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Spent embryo culture medium metabolites are related to the in vitro attachment ability of blastocysts.

ABSTRACT: The metabolomic profile of an embryo culture medium can aid in the advanced prediction of embryonic developmental potential and genetic integrity. But it is not known if this technology can be used to determine the in vitro potential of inner cell mass (ICM) in adherence and proliferation. Here, we investigated the developmental potential of mouse 2-cell embryos carrying cisplatin-induced DNA lesions (IDL), beyond blastocyst stage using ICM outgrowth assay. The genetic integrity of ICM cells was determined by comet assay. The metabolic signatures of spent medium were recorded 84 hours post injection of hCG (hpi-hCG), and after 96 hours of extended in vitro culture (Ex 96) by NMR spectroscopy. We observed that blastocysts that lack the ability to adhere in vitro had an increased requirement of pyruvate (p < 0.01), lactate (p < 0.01), and were accompanied by a significant reduction of pyruvate-alanine ratio in the culture medium. We propose that the aforementioned metabolites from 84 hpi-hCG spent medium be further explored using appropriate experimental models, to prove their potential as biomarkers in the prediction of implantation ability of in vitro derived human embryos in clinical settings.

SUBMITTER: D'Souza F

PROVIDER: S-EPMC6242932 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Prediction model for day 3 embryo implantation potential based on metabolites in spent embryo culture medium.

Project description:BackgroundMetabolites in spent embryo culture medium correlate with the embryo's viability. However, there is no widely accepted method using metabolite dada to predict successful implantation. We sought to combine metabolomic profiling of spent embryo culture medium and clinical variables to create an implantation prediction model as an adjunct to morphological screening of day 3 embryos.MethodsThis investigation was a prospective, nested case-control study. Forty-two day 3 embryos from 34 patients were transferred, and the spent embryo culture medium was collected. Twenty-two embryos implanted successfully, and the others failed. Metabolites in the medium relevant to implantation were detected and measured by Liquid Chromatography-Mass Spectrometry. Clinical signatures relevant to embryo implantation were subjected to univariate analysis to select candidates for a prediction model. Multivariate logistical regression of the clinical and metabolomic candidates was used to construct a prediction model for embryo implantation potential.ResultsThe levels of 13 metabolites were significantly different between the successful and failed groups, among which five were most relevant and interpretable selected by Least Absolute Shrinkage and Selection Operator regression analysis. None of the clinical variables significantly affected day 3 embryo implantation. The most relevant and interpretable set of metabolites was used to construct a prediction model for day 3 embryo implantation potential with an accuracy of 0.88.ConclusionsDay 3 embryos'implantation potential could be noninvasively predicted by the spent embryo culture medium's metabolites measured by LC-MS. This approach may become a useful adjunct to morphological evaluation of day 3 embryos.

| S-EPMC10249307 | biostudies-literature

Characterization of microRNAs in spent culture medium associated with human embryo quality and development.

Project description:BackgroundGiven implantation failure limits the improvement of in vitro fertilization (IVF) success rates, there is an urgency to identify potential biomarkers for embryo quality and predict the outcomes of IVF-embryo transfer (IVF-ET).MethodsUsing RNA-sequencing, we identified the expression profiles of 16 spent culture medium (SCM) collected from embryos at the cleavage on day 3 (D3 cleavage) and blastocyst stages on day 5 (D5 blastocyst) during IVF cycles. Differentially expressed miRNAs (DEmiRNAs) were then identified, and microRNA (miRNA)-messenger RNA (mRNA) interaction networks were constructed. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) confirmation and validation in the Gene Expression Omnibus (GEO) database were performed.ResultsCompared with the pregnant group, 29 DEmiRNAs were detected in the non-pregnant group at D3 cleavage, and 26 were detected in the non-pregnant group at D5 blastocyst. Among them, a total of six known miRNAs, including hsa-miR-199a-3p>hsa-miR-199b-3p, hsa-miR-199a-5p, hsa-miR-379-5p, hsa-miR-432-5p, hsa-miR-99a-5p, and hsa-miR-483-5p, were identified. The results of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that these target genes of DEmiRNAs were associated with various biological processes (BPs). The results of validation in qRT-PCR and the GEO database suggested the reliability of our RNA-sequencing results.ConclusionsIn conclusion, we identified three miRNAs, including hsa-miR-199a-5p, hsa-miR-483-5p, and hsa-miR-432-5p, which may serve as biomarkers for embryo quality during IVF cycles.

| S-EPMC8667117 | biostudies-literature

Abundance of cell-free mitochondrial DNA in spent culture medium associated with morphokinetics and blastocyst collapse of expanded blastocysts.

Project description:Purpose:This retrospective observational study investigated relationships between the abundance of cell-free mitochondrial DNA (cf-mtDNA) in spent culture medium (SCM) of human-expanded blastocysts and their morphokinetics to address the question of whether the abundance of cf-mtDNA in SCM could predict the quality of blastocysts. Methods:Embryos (n = 53) were individually cultured in a time-lapse incubator until they reached the expanded blastocyst stage (5 or 6 days), following which copy numbers of cf-mtDNA in SCM (20 ?L) of expanded blastocysts were determined using real-time PCR. Results:The duration between start of blastulation to expanded blastocyst (tEB-tSB) and between that of the blastocyst stage to expanded blastocyst (tEB-tB) significantly and positively correlated with the abundance of cf-mtDNA in the SCM (tEB-tSB: r = .46; P < .01; tEB-tB: r = .47; P < .01). The abundance of cf-mtDNA in the SCM was significantly greater in blastocysts with blastocyst collapse (BC), than without BC, and significantly and positively correlated with the number of BC. Conclusions:The abundance of cf-mtDNA in the SCM was associated with expansion duration and BC. Thus, cf-mtDNA abundance in the SCM serves as a marker to predict the quality of expanded blastocysts.

| S-EPMC7542021 | biostudies-literature

Preimplantation Genetic Screening with Spent Culture Medium/Blastocoel Fluid for in Vitro Fertilization.

Project description:Preimplantation genetic screening (PGS) detects chromosomal aneuploidy from DNA extracted from trophectodermal biopsy of the embryos before implantation. Although a controlled study showed no difference in pregnancy rates between this invasive cell biopsy technique and a non-biopsied control group, the potential long-term damage by the current PGS method has not be completely ruled out. We therefore tested a less-invasive protocol which utilizes spent culture medium combining with blastocoel fluid (ECB) to assess chromosomal aneuploidy. We compared the new protocol with the currently employed trophectodermal biopsy method against chromosomal information obtained from the remaining embryo. We found that the new technique generated information about aneuploidy that was not entirely identical to obtained from the biopsied trophectoderm or the remaining embryo. As the origins of the DNA extracted from the three sample types were not the same, the significance and interpretation of each result would have its own meaning. The possible implications derived from the ECB results as well as those from cell biopsy were discussed. The effectiveness of this new approach in selecting the best embryo for uterine implantation awaits further long term evaluation.

| S-EPMC6006313 | biostudies-literature

Transcriptional Profiling of Porcine Blastocysts Produced In Vitro in a Chemically Defined Culture Medium.

Project description:The development of chemically defined media is a growing trend in in vitro embryo production (IVP). Recently, traditional undefined culture medium with bovine serum albumin (BSA) has been successfully replaced by a chemically defined medium using substances with embryotrophic properties such as platelet factor 4 (PF4). Although the use of this medium sustains IVP, the impact of defined media on the embryonic transcriptome has not been fully elucidated. This study analyzed the transcriptome of porcine IVP blastocysts, cultured in defined (PF4 group) and undefined media (BSA group) by microarrays. In vivo-derived blastocysts (IVV group) were used as a standard of maximum embryo quality. The results showed no differentially expressed genes (DEG) between the PF4 and BSA groups. However, a total of 2780 and 2577 DEGs were detected when comparing the PF4 or the BSA group with the IVV group, respectively. Most of these genes were common in both in vitro groups (2132) and present in some enriched pathways, such as cell cycle, lysosome and/or metabolic pathways. These results show that IVP conditions strongly affect embryo transcriptome and that the defined culture medium with PF4 is a guaranteed replacement for traditional culture with BSA.

| S-EPMC8156047 | biostudies-literature

Improved Non-Invasive Preimplantation Genetic Testing for Beta-Thalassemia Using Spent Embryo Culture Medium Containing Blastocoelic Fluid.

Project description:ObjectivesTo compare successful beta-thalassemia (β-thalassemia) detection rates obtained using spent culture medium and spent culture medium containing blastocoelic fluid (BF).MethodThis study involved data from 10 couples who underwent preimplantation genetic testing (PGT) for β-thalassemia. A total of 26 samples of spent culture medium containing BF (group A) and 33 samples without BF (group B) were collected and analyzed. The DNA concentration and β-thalassemia detection rates were evaluated.ResultsThe HBB mutation analysis results of 34 samples were concordant with the biopsy results (34/59, 57.6%). In group A, the HBB mutation analysis results of 19 of 26 samples (73.1%) were concordant with the biopsy results. The concordance rate in group A was higher than that in group B (15/33, 45.5%; P < 0.05). The haplotyping results of 38 samples were concordant with the biopsy results (38/59, 64.4%). The concordance rate in group B was 17/33 (51.5%), which was significantly lower than that in group A (21/26, 80.8%) (P < 0.05). In group A, the mean DNA concentration of samples with <10% fragmentation was 107.3 ± 70.1 ng/μL, which was lower than that of samples with ≥10% fragmentation (194.6 ± 28.0 ng/μL) (P < 0.05). However, the detection rates of <10% and ≥10% fragmentation were not significantly different (P > 0.05).ConclusionThe β-thalassemia detection rate with non-invasive PGT using the spent culture medium containing BF was higher than that using the spent culture medium alone. Fragmentation is associated with DNA concentration in the spent culture medium containing BF.

| S-EPMC8810495 | biostudies-literature

Characterization of microRNAs in spent culture medium associated with human embryo quality and development

Project description:Purpose: Given implantation failure limited the improvement of the in vitro fertilization (IVF) success rate, it was urgent to identify potential biomarkers for embryo quality and predicting the outcomes of IVF-ET. Methods: The expression profiles of 16 spent culture media (SCM) collected from embryos at the cleavage on Day 3 (D3 cleavage) and blastocyst stages on Day 5 (D5 blastocyst) during IVF cycles were determined with RNA-sequencing. Differentially expressed miRNAs (DEmiRNAs) were identified with p-value < 0.05 and |log2FC|> 1. Then, the miRNA-mRNA interaction networks were constructed by using Cytoscape software. Finally, the GO and KEGG pathway analysis of target genes of DEmiRNAs were conducted. Results: Compared with pregnant groups, 29 DEmiRNA were detected in non-pregnant groups at D3 cleavage, and 26 DEmiRNA were detected in non-pregnant group at D5 blastocyst. Among them, a total of six known miRNAs, including hsa-miR-199a-3p>hsa-miR-199b-3p, hsa-miR-199a-5p, hsa-miR-379-5p, hsa-miR-432-5p, hsa-miR-99a-5p and hsa-miR-483-5p, were identified. The results of GO and KEGG pathway analysis indicated that these target genes of DEmiRNAs were associated with various biological processes. Conclusion: In conclusion, we identified three miRNAs, including hsa-miR-199a-5p, hsa-miR-483-5p and hsa-miR-432-5p, may serve as biomarkers for embryo quality during IVF cycles.

2023-10-31 | GSE167961 | GEO

Minimally Invasive Cell-Free Human Embryo Aneuploidy Testing (miPGT-A) Utilizing Combined Spent Embryo Culture Medium and Blastocoel Fluid -Towards Development of a Clinical Assay.

Project description:Preimplantation genetic testing for aneuploidies (PGT-A) using trophectoderm (TE) biopsy samples is labour intensive, invasive, and subject to sampling bias. In this study, we report on the efficacy and factors affecting accuracy of a technique we pioneered for minimally invasive preimplantation genetic testing for aneuploidy (miPGT-A). Our technique uses cell-free embryonic DNA (cfeDNA) in spent embryo culture medium (SEM) combined with blastocoel fluid (BF) to increase the amount of assayable cfeDNA. We compared miPGT-A results (n?=?145 embryos) with standard PGT-A analysis of the corresponding trophectoderm biopsy. We found that accuracy of miPGT was not related to blastocyst morphological grade. The overall concordance rate per sample for euploidy/aneuploidy status between miPGT-A and TE biopsy samples was 88/90 (97.8%), and was not different between good 47/48 (97.9%) and moderate/low quality blastocysts 41/42 (97.9%) (p?>?0.05). Importantly, we also discovered that for cfeDNA analysis, the SurePlex whole genome amplification (WGA) kit can be utilized without an additional cell lysis/extraction DNA step; this efficiency likely reduces the risk of maternal contamination. Regarding origin of embryonic cfeDNA, the average amount of miPGT-A WGA-DNA we obtained from blastocysts with different morphological grades, as well as the size miPGT-A WGA-DNA fragments, suggest that it is unlikely that apoptosis and necrosis are only mechanisms of DNA release from the inner cell mass (ICM) and TE into BF and SEM.

| S-EPMC7190856 | biostudies-literature

Apoptosis triggers the release of microRNA miR-294 in spent culture media of blastocysts.

Project description:PurposeTo study whether members of the miR-290-295 cluster in spent culture medium (SCM) of embryos are correlated with morphokinetics and apoptosis.MethodsCryopreserved 1-cell stage mouse embryos were cultured to the blastocyst stage, development was monitored by time-lapse, 59 SCM were collected, and miR-291a and miR-294 were detected with polymerase chain reaction (PCR). Blastocysts were immuno-stained for sexing (H2AK119ub) and for apoptosis (TUNEL). Each embryo and SCM were individually processed. Correlations were run between the miRNAs and developmental events (t2, t3, t4, t5, t8, tSB, tB, ECC2, ECC3, s2, s3, dB) and apoptosis (apoptotic cells/total cell number %). MiR-294 SCM and cell levels were compared in 40 blastocysts. Apoptosis was induced in 15 blastocysts with UV radiation and SCM samples were analyzed for miR-294.ResultsMiR-291a and miR-294 are released in variable levels by mouse blastocysts. Their release is similar between male and female embryos. No significant correlations were found between these miRNAs and development. MiR-294 was significantly positively correlated with apoptosis (r = 0.560, p < 0.001). Cellular expression was lower in blastocysts that released miR-294 in high levels compared with null, low, and medium release embryos (p < 0.01). UV radiation caused apoptosis which triggered higher secretion of miR-294 in 15 blastocysts versus 13 control embryos (p < 0.01).Conclusion(s)MicroRNAs are important regulators of preimplantation development. Apoptosis triggers the release of miR-294 by blastocysts which possibly serves a secretory role for embryo-maternal communication. SCM miRNA analysis is possible for individually cultured embryos and future studies can investigate miRNAs as noninvasive markers of embryo quality.

| S-EPMC7376808 | biostudies-literature

Does Embryo Culture Medium Influence the Health and Development of Children Born after In Vitro Fertilization?

Project description:In animal studies, extensive data revealed the influence of culture medium on embryonic development, foetal growth and the behaviour of offspring. However, this impact has never been investigated in humans. For the first time, we investigated in depth the effects of embryo culture media on health, growth and development of infants conceived by In Vitro Fertilization until the age of 5 years old. This single-centre cohort study was based on an earlier randomized study. During six months, in vitro fertilization attempts (No. 371) were randomized according to two media (Single Step Medium--SSM group) or Global medium (Global group). This randomized study was stopped prematurely as significantly lower pregnancy and implantation rates were observed in the SSM group. Singletons (No. 73) conceived in the randomized study were included (42 for Global and 31 for SSM). The medical data for gestational, neonatal and early childhood periods were extracted from medical records and parental interviews (256 variables recorded). The developmental profiles of the children in eight domains (social, self-help, gross motor, fine motor, expressive language, language comprehension, letter knowledge and number knowledge--270 items) were compared in relation to the culture medium. The delivery rate was significantly lower in the SSM group than in the Global group (p<0.05). The culture medium had no significant effect on birthweight, risk of malformation (minor and major), growth and the frequency of medical concerns. However, the children of the Global group were less likely than those of the SSM group to show developmental problems (p = 0.002), irrespective of the different domains. In conclusion, our findings showed that the embryo culture medium may have an impact on further development.

| S-EPMC4805279 | biostudies-literature

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