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

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.

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

Reproduction Fertility and Development ◽

10.1071/rdv32n2ab80 ◽

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.

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P–275 Development of a prediction model using machine learning on small noncoding RNA biomarkers for non-invasive selection of high-quality embryos for the in vitro fertilization process

Human Reproduction ◽

10.1093/humrep/deab130.274 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

M Rabajdova ◽

K Šoltys ◽

M Kloc ◽

O Slaby ◽

S Toporcerova ◽

...

Keyword(s):

Culture Medium ◽

Embryo Quality ◽

Massively Parallel Sequencing ◽

Culture Media ◽

Predictive Biomarkers ◽

High Quality ◽

Non Invasive ◽

Embryo Culture Medium ◽

Selection Of

Abstract Study question The aim of the study was to identify molecules in the embryo culture medium as important predictive biomarkers of high-quality embryos Summary answer The study identified 14 canonical iso-miRNA molecules that prognostically determine the quality of the embryo with a prediction accuracy with 95% sensitivity and 80% specificity. What is known already The quality of the embryo for the success of the IVF process is not specifically diagnosed, only morphological features (monitoring in the embryoscope) are considered. Embryo quality selection systems have likely reached their peak. The success rate of the IVF process is only 29%.; it is therefore necessary to look for other biomarkers. The oocyte itself can significantly predict the development of the early embryo,as it is a supplier of RNA and cellular mechanisms. However, collection follicular fluid is technically demanding. The probability of oocyte fertilization does not reach the required percentage therefore other embryological techniques multiply the economic costs. Study design, size, duration Women (n = 734) who visited the IVF centre were recruited for the study. Oocytes were collected from 54 of them and used for IVF. After 4/5-day embryo cultivation, the best quality embryo was selected and used for implantation into the uterus. The culture medium has been collected from 60 embryos during 3 years (2018–2020). Written informed consent was obtained from all patients. The study has been approved by the Ethical committee of the Košice governing region Participants/materials, setting, methods We used fresh/frozen culture media of embryos selected using an embryoscope. Further, information regarding the success of IVF, pregnancy and IVF failure was collected. Culture media libraries of noncoding small RNAs (miRNAs) were examined using massively parallel sequencing on the Illumina platform. Obtained data was processed with freely available bioinformatic tools and machine learning. For methods with different models, the number of predictive biomarkers and specific prognostic-predictive molecules were selected. Main results and the role of chance The main results of the study specifically identify ncRNA molecules that prognostically and predictively select a high-quality embryo suitable for IVF transmission from a low-quality embryo with 95% sensitivity and 80% specificity with an average accuracy of 85% in 4 different models. We also determined the minimum of 14 miRNA as prediction biomarkers. The developed model can predict embryo quality from the culture medium based on ncRNA results from sequence data and set the cut-off value for the expression and significance of individual miRNA molecules with respect to embryo quality. Furthermore, positive and negative correlations of miRNA molecules with different distributions in a high-quality embryo compared to a low-quality embryo were determined. The molecules identified in the embryo culture medium were organized according to their importance, resp. significance based on their significance coefficient. So far, there is no evidence of pending patents regarding the distribution of specific canonical miRNAs and iso-miRNA molecules analysed by massively parallel sequencing in terms of biological competence and embryo quality determination with multifactorial consideration of its variation. This is the first study focused on the success of the IVF process based on embryo quality prediction. Limitations, reasons for caution Exploratory data need to be validated in a larger scale study. Wider implications of the findings: The given miRNA molecules and the software model can be used as a safe,non-invasive diagnostic test for the selection of a highly competent embryo. Canonical and iso-miRNA molecules from the study can be used in other forms of diagnostic assays, such as specific embryo selection probes and, plate hybridization assay. Trial registration number non clinical trials

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108-PA10 Ultrasonography (US): a non-invasive method of assessment of the character of pleural effusion (PE)

Tubercle and Lung Disease ◽

10.1016/0962-8479(95)90102-7 ◽

1995 ◽

Vol 76 ◽

pp. 23

Author(s):

J. Gryminski ◽

L. Pawlicka ◽

M. Janowiec ◽

Z. Piasecki

Keyword(s):

Pleural Effusion ◽

Non Invasive ◽

Invasive Method ◽

Method Of Assessment

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Embryo sHLA-G secretion is related to pregnancy rate

Zygote ◽

10.1017/s0967199419000054 ◽

2019 ◽

Vol 27 (02) ◽

pp. 78-81

Author(s):

R. Rodríguez Díaz ◽

R. Blanes Zamora ◽

R. Vaca Sánchez ◽

J. González Pérez ◽

J.C. Alberto Bethencourt

Keyword(s):

Pregnancy Rate ◽

Culture Medium ◽

Embryo Quality ◽

Statistical Significance ◽

Preimplantation Embryos ◽

Enzyme Linked Immunosorbent Assay ◽

Significant Difference ◽

Invasive Method ◽

Group A

SummaryHLA-G expression has been detected in early preimplantation embryos and it has been postulated that a relationship between embryonic expression of this factor and successful pregnancy may exist. Forty-six patients were prospectively selected from our centre ‘Unidad de Reproducción Humana, Hospital Universitario de Canarias’ for conducting this study. In all cases, metaphase II (MII) oocytes were fertilized using intracytoplasmic sperm injection 2–4 h after retrieval. Embryos were cultured individually in 20 µl droplets of G-1 medium (VitroLife) under oil at 37°C and a 6% CO2environment. Fertilization was assessed at 18 h postinsemination and all oocytes fertilized were passed into a new culture plaque individually in 300 µl culture medium until day 3 of culture. The culture medium was examined for the expression and secretion of sHLA-G with a sandwich enzyme-linked immunosorbent assay kit (BioVendor, Heidelberg, Germany) according to the manufacturer’s instructions. We found statistical significance between higher levels of sHLA-G secretion and pregnancy rate. When both groups were compared there was no difference in embryo quality of transferred embryos, but a significant difference in the number of oocytes and the embryo quality of the cohort existed that was greater in the pregnant group. A standardized sHLA-G assay with a specifically defined range and standard units provides a non-invasive method to identify the most competent embryos for transfer.

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