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DNA methylome reveals cellular origin of cell-free DNA in spent medium of human preimplantation embryos.

ABSTRACT: The discovery of embryonic cell-free DNA (cfDNA) in spent embryo culture media (SECM) has brought hope for noninvasive preimplantation genetic testing. However, the cellular origins of SECM cfDNA are not sufficiently understood, and methods for determining maternal DNA contamination are limited. Here, we performed whole-genome DNA methylation sequencing for SECM cfDNA. Our results demonstrated that SECM cfDNA was derived from blastocysts, cumulus cells, and polar bodies. We identified the cumulus-specific differentially methylated regions (DMRs) and oocyte/polar body-specific DMRs, and established an algorithm for deducing the cumulus, polar body, and net maternal DNA contamination ratios in SECM. We showed that DNA methylation sequencing accurately detected chromosome aneuploidy in SECM and distinguished SECM samples with low and high false negative rates and gender discordance rates, after integrating the origin analysis. Our work provides insights into the characterization of embryonic DNA in SECM and provides a perspective for noninvasive preimplantation genetic testing in reproductive medicine.

SUBMITTER: Chen Y

PROVIDER: S-EPMC8203451 | biostudies-literature |

REPOSITORIES: biostudies-literature

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DNA methylation protocol for analyzing cell-free DNA in the spent culture medium of human preimplantation embryos.

Project description:Cell-free DNA (cfDNA) in spent embryo culture media (SECM) provides prospects for noninvasive preimplantation genetic testing. Here, we present a post-bisulfite-adapter-tagging (PBAT)-based whole-genome DNA methylation sequencing protocol (SECM-PBAT) for human SECM cfDNA analysis. We describe steps for SECM lysis, bisulfite conversion and purification, preamplification by random priming, tagging adapter II, and library establishment. We then detail library quality control, sequencing, and bioinformatics analysis. This approach simultaneously detects chromosome aneuploidy and deduces the proportional contributions of cellular components. For complete details on the use and execution of this protocol, please refer to Chen et al. (2021).1.

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Cell-free DNA in spent culture medium effectively reflects the chromosomal status of embryos following culturing beyond implantation compared to trophectoderm biopsy.

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| S-EPMC7877764 | biostudies-literature

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

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Single Cell DNA Methylome Sequencing of Human Preimplantation Embryos

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2017-12-19 | GSE81233 | GEO

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Species and embryo genome origin affect lipid droplets in preimplantation embryos.

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| S-EPMC10217358 | 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

Aberrant DNA methylation reprogramming in bovine SCNT preimplantation embryos.

Project description:DNA methylation reprogramming plays important roles in mammalian embryogenesis. Mammalian somatic cell nuclear transfer (SCNT) embryos with reprogramming defects fail to develop. Thus, we compared DNA methylation reprogramming in preimplantation embryos from bovine SCNT and in vitro fertilization (IVF) and analyzed the influence of vitamin C (VC) on the reprogramming of DNA methylation. The results showed that global DNA methylation followed a typical pattern of demethylation and remethylation in IVF preimplantation embryos; however, the global genome remained hypermethylated in SCNT preimplantation embryos. Compared with the IVF group, locus DNA methylation reprogramming showed three patterns in the SCNT group. First, some pluripotency genes (POU5F1 and NANOG) and repeated elements (satellite I and α-satellite) showed insufficient demethylation and hypermethylation in the SCNT group. Second, a differentially methylated region (DMR) of an imprint control region (ICR) in H19 exhibited excessive demethylation and hypomethylation. Third, some pluripotency genes (CDX2 and SOX2) were hypomethylated in both the IVF and SCNT groups. Additionally, VC improved the DNA methylation reprogramming of satellite I, α-satellite and H19 but not that of POU5F1 and NANOG in SCNT preimplantation embryos. These results indicate that DNA methylation reprogramming was aberrant and that VC influenced DNA methylation reprogramming in SCNT embryos in a locus-specific manner.

| S-EPMC4960566 | 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

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