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Preimplantation genetic screening of blastocysts by multiplex qPCR followed by fresh embryo transfer: validation and verification.

Yang YS, Chang SP, Chen HF, Ma GC, Lin WH, Lin CF, Tsai FP, Wu CH, Tsai HD, Lee TH, Chen M - Mol Cytogenet (2015)

Bottom Line: Successful diagnoses were established in all embryos and the rate of successful diagnosis was 100 %.This study verified the favorable outcome of adopting PGS with qPCR + FET in our own setting.Expanding the repertoire of aneuploidies being investigated (from a limited set to all 24 chromosomes) is underway and a randomized study by comparing qPCR and other PGS technologies is warranted.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, College of Medicine, National Taiwan University, Taipei, Taiwan.

ABSTRACT

Background: Aneuploidy is an important etiology of implantation failure and quantitative real-time polymerase chain reaction (qPCR) seems a promising preimplantation genetic screening (PGS) technology to detect aneuploidies. This verification study aimed at verifying the impact on reproductive outcomes in in vitro fertilization (IVF) cycles using fresh embryo transfer (FET) in which the embryos were selected by blastocyst biopsy with qPCR-based PGS in our settings.

Results: A total of 13 infertile couples with more than once failed in vitro fertilization were enrolled during July to October of 2014. PGS was conducted by qPCR with selectively amplified markers to detect common aneuploidies (chromosomes 13, 18, 21, X, and Y). The design of the qPCR molecular markers adopted the locked nucleic acid (LNA) strategy. The blastocyst biopsy was performed on Day 5/6 and the PGS was done on the same day, which enabled FET. A total of 72 blastocysts were biopsied. Successful diagnoses were established in all embryos and the rate of successful diagnosis was 100 %. The aneuploidy rate was 38.9 % (28/72). 28 embryos were transferred. The clinical pregnancy rate was 61.5 % (8/13) per cycle. Early first trimester abortion was encountered in 1 and the ongoing pregnancy rate was 53.8 % (7/13) per cycle.

Conclusion: This study verified the favorable outcome of adopting PGS with qPCR + FET in our own setting. Expanding the repertoire of aneuploidies being investigated (from a limited set to all 24 chromosomes) is underway and a randomized study by comparing qPCR and other PGS technologies is warranted.

No MeSH data available.


Related in: MedlinePlus

The diagram of the in-house qPCR PGS system. The flowchart of (a) detection of common aneuploidies, and (b) signal normalization and data analysis
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Fig1: The diagram of the in-house qPCR PGS system. The flowchart of (a) detection of common aneuploidies, and (b) signal normalization and data analysis

Mentions: Each 5 cells separated from the cell lines of known common aneuploidies (including trisomy 21, trisomy 13, trisomy 18, 47,XXY, and 45,X) were processed by cell lysis of proteinase K, and the products were subjected to a 50-μL reaction volume of multiplex nested-PCR amplication for 18 cycles using an Applied Biosystems Veriti thermal cycler (Life Technologies, California, USA), and then the PCR products were purified using Agencourt AMPure XP system (Beckman Coulter, California, USA). Dual color hydrolysis probe assays were performed in triplicate to normalize and simpify calculation and to evaluate chromosomal copies, using Lightcycler 480 probes Master (Roche, Mannheim, Germany), a 20-μL reaction volume, a 96-well plate, and a 7 Light Cycler 480 Real-Time PCR System, as recommended by the supplier (Roche, Mannheim, Germany). Each well contains a particular target, and a common control reaction. A unique method of the standard delta delta threshold cycle (ΔΔCp) method was used for relative quantification. In our experiments, the Cp variation of all HEX™ reactions obtained for each well of the same sample will be controlled and ranged in less than 0.2, indicating the test sample was evenly distributed to each well. Each chromosome-specific ΔCp was calculated from the Cp of the FAM™ reactions targeting a specific chromosome minus the control Cp of the HEX™ reactions targeting the chromosome 1 within the same well. The same process was applied to individually determine the ΔCp for each targeted chromosome of the test sample, including reference set of normal male cell lines [BCRC number: 08C0011, 08C0012, 08C0013, 08C0021 and 08C0025]. Each chromosome- specific ΔCp was then normalized to the average chromosome-specific ΔCp values derived from the same evaluation of the reference set, which had been confirmed by FISH method. The calibrated chromosome-specific ΔCp values were used to calculate fold change by considering the ΔΔCp values as the negative exponent of 2, as previously described [18, 23]. The methodology was designed to specifically identify whole-chromosome but not segmental aneuploidy. The flowchart and diagram of the in-house qPCR PGS system were illustrated in Fig. 1. This qPCR was capable of accurate aneuploidy screening in 4 h, which allowed rapid evaluation of the trophectoderm biopsies and therefore provided a feasible opportunity for subsequent FET.Fig. 1


Preimplantation genetic screening of blastocysts by multiplex qPCR followed by fresh embryo transfer: validation and verification.

Yang YS, Chang SP, Chen HF, Ma GC, Lin WH, Lin CF, Tsai FP, Wu CH, Tsai HD, Lee TH, Chen M - Mol Cytogenet (2015)

The diagram of the in-house qPCR PGS system. The flowchart of (a) detection of common aneuploidies, and (b) signal normalization and data analysis
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4495615&req=5

Fig1: The diagram of the in-house qPCR PGS system. The flowchart of (a) detection of common aneuploidies, and (b) signal normalization and data analysis
Mentions: Each 5 cells separated from the cell lines of known common aneuploidies (including trisomy 21, trisomy 13, trisomy 18, 47,XXY, and 45,X) were processed by cell lysis of proteinase K, and the products were subjected to a 50-μL reaction volume of multiplex nested-PCR amplication for 18 cycles using an Applied Biosystems Veriti thermal cycler (Life Technologies, California, USA), and then the PCR products were purified using Agencourt AMPure XP system (Beckman Coulter, California, USA). Dual color hydrolysis probe assays were performed in triplicate to normalize and simpify calculation and to evaluate chromosomal copies, using Lightcycler 480 probes Master (Roche, Mannheim, Germany), a 20-μL reaction volume, a 96-well plate, and a 7 Light Cycler 480 Real-Time PCR System, as recommended by the supplier (Roche, Mannheim, Germany). Each well contains a particular target, and a common control reaction. A unique method of the standard delta delta threshold cycle (ΔΔCp) method was used for relative quantification. In our experiments, the Cp variation of all HEX™ reactions obtained for each well of the same sample will be controlled and ranged in less than 0.2, indicating the test sample was evenly distributed to each well. Each chromosome-specific ΔCp was calculated from the Cp of the FAM™ reactions targeting a specific chromosome minus the control Cp of the HEX™ reactions targeting the chromosome 1 within the same well. The same process was applied to individually determine the ΔCp for each targeted chromosome of the test sample, including reference set of normal male cell lines [BCRC number: 08C0011, 08C0012, 08C0013, 08C0021 and 08C0025]. Each chromosome- specific ΔCp was then normalized to the average chromosome-specific ΔCp values derived from the same evaluation of the reference set, which had been confirmed by FISH method. The calibrated chromosome-specific ΔCp values were used to calculate fold change by considering the ΔΔCp values as the negative exponent of 2, as previously described [18, 23]. The methodology was designed to specifically identify whole-chromosome but not segmental aneuploidy. The flowchart and diagram of the in-house qPCR PGS system were illustrated in Fig. 1. This qPCR was capable of accurate aneuploidy screening in 4 h, which allowed rapid evaluation of the trophectoderm biopsies and therefore provided a feasible opportunity for subsequent FET.Fig. 1

Bottom Line: Successful diagnoses were established in all embryos and the rate of successful diagnosis was 100 %.This study verified the favorable outcome of adopting PGS with qPCR + FET in our own setting.Expanding the repertoire of aneuploidies being investigated (from a limited set to all 24 chromosomes) is underway and a randomized study by comparing qPCR and other PGS technologies is warranted.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, College of Medicine, National Taiwan University, Taipei, Taiwan.

ABSTRACT

Background: Aneuploidy is an important etiology of implantation failure and quantitative real-time polymerase chain reaction (qPCR) seems a promising preimplantation genetic screening (PGS) technology to detect aneuploidies. This verification study aimed at verifying the impact on reproductive outcomes in in vitro fertilization (IVF) cycles using fresh embryo transfer (FET) in which the embryos were selected by blastocyst biopsy with qPCR-based PGS in our settings.

Results: A total of 13 infertile couples with more than once failed in vitro fertilization were enrolled during July to October of 2014. PGS was conducted by qPCR with selectively amplified markers to detect common aneuploidies (chromosomes 13, 18, 21, X, and Y). The design of the qPCR molecular markers adopted the locked nucleic acid (LNA) strategy. The blastocyst biopsy was performed on Day 5/6 and the PGS was done on the same day, which enabled FET. A total of 72 blastocysts were biopsied. Successful diagnoses were established in all embryos and the rate of successful diagnosis was 100 %. The aneuploidy rate was 38.9 % (28/72). 28 embryos were transferred. The clinical pregnancy rate was 61.5 % (8/13) per cycle. Early first trimester abortion was encountered in 1 and the ongoing pregnancy rate was 53.8 % (7/13) per cycle.

Conclusion: This study verified the favorable outcome of adopting PGS with qPCR + FET in our own setting. Expanding the repertoire of aneuploidies being investigated (from a limited set to all 24 chromosomes) is underway and a randomized study by comparing qPCR and other PGS technologies is warranted.

No MeSH data available.


Related in: MedlinePlus