Detection and analysis of DNA material in human blastocoel fluid

Shangguan, Tao; He, Wei; Li, Hongmei; Shang, Xiaoyun; Liu, Yonggang; Bai, Xueyao; Li, Mingduo; Xie, Jiafei

doi:10.15761/bgg.1000128

Cited by 8 publications

(3 citation statements)

References 14 publications

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“…also used similar methods, with an amplification success rate of 72.7% (8/11); the amplification efficiency of hotspot mutation regions of ten randomly selected genes was 84.0% (42/50). In spinal muscular atrophy and phenylpropionate ketoneuria, a total of seven samples had 100% WGA success rates, and the sequences of six BF samples were consistent with their paired TE results [33] . However, in a study by Capalbo et al [26] ,.…”

Section: Cfdna Research Based On Bfmentioning

confidence: 65%

Non-invasive chromosome screening for embryo preimplantation using cell-free DNA

Yao²,

Wang³

et al. 2022

Reproductive and Developmental Medicine

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Preimplantation genetic testing (PGT) is a widely adopted screening method that can be performed to identify and select embryos with normal ploidy; however, PGT relies on embryo biopsy, that is, polar body, embryo cells, or trophectoderm biopsy, to obtain embryonic DNA, increase its technical limitations. Studies have indicated that biopsy may have an influence on the quality and development of embryos, and increase the chance of abnormal epigenetic modifications. Therefore, non-invasive PGT (niPGT) detection of cell-free DNA (cfDNA) has gradually become a hot research topic in the field of assisted reproduction. Studies showed cfDNA could be detected in blastocyst fluid and spent culture medium (SCM) in vitro cultured embryos. The cfDNA collection requires less skill and makes lower risk to embryos. Some studies have been conducted to evaluate the feasibility of SCM-based niPGT approaches. When comparing the ploidy consistency of cfDNA in SCM, its consistency to the conventional PGT for aneuploidies results fluctuated widely, it is critical to recognize the factors influencing accuracy. These contradictory results may be related to factors such as the difference in SCM sampling methods and sampling time, and the definition of consistency. In this review, we aimed to comprehensively summarize how researchers use embryonic cfDNA to conduct niPGT detection. It also systematically reviews the factors affecting the accuracy of the test and its underlying issues, as well as prospective applications. We hope to provide a basis for future niPGT research and a useful reference for the standardized operation of niPGT that can be widely applied in clinical practice.

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Section: Cfdna Research Based On Bfmentioning

confidence: 65%

Non-invasive chromosome screening for embryo preimplantation using cell-free DNA

Yao²,

Wang³

et al. 2022

Reproductive and Developmental Medicine

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“…The few available studies have assessed cfDNA on a very limited number of samples, either directly with the use of quantitative PCR (custom or validated TaqMan genotype assays) for the amplification of multicopy genes on chromosomes 17 and Y or the combined detection of specific gene variants and multiple SNPs surrounding them [ 61 , 82 ]. In other studies, whole genome amplification (PicoPlex or RepliG multiple displacement amplification, MDA) was followed by PCR for the amplification of specific regions of selected genes ( TCIRG1, SCN5A, RHO, EXTL1, SLC4A1, VWF, HSF4, NPC1, PTCH1, EPS8L3, SMN1, SRY, ACTB, PAH ) and validation of a couple of these by sequencing [ 58 , 82 , 83 ]. These studies, reviewed and summarized in a table in Leaver and Wells 2020, have demonstrated the possibility of cfDNA detection in BF but also underlined the limitations associated with variable and low detection rate, even after WGA, ranging from 42.9-84% (much lower in comparison to the ~95% efficiency WGA efficiency achieved from biopsied cells), increased allele dropout, risk of maternal contamination, lack of consistency and reliability [ 84 ].…”

Section: A Look Into the Strategies Employed For Nipgtmentioning

confidence: 99%

An Update on Non-invasive Approaches for Genetic Testing of the Preimplantation Embryo

Kakourou

Mamasa

Vrettou

et al. 2022

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Preimplantation Genetic Testing (PGT) aims to reduce the chance of an affected pregnancy or improve success in an assisted reproduction cycle. Since the first established pregnancies in 1990, methodological approaches have greatly evolved, combined with significant advances in the embryological laboratory. The application of preimplantation testing has expanded, while accuracy and reliability of monogenic and chromosomal analysis has improved. The procedure traditionally employs an invasive approach to assess the nucleic acid content of embryos. All biopsy procedures require high technical skill, costly equipment, and may impact both the accuracy of genetic testing and embryo viability. To overcome these limitations, many researchers have focused on the analysis of cell-free DNA (cfDNA) at the preimplantation stage, sampled either from the blastocoel or from embryo culture media, to determine the genetic status of the embryo non-invasively. Studies have assessed the origin of cfDNA and application in non-invasive testing for monogenic disease and chromosomal aneuploidies. Herein, we discuss the state-of-the-art for modern non-invasive embryonic genetic material assessment, in the context of PGT. The results are difficult to integrate due to numerous methodological differences between the studies, while further work is required to assess the suitability of cfDNA analysis for clinical application.

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“…Whilst it could be argued that the relatively disappointing amplification rates in both studies could be associated with the use of the same WGA technology (PicoPLEX, Rubicon Genomics), PCR amplification rates were no better in a report following a multiple displacement amplification (MDA) based WGA approach (REPLI-g, Qiagen) applied to seven paired BF and trophectoderm biopsies (Shangguan et al, 2017). An attempt was made to amplify regions of the spinal muscular atrophy, phenylketonuria, SRY and ß-actin genes from WGA products.…”

Section: Pgt-m Using Blastocentesismentioning

confidence: 99%

Non-invasive preimplantation genetic testing (niPGT): the next revolution in reproductive genetics?

Leaver

Wells

2019

Human Reproduction Update

121

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BACKGROUND Preimplantation genetic testing (PGT) encompasses methods that allow embryos to be tested for severe inherited conditions or for chromosome abnormalities, relevant to embryo health and viability. In order to obtain embryonic genetic material for analysis, a biopsy is required, involving the removal of one or more cells. This invasive procedure greatly increases the costs of PGT and there have been concerns that embryo viability could be compromised in some cases. The recent discovery of DNA within the blastocoele fluid (BF) of blastocysts and in spent embryo culture media (SCM) has led to interest in the development of non-invasive methods of PGT (niPGT). OBJECTIVE AND RATIONALE This review evaluates the current scientific evidence regarding non-invasive genetic assessment of preimplantation embryos. The success of different PGT methodologies in collecting and analysing extra-embryonic DNA is evaluated, and consideration is given to the potential biological and technical hindrances to obtaining a reliable clinical diagnosis. SEARCH METHODS Original research and review papers concerning niPGT were sourced by searching PubMed and Google Scholar databases until July 2019. Searches comprised the keywords: ‘non-invasive’; ‘cell-free DNA’; ‘blastocentesis’; ‘blastocoel fluid’; ‘spent culture media’; ‘embryo culture medium’; ‘preimplantation genetic testing’; ‘preimplantation genetic diagnosis’; ‘preimplantation genetic screening’; and ‘aneuploidy’. OUTCOMES Embryonic DNA is frequently detectable in BF and SCM of embryos produced during IVF treatment. Initial studies have achieved some success when performing cytogenetic and molecular genetic analysis. However, in many cases, the efficiency has been restricted by technical complications associated with the low quantity and quality of the DNA. Reported levels of ploidy agreement between SCM/BF samples and biopsied embryonic cells vary widely. In some cases, a discrepancy with respect to cytogenetic data obtained after trophectoderm biopsy may be attributable to embryonic mosaicism or DNA contamination (usually of maternal origin). Some research indicates that aneuploid cells are preferentially eliminated from the embryo, suggesting that their DNA might be over-represented in SCM and BF samples; this hypothesis requires further investigation. WIDER IMPLICATIONS Available data suggest that BF and SCM samples frequently provide DNA templates suitable for genetic analyses, offering a potential means of PGT that is less expensive than traditional methods, requires less micromanipulation skill and poses a lower risk to embryos. Critically, DNA isolation and amplification protocols must be optimised to reproducibly obtain an accurate clinical diagnosis, whilst minimising the impact of confounding factors such as contamination. Further investigations are required to understand the mechanisms underlying the release of embryonic DNA and to determine the extent to which this material reflects the true genetic status of the corresponding embryo. Currently, the clinic al potential of niPGT remains unknown.

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Detection and analysis of DNA material in human blastocoel fluid

Cited by 8 publications

References 14 publications

Non-invasive chromosome screening for embryo preimplantation using cell-free DNA

Non-invasive chromosome screening for embryo preimplantation using cell-free DNA

An Update on Non-invasive Approaches for Genetic Testing of the Preimplantation Embryo

Non-invasive preimplantation genetic testing (niPGT): the next revolution in reproductive genetics?

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