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

2020 ◽  
Vol 32 (2) ◽  
pp. 166
Author(s):  
C. Aguilera ◽  
D. Veraguas ◽  
C. Henriquez ◽  
A. Velasquez ◽  
F. O. Castro ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Embryo Culture ◽  
Culture Medium ◽  
Chromosomal Abnormalities ◽  
Genetic Diagnosis ◽  
Poor Quality ◽  
Human Embryos ◽  
Acgh Analysis ◽  
Embryo Culture Medium ◽  
Efficiency Of Selection

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.

Evaluation of extracellular vesicles and gDNA from culture medium as a possible indicator of developmental competence in human embryos

Zygote ◽  
2020 ◽  
pp. 1-12
Author(s):  
Daniel Veraguas ◽  
Constanza Aguilera ◽  
Carlos Henriquez ◽  
Alejandra E. Velasquez ◽  
Barbara Melo-Baez ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Culture Medium ◽  
Chromosomal Abnormalities ◽  
Poor Quality ◽  
Developmental Competence ◽  
Human Embryos ◽  
Comparative Genomic ◽  
Acgh Analysis ◽  
High Incidence

Summary Human embryos generated in vitro have a high incidence of chromosomal abnormalities that negatively affect pregnancy rate. Embryos generated in vitro secrete extracellular vesicles (EVs) into the culture medium that could be used potentially as indicators of embryo competence. This research aimed to evaluate the concentration and size of EVs and their gDNA content as an indicator of developmental competence in human embryos. Human embryos generated by intracytoplasmic sperm injection (ICSI) were classified morphologically as of either TOP, FAIR or POOR quality. Culture medium and developmentally arrested embryos (which were not able to be used for embryo transfer) were collected. Microvesicles, exosomes (MV/Exo) and apoptotic bodies (ABs) were isolated from culture medium. Nanoparticle tracking analysis (NTA) and array comparative genomic hybridization (aCGH) analysis were performed to evaluate EVs and their gDNA content. From NTA, the diameter (mean) of MVs/Exo from TOP quality embryos was higher (112.17 nm) compared with that of FAIR (108.02) and POOR quality embryos (102.78 nm) (P < 0.05). aCGH analysis indicated that MVs/Exo and ABs carried gDNA with the presence of 23 chromosome pairs. However, when arrested embryos were compared with their respective MVs/Exo and ABs, the latter had an increased rate of chromosomal abnormalities (24.9%) compared with embryos (8.7%) (P < 0.05). In conclusion, the size of EVs from culture medium might be an alternative for evaluating competence of human embryos, however more studies are needed to validate the use of gDNA from EVs as an indicator of embryo competence.

scholarly journals GC-MS/MS analysis of metabolites derived from a single human blastocyst

Metabolomics ◽  
2021 ◽  
Vol 17 (2) ◽  
Author(s):  
Naomi Inoue ◽  
Yoshihiro Nishida ◽  
Emi Harada ◽  
Kumiko Sakai ◽  
Hisashi Narahara
Keyword(s):  
Culture Medium ◽  
Embryo Quality ◽  
Poor Quality ◽  
Human Embryos ◽  
Non Invasive ◽  
Embryo Culture Medium ◽  
Invasive Method ◽  
Acid Metabolites ◽  
Branched Chain ◽  
Method Of Assessment

Abstract Introduction The field of assisted reproductive technology (ART) has significantly advanced; however, morphological evaluation remains as the chosen method of assessment of embryo quality. Objective We aimed to examine metabolic changes in embryo culture medium to develop a non-invasive method for evaluation of embryo quality. Methods We performed metabolic analysis of culture medium obtained from a single blastocyst cultured for freezing. Results In total, 187 (39.8%) of the 469 detectable organic acid metabolites were identified. A significant change (p < 0.05) was observed in eight metabolites between the good-quality and poor-quality embryo groups. Differences were observed in several metabolic pathways between the good-quality and poor-quality embryo groups. Metabolites that showed significant changes were primarily involved in the metabolism of branched-chain amino acids. Conclusion The quantification of metabolism in human embryos may assist in identification and selection of good-quality embryos with high rates of survival before freezing and implantation in conjunction with morphological classification. This may help to identify embryos with high rates of survival.

Oxygen tension modulates extracellular vesicles and its miRNA contents in bovine embryo culture medium

2019 ◽  
Vol 86 (8) ◽  
pp. 1067-1080 ◽  
Author(s):  
Gabriella Mamede Andrade ◽  
Monalisa Medrado Bomfim ◽  
Maite del Collado ◽  
Flávio Vieira Meirelles ◽  
Felipe Perecin ◽  
...  
Keyword(s):  
Oxygen Tension ◽  
Extracellular Vesicles ◽  
Embryo Culture ◽  
Culture Medium ◽  
Bovine Embryo ◽  
Embryo Culture Medium

scholarly journals Isolation and Characterization of Functionally Active Extracellular Vesicles from Culture Medium Conditioned by Bovine Embryos In Vitro

2018 ◽  
Vol 20 (1) ◽  
pp. 38 ◽  
Author(s):  
Krishna Pavani ◽  
An Hendrix ◽  
Wim Van Den Broeck ◽  
Liesbeth Couck ◽  
Katarzyna Szymanska ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Embryo Culture ◽  
Culture Medium ◽  
Culture Media ◽  
Apoptotic Cell ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Isolation And Characterization ◽  
Embryo Culture Medium

Extracellular vesicles (EVs) play a possible role in cell–cell communication and are found in various body fluids and cell conditioned culture media. The aim of this study was to isolate and characterize EVs in culture medium conditioned by bovine embryos in group and to verify if these EVs are functionally active. Initially, ultracentrifuged bovine serum albumin (BSA) containing medium was selected as suitable EV-free embryo culture medium. Next, EVs were isolated from embryo conditioned culture medium by OptiPrepTM density gradient ultracentrifugation. Isolated EVs were characterized by nanoparticle tracking analysis, western blotting, transmission, and immunoelectron microscopy. Bovine embryo-derived EVs were sizing between 25–230 nm with an average concentration of 236.5 ± 1.27 × 108 particles/mL. Moreover, PKH67 EV pre-labeling showed that embryo-secreted EVs were uptaken by zona-intact bovine embryos. Since BSA did not appear to be a contaminating EV source in culture medium, EV functionality was tested in BSA containing medium. Individual embryo culture in BSA medium enriched with EVs derived from conditioned embryo culture medium showed significantly higher blastocyst rates at day 7 and 8 together with a significantly lower apoptotic cell ratio. In conclusion, our study shows that EVs play an important role in inter embryo communication during bovine embryo culture in group.

scholarly journals Noninvasive chromosome screening of human embryos by genome sequencing of embryo culture medium for in vitro fertilization

2016 ◽  
Vol 113 (42) ◽  
pp. 11907-11912 ◽  
Author(s):  
Juanjuan Xu ◽  
Rui Fang ◽  
Li Chen ◽  
Daozhen Chen ◽  
Jian-Ping Xiao ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Culture Medium ◽  
Chromosomal Abnormalities ◽  
Preimplantation Genetic Screening ◽  
Human Embryos ◽  
Balanced Translocation ◽  
Vitro Fertilization ◽  
Embryo Culture Medium ◽  
Next Generation Sequencing Ngs

Preimplantation genetic screening (PGS) is widely used to select in vitro-fertilized embryos free of chromosomal abnormalities and to improve the clinical outcome of in vitro fertilization (IVF). A disadvantage of PGS is that it requires biopsy of the preimplantation human embryo, which can limit the clinical applicability of PGS due to the invasiveness and complexity of the process. Here, we present and validate a noninvasive chromosome screening (NICS) method based on sequencing the genomic DNA secreted into the culture medium from the human blastocyst. By using multiple annealing and looping-based amplification cycles (MALBAC) for whole-genome amplification (WGA), we performed next-generation sequencing (NGS) on the spent culture medium used to culture human blastocysts (n = 42) and obtained the ploidy information of all 24 chromosomes. We validated these results by comparing each with their corresponding whole donated embryo and obtained a high correlation for identification of chromosomal abnormalities (sensitivity, 0.882, and specificity, 0.840). With this validated NICS method, we performed chromosome screening on IVF embryos from seven couples with balanced translocation, azoospermia, or recurrent pregnancy loss. Six of them achieved successful clinical pregnancies, and five have already achieved healthy live births thus far. The NICS method avoids the need for embryo biopsy and therefore substantially increases the safety of its use. The method has the potential of much wider chromosome screening applicability in clinical IVF, due to its high accuracy and noninvasiveness.

Infection in embryo culture medium

2021 ◽  
pp. 509-514
Author(s):  
Alison Campbell ◽  
Louise Best
Keyword(s):  
Embryo Culture ◽  
Culture Medium ◽  
Embryo Culture Medium

Sample preparations are important for fluorescence in situ hybridization of cells biopsied from preimplantation embryos

Zygote ◽  
2013 ◽  
Vol 22 (3) ◽  
pp. 300-304
Author(s):  
Lifei Li ◽  
Xuehong Zhang ◽  
Weihua Wang
Keyword(s):  
Sample Preparation ◽  
Chromosomal Abnormalities ◽  
Genetic Diagnosis ◽  
Preimplantation Embryos ◽  
Human Embryos ◽  
Fixation Time ◽  
Preparation Technique ◽  
Hypotonic Solution ◽  
Modified Method

SummaryFluorescence in situ hybridization (FISH) is a cytogenetic technology used to detect chromosomal abnormalities in preimplantation human embryos. However, its efficiency is not stable due to improper sample preparation. The present study was designed to modify the current sample preparation technique and then to evaluate its efficiency in human preimplantation genetic diagnosis (PGD). Day 3 cleavage embryos as well as day 5 and 6 blastocysts were biopsied by mechanical aspiration method. In the present study, two methods were used for sample preparation of the biopsied cells. Method I was the traditional method, in which each blastomere was placed in a hypotonic solution for 5 min and then fixed on glass slides. The slides were kept at room temperature before the FISH procedures. Method II was a modified method, in which all blastomeres were placed individually in hypotonic solution drops covered by oil for at least 5 min and then fixed on slides with 0.1% Tween/HCl. After fixation, the slides were kept at –20°C for at least 30 min before the FISH procedures. The two methods were compared in terms of time consumption and proportions of blastomeres with FISH signals. In total, 329 blastomeres from day 3 embryos were fixed by Method I with an average fixation time of 8–10 min for each blastomere. By contrast, with Method II, 362 blastomeres were fixed and the average time was 3–4 min for each blastomere. After FISH, more nuclei had signals with Method II (97.2%) than with Method I (86.9%). All cells that were biopsied from blastocysts and prepared with Method II had FISH signals. However, Method I was not suitable for the fixation of multiple cells biopsied from blastocysts as cells were not traceable during the fixation. The present study indicates that proper sample preparation is critical for obtaining FISH signals in cells biopsied from preimplantation human embryos; hence these modifications can increase the efficiency of human PGD.

Sign in / Sign up

Export Citation Format

Share Document