)(104)(105)(106). However, only a few studies have investigated the role of EVs as a pre-implantation embryo quality marker (9,(107)(108)(109)(110)(111)(112)(113). It is important to note that, to date, the only method for investigating mammalian embryo-derived EVs is by using in vitro embryo production and analyzing its culture media.…”

“…Embryonic EVs were also used as a non-invasive method of performing pre-implantation genomic testing (113), as they were shown to contain genomic DNA (gDNA). By using microarraybased comparative genomic hybridization, EV gDNA was evaluated, and a representation of all 23 pairs of chromosomes was present (113).…”

“…Embryonic EVs were also used as a non-invasive method of performing pre-implantation genomic testing (113), as they were shown to contain genomic DNA (gDNA). By using microarraybased comparative genomic hybridization, EV gDNA was evaluated, and a representation of all 23 pairs of chromosomes was present (113). However, when comparing with embryonic biopsy (cell extracted gDNA), the rate of similitude between chromosome abnormalities detected in individually cultured embryos and their respective EVs was 70-80% (113).…”

“…By using microarraybased comparative genomic hybridization, EV gDNA was evaluated, and a representation of all 23 pairs of chromosomes was present (113). However, when comparing with embryonic biopsy (cell extracted gDNA), the rate of similitude between chromosome abnormalities detected in individually cultured embryos and their respective EVs was 70-80% (113). In addition to gDNA and miRNA analysis, the characterization of embryonic EV proteins can also serve as a quality biomarker.…”