Next-generation sequencing: challenges in reproductive genetics Since the development of preimplantation genetic diagnosis and screening, technological development has been its main driving force. Now, on the 20th anniversary of preimplantation genetic diagnosis, next-generation sequencing (NGS) represents the next challenge in this field. This technology has opened up a myriad of possibilities, not only for clinical applications, but also in helping us to obtain a deeper understanding of human embryology. Next-generation sequencing was initially developed for use with genomic DNA, but it was later applied to the analysis of the very small quantities of free fetal DNA found in circulating maternal blood, and then to the analysis of trophectoderm biopsies. In their article, Fiorentino et al. (1) demonstrate, for the first time, a robust high-throughput methodology for the use of NGS technology at the single blastomere level. This protocol will broaden the application of NGS technology, not only for good prognosis patients, but also for those with a bad prognosis in which the use of cleavage-stage embryo biopsies may offer new possibilities for a successful clinical outcome (2). Fiorentino and co-workers (1) performed a large, blinded, preclinical validation study to determine the accuracy and prediction power of a NGS-based 24-aneuploidy screening protocol in assessing DNA copy number in single cells. First, the protocol was validated on single cells from cultured amniotic fluids or the products of conception that had known chromosomal abnormalities. Next, whole genome amplification products from single blastomeres obtained from cleavage-stage embryos were analyzed. Successful results were obtained using NGS in all the samples analyzed, and there was a high rate of concordance between NGS and array comparative genomic hybridization results. One sample produced discordant results, and was the result of a NGS false-positive call for trisomy 18. All of the remaining chromosomes in the samples were consistent between NGS and array comparative genomic hybridization, including segmental aneusomies, which were reliably identified with a segmental imbalance as small as 14 Mb. There were no false-negative diagnoses for aneuploid chromosomes or embryos, and there was a high level of specificity and sensitivity. In addition, concordance analysis between HiSeq and MiSeq sequencing demonstrated overlapping results, indicating that the NGS-based method for 24-chromosome screening can be used with both instruments. The copy number assessment method that they describe is therefore platform and alignment-software independent. Yin et al. (3) previously reported a low coverage application for NGS in the identification of aneuploidies and unbalanced chromosomal rearrangements in trophectoderm biopsies. The efficiency of their approach was estimated by comparing the analysis of the same whole genome amplification products using single nucleotide polymorphism arrays. The concordance rate was very high, except for one sample with an unbalanced chromosome rearrangement in which a different size was found by each of the technologies. This was the first study demonstrating that NGS could accurately 1252
detect aneuploidy and chromosome imbalances in trophectoderm biopsies with high accuracy using a flexible and costeffective strategy. Yin et al. (3) reported the possibility of correcting the whole genome amplification bias during data analysis as a major advantage of the approach. However, in this study the turn-around time and the need for embryo vitrification and deferred blastocyst transfer were limiting factors. However, Fiorentino and co-workers (1) show that this limitation can be partially overcome by using a shorter protocol and day 3 biopsies, allowing 2 days for the analysis before fresh blastocyst transfer. The incorporation of NGS to the field of reproductive genetics opens up many near-future possibilities, such as the detection of mitochondrial mutations, Mendelian diseases, translocations, and microdeletion syndromes, all in a single experiment from a single sample. This type of genetic analysis on cleavage-stage embryos or blastocysts could also result in the development of new genetic viability markers for embryo selection in IVF cycles. In addition, the possibility of running many samples on a single lane, with an accompanying decrease in the cost of comprehensive chromosome screening, is tantalizing as several prospective randomized trials using 24-chromosome screening with other technologies have already shown improved delivery and live birth rates for good-prognosis patients (4) and in couples with different ages and indications (5). The possibility of screening embryos before transferring them to the uterus is changing the reproductive field. We are moving toward higher pregnancy rates (PR) using single embryo transfers (ETs). This decreased cost raises two important issues in this field: the need to centralize genetic analysis, and the impact that the automation of several processes, such as library preparation, may have. Bioinformatics and data processing also represent an important challenge in uncovering new genetic markers that affect implantation ability and disease susceptibility. Despite the fast development of NGS we should bear in mind the need to validate each protocol, new sequencing machine, or bioinformatic analysis technique. Different strategies for whole genome amplification might appear in the near future that can improve the accuracy and types of genetic defects that NGS can detect. Taylored filtering and bioinformatic analysis could even improve the ability of the new platforms to detect different levels of mosaicism in trophectoderm cells, and NGS, in combination with bioinformatics, might also improve our ability to diagnose segmental aneuploidies. In conclusion, recent developments in benchtop sequencing platforms, combined with existing bioinformatics tools, mean that the target of sequencing individual cells for embryo diagnosis is now a realistic and feasible near-future goal. With the advent of NGS techniques, a new opportunity to perform cost-efficient genetic testing has emerged by sequencing in different clinical scenarios. In the comprehensive chromosome screening field, this may contribute to definitive improvements in the genetic assessment of embryos before transferring them to the uterus. Nevertheless, further studies with large sample sizes will be needed to establish the real value of this technology and to outline its potential for routine clinical applications.
VOL. 101 NO. 5 / MAY 2014
Fertility and Sterility® Carmen Rubio, Ph.D. IVIOMICS and Instituto Universitario IVI Valencia University; Fundaci on Instituto Valenciano de Infertilidad (FIVI)/ INCLIVA, Valencia University, Valencia, Spain http://dx.doi.org/10.1016/j.fertnstert.2014.03.005 You can discuss this article with its authors and other ASRM members at http://fertstertforum.com/rubioc-next-generationsequencing-challenges-reproductive-genetics/
REFERENCES 1.
2.
3.
4.
Use your smartphone to scan this QR code and connect to the discussion forum for this article now.* * Download a free QR code scanner by searching for “QR scanner” in your smartphone’s app store or app marketplace.
VOL. 101 NO. 5 / MAY 2014
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Fiorentino F, Biricik A, Bono S, Spizzichino L, Cotroneo E, Cottone G, et al. Development and validation of a next-generation sequencing-based protocol for 24-chromosome aneuploidy screening of embryos. Fertil Steril 2014;101: 1375–82. Rodrigo L, Mateu E, Mercader A, Cobo AC, Peinado V, Milan M, et al. New tools for embryo selection: comprehensive chromosome screening by array comparative genomic hybridization. Biomed Res Int 2014. In press. Yin X, Tan K, Vajta G, Jiang H, Tan Y, Zhang C, et al. Massively parallel sequencing for chromosomal abnormality testing in trophectoderm cells of human blastocysts. Biol Reprod 2013;88:69. Yang Z, Liu J, Collins GS, Salem SA, Liu X, Lyle SS, et al. Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study. Mol Cytogenet 2012;5:24. Scott RT Jr, Upham KM, Forman EJ, Hong KH, Scott KL, Taylor D, et al. Blastocyst biopsy with comprehensive chromosome screening and fresh embryo transfer significantly increases in vitro fertilization implantation and delivery rates: a randomized controlled trial. Fertil Steril 2013;100:697–703.
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detect aneuploidy and chromosome imbalances in trophectoderm biopsies with high accuracy using a flexible and costeffective strategy. Yin et al. (3) reported the possibility of correcting the whole genome amplification bias during data analysis as a major advantage of the approach. However, in this study the turn-around time and the need for embryo vitrification and deferred blastocyst transfer were limiting factors. However, Fiorentino and co-workers (1) show that this limitation can be partially overcome by using a shorter protocol and day 3 biopsies, allowing 2 days for the analysis before fresh blastocyst transfer. The incorporation of NGS to the field of reproductive genetics opens up many near-future possibilities, such as the detection of mitochondrial mutations, Mendelian diseases, translocations, and microdeletion syndromes, all in a single experiment from a single sample. This type of genetic analysis on cleavage-stage embryos or blastocysts could also result in the development of new genetic viability markers for embryo selection in IVF cycles. In addition, the possibility of running many samples on a single lane, with an accompanying decrease in the cost of comprehensive chromosome screening, is tantalizing as several prospective randomized trials using 24-chromosome screening with other technologies have already shown improved delivery and live birth rates for good-prognosis patients (4) and in couples with different ages and indications (5). The possibility of screening embryos before transferring them to the uterus is changing the reproductive field. We are moving toward higher pregnancy rates (PR) using single embryo transfers (ETs). This decreased cost raises two important issues in this field: the need to centralize genetic analysis, and the impact that the automation of several processes, such as library preparation, may have. Bioinformatics and data processing also represent an important challenge in uncovering new genetic markers that affect implantation ability and disease susceptibility. Despite the fast development of NGS we should bear in mind the need to validate each protocol, new sequencing machine, or bioinformatic analysis technique. Different strategies for whole genome amplification might appear in the near future that can improve the accuracy and types of genetic defects that NGS can detect. Taylored filtering and bioinformatic analysis could even improve the ability of the new platforms to detect different levels of mosaicism in trophectoderm cells, and NGS, in combination with bioinformatics, might also improve our ability to diagnose segmental aneuploidies. In conclusion, recent developments in benchtop sequencing platforms, combined with existing bioinformatics tools, mean that the target of sequencing individual cells for embryo diagnosis is now a realistic and feasible near-future goal. With the advent of NGS techniques, a new opportunity to perform cost-efficient genetic testing has emerged by sequencing in different clinical scenarios. In the comprehensive chromosome screening field, this may contribute to definitive improvements in the genetic assessment of embryos before transferring them to the uterus. Nevertheless, further studies with large sample sizes will be needed to establish the real value of this technology and to outline its potential for routine clinical applications.
VOL. 101 NO. 5 / MAY 2014
Fertility and Sterility® Carmen Rubio, Ph.D. IVIOMICS and Instituto Universitario IVI Valencia University; Fundaci on Instituto Valenciano de Infertilidad (FIVI)/ INCLIVA, Valencia University, Valencia, Spain http://dx.doi.org/10.1016/j.fertnstert.2014.03.005 You can discuss this article with its authors and other ASRM members at http://fertstertforum.com/rubioc-next-generationsequencing-challenges-reproductive-genetics/
REFERENCES 1.
2.
3.
4.
Use your smartphone to scan this QR code and connect to the discussion forum for this article now.* * Download a free QR code scanner by searching for “QR scanner” in your smartphone’s app store or app marketplace.
VOL. 101 NO. 5 / MAY 2014
5.
Fiorentino F, Biricik A, Bono S, Spizzichino L, Cotroneo E, Cottone G, et al. Development and validation of a next-generation sequencing-based protocol for 24-chromosome aneuploidy screening of embryos. Fertil Steril 2014;101: 1375–82. Rodrigo L, Mateu E, Mercader A, Cobo AC, Peinado V, Milan M, et al. New tools for embryo selection: comprehensive chromosome screening by array comparative genomic hybridization. Biomed Res Int 2014. In press. Yin X, Tan K, Vajta G, Jiang H, Tan Y, Zhang C, et al. Massively parallel sequencing for chromosomal abnormality testing in trophectoderm cells of human blastocysts. Biol Reprod 2013;88:69. Yang Z, Liu J, Collins GS, Salem SA, Liu X, Lyle SS, et al. Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study. Mol Cytogenet 2012;5:24. Scott RT Jr, Upham KM, Forman EJ, Hong KH, Scott KL, Taylor D, et al. Blastocyst biopsy with comprehensive chromosome screening and fresh embryo transfer significantly increases in vitro fertilization implantation and delivery rates: a randomized controlled trial. Fertil Steril 2013;100:697–703.
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