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• W2512095034 abstract "ObjectivePre-implantation genetic screening (PGS) enables the selection of in vitro-fertilized embryos to avoid chromosomal imbalance, and is currently widely used to improve the clinical outcome of in-vitro fertilization (IVF). However, PGS requires embryo biopsy before implantation, which limits the clinical applicability of PGS due to the invasiveness and complexity in the embryo biopsy process. Here we present and validate a non-invasive chromosome screening (NICSTM) method using blastocyst culture medium.DesignBy performing whole genome amplification and sequencing on the blastocyst culture medium, we obtained the ploidy information on all 24 chromosomes. We then validated the ploidy information obtained from the culture medium by comparing with the corresponding donated blastocyst embryos.Materials and MethodsAll embryos were fertilized by ICSI. After warming, embryos were placed in 30 μl droplets of blastocyst medium (SAGE, US) under washed and pre-gassed mineral oil (SAGE) and then further cultured to the blastocysts under 5.5% CO2, 5% O2, and balance N2 at37°C in Labotect C16 incubators (Labotect, Germany). To prevent media contamination, individual Pasteur pipettes were used for each embryo. The blastocysts for final confirmation were transferred into RNAse-DNAse-free polymerase chain reaction (PCR) tubes that contained 5μl of cell lysis buffer (Yikon Genomics, China). The same quantity of blastocyst culture medium without embryo culture was collected as a negative control. All collected samples were frozen immediately by liquid nitrogen and stored under -80°Cuntil NICSTM assay. The MALBACTM single-cell WGA method was used to amplify the culture medium as well as the embryos, following the protocol provided by the manufacturer (Cat. No. YK001B; Yikon Genomics, China). The amplifications products were then used for constructing sequencing libraries for sequencing on an Illumina HiSeq 2500 platform, yielding ∼2 million sequencing reads on each sample. The read numbers were counted along the whole genome with a bin size of 1 Mb. A copy number gain from 2 to 3 copies results in a 50% increase in read counts, while a copy number loss from 2 copies to 1 copy results in a 50% decrease in read counts.ResultsWith the NICSTM method, we perform chromosome screening on the IVF embryos from a patient carrying balanced chromosome translocation, and obtained a successful pregnancy and live birth with balanced chromosomes, which was confirmed by amniocentesis. We then applied the NICSTM essay on 15 patients and here we report the clinical outcome of our first 15 patients. We obtained a sensitivity of 0.882 and a specificity of 0.840 for identifying chromosomal abnormalities.ConclusionsThe NICSTM method avoids invasive and sophisticated embryo biopsy procedure and can potentially enables the wide applicability of chromosome screening in the clinical practice of IVF. ObjectivePre-implantation genetic screening (PGS) enables the selection of in vitro-fertilized embryos to avoid chromosomal imbalance, and is currently widely used to improve the clinical outcome of in-vitro fertilization (IVF). However, PGS requires embryo biopsy before implantation, which limits the clinical applicability of PGS due to the invasiveness and complexity in the embryo biopsy process. Here we present and validate a non-invasive chromosome screening (NICSTM) method using blastocyst culture medium. Pre-implantation genetic screening (PGS) enables the selection of in vitro-fertilized embryos to avoid chromosomal imbalance, and is currently widely used to improve the clinical outcome of in-vitro fertilization (IVF). However, PGS requires embryo biopsy before implantation, which limits the clinical applicability of PGS due to the invasiveness and complexity in the embryo biopsy process. Here we present and validate a non-invasive chromosome screening (NICSTM) method using blastocyst culture medium. DesignBy performing whole genome amplification and sequencing on the blastocyst culture medium, we obtained the ploidy information on all 24 chromosomes. We then validated the ploidy information obtained from the culture medium by comparing with the corresponding donated blastocyst embryos. By performing whole genome amplification and sequencing on the blastocyst culture medium, we obtained the ploidy information on all 24 chromosomes. We then validated the ploidy information obtained from the culture medium by comparing with the corresponding donated blastocyst embryos. Materials and MethodsAll embryos were fertilized by ICSI. After warming, embryos were placed in 30 μl droplets of blastocyst medium (SAGE, US) under washed and pre-gassed mineral oil (SAGE) and then further cultured to the blastocysts under 5.5% CO2, 5% O2, and balance N2 at37°C in Labotect C16 incubators (Labotect, Germany). To prevent media contamination, individual Pasteur pipettes were used for each embryo. The blastocysts for final confirmation were transferred into RNAse-DNAse-free polymerase chain reaction (PCR) tubes that contained 5μl of cell lysis buffer (Yikon Genomics, China). The same quantity of blastocyst culture medium without embryo culture was collected as a negative control. All collected samples were frozen immediately by liquid nitrogen and stored under -80°Cuntil NICSTM assay. The MALBACTM single-cell WGA method was used to amplify the culture medium as well as the embryos, following the protocol provided by the manufacturer (Cat. No. YK001B; Yikon Genomics, China). The amplifications products were then used for constructing sequencing libraries for sequencing on an Illumina HiSeq 2500 platform, yielding ∼2 million sequencing reads on each sample. The read numbers were counted along the whole genome with a bin size of 1 Mb. A copy number gain from 2 to 3 copies results in a 50% increase in read counts, while a copy number loss from 2 copies to 1 copy results in a 50% decrease in read counts. All embryos were fertilized by ICSI. After warming, embryos were placed in 30 μl droplets of blastocyst medium (SAGE, US) under washed and pre-gassed mineral oil (SAGE) and then further cultured to the blastocysts under 5.5% CO2, 5% O2, and balance N2 at37°C in Labotect C16 incubators (Labotect, Germany). To prevent media contamination, individual Pasteur pipettes were used for each embryo. The blastocysts for final confirmation were transferred into RNAse-DNAse-free polymerase chain reaction (PCR) tubes that contained 5μl of cell lysis buffer (Yikon Genomics, China). The same quantity of blastocyst culture medium without embryo culture was collected as a negative control. All collected samples were frozen immediately by liquid nitrogen and stored under -80°Cuntil NICSTM assay. The MALBACTM single-cell WGA method was used to amplify the culture medium as well as the embryos, following the protocol provided by the manufacturer (Cat. No. YK001B; Yikon Genomics, China). The amplifications products were then used for constructing sequencing libraries for sequencing on an Illumina HiSeq 2500 platform, yielding ∼2 million sequencing reads on each sample. The read numbers were counted along the whole genome with a bin size of 1 Mb. A copy number gain from 2 to 3 copies results in a 50% increase in read counts, while a copy number loss from 2 copies to 1 copy results in a 50% decrease in read counts. ResultsWith the NICSTM method, we perform chromosome screening on the IVF embryos from a patient carrying balanced chromosome translocation, and obtained a successful pregnancy and live birth with balanced chromosomes, which was confirmed by amniocentesis. We then applied the NICSTM essay on 15 patients and here we report the clinical outcome of our first 15 patients. We obtained a sensitivity of 0.882 and a specificity of 0.840 for identifying chromosomal abnormalities. With the NICSTM method, we perform chromosome screening on the IVF embryos from a patient carrying balanced chromosome translocation, and obtained a successful pregnancy and live birth with balanced chromosomes, which was confirmed by amniocentesis. We then applied the NICSTM essay on 15 patients and here we report the clinical outcome of our first 15 patients. We obtained a sensitivity of 0.882 and a specificity of 0.840 for identifying chromosomal abnormalities. ConclusionsThe NICSTM method avoids invasive and sophisticated embryo biopsy procedure and can potentially enables the wide applicability of chromosome screening in the clinical practice of IVF. The NICSTM method avoids invasive and sophisticated embryo biopsy procedure and can potentially enables the wide applicability of chromosome screening in the clinical practice of IVF." @default.
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• W2512095034 date "2016-09-01" @default.
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• W2512095034 title "Non-invasive chromosome screening of human embryos by genome sequencing from blastocyst culture medium: validation & a pilot study" @default.
• W2512095034 doi "https://doi.org/10.1016/j.fertnstert.2016.07.461" @default.
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