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Randomized comparison of next-generation sequencing and array comparative genomic hybridization for preimplantation genetic screening: a pilot study.

Yang Z, Lin J, Zhang J, Fong WI, Li P, Zhao R, Liu X, Podevin W, Kuang Y, Liu J - BMC Med Genomics (2015)

Bottom Line: The NGS results were then compared with those of aCGH.Moreover, NGS screening identified euploid blastocysts for transfer and resulted in similarly high ongoing pregnancy rates for PGS patients compared to aCGH screening (74.7 % vs. 69.2 %, respectively, p >0.05).With the observed high accuracy of 24-chromosome diagnosis and the resulting high ongoing pregnancy and implantation rates, NGS has demonstrated an efficient, robust high-throughput technology for PGS.

View Article: PubMed Central - PubMed

Affiliation: ZytoGen Global Genetics Institute, Timonium, MD, USA. sunmiy31@hotmail.com.

ABSTRACT

Background: Recent advances in next-generation sequencing (NGS) have provided new methods for preimplantation genetic screening (PGS) of human embryos from in vitro fertilization (IVF) cycles. However, there is still limited information about clinical applications of NGS in IVF and PGS (IVF-PGS) treatments. The present study aimed to investigate the effects of NGS screening on clinical pregnancy and implantation outcomes for PGS patients in comparison to array comparative genomic hybridization (aCGH) screening.

Methods: This study was performed in two phases. Phase I study evaluated the accuracy of NGS for aneuploidy screening in comparison to aCGH. Whole-genome amplification (WGA) products (n = 164) derived from previous IVF-PGS cycles (n = 38) were retrospectively analyzed with NGS. The NGS results were then compared with those of aCGH. Phase II study further compared clinical pregnancy and implantation outcomes between NGS and aCGH for IVF-PGS patients. A total of 172 patients at mean age 35.2 ± 3.5 years were randomized into two groups: 1) NGS (Group A): patients (n = 86) had embryos screened with NGS and 2) aCGH (Group B): patients (n = 86) had embryos screened with aCGH. For both groups, blastocysts were vitrified after trophectoderm biopsy. One to two euploid blastocysts were thawed and transferred to individual patients primarily based on the PGS results. Ongoing pregnancy and implantation rates were compared between the two study groups.

Results: NGS detected all types of aneuploidies of human blastocysts accurately and provided a 100 % 24-chromosome diagnosis consistency with the highly validated aCGH method. Moreover, NGS screening identified euploid blastocysts for transfer and resulted in similarly high ongoing pregnancy rates for PGS patients compared to aCGH screening (74.7 % vs. 69.2 %, respectively, p >0.05). The observed implantation rates were also comparable between the NGS and aCGH groups (70.5 % vs. 66.2 %, respectively, p >0.05).

Conclusions: While NGS screening has been recently introduced to assist IVF patients, this is the first randomized clinical study on the efficiency of NGS for preimplantation genetic screening in comparison to aCGH. With the observed high accuracy of 24-chromosome diagnosis and the resulting high ongoing pregnancy and implantation rates, NGS has demonstrated an efficient, robust high-throughput technology for PGS.

No MeSH data available.


Related in: MedlinePlus

Representative profiles showing mosaicism resulted from NGS (the upper profile) and aCGH (the lower profile) screening of the same WGA product. The upper profile (a) was resulted from NGS screening which revealed a 46 % mosaicism of chromosome 12 accurately. The lower profile (b) was resulted from aCGH screening of the same WGA product, which was unable to detect the mosaicism of chromosome 12
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Fig3: Representative profiles showing mosaicism resulted from NGS (the upper profile) and aCGH (the lower profile) screening of the same WGA product. The upper profile (a) was resulted from NGS screening which revealed a 46 % mosaicism of chromosome 12 accurately. The lower profile (b) was resulted from aCGH screening of the same WGA product, which was unable to detect the mosaicism of chromosome 12

Mentions: Whole genomic amplification of the biopsy samples in the NGS group was performed using the same method as the aCGH group. WGA products were then quantified using the QuanTit dsDNA HS Assay Kit (Life Technologies Corporation, Grand Island, NY, USA). Dual-indexed libraries were prepared using the Nextera XT DNA Sample Preparation Kit and Index Kits with the input sample DNA at 0.2 ng/μl (1 ng total) (Illumina, San Diego, USA). The quality of a subset of libraries was assessed using the Agilent High Sensitivity DNA Kit (Agilent Technologies Inc, Santa Clara, CA, USA) and by sequencing with the MiSeq Reagent Kit v3 (Illumina Inc., San Diego, USA). Paired-end, dual index 2x36bp sequencing was performed using the Illumina workflow on a HiSeq 2000 with 96-plex per lane (Illumina, San Diego, USA). Reads were aligned to the human genome hg19 using iSAAC within the HiSeq Analysis Software. Bash scripting, BEDtools and SAMtools were used to remove unmapped reads, duplicate reads, reads with low mapping scores and reads with an edit distance greater than one. Each chromosome was divided into intervals each approximately covering 1 Mb of sequence. Filtered reads from each sample were then mapped into the corresponding chromosome interval or bin. The count data in each bin was normalized using GC content, and in-silico reference data in order to remove bias. The normalized bin counts were then re-expressed as copy number by assuming the median autosomal read count corresponds to copy number two. The bin-wise copy number values for each chromosome were smoothed with a 13-bin sliding median. Automated copy-number status for each chromosome was determined using the median of smoothed copy-number values across the chromosome as described elsewhere [45]. In particular, the analysis pipeline expected a default copy number of 2 for autosomes; the sample sex and sex chromosome copy numbers were determined by an initial calling algorithm. Embryos were diagnosed as abnormal or aneuploid if the median chromosomal copy number measures deviated from the default copy number. Chromosomal gain or trisomy (copy number >2) and chromosomal loss or monosomy (copy number <2) are seen as horizontal green bars above and below, respectively in Figs. 1, 2 and 3, the copy number state of 2. The method also allows a specific copy number (1, 2, 3, or 4) to be directly assigned. Embryos were diagnosed as normal or euploid if the generated plot showed no gain or loss.Fig. 1


Randomized comparison of next-generation sequencing and array comparative genomic hybridization for preimplantation genetic screening: a pilot study.

Yang Z, Lin J, Zhang J, Fong WI, Li P, Zhao R, Liu X, Podevin W, Kuang Y, Liu J - BMC Med Genomics (2015)

Representative profiles showing mosaicism resulted from NGS (the upper profile) and aCGH (the lower profile) screening of the same WGA product. The upper profile (a) was resulted from NGS screening which revealed a 46 % mosaicism of chromosome 12 accurately. The lower profile (b) was resulted from aCGH screening of the same WGA product, which was unable to detect the mosaicism of chromosome 12
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4477308&req=5

Fig3: Representative profiles showing mosaicism resulted from NGS (the upper profile) and aCGH (the lower profile) screening of the same WGA product. The upper profile (a) was resulted from NGS screening which revealed a 46 % mosaicism of chromosome 12 accurately. The lower profile (b) was resulted from aCGH screening of the same WGA product, which was unable to detect the mosaicism of chromosome 12
Mentions: Whole genomic amplification of the biopsy samples in the NGS group was performed using the same method as the aCGH group. WGA products were then quantified using the QuanTit dsDNA HS Assay Kit (Life Technologies Corporation, Grand Island, NY, USA). Dual-indexed libraries were prepared using the Nextera XT DNA Sample Preparation Kit and Index Kits with the input sample DNA at 0.2 ng/μl (1 ng total) (Illumina, San Diego, USA). The quality of a subset of libraries was assessed using the Agilent High Sensitivity DNA Kit (Agilent Technologies Inc, Santa Clara, CA, USA) and by sequencing with the MiSeq Reagent Kit v3 (Illumina Inc., San Diego, USA). Paired-end, dual index 2x36bp sequencing was performed using the Illumina workflow on a HiSeq 2000 with 96-plex per lane (Illumina, San Diego, USA). Reads were aligned to the human genome hg19 using iSAAC within the HiSeq Analysis Software. Bash scripting, BEDtools and SAMtools were used to remove unmapped reads, duplicate reads, reads with low mapping scores and reads with an edit distance greater than one. Each chromosome was divided into intervals each approximately covering 1 Mb of sequence. Filtered reads from each sample were then mapped into the corresponding chromosome interval or bin. The count data in each bin was normalized using GC content, and in-silico reference data in order to remove bias. The normalized bin counts were then re-expressed as copy number by assuming the median autosomal read count corresponds to copy number two. The bin-wise copy number values for each chromosome were smoothed with a 13-bin sliding median. Automated copy-number status for each chromosome was determined using the median of smoothed copy-number values across the chromosome as described elsewhere [45]. In particular, the analysis pipeline expected a default copy number of 2 for autosomes; the sample sex and sex chromosome copy numbers were determined by an initial calling algorithm. Embryos were diagnosed as abnormal or aneuploid if the median chromosomal copy number measures deviated from the default copy number. Chromosomal gain or trisomy (copy number >2) and chromosomal loss or monosomy (copy number <2) are seen as horizontal green bars above and below, respectively in Figs. 1, 2 and 3, the copy number state of 2. The method also allows a specific copy number (1, 2, 3, or 4) to be directly assigned. Embryos were diagnosed as normal or euploid if the generated plot showed no gain or loss.Fig. 1

Bottom Line: The NGS results were then compared with those of aCGH.Moreover, NGS screening identified euploid blastocysts for transfer and resulted in similarly high ongoing pregnancy rates for PGS patients compared to aCGH screening (74.7 % vs. 69.2 %, respectively, p >0.05).With the observed high accuracy of 24-chromosome diagnosis and the resulting high ongoing pregnancy and implantation rates, NGS has demonstrated an efficient, robust high-throughput technology for PGS.

View Article: PubMed Central - PubMed

Affiliation: ZytoGen Global Genetics Institute, Timonium, MD, USA. sunmiy31@hotmail.com.

ABSTRACT

Background: Recent advances in next-generation sequencing (NGS) have provided new methods for preimplantation genetic screening (PGS) of human embryos from in vitro fertilization (IVF) cycles. However, there is still limited information about clinical applications of NGS in IVF and PGS (IVF-PGS) treatments. The present study aimed to investigate the effects of NGS screening on clinical pregnancy and implantation outcomes for PGS patients in comparison to array comparative genomic hybridization (aCGH) screening.

Methods: This study was performed in two phases. Phase I study evaluated the accuracy of NGS for aneuploidy screening in comparison to aCGH. Whole-genome amplification (WGA) products (n = 164) derived from previous IVF-PGS cycles (n = 38) were retrospectively analyzed with NGS. The NGS results were then compared with those of aCGH. Phase II study further compared clinical pregnancy and implantation outcomes between NGS and aCGH for IVF-PGS patients. A total of 172 patients at mean age 35.2 ± 3.5 years were randomized into two groups: 1) NGS (Group A): patients (n = 86) had embryos screened with NGS and 2) aCGH (Group B): patients (n = 86) had embryos screened with aCGH. For both groups, blastocysts were vitrified after trophectoderm biopsy. One to two euploid blastocysts were thawed and transferred to individual patients primarily based on the PGS results. Ongoing pregnancy and implantation rates were compared between the two study groups.

Results: NGS detected all types of aneuploidies of human blastocysts accurately and provided a 100 % 24-chromosome diagnosis consistency with the highly validated aCGH method. Moreover, NGS screening identified euploid blastocysts for transfer and resulted in similarly high ongoing pregnancy rates for PGS patients compared to aCGH screening (74.7 % vs. 69.2 %, respectively, p >0.05). The observed implantation rates were also comparable between the NGS and aCGH groups (70.5 % vs. 66.2 %, respectively, p >0.05).

Conclusions: While NGS screening has been recently introduced to assist IVF patients, this is the first randomized clinical study on the efficiency of NGS for preimplantation genetic screening in comparison to aCGH. With the observed high accuracy of 24-chromosome diagnosis and the resulting high ongoing pregnancy and implantation rates, NGS has demonstrated an efficient, robust high-throughput technology for PGS.

No MeSH data available.


Related in: MedlinePlus