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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.

ABSTRACT: This prospective study evaluated the accuracy of non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) using cell-free DNA in spent culture medium, as well as that of preimplantation genetic testing for aneuploidy (PGT-A) using trophectoderm (TE) biopsy after culturing beyond implantation. Twenty frozen blastocysts donated by 12 patients who underwent IVF at our institution were investigated. Of these, 10 were frozen on day 5 and 10 on day 6. Spent culture medium and TE cells were collected from each blastocyst after thawing, and the embryos were cultured in vitro for up to 10 days. The outgrowths after culturing beyond implantation were sampled and subjected to chromosome analysis using next-generation sequencing. Chromosomal concordance rate, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), false-positive rate (FPR), and false-negative rate (FNR) of niPGT-A and PGT-A against each outgrowth were analyzed. The concordance rate between the niPGT-A and outgrowth samples was 9/16 (56.3%), and the concordance rate between the PGT-A and outgrowth samples was 7/16 (43.8%). NiPGT-A exhibited 100% sensitivity, 87.5% specificity, 88.9% PPV, 100% NPV, 12.5% FPR, and 0% FNR. PGT-A exhibited 87.5% sensitivity, 77.8% specificity, 87.5% PPV, 75% NPV, 14.3% FPR, and 22.2% FNR. NiPGT-A may be more accurate than PGT-A in terms of ploidy diagnostic accuracy in outgrowths.

SUBMITTER: Shitara A

PROVIDER: S-EPMC7877764 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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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.

Shitara Akihiro A Takahashi Kazumasa K Goto Mayumi M Takahashi Harunori H Iwasawa Takuya T Onodera Yohei Y Makino Kenichi K Miura Hiroshi H Shirasawa Hiromitsu H Sato Wataru W Kumazawa Yukiyo Y Terada Yukihiro Y

PloS one 20210211 2

This prospective study evaluated the accuracy of non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) using cell-free DNA in spent culture medium, as well as that of preimplantation genetic testing for aneuploidy (PGT-A) using trophectoderm (TE) biopsy after culturing beyond implantation. Twenty frozen blastocysts donated by 12 patients who underwent IVF at our institution were investigated. Of these, 10 were frozen on day 5 and 10 on day 6. Spent culture medium and TE cells wer ...[more]

PMID: 33571233

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Project description:Study questionIn PGS, does chromosomal constitution differ among trophectoderm (TE) biopsy sites and between them and the inner cell mass (ICM)?Summary answerThe ploidy concordance between ICM and TE was independent of whether the biopsy site in the TE was near to or far from the ICM.What is known alreadyTE biopsies are considered less harmful to developing embryos than blastomere biopsies. Removal of multi-cellular samples permits high-resolution next-generation sequencing (Veriseq NGS) to detect aneuploidy present in a minority of cells (mosaicism of diploid and aneuploid cells). However, the prevalence of ploidy discrepancies between different TE biopsy sites and the ICM, as well as confined mosaicism (aneuploidy only in a particular area), has not been established.Study design, size, durationBiopsies were taken from a site opposite to the ICM (TE1), near the ICM (TE2) and within the ICM of the same embryo in 33 donated blastocysts obtained from 12 volunteer patients. The samples were analyzed by the Veriseq NGS to assess ploidy concordance.Participants/materials, setting, methodsThe mean age of the patients was 34.4 years, and samples from all three biopsy sites were achieved in 29 frozen thawed blastocysts. The aneuploid percentage in each sample was interpreted by Veriseq NGS at the finest resolution involving the number of reads after filtering, sample overall noise score, and average quality/alignment scores according to the Veriseq quality control assessment. Ploidy concordance was then assessed between different TE fractions, and between the TE and ICM.Main results and the role of chanceThe euploid rates were similar in the TEs and ICM, and no preferential allocation of euploid lineage within a blastocyst was demonstrated. Whether the biopsy site in the TE was near to or far from the ICM, the chromosomal consistency rate was similar [TE1-to-ICM, 86.2% (25/29) versus TE2-to-ICM, 89.7% (26/29); P = 1.0], suggesting that the cells with different chromosomal components may spread randomly throughout the TE. The following two types of inconsistent PGS conclusions between TE and ICM due to confined mosaicism were observed: (i) euploid TE with mosaic ICM (3%) (1/29); and (ii) mosaic TE with euploid ICM (3%) (1/29) or with aneuploid ICM (7%) (2/29). Thus, the overall rate of confined mosaicism was 14% (4/29).Large scale dataN/A.Limitation, reasons for cautionThe approach used in the present study was affected by biopsy manipulation limitations involving possible cell contamination and the technical challenge of comprehensive chromosomal screening (CCS) procedures.Wider implications of the findingsThe rate of confined mosaicism in the blastocysts was estimated in this preliminary study, thus, specifying the incidence of biological sampling biases. The results also verified the random distribution of different cell lineages, and the representative value of a single biopsied sample from the TE.Study funding and conflict of interest(s)No external funding was obtained; all the authors declare no conflicts of interest regarding this study.

Project description:BackgroundIdentification of a non-invasive technique to assess embryo implantation potential in assisted reproduction would greatly increase success rates and lead more efficiently to single embryo transfer. Early studies suggested metabonomic analysis of spent culture media could improve embryo selection. The goal of this study is to assess if embryo implantation can be predicted based on proton nuclear magnetic resonance ((1)H NMR) profiles of spent embryo culture media from patients undergoing transfer of multiple embryos on cycle day 3.MethodWe conducted a retrospective study in an academic assisted reproduction technology (ART) program and analyzed the data in a university research center. Two hundred twenty-eight spent culture media samples originating from 108 patients were individually analyzed. Specifically, five distinct sets (1 to 5) of different types of spent media samples (volume ~14 μL) from embryos that resulted in clinical pregnancy (positive heart rate at 6 weeks gestation) (n (1) = 29; n (2) = 19; n (3) = 9; n (4) = 12; n (5) = 33; n (total) = 102) and from embryos that did not implant (n (1) = 28; n (2) = 29; n (3) = 18; n (4) = 15; n (5) = 36; n (total) = 126) were collected on day 3 of embryo growth. The media samples were profiled using (1)H NMR spectroscopy, and the NMR profiles of sets 1 to 5 were subject to standard uni- and multi-variate data analyses in order to evaluate potential correlation of profiles with implantation success.ResultsFor set 1 of the media samples, a borderline class separation of NMR profiles was obtained by use of principal component analysis (PCA) and logistic regression. This tentative class separation could not be repeated and validated in any of the other media sets 2 to 5.ConclusionsDespite the rigorous technical approach, (1)H NMR based profiling of spent culture media cannot predict success of implantation for day 3 human embryos.

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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.

Publications

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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