This document describes the development of a rapid and low-cost next-generation sequencing (NGS) protocol for diagnosing aneuploidy in single cells from human preimplantation embryos. The authors optimized an NGS method that takes less than 15 hours and has consumable costs that are only two-thirds of existing methods. They validated the method on 54 cells with 100% sensitivity and specificity for detecting aneuploidy. The method was then applied clinically in two IVF cycles that resulted in healthy pregnancies. The NGS approach could also detect specified mutations and found an association between elevated mitochondrial DNA and aneuploidy.
Application of NGS technologies to Preimplantation Genetic Diagnosis (PGD)Andreu Paytuví
This document discusses the application of next generation sequencing (NGS) technologies to preimplantation genetic diagnosis (PGD). PGD is used to test embryos for genetic abnormalities prior to implantation to avoid miscarriages. NGS allows for PGD with sequencing of entire genomes from single cells. Data analysis uses hidden Markov models to determine ploidy status based on coverage levels. NGS-PGD costs approximately $70 per embryo and takes around 15 hours. Case studies demonstrate detection of cystic fibrosis mutations and aneuploidies from embryo biopsies.
This document summarizes a study that assessed the correlation between day 5 and day 6 blastocyst morphology and ploidy status using next-generation sequencing. The study found that slower developing blastocysts biopsied on day 6, but at the same morphological stage as day 5 blastocysts, did not have a similar chromosomal status and provided a lower chance of achieving pregnancy. Specifically, the study analyzed 168 blastocysts biopsied on either day 5 or day 6 that underwent trophectoderm biopsy and next-generation sequencing. It found that day 5 blastocysts had a higher euploidy rate than day 6 blastocysts of the same morphological stage.
Preimplantation Genetic Diagnosis using Next Generation Sequencing for Social...Maryam Rafati
The document discusses techniques for preimplantation genetic diagnosis (PGD), including PCR-based techniques, fluorescence in situ hybridization (FISH), and next generation sequencing (NGS). It summarizes misdiagnosis rates for different techniques and applications. NGS is presented as a rapid and low-cost method for comprehensive aneuploidy screening and simultaneous investigation of single gene disorders. Clinical experience using NGS-PGD is discussed, showing transfer of a single euploid embryo can increase pregnancy rates for patients with recurrent implantation failure. The European Society of Human Reproduction and Embryology (ESHRE) guidelines for PGD are also summarized.
Open Frame Sequencing™ is a universal tool that allows planning comprehensive genetic diagnostics personalized for each Patient. This solution is dedicated to specialists who expect flexible approach, efficient cooperation and “tailor made” solutions in their daily work.
This document summarizes a study applying post-light semiconductor-based next-generation sequencing (PLS-NGS) in preimplantation genetic screening with fresh embryo transfers. The study found that PLS-NGS analysis of biopsied blastomeres could be completed within 12 hours, allowing selection of normal embryos for fresh transfer without needing blastocyst vitrification. Patients undergoing PGS and fresh transfer based on PLS-NGS results had higher pregnancy and implantation rates and lower miscarriage rates than a control group without PGS. The results suggest PLS-NGS may be an effective tool for embryo selection in IVF when used to allow fresh embryo transfers.
This document discusses preimplantation genetic screening (PGS) using comprehensive chromosome screening (CCS) techniques like array comparative genomic hybridization (aCGH) to test embryos for chromosome abnormalities. It provides data from over 39,000 PGS procedures performed by Reprogenetics Laboratories showing that CCS-based PGS can double implantation rates and eliminate the negative impact of maternal age on implantation by selecting only euploid embryos for transfer. Randomized controlled trials show pregnancy rates are 20-30% higher with CCS-based PGS compared to untested embryos. The document concludes that CCS-based PGS has proven its ability to improve IVF outcomes by selecting embryos without chromosome abnormalities.
Application of NGS technologies to Preimplantation Genetic Diagnosis (PGD)Andreu Paytuví
This document discusses the application of next generation sequencing (NGS) technologies to preimplantation genetic diagnosis (PGD). PGD is used to test embryos for genetic abnormalities prior to implantation to avoid miscarriages. NGS allows for PGD with sequencing of entire genomes from single cells. Data analysis uses hidden Markov models to determine ploidy status based on coverage levels. NGS-PGD costs approximately $70 per embryo and takes around 15 hours. Case studies demonstrate detection of cystic fibrosis mutations and aneuploidies from embryo biopsies.
This document summarizes a study that assessed the correlation between day 5 and day 6 blastocyst morphology and ploidy status using next-generation sequencing. The study found that slower developing blastocysts biopsied on day 6, but at the same morphological stage as day 5 blastocysts, did not have a similar chromosomal status and provided a lower chance of achieving pregnancy. Specifically, the study analyzed 168 blastocysts biopsied on either day 5 or day 6 that underwent trophectoderm biopsy and next-generation sequencing. It found that day 5 blastocysts had a higher euploidy rate than day 6 blastocysts of the same morphological stage.
Preimplantation Genetic Diagnosis using Next Generation Sequencing for Social...Maryam Rafati
The document discusses techniques for preimplantation genetic diagnosis (PGD), including PCR-based techniques, fluorescence in situ hybridization (FISH), and next generation sequencing (NGS). It summarizes misdiagnosis rates for different techniques and applications. NGS is presented as a rapid and low-cost method for comprehensive aneuploidy screening and simultaneous investigation of single gene disorders. Clinical experience using NGS-PGD is discussed, showing transfer of a single euploid embryo can increase pregnancy rates for patients with recurrent implantation failure. The European Society of Human Reproduction and Embryology (ESHRE) guidelines for PGD are also summarized.
Open Frame Sequencing™ is a universal tool that allows planning comprehensive genetic diagnostics personalized for each Patient. This solution is dedicated to specialists who expect flexible approach, efficient cooperation and “tailor made” solutions in their daily work.
This document summarizes a study applying post-light semiconductor-based next-generation sequencing (PLS-NGS) in preimplantation genetic screening with fresh embryo transfers. The study found that PLS-NGS analysis of biopsied blastomeres could be completed within 12 hours, allowing selection of normal embryos for fresh transfer without needing blastocyst vitrification. Patients undergoing PGS and fresh transfer based on PLS-NGS results had higher pregnancy and implantation rates and lower miscarriage rates than a control group without PGS. The results suggest PLS-NGS may be an effective tool for embryo selection in IVF when used to allow fresh embryo transfers.
This document discusses preimplantation genetic screening (PGS) using comprehensive chromosome screening (CCS) techniques like array comparative genomic hybridization (aCGH) to test embryos for chromosome abnormalities. It provides data from over 39,000 PGS procedures performed by Reprogenetics Laboratories showing that CCS-based PGS can double implantation rates and eliminate the negative impact of maternal age on implantation by selecting only euploid embryos for transfer. Randomized controlled trials show pregnancy rates are 20-30% higher with CCS-based PGS compared to untested embryos. The document concludes that CCS-based PGS has proven its ability to improve IVF outcomes by selecting embryos without chromosome abnormalities.
1. The document discusses preimplantation genetic screening (PGS) using next generation sequencing to test embryos for aneuploidies.
2. Studies show that PGS improves IVF outcomes like implantation rates, pregnancy rates, and live birth rates while reducing miscarriage rates. It helps mitigate the effects of advanced maternal age.
3. PGS enables more efficient single embryo transfers, reducing multiple pregnancies and births. Next generation sequencing methods for PGS may provide advantages over previous technologies in detecting mosaicism and subchromosomal changes.
This randomized controlled trial tested whether performing blastocyst biopsy with comprehensive chromosome screening (CCS) improves in vitro fertilization (IVF) outcomes compared to routine care. They found:
1) Sustained implantation rates (probability of embryo implanting and resulting in delivery) and delivery rates per cycle were significantly higher in the CCS group compared to the routine care group.
2) In the CCS group, 61 of 72 treatment cycles led to delivery (84.7%) compared to 56 of 83 (67.5%) in the routine care group.
3) Use of CCS with blastocyst biopsy and rapid quantitative PCR-based screening resulted in statistically significantly improved IVF outcomes, with
Introduction: Preimplantation genetic screening is alive and very well. Meldr...鋒博 蔡
This document compares different technologies for 24-chromosome copy number analysis in preimplantation genetic screening and diagnosis. It discusses the differences between screening and diagnostic tests, with screening tests being noninvasive, rapid and lower cost to select embryos, while diagnostic tests require higher accuracy. Technologies reviewed include fluorescence in situ hybridization, comparative genomic hybridization, array comparative genomic hybridization and next generation sequencing, with array CGH currently being the most widely used due to its accuracy and ability to analyze all chromosomes.
This document discusses next steps in preimplantation genetic testing (PGT). It compares different biopsy stages such as polar body, cleavage-stage embryo, and blastocyst biopsies. Blastocyst biopsy has advantages of being non-invasive, having high concordance with trophectoderm biopsy, and reducing impact of embryo biopsy. The document also discusses whole genome amplification methods like array comparative genomic hybridization and next generation sequencing that are used for PGT and their advantages and limitations. Finally, it reviews studies on the possibility of non-invasive PGT using spent culture media and highlights variables like culture conditions, amplification methods, and PGT platforms that impact the results of non-invasive testing.
July 2015 is the 25th anniversary of the first births following preimplantation genetic diagnosis (PGD) world-wide. In this presentation, landmark developments are briefly reviewed and future developments are outlined which I believe will revolutionise the practice of IVF.
The document discusses a genetic test called iMGE Test that analyzes miscarriage material to identify chromosomal defects that may cause pregnancy loss. The test uses next generation sequencing to analyze all chromosomes simultaneously and does not require cell culture, providing results for over 95% of cases. Identifying genetic causes can help guide family planning decisions and determine if preimplantation genetic diagnosis would be beneficial for couples seeking to avoid miscarriage in future pregnancies. The test is recommended after miscarriage for women over 35, those with a family history of genetic defects, or couples with a history of infertility or recurrent miscarriage.
This document describes PGS-NGS 360°, a preimplantation genetic screening technique that uses next generation sequencing to examine embryos for genetic defects. It analyzes all chromosomes to screen for common defects like Down syndrome. PGS-NGS 360° is said to increase embryo implantation rates, reduce miscarriage risk, and allow for single embryo transfers, decreasing multiple pregnancies. The document provides indications for using this technique, such as advanced maternal age or previous miscarriages. It also describes the methodology, accuracy, and limitations of next generation sequencing for preimplantation genetic screening.
PGD ONE is a preimplantation genetic diagnosis test that detects genetic defects in embryos prior to pregnancy by analyzing DNA material collected through embryo biopsy. It can diagnose many known monogenic genetic diseases and reduce the risk of occurrence in offspring. The test uses next generation sequencing, the most accurate DNA analysis method available. It is a customized test designed for each patient based on their specific genetic diagnoses or family histories.
This study examined how often comprehensive chromosome screening (CCS) would alter the selection of embryos for transfer compared to traditional day 5 morphology-based selection. Out of 100 consecutive cycles:
- 22% of embryos selected based on day 5 morphology alone would have been aneuploid according to CCS results. This was lower than the 32% aneuploidy rate of all biopsied embryos.
- Patients aged 35 or older had a higher risk (31%) of an aneuploid best quality day 5 embryo being selected than younger patients (14%).
- Among cycles where CCS altered selection, 74% resulted in delivery including 77% for elective single embryo transfer cycles. Most patients
This study compared two methods for screening embryo cells for chromosomal abnormalities: fluorescence in situ hybridization (FISH) and single-nucleotide polymorphism (SNP) microarray analysis. Thirteen arrested embryos were each biopsied into individual cells, with 160 cells total randomized into the two screening methods. Microarray analysis provided interpretable results for more cells (96% vs 83% for FISH) and detected mosaicism (differences between cells of the same embryo) significantly less often than FISH (31% vs 100%). Direct comparison found FISH detected more unique genetic diagnoses per embryo on average. This is the first study to directly compare these two screening methods using paired cells from the same embryos, suggesting FISH may
This document discusses preimplantation genetic testing (PGT), which includes preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS). PGD determines an embryo's genotype to test for genetic disorders, while PGS assesses the embryo's chromosome number. The document outlines the history and development of PGT, including key milestones. It also describes current technologies used for PGT, such as fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR). The document provides an example of how one clinic uses PGT to screen for chromosomal abnormalities and genetic disorders.
This method accurately detected sex chromosome aneuploidies (45,X, 47,XXY, 47,XYY) in cell-free DNA isolated from maternal plasma. It analyzed 201 pregnancies including 16 with sex chromosome aneuploidies and 185 normal controls. The method involved massively multiplexed PCR and sequencing of 19,488 SNPs across chromosomes 13, 18, 21, X and Y. Using a statistical algorithm to analyze the SNP data, it correctly identified the copy number at all five chromosomes in 93% of samples, detecting sex chromosome aneuploidies with high sensitivity and specificity.
This document discusses the process of embryo transfer in beef cattle. It involves collecting embryos from a superovulated donor cow through artificial insemination and flushing, and then transferring the embryos to synchronized recipient cows to complete gestation. The key steps are superovulating the donor cow, artificially inseminating her, flushing her uterus 7 days later to collect embryos, processing and evaluating the embryos, and then transferring high quality embryos into synchronized recipient cows 16 days after their estrus cycles have been aligned through hormone treatments.
Clinical manaement of in vitrofertilizatonwithpreimplantation geneticdiagnosi...t7260678
This document discusses clinical management of in vitro fertilization with preimplantation genetic diagnosis (PGD). It covers:
1. PGD was introduced in 1990 to test embryos for genetic diseases before implantation, reducing risks of terminating or delivering sick children. It has helped couples at high risk of passing on genetic diseases.
2. Studies show PGD is safe when performed by experienced labs, with similar outcomes to regular IVF. The biopsy should remove one cell from day 3 embryos.
3. Optimizing PGD success requires an experienced clinic, skilled embryologists, removing one cell, and transferring high-quality embryos one at a time to avoid multiples. Number of eggs retrieved is a key factor.
This document discusses the use of time-lapse imaging to quantify the exact timing of cell divisions during embryo development. It notes that conventional grading may miss subtle differences between embryos, but time-lapse allows for collection of data on individual embryos over time. This provides precise definitions of timing checkpoints from fertilization to blastocyst.
Other potential definitions of success in art itt, et,t7260678
This document discusses success rates for IVF/ICSI treatment and strategies to improve the patient experience. It notes that only 50% of couples will achieve an ongoing pregnancy within 1 year of starting treatment. Risks like ovarian hyperstimulation syndrome are discussed. The importance of patient selection, stimulation protocols, and embryo transfer techniques are covered. The document advocates for single embryo transfer to reduce multiples without negatively impacting success rates. It also stresses providing patients with information to manage expectations and outcomes.
This document summarizes research on replacing the mitochondrial DNA (mtDNA) in human eggs to prevent inherited mtDNA diseases. Key points include:
- Over 700 mtDNA mutations can cause inherited diseases affecting thousands born each year in the US. Current treatments don't exist.
- Researchers developed a technique called spindle transfer to replace the entire mtDNA in an egg by transferring its nuclear DNA to a donor egg, eliminating future risk of transmission.
- Studies in monkey eggs showed minimal mtDNA carryover and normal development of offspring. Similar success was found replacing mtDNA in human eggs.
- Some manipulated human eggs showed abnormal fertilization but normally fertilized eggs developed into embryos and stem cell lines without detected mt
This document summarizes several studies on preimplantation genetic screening (PGS) using array comparative genomic hybridization (aCGH). Key findings include:
1) A pilot study of PGS using aCGH in "good prognosis" IVF patients under 35 found a 49.4% aneuploidy rate and an ongoing clinical pregnancy rate of 69%.
2) A randomized controlled trial in patients under 35 found higher pregnancy and ongoing pregnancy rates with day 5 biopsy and day 6 transfer plus aCGH (70.9% and 69.1%) compared to the control group without aCGH (45.8% and 41.7%).
3) Data from over 2000 patients showed eup
1. The document discusses preimplantation genetic screening (PGS) using next generation sequencing to test embryos for aneuploidies.
2. Studies show that PGS improves IVF outcomes like implantation rates, pregnancy rates, and live birth rates while reducing miscarriage rates. It helps mitigate the effects of advanced maternal age.
3. PGS enables more efficient single embryo transfers, reducing multiple pregnancies and births. Next generation sequencing methods for PGS may provide advantages over previous technologies in detecting mosaicism and subchromosomal changes.
This randomized controlled trial tested whether performing blastocyst biopsy with comprehensive chromosome screening (CCS) improves in vitro fertilization (IVF) outcomes compared to routine care. They found:
1) Sustained implantation rates (probability of embryo implanting and resulting in delivery) and delivery rates per cycle were significantly higher in the CCS group compared to the routine care group.
2) In the CCS group, 61 of 72 treatment cycles led to delivery (84.7%) compared to 56 of 83 (67.5%) in the routine care group.
3) Use of CCS with blastocyst biopsy and rapid quantitative PCR-based screening resulted in statistically significantly improved IVF outcomes, with
Introduction: Preimplantation genetic screening is alive and very well. Meldr...鋒博 蔡
This document compares different technologies for 24-chromosome copy number analysis in preimplantation genetic screening and diagnosis. It discusses the differences between screening and diagnostic tests, with screening tests being noninvasive, rapid and lower cost to select embryos, while diagnostic tests require higher accuracy. Technologies reviewed include fluorescence in situ hybridization, comparative genomic hybridization, array comparative genomic hybridization and next generation sequencing, with array CGH currently being the most widely used due to its accuracy and ability to analyze all chromosomes.
This document discusses next steps in preimplantation genetic testing (PGT). It compares different biopsy stages such as polar body, cleavage-stage embryo, and blastocyst biopsies. Blastocyst biopsy has advantages of being non-invasive, having high concordance with trophectoderm biopsy, and reducing impact of embryo biopsy. The document also discusses whole genome amplification methods like array comparative genomic hybridization and next generation sequencing that are used for PGT and their advantages and limitations. Finally, it reviews studies on the possibility of non-invasive PGT using spent culture media and highlights variables like culture conditions, amplification methods, and PGT platforms that impact the results of non-invasive testing.
July 2015 is the 25th anniversary of the first births following preimplantation genetic diagnosis (PGD) world-wide. In this presentation, landmark developments are briefly reviewed and future developments are outlined which I believe will revolutionise the practice of IVF.
The document discusses a genetic test called iMGE Test that analyzes miscarriage material to identify chromosomal defects that may cause pregnancy loss. The test uses next generation sequencing to analyze all chromosomes simultaneously and does not require cell culture, providing results for over 95% of cases. Identifying genetic causes can help guide family planning decisions and determine if preimplantation genetic diagnosis would be beneficial for couples seeking to avoid miscarriage in future pregnancies. The test is recommended after miscarriage for women over 35, those with a family history of genetic defects, or couples with a history of infertility or recurrent miscarriage.
This document describes PGS-NGS 360°, a preimplantation genetic screening technique that uses next generation sequencing to examine embryos for genetic defects. It analyzes all chromosomes to screen for common defects like Down syndrome. PGS-NGS 360° is said to increase embryo implantation rates, reduce miscarriage risk, and allow for single embryo transfers, decreasing multiple pregnancies. The document provides indications for using this technique, such as advanced maternal age or previous miscarriages. It also describes the methodology, accuracy, and limitations of next generation sequencing for preimplantation genetic screening.
PGD ONE is a preimplantation genetic diagnosis test that detects genetic defects in embryos prior to pregnancy by analyzing DNA material collected through embryo biopsy. It can diagnose many known monogenic genetic diseases and reduce the risk of occurrence in offspring. The test uses next generation sequencing, the most accurate DNA analysis method available. It is a customized test designed for each patient based on their specific genetic diagnoses or family histories.
This study examined how often comprehensive chromosome screening (CCS) would alter the selection of embryos for transfer compared to traditional day 5 morphology-based selection. Out of 100 consecutive cycles:
- 22% of embryos selected based on day 5 morphology alone would have been aneuploid according to CCS results. This was lower than the 32% aneuploidy rate of all biopsied embryos.
- Patients aged 35 or older had a higher risk (31%) of an aneuploid best quality day 5 embryo being selected than younger patients (14%).
- Among cycles where CCS altered selection, 74% resulted in delivery including 77% for elective single embryo transfer cycles. Most patients
This study compared two methods for screening embryo cells for chromosomal abnormalities: fluorescence in situ hybridization (FISH) and single-nucleotide polymorphism (SNP) microarray analysis. Thirteen arrested embryos were each biopsied into individual cells, with 160 cells total randomized into the two screening methods. Microarray analysis provided interpretable results for more cells (96% vs 83% for FISH) and detected mosaicism (differences between cells of the same embryo) significantly less often than FISH (31% vs 100%). Direct comparison found FISH detected more unique genetic diagnoses per embryo on average. This is the first study to directly compare these two screening methods using paired cells from the same embryos, suggesting FISH may
This document discusses preimplantation genetic testing (PGT), which includes preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS). PGD determines an embryo's genotype to test for genetic disorders, while PGS assesses the embryo's chromosome number. The document outlines the history and development of PGT, including key milestones. It also describes current technologies used for PGT, such as fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR). The document provides an example of how one clinic uses PGT to screen for chromosomal abnormalities and genetic disorders.
This method accurately detected sex chromosome aneuploidies (45,X, 47,XXY, 47,XYY) in cell-free DNA isolated from maternal plasma. It analyzed 201 pregnancies including 16 with sex chromosome aneuploidies and 185 normal controls. The method involved massively multiplexed PCR and sequencing of 19,488 SNPs across chromosomes 13, 18, 21, X and Y. Using a statistical algorithm to analyze the SNP data, it correctly identified the copy number at all five chromosomes in 93% of samples, detecting sex chromosome aneuploidies with high sensitivity and specificity.
This document discusses the process of embryo transfer in beef cattle. It involves collecting embryos from a superovulated donor cow through artificial insemination and flushing, and then transferring the embryos to synchronized recipient cows to complete gestation. The key steps are superovulating the donor cow, artificially inseminating her, flushing her uterus 7 days later to collect embryos, processing and evaluating the embryos, and then transferring high quality embryos into synchronized recipient cows 16 days after their estrus cycles have been aligned through hormone treatments.
Clinical manaement of in vitrofertilizatonwithpreimplantation geneticdiagnosi...t7260678
This document discusses clinical management of in vitro fertilization with preimplantation genetic diagnosis (PGD). It covers:
1. PGD was introduced in 1990 to test embryos for genetic diseases before implantation, reducing risks of terminating or delivering sick children. It has helped couples at high risk of passing on genetic diseases.
2. Studies show PGD is safe when performed by experienced labs, with similar outcomes to regular IVF. The biopsy should remove one cell from day 3 embryos.
3. Optimizing PGD success requires an experienced clinic, skilled embryologists, removing one cell, and transferring high-quality embryos one at a time to avoid multiples. Number of eggs retrieved is a key factor.
This document discusses the use of time-lapse imaging to quantify the exact timing of cell divisions during embryo development. It notes that conventional grading may miss subtle differences between embryos, but time-lapse allows for collection of data on individual embryos over time. This provides precise definitions of timing checkpoints from fertilization to blastocyst.
Other potential definitions of success in art itt, et,t7260678
This document discusses success rates for IVF/ICSI treatment and strategies to improve the patient experience. It notes that only 50% of couples will achieve an ongoing pregnancy within 1 year of starting treatment. Risks like ovarian hyperstimulation syndrome are discussed. The importance of patient selection, stimulation protocols, and embryo transfer techniques are covered. The document advocates for single embryo transfer to reduce multiples without negatively impacting success rates. It also stresses providing patients with information to manage expectations and outcomes.
This document summarizes research on replacing the mitochondrial DNA (mtDNA) in human eggs to prevent inherited mtDNA diseases. Key points include:
- Over 700 mtDNA mutations can cause inherited diseases affecting thousands born each year in the US. Current treatments don't exist.
- Researchers developed a technique called spindle transfer to replace the entire mtDNA in an egg by transferring its nuclear DNA to a donor egg, eliminating future risk of transmission.
- Studies in monkey eggs showed minimal mtDNA carryover and normal development of offspring. Similar success was found replacing mtDNA in human eggs.
- Some manipulated human eggs showed abnormal fertilization but normally fertilized eggs developed into embryos and stem cell lines without detected mt
This document summarizes several studies on preimplantation genetic screening (PGS) using array comparative genomic hybridization (aCGH). Key findings include:
1) A pilot study of PGS using aCGH in "good prognosis" IVF patients under 35 found a 49.4% aneuploidy rate and an ongoing clinical pregnancy rate of 69%.
2) A randomized controlled trial in patients under 35 found higher pregnancy and ongoing pregnancy rates with day 5 biopsy and day 6 transfer plus aCGH (70.9% and 69.1%) compared to the control group without aCGH (45.8% and 41.7%).
3) Data from over 2000 patients showed eup
1) A couple underwent preimplantation genetic diagnosis (PGD) for Dyskeratosis Congenita caused by a mutation in the RTEL1 gene, as they previously had an affected child.
2) Conventional PGD using short tandem repeat (STR) markers near the gene found high rates of allele drop out and no results for some embryos.
3) Reanalysis using quantitative PCR (qPCR) of single nucleotide polymorphisms (SNPs) closer to the mutation found the STR-based method misdiagnosed 21% of embryos due to undetected recombinations between the STRs and mutation.
4) qPCR allowed simultaneous analysis of multiple linked SNPs and the mutation, avoiding
This study evaluated the impact of standard- and high-dose GnRH antagonists compared to a GnRH agonist on endometrial development in women undergoing controlled ovarian stimulation for oocyte donation. Thirty-one women were treated with either a standard dose of ganirelix, a high dose of ganirelix, or buserelin. Endometrial biopsies on days 2 and 7 after HCG administration found that development was similar in the standard- and high-dose ganirelix groups and comparable to natural cycles, but development was arrested in the buserelin group. Gene expression patterns after ganirelix more closely matched natural cycles than after buserelin. The study concluded that
This document discusses comprehensive chromosomal screening at the blastocyst stage using comparative genomic hybridization (CGH). CGH allows testing of all chromosomes and avoids issues with mosaicism seen at earlier stages. Clinical results showed high pregnancy and birth rates even in older patients when euploid embryos were transferred, though pregnancy rates per cycle declined with age as more patients had only aneuploid embryos. While screening improved outcomes, age still reduced pregnancy chances due to increased aneuploidy frequency. Further randomized trials are needed to validate the approach.
This document discusses preimplantation genetic diagnosis (PGD), which involves in vitro fertilization combined with genetic testing of embryos to select embryos without specific genetic defects or diseases. It notes that PGD requires expertise across fertility medicine, genetics, embryology and molecular analysis. While PGD can reduce health risks for offspring and minimize inheritance of disabilities, children conceived through assisted reproductive technologies like IVF may have a slightly higher risk of birth defects. The use of PGD is increasing for conditions like single gene disorders but it has limitations such as mosaicism and laboratory errors. Guidelines and laws regulate the application of PGD and prohibit its use for non-medical sex selection.
Development and validation of an accurate quantitative real time polymerase c...t7260678
This document describes the development and validation of a quantitative real-time polymerase chain reaction (qPCR) method for comprehensive chromosomal aneuploidy screening of human blastocysts. The method was found to be highly accurate, correctly diagnosing aneuploidies in 97.6% of cell line samples and 98.6% of human blastocyst samples compared to conventional methods. The qPCR method can provide a diagnosis for all 24 chromosomes in only 4 hours, making it suitable for screening of blastocyst biopsies without the need for cryopreservation. This rapid method could allow for fresh euploid embryo transfers and improve outcomes for couples undergoing in vitro fertilization.
New developments in reproductive medicine (1)t7260678
1. Approximately 15-20% of couples in Germany experience infertility issues. New developments in reproductive medicine include GnRH-antagonists for ovarian stimulation, elective single embryo transfer (eSET) to reduce multiple pregnancies, blastocyst transfer, in-vitro maturation, and vitrification for cryopreservation.
2. Studies show eSET results in similar pregnancy rates as double embryo transfer but significantly reduces multiple pregnancy risks. Vitrification is an improved cryopreservation technique with higher post-thaw survival and pregnancy rates compared to slow freezing.
3. In-vitro maturation of oocytes is a promising new technique that could help avoid ovarian hyperstimulation syndrome and enable fertility preservation for cancer patients.
1. The document discusses human reproductive cloning and whether it should be pursued as a way to help infertile patients have biologically related children.
2. Studies showed that cloning techniques developed for animals can be applied to human cells, successfully generating human-animal hybrid embryos.
3. However, critics argue that cloning is inefficient and risky, and may produce unhealthy offspring, while proponents counter that success rates are improving and critics misrepresent facts.
1. The document discusses various techniques that have been proposed to modify the embryo transfer procedure in order to optimize results.
2. Randomized controlled trials have found that performing a trial embryo transfer before the actual procedure, using ultrasound guidance for the transfer, and depositing embryos 2cm below the uterine fundus can significantly increase pregnancy rates.
3. However, randomized trials have also found that flushing the cervical canal, catheter withdrawal time, use of a fibrin sealant, bed rest duration after transfer, and catheter type do not affect pregnancy outcomes. The effects of some other proposed techniques, such as transfer with a full bladder or antibiotic administration, require further study.
This document discusses several potential technologies for identifying viable oocytes, including polar body biopsy, spindle imaging, and zona pellucida birefringence. Polar body biopsy and array comparative genomic hybridization can be used to assess chromosomal disorders in oocytes. Spindle imaging using polarization microscopy can identify the meiotic stage of an oocyte. Zona pellucida birefringence imaging using polarization microscopy provides information on cytoplasmic maturity, with more structured zonae correlating with better developmental potential. These technologies, especially polar body biopsy, spindle imaging, and zona imaging, may help select the most implantation-competent embryos.
The document discusses various aspects of embryo transfer techniques in IVF. It describes the typical steps, including selection of the best embryos for transfer. It notes that factors like the transferring physician's skill, catheter type, trial embryo transfer accuracy, ultrasound guidance, and uterine contractions can influence the success of embryo transfer. Research studies are summarized that found around 30% of patients had a uterine measurement discrepancy between trial transfer and ultrasound-guided transfer, and soft catheters were associated with higher pregnancy rates than firm catheters.
Dr. irene souter pgd stickler foundation (5)t7260678
This document discusses preimplantation genetic diagnosis (PGD), which involves biopsy of a single cell from each embryo followed by genetic analysis to identify normal embryos for implantation. PGD is offered to couples at risk of passing on genetic disorders, chromosomal issues, or with recurrent pregnancy loss. The process involves ovarian stimulation, egg retrieval, fertilization, embryo biopsy on day 3, genetic analysis, and embryo transfer. Common indications for PGD include single gene disorders, translocations, aneuploidy screening, and HLA matching. While mistakes can occur, studies show delivery outcomes and malformation rates are similar to ICSI. PGD has allowed many to have healthy children who would otherwise be at high risk of genetic conditions.
This document discusses the application of next-generation sequencing (NGS) technologies to preimplantation genetic diagnosis (PGD). It provides a brief history of PGD and explains the use of NGS-based PGD to detect chromosomal abnormalities in embryos before implantation. The document outlines the PGD workflow using NGS, which involves sequencing embryos and analyzing the data using hidden Markov models and other statistical methods to determine ploidy status and identify chromosomal abnormalities. Benefits of NGS-based PGD include lower costs relative to array-based methods and the ability to assess more embryos.
This randomized controlled trial compared outcomes of in vitro fertilization (IVF) when comprehensive chromosome screening (CCS) of blastocysts was used versus the standard of care. They found that using CCS resulted in significantly higher sustained implantation rates (66.4% vs 47.9%) and delivery rates per cycle (84.7% vs 67.5%) compared to the control group. CCS improved IVF outcomes by enabling selection of euploid embryos for transfer, leading to meaningful increases in the likelihood of successful implantation and delivery.
This document compares different technologies for 24-chromosome copy number analysis in preimplantation genetic screening and diagnosis. It discusses the differences between screening and diagnostic tests, with screening tests being noninvasive, low-cost and allowing analysis of all patients to prioritize embryos, while diagnostic tests require high accuracy. It reviews technologies for copy number analysis including chromosome spreading, array comparative genomic hybridization, quantitative PCR and next generation sequencing, discussing their advantages and limitations for screening and diagnosis.
The document compares euploidy rates between blastomere biopsies on day 3 embryos and trophectoderm biopsies on day 5-7 blastocysts. Of the 1603 embryos biopsied, 31% were euploid, 62% were aneuploid, and 7% were unanalyzable. A significantly higher proportion of embryos were euploid with trophectoderm biopsy on day 5-7 (42%) compared to blastomere biopsy on day 3 (24%). Combining blastocyst culture, trophectoderm biopsy, and aneuploidy screening using aCGH provides a more efficient means of achieving euploid pregnancies in IVF.
This study evaluated the use of blastocyst biopsy and array comparative genomic hybridization (aCGH) for preimplantation genetic diagnosis in 12 patients with chromosomal translocations. The diagnostic efficiency was 90.2% and euploidy rate was 32.7%. Ten cycles of thawed embryo transfer resulted in three live births and three ongoing pregnancies, for an ongoing pregnancy rate of 60% per transfer cycle. Prenatal diagnoses confirmed the PGD/aCGH results. The strategy demonstrates promising outcomes and may provide a more effective approach than traditional methods like fluorescence in situ hybridization. Larger studies are still needed to verify the results.
This document summarizes several adjunct techniques used in IVF laboratories including sperm DNA fragmentation testing, advanced sperm selection methods like IMSI and pICSI, embryo selection techniques like time-lapse imaging and PGS, and mitochondrial DNA load measurement. It reviews the current evidence for each technique, noting that while some like TL imaging show promise, the evidence is still limited and inconclusive for many techniques to recommend their routine use to improve IVF outcomes. Larger randomized controlled trials are still needed to prove effectiveness.
This study prospectively compared pregnancy and implantation outcomes between two groups of patients undergoing preimplantation genetic screening (PGS). Group A embryos (n=582) were cultured and monitored using a time-lapse system, while Group B embryos (n=581) were cultured conventionally. Both groups underwent trophectoderm biopsy and array comparative genomic hybridization (aCGH) testing on day 5. Euploid blastocysts displaying the most predictive morphokinetic parameters (Group A) or best morphology (Group B) were transferred. Clinical pregnancy, implantation, and ongoing pregnancy rates were significantly higher in Group A compared to Group B, demonstrating improved outcomes when selecting competent blastocysts combining time-lapse monitoring
This study compared pregnancy outcomes of in vitro fertilization (IVF) patients who underwent single embryo transfer where the embryo was selected based on (1) morphology alone or (2) morphology assessed with array comparative genomic hybridization (aCGH). Patients were randomly assigned to one of the two selection methods. The clinical pregnancy and ongoing pregnancy rates were significantly higher in the group where aCGH was used in addition to morphology to select the embryo. No twin pregnancies occurred. The results suggest that aCGH may improve pregnancy outcomes compared to morphology alone by detecting chromosomal abnormalities.
This document discusses the potential for using time-lapse embryo imaging to non-invasively determine embryonic aneuploidy (chromosomal abnormalities) through examination of embryo morphology and timing of developmental events. Recent studies have found that early cleavage timings observed through time-lapse imaging can provide insight into chromosomal status. However, the predictive ability is limited and embryo biopsy with preimplantation genetic screening remains the most reliable method to assess chromosomal complement. Continued research aiming to improve modeling may enhance the ability to detect aneuploidy without biopsy using morphokinetic data.
This document discusses the potential for using time-lapse embryo imaging to non-invasively determine embryonic aneuploidy (chromosomal abnormalities) through examination of embryo morphology and timing of developmental events. Recent studies have found that early cleavage timings observed through time-lapse imaging can provide insight into chromosomal status. However, the predictive ability is limited and embryo biopsy with preimplantation genetic screening remains the most reliable method to assess chromosomal complement. Continued research aiming to improve predictive models through analysis of multiple morphological features and timings may help select embryos less likely to be aneuploid.
This document reviews studies examining the relationship between embryo development patterns observed via time-lapse imaging and embryonic aneuploidy. It finds that while some early studies show timing of cleavages may indicate chromosomal complement, the predictive power is limited. Continued refinement of modeling may help improve ability to determine aneuploidy non-invasively, but biopsy with preimplantation genetic screening remains the most reliable method.
Illumina is a leader in genomics technologies that provide reliable answers to guide reproductive and genetic health choices. Their portfolio includes technologies for preimplantation genetic screening and diagnosis, non-invasive prenatal testing, cytogenetics, carrier screening, and identifying inherited conditions. These solutions deliver accurate information through next-generation sequencing and microarrays to empower informed choices and transform lives along the healthcare continuum.
Preimplantation genetic diagnosis (PGD) allows embryos created through in vitro fertilization to be tested for genetic defects before implantation. It is primarily used for two groups - individuals at high risk of passing on genetic diseases to prevent disease or termination of pregnancies, and to screen embryos for chromosomal abnormalities to improve IVF success rates. The techniques used include biopsy of polar bodies or blastomeres from embryos, followed by analysis using polymerase chain reaction, fluorescence in situ hybridization, or comparative genomic hybridization. PGD is most commonly used for common single gene disorders and chromosomal translocations but requires specialized expertise and is not feasible for rare genetic conditions. It has helped many families avoid transmission of inherited diseases and improve outcomes of
Preimplantation genetic diagnosis (PGD) was introduced in the 1990s to test embryos created through in vitro fertilization for genetic defects before implantation. Techniques like fluorescence in situ hybridization and polymerase chain reaction allow for analysis of chromosomes and genes in single cells from embryos. PGD is used for couples at high risk of passing on genetic diseases and for in vitro fertilization patients undergoing screening of embryos for chromosomal abnormalities. The techniques and indications for PGD are discussed along with results and outcomes of pregnancies achieved through the procedure.
Preimplantation genetic diagnosis (PGD) allows embryos created through in vitro fertilization to be tested for genetic defects before implantation. It is primarily used for two groups - individuals at high risk of passing on genetic diseases to prevent disease or termination of pregnancies, and to screen embryos for chromosomal abnormalities to improve IVF success rates. The techniques used include biopsy of polar bodies or blastomeres from embryos, followed by analysis using polymerase chain reaction, fluorescence in situ hybridization, or comparative genomic hybridization. PGD is most commonly used for common single gene disorders and chromosomal translocations but is limited by the technical challenges of testing single cells.
This document summarizes a study that developed a new microarray platform capable of simultaneously assessing aneuploidy, mitochondrial DNA content, and single-nucleotide polymorphisms in human polar bodies and embryos. The microarray was optimized and validated using cell lines and clinical samples. Results found the microarray detected aneuploidies with 97% accuracy and could accurately determine relative mitochondrial DNA quantities and genotypes, allowing confirmation of parental origin. The microarray provides information beyond chromosomal analysis alone that could improve embryo assessment and selection.
This document provides a cautionary commentary on recent publications claiming that time-lapse imaging can be used to assess embryo aneuploidy risk and increase pregnancy rates. The authors believe the claims are premature and unsubstantiated. They argue that the studies are underpowered and likely confounded by maternal age rather than embryo aneuploidy alone. Larger, age-adjusted datasets or randomized controlled trials are needed to validate the findings before clinical application.
This document provides a cautionary commentary on recent publications claiming that time-lapse imaging can be used to assess embryo aneuploidy risk and increase pregnancy rates. The authors believe the claims are premature and unsubstantiated. They argue that the studies are underpowered and likely confounded by maternal age rather than embryo aneuploidy alone. Larger, age-adjusted datasets or randomized controlled trials are needed to validate the findings before clinical application.
This study investigated the incidence and clinical implications of multinucleated (MN) blastomeres in embryos undergoing preimplantation genetic screening (PGS) or preimplantation genetic diagnosis (PGD). The study found that 41.3% of cycles involved at least one MN embryo. While the majority of MN blastomeres showed chromosomal abnormalities, some embryos with MN blastomeres free of genetic abnormalities tested resulted in three healthy deliveries. This suggests that genetic analysis of MN embryos can identify some that may result in healthy births.
The document discusses preimplantation genetic testing for aneuploidy (PGT-A), which screens embryos for chromosomal abnormalities during in vitro fertilization (IVF) treatment. It notes that while PGT-A is purported to improve live birth rates and reduce miscarriage risks, two recent large randomized controlled trials found no significant benefits. A study analyzed in the document found PGT-A to be not cost-effective due to the high costs associated with preventing one miscarriage. Overall, the document concludes that the current evidence is insufficient to support the routine use of PGT-A in clinical practice.
Preimplantation genetic screening (pgs) current ppt鋒博 蔡
This document summarizes preimplantation genetic testing techniques used to screen embryos for genetic disorders prior to implantation. It discusses the current status and future prospects of preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS). PGD determines an embryo's genotype to test for specific genetic disorders, while PGS assesses the embryo's chromosome number (karyotype) to screen for chromosomal abnormalities. The document outlines several techniques used for PGD and PGS, including multiplex PCR, fluorescence in situ hybridization (FISH), and whole genome amplification from single cells. It provides examples of how these techniques are applied to test for conditions like spinal muscular atrophy, sickle cell an
This document summarizes recent research on embryo implantation and selection techniques presented at the 2006 ESHRE conference. It discusses factors that influence implantation rates, such as embryo morphology, endometrial receptivity, preimplantation genetic diagnosis (PGD), and blastocyst culture. Several studies presented found that morphological features like early cleavage and blastocyst formation correlated with successful implantation. PGD and blastocyst biopsy were shown to improve implantation and birth rates compared to cleavage-stage biopsy. However, mosaicism remains a challenge for PGD accuracy. Overall, the goal of this research is to better understand factors influencing implantation and develop techniques to select the most viable embryos.
The document outlines the key stages of human embryonic development from fertilization through the fetal period. It begins with fertilization and the early cleavage stages. Around 3 days post-fertilization, the embryo reaches the morula stage and enters the uterus. It then forms a blastocyst with an inner cell mass and trophoblast cells. Around day 6, the blastocyst implants into the uterine wall and the trophoblasts begin to produce hCG. Organ systems rapidly develop through week 8, after which the fetal period of growth begins until birth.
Implantation begins around 6 days after fertilization and is usually complete by 11-12 days. The blastocyst implants in the endometrium through enzymes produced by the trophoblast. Trophoblast cells penetrate the endometrium and develop into two layers. By 10 days the conceptus is fully embedded and a blood supply is established. The formation of the bilaminar embryonic disc and primary chorionic villi occurs around 13 days. Ectopic pregnancies can occur if implantation is outside the uterus, most commonly in the fallopian tubes.
1. The stages of pregnancy and development include fertilization, embryonic development, fetal development, growth, and childbirth.
2. Fertilization occurs when a sperm cell fertilizes an egg cell in the fallopian tubes. The zygote then undergoes cell division and implants in the uterus.
3. During embryonic development, the embryo undergoes differentiation and formation of the three primary layers and organs.
4. The fetal stage involves continued growth and maturation of all body systems until birth.
1) The document describes the key stages of human embryonic development from fertilization through the formation of the basic body plan and extraembryonic membranes over the first 8 weeks.
2) It explains how the three primary germ layers (ectoderm, mesoderm and endoderm) form and give rise to the major tissues and organ systems.
3) The role and formation of the embryonic membranes - amnion, yolk sac and allantois - as well as the placenta for nutrient exchange are covered.
Day 0-1: Fertilization and early zygotic transcription.
Day 1-3: Cleavage stages and compaction to morula.
Day 3-5: Blastocyst formation with inner cell mass and trophectoderm, hatching from zona pellucida.
Day 6-9: Implantation into endometrium and initiation of placenta formation through lacunae and trophoblast invasion.
Pre-implantation genetic diagnosis (PGD) involves testing a single cell from an 8-cell embryo during in vitro fertilization (IVF) to screen for genetic disorders and improve the chances of a normal pregnancy. A cell is removed from the embryo and tested using fluorescence in situ hybridization (FISH) to check chromosome number and size, or polymerase chain reaction (PCR) to test for specific genetic mutations. Embryos found to be free of genetic disorders based on testing are then implanted into the uterus, while affected embryos are not transferred. PGD allows couples at risk of passing on genetic diseases to potentially have healthy children.
Embryo transfer involves removing embryos from female cattle of superior genetics and implanting them into recipient females. This allows one donor cow to produce many offspring, improving herd genetics faster. The process begins by synchronizing donor and recipient estrous cycles then flushing embryos from the donor 7 days after breeding. Embryos are examined before transferring viable ones into recipients. While expensive, embryo transfer can increase genetic gains if used with donor cows having desirable traits.
Embryo transfer is a process where an embryo is collected from a donor female and transferred to a recipient female to complete its development. It allows genetically superior females to produce more offspring than through natural reproduction. Embryo transfer is used in cattle, horses, goats, sheep and other domestic and non-domestic species. The process involves superovulating the donor female, collecting embryos 6-9 days after breeding, and transferring high quality embryos into a synchronized recipient female. Embryo transfer maximizes genetic gains and production from elite females.
This document provides an overview of embryo transfer (ET) in cattle. It describes the ET process, which involves removing embryos from a genetically superior donor cow and transferring them to recipient cows. The goal of ET is to efficiently produce genetically superior offspring. The document outlines the steps of synchronizing donor and recipient cows, flushing embryos from the donor, examining and transferring high quality embryos to recipients. It notes that ET is an expensive but effective way to introduce superior genetics into a herd within a short time period.
The document discusses embryo transfer, which is a process where an embryo is collected from a donor female and transferred to a recipient female to complete its development. Embryo transfer allows a genetically superior female to produce more offspring than through natural reproduction. Key aspects discussed include selecting donor females, inducing superovulation in donors to release multiple eggs, inseminating donors, non-surgical and surgical embryo recovery methods, evaluating and storing embryos, and transferring embryos into recipient females through non-surgical or surgical methods.
This document discusses the process of embryo transfer in beef cattle. It involves collecting embryos from a superovulated donor cow through artificial insemination and flushing, and then transferring the embryos to synchronized recipient cows to complete gestation. The key steps are superovulating the donor cow, artificially inseminating her, flushing her uterus 7 days later to collect embryos, processing and evaluating the embryos, and then transferring high quality embryos into synchronized recipient cows 16 days after their estrus cycles have been aligned through hormone treatments. While expensive, embryo transfer allows for increasing the number of offspring from genetically superior cows and marketing their embryos.
This document discusses the process of embryo transfer in beef cattle. It involves collecting embryos from a superovulated donor cow through artificial insemination and flushing, and then transferring the embryos to synchronized recipient cows to complete gestation. The key steps are superovulating the donor cow, artificially inseminating her, flushing her uterus 7 days later to collect embryos, processing and evaluating the embryos, and then transferring high quality embryos into synchronized recipient cows 16 days after their estrus cycles have been aligned through hormone treatments.
This document provides an overview of embryo transfer (ET) in cattle. It describes the ET process, which involves removing embryos from a genetically superior donor cow and transferring them to recipient cows. The goal of ET is to efficiently produce genetically superior offspring. The document outlines the steps of synchronizing donor and recipient cows, flushing embryos from the donor, examining and transferring high quality embryos to recipients. It notes that ET is an expensive but effective way to introduce superior genetics into a herd within a short time period.
Embryo transfer involves removing embryos from female cattle of superior genetics and implanting them into recipient females. This allows one donor cow to produce many offspring, improving herd genetics faster. The process is complicated, requiring synchronization of donor and recipient estrous cycles, flushing embryos from the donor one week after breeding, and transferring high quality embryos. While expensive, embryo transfer can increase genetic gains if used with donor cows that have desirable traits worth propagating.
1. Methods
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ORIGINAL ARTICLE
Clinical utilisation of a rapid low-pass whole
genome sequencing technique for the diagnosis of
aneuploidy in human embryos prior to implantation
Dagan Wells,1 Kulvinder Kaur,2 Jamie Grifo,3 Michael Glassner,4 Jenny C Taylor,2
Elpida Fragouli,5 Santiago Munne6
▸ Additional material is
published online only. To view
please visit the journal online
(http://dx.doi.org/10.1136/
jmedgenet-2014-102497).
1Nuffield Department of
Obstetrics and Gynaecology,
John Radcliffe Hospital,
University of Oxford, Oxford,
UK
2NIHR Oxford Biomedical
Research Centre, Wellcome
Trust Centre for Human
Genetics, Oxford, UK
3New York University Fertility
Center, New York, New York,
USA
4Main Line Fertility, Bryn
Mawr, Pennsylvania, USA
5Reprogenetics UK, Institute of
Reproductive Sciences, Oxford,
UK
6Reprogenetics LLC, Livingston,
New Jersey, USA
Correspondence to
Dr Dagan Wells,
University of Oxford,
Department of Obstetrics and
Gynaecology, John Radcliffe
Hospital, Women’s Centre,
Oxford, OX3 9DU, UK;
dagan.wells@obs-gyn.ox.ac.uk
Received 29 April 2014
Revised 4 June 2014
Accepted 20 June 2014
To cite: Wells D, Kaur K,
Grifo J, et al. J Med Genet
2014;51:553–562.
ABSTRACT
Background The majority of human embryos created
using in vitro fertilisation (IVF) techniques are aneuploid.
Comprehensive chromosome screening methods,
applicable to single cells biopsied from preimplantation
embryos, allow reliable identification and transfer of
euploid embryos. Recently, randomised trials using such
methods have indicated that aneuploidy screening
improves IVF success rates. However, the high cost of
testing has restricted the availability of this potentially
beneficial strategy. This study aimed to harness next-generation
sequencing (NGS) technology, with the
intention of lowering the costs of preimplantation
aneuploidy screening.
Methods Embryo biopsy, whole genome amplification
and semiconductor sequencing.
Results A rapid (<15 h) NGS protocol was developed,
with consumable cost only two-thirds that of the most
widely used method for embryo aneuploidy detection.
Validation involved blinded analysis of 54 cells from cell
lines or biopsies from human embryos. Sensitivity and
specificity were 100%. The method was applied
clinically, assisting in the selection of euploid embryos in
two IVF cycles, producing healthy children in both cases.
The NGS approach was also able to reveal specified
mutations in the nuclear or mitochondrial genomes in
parallel with chromosome assessment. Interestingly,
elevated mitochondrial DNA content was associated with
aneuploidy ( p<0.05), a finding suggestive of a link
between mitochondria and chromosomal malsegregation.
Conclusions This study demonstrates that NGS
provides highly accurate, low-cost diagnosis of
aneuploidy in cells from human preimplantation embryos
and is rapid enough to allow testing without embryo
cryopreservation. The method described also has the
potential to shed light on other aspects of embryo
genetics of relevance to health and viability.
INTRODUCTION
Chromosome segregation during female meiosis is
particularly error prone in humans, a problem
that worsens with advancing age. Recent studies
have demonstrated that approximately a quarter
of oocytes from women in their early 30s are
chromosomally abnormal, with aneuploidy rates
increasing to over 75% in the oocytes of women
over 40.1 It has been shown that most of the
aneuploid embryos produced from such oocytes
fail to implant in the uterus, although a minority
do succeed in forming a pregnancy only to later
miscarry.2 The high frequency of chromosome
abnormality during the first few days of life is of
great relevance to infertility treatments such as in
vitro fertilisation (IVF). Typically, IVF involves
the fertilisation of several oocytes, but in order to
avoid multiple pregnancy and the associated risks
of complications for the mother and children, it
is recommended that the number of embryos
transferred to the uterus is restricted, ideally to a
single embryo.3 Maximising the likelihood of
obtaining a pregnancy depends on accurate deter-mination
of the embryo with the greatest capacity
for producing a child, ensuring that it is priori-tised
for transfer. Currently, the decision of
which embryo should be transferred is primarily
based on a simple evaluation of morphology.
However, the appearance of an embryo is only
weakly correlated with its potential to form a
pregnancy and reveals no useful information
about its chromosomal status. In theory, screening
for aneuploidy, a problem which has a more
definitive impact on the ability of an embryo to
produce a healthy baby, could provide a valuable
means of identifying viable embryos.
The main obstacle to testing human embryos for
aneuploidy is the extremely limited amount of
tissue available for analysis. At the time when most
genetic testing has traditionally been carried out,
embryos are composed of just 6–10 cells and only
one cell (blastomere) may be safely removed for
analysis. Although obtaining accurate genetic infor-mation
from a single cell is challenging, recent
years have seen several methods developed for this
purpose. Some protocols are based on the use of
microarrays (eg, comparative genomic hybridisation
or analysis of single nucleotide polymorphisms),4 5
while others involve quantitative PCR.6 In the last
2 years, several randomised trials using these tech-niques
have been undertaken, producing clinical
data supporting the hypothesis that screening of
embryos for aneuploidy can improve IVF out-comes,
increasing pregnancy rates and reducing
miscarriages.7–10 However, all methods currently
available for the genetic analysis of preimplantation
embryos suffer from shortcomings which limit
their clinical applicability.
Cost is an important consideration for both clin-ical
and scientific applications of single cell analysis.
In the context of IVF treatment, the cost of aneu-ploidy
screening is multiplied by the number of
embryos produced by the couple (averaging
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2. Methods
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approximately six, but in some cases exceeding 20). The
expense associated with the testing of multiple samples puts
chromosome analysis beyond the reach of many patients. The
potential for producing a large number of embryos in a single
cycle of IVF treatment also means that any test must be scalable,
allowing multiple samples to be assessed simultaneously. For any
clinically applied diagnostic assay, it is advantageous if results
can be provided rapidly. However, in the case of preimplanta-tion
embryo testing the speed of the procedure assumes even
greater importance since the window of time during which the
embryo has the capacity to implant and the mother’s endomet-rium
is receptive is narrow. Depending on the exact embryo
biopsy strategy employed, there may be less than 24 h available
for genetic analysis.
This study aimed to develop a rapid, scalable, cost-effective
method for the genetic analysis of single cells (blastomeres) or
trophectoderm biopsies derived from human preimplantation
embryos. For this purpose, we chose to employ next-generation
sequencing (NGS) technology. NGS is a term that encompasses
a variety of different methods capable of generating large
quantities of DNA sequence information, rapidly and at a low
cost per base. Our data confirm that NGS can be successfully
applied to the diagnosis of a variety of genetic abnormalities
in single cells from human embryos and has various advantages
over traditional technologies used for preimplantation genetic
diagnosis and screening. Clinical applicability was
demonstrated.
RESULTS
Optimisation of an NGS protocol for use with whole
genome amplification products from single cells
Optimisation of the NGS protocol was performed in order to
maximise the quantity of data (ie, number of reads) obtained
per sequencing run. The number of reads per run is an
important consideration, as the greater the amount of
sequence data produced the larger the number of samples that
can be simultaneously tested. Multiplex analysis of samples is
an essential factor in lowering the per-embryo costs of NGS.
Efforts were also made to reduce the time from sample acqui-sition
to results, which is important given the extremely
limited time available for the assessment of preimplantation
embryos. Protocol improvements increased the amount of
sequence data obtained more than threefold compared with
initial results, producing an average per chip of 356.62 Mb.
A time series of incubations was performed on a range of
input DNA concentrations (100–500 ng) of whole genome
amplification (WGA) product to establish the optimal frag-mentation
conditions for samples derived from single cells.
An amount of 100 ng of input DNA was found to be suffi-cient
for generating successful sequencing libraries and was
used for all subsequent samples in order to minimise the
amount of WGA necessary. Reducing the amount of amplifi-cation
decreases the risk of artefacts and allows for an acceler-ated
protocol. Accurate quantification of the input material
was essential for enabling this lower range of WGA DNA to
be used. Quantification using a Nanodrop spectrophotomer
consistently resulted in an overestimation of the DNA concen-tration
resulting in insufficient input material being used for
library generation. The Qubit high sensitivity double stranded
DNA (HS dsDNA) quantification method was found to give a
much more accurate estimation of the amount of DNA
present in the sample. Incubation times of 15 and 17.5 min
provided the optimal size fragment distribution for Ion
Torrent 100 and 200 bp chemistries, respectively.
Agarose and solid phase reversible immobilisation (SPRI)
bead-based methods were both tested for size selection of the
sequencing libraries. For the SPRI bead size selection, the
AxyPrep FragmentSelect-I kit (Appleton Woods) was trialled. A
ratio of 2× input sample volume to 1.8× input volume of
binding to wash off was used to select the fragment at 300 bp.
This method was reproducible, rapid and effective for fully
removing the upper and lower fragments of DNA, but resulted
in a relatively broad size range distribution. This negatively
affected the amount of data generated on the Ion Torrent
PGM sequencer, as the shorter fragments were preferentially
amplified. Gel excision was found to give a much tighter distri-bution
of read lengths. The sample fraction at 300 bp was
excised using E-Gel SizeSelect Gels (Life Technologies).
Both 100 and 200 bp sequencing chemistry were found to
be appropriate for analysis of aneuploidy and DNA sequence
in single cells. Ultimately, in order to generate as much data
as possible for each sample, the 200 bp chemistry was
selected. A minimum of five cycles of adaptor mediated amp-lification
was required to generate quantifiable sequence-ready
libraries. The samples were quantified on a 2100 Bioanalyser
High Sensitivity Chip (Agilent Technologies, Santa Clara,
California, USA). Clonal amplification for template prepar-ation
was performed on the Ion OneTouch system. Input con-centrations
of 18 and 24 pM were tested to establish the
optimal sample to bead ratio for template preparation.
An amount of 24 pM input concentration resulted in a
greater yield of data without generating significantly more
polyclonal reads.
Aneuploidy can be detected in single cells from embryos
using a rapid low-pass genome sequencing methodology
The current research focused on the use of multiple displace-ment
amplification (MDA) in order to generate sufficient
DNA for subsequent NGS. Amplification was obtained
from 61/61 (100%) samples. Initially, NGS was applied to
single cells isolated from six well characterised, karyotypically
stable cell lines (samples 1–18 in table 1).
From the analysis of single cells of known karyotype, it was
clear that WGA introduced distortions in the relationship
between the number of sequence reads and chromosome
length, complicating attempts to assess aneuploidy based on
reads per chromosome. However, these deviations were found
to be highly reproducible from one sample to the next, pre-sumably
a consequence of preferential DNA amplification
associated with differences in the base composition and chro-matin
structure of individual chromosomes. The consistency
of the distortions meant that it was relatively simple to com-pensate
for amplification bias. This was achieved by compar-ing
results obtained from cell line and embryo cells with
those obtained from a series of chromosomally normal refer-ence
samples. Essentially, a set of 24 reference values were
created by averaging the percentage of mapped reads attribut-able
to each chromosome in a series of euploid samples (see
online supplementary table S1). For embryo (test) samples,
the percentage of reads derived from each chromosome was
divided by the reference value for the same chromosome.
Chromosomes present in a normal (disomic) state displayed a
test to reference ratio ranging from 0.7 to 1.2, whereas
chromosomal gain (eg, trisomy) and loss (eg, monosomy)
were associated with ratios >1.2 and <0.7, respectively. Using
this approach, the karyotypes of all single cells derived from
cell lines were correctly defined using NGS (summarised in
table 1).
554 Wells D, et al. J Med Genet 2014;51:553–562. doi:10.1136/jmedgenet-2014-102497
3. Methods
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Table 1 Samples assessed and cytogenetic results obtained
Sample number Predicted karyotype based on NGS result Source of cells Confirmatory result (method)
1–3 47,XY,+21 Single cell from cell line 47,XY,+21 (G-banding)
4–6 45,X Single cell from cell line 45,X (G-banding)
7 and 8 47,XY,+16 Single cell from cell line 47,XY,+16 (G-banding)
9 and 10 47,XY,+18 Single cell from cell line 47,XY,+18 (G-banding)
11–14 46,XY Single cell from cell line 46,XY (G-banding)
15–18 46,XX Single cell from cell line 46,XX (G-banding)
19 47,XX,+12 Embryo (blastomere) 47,XX,+12 (aCGH)
20 47,XX,+6 Embryo (blastomere) 47,XX,+6 (aCGH)
21 48,XX,+8,+9 Embryo (blastomere) 48,XX,+8,+9 (aCGH)
22 43,XY,−17,−21,−22 Embryo (blastomere) 43,XY,−17,−21,−22 (aCGH)
23 46,XY Embryo (trophectoderm) 46,XY (aCGH)
24 46,XY Embryo (trophectoderm) 46,XY (aCGH)
25 46,XY Embryo (trophectoderm) 46,XY (aCGH)
26 46,XY,−13,+21 Embryo (trophectoderm) 46,XY,−13,+21 (aCGH)
27 46,XX,+14,−16 Embryo (trophectoderm) 46,XX,+14,−16 (aCGH)
28 46,XX,+19,−22 Embryo (trophectoderm) 46,XX,+19,−22 (aCGH)
29 45,XX,−9 Embryo (trophectoderm) 45,XX,−9 (aCGH)
30 46,XX Embryo (trophectoderm) 46,XX (aCGH)
31 48,XX,+11,+19 Embryo (trophectoderm) 48,XX,+11,+19 (aCGH)
32 44,XY,−10,−18 Embryo (trophectoderm) 44,XY,−10,−18 (aCGH)
33 46,XX,-2,+16 Embryo (trophectoderm) 46,XX,−2,+16 (aCGH)
34 46,XY,+1,+9,−10,+11,−12,−22 Embryo (trophectoderm) 44,XY,+9,−10,−12,−22 (aCGH)
35 47,XX,+9,+10,−21 Embryo (trophectoderm) 47,XX,+9,+10,−21 (aCGH)
36 46,XX,+13,−17 Embryo (trophectoderm) 46,XX,+13,−17 (aCGH)
37 45,XXY,−15,−17 Embryo (trophectoderm) 45,XXY,−13,−15,+16,−17 (aCGH)
38 45,XY,−18 Embryo (trophectoderm) 45,XY,−18 (aCGH)
39 44,XY,−12,−16 Embryo (trophectoderm) 44,XY,−12,−16 (aCGH)
40 47,XY,+14 Embryo (trophectoderm) 47,XY,+14 (aCGH)
41 47,XX,+21 Embryo (trophectoderm) 47,XX,+21 (aCGH)
42 47,XX,+22 Embryo (trophectoderm) 47,XX,+22 (aCGH)
43 47,XX,+16 Embryo (trophectoderm) 47,XX,+16 (aCGH)
44 47,XX,+18 Embryo (trophectoderm) 47,XX,+18 (aCGH)
45 47,XY,−7 Embryo (trophectoderm) 47,XY,−7 (aCGH)
46 45,X Embryo (trophectoderm) 45,X (aCGH)
47 45,XY,−22 Embryo (trophectoderm) 45,XY,−22 (aCGH)
48a 45,XX,−22 Embryo (trophectoderm) 45,XX−22 (aCGH)
48b 45,XX,−22 Embryo (trophectoderm)
48c 45,XX,−22 Embryo (trophectoderm)
49 46,XX Embryo (trophectoderm) 46,XX (aCGH)
50 46,XY Embryo (trophectoderm) 46,XY (aCGH)
51 46,XY Embryo (trophectoderm) 46,XY (aCGH)
52 46,XY Embryo (trophectoderm) 46,XY (aCGH)
53 44,XY,−15,−19 Embryo (trophectoderm) 44,XY,−15,−19 (aCGH)
54 47,XY,+22 Embryo (trophectoderm) 47,XY,+22 (aCGH)
55 46,XX Clinical embryo biopsy (trophectoderm) Not applicable
56 46,XY Clinical embryo biopsy (trophectoderm) Not applicable
57 45,XY,−12 Clinical embryo biopsy (trophectoderm) Not applicable
58 45,XX−2 Clinical embryo biopsy (trophectoderm) Not applicable
59 46,XX Clinical embryo biopsy (trophectoderm) Not applicable
60 46,XY Clinical embryo biopsy (trophectoderm) Not applicable
61 46,XY Clinical embryo biopsy (trophectoderm) Not applicable
Samples 23–54 (excluding 48b and 48c) were tested a second time as part of a large barcoded multiplex (a total of 32 samples) and yielded identical cytogenetic results. Samples 48a,
48b and 48c represent three separate trophectoderm biopsies performed on the same embryo.
aCGH, microarray comparative genomic hybridisation; NGS, next-generation sequencing.
Wells D, et al. J Med Genet 2014;51:553–562. doi:10.1136/jmedgenet-2014-102497 555
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NGS provides a highly accurate detection of aneuploidy and
confirms that monosomy and other unusual forms of
aneuploidy remain common at the final stage of
preimplantation development
During the initial validation phase of this study, 38 samples
from human embryos were analysed using NGS. Results were
obtained from all embryos tested (100%). The samples assessed
included cells from 32 blastocysts, the final stage of preimplan-tation
development. The blastocysts were derived from five
infertile couples of advanced reproductive age (average female
age 42 years). Overall, 24 of the embryos were diagnosed abnor-mal
(75%), emphasising the high risk of transferring an aneu-ploid
embryo to the uterus when such patients undergo IVF
treatment. A total of 51 chromosome errors were detected
(table 1; see online supplementary figure S1). With the excep-tion
of chromosomes 3, 4, 5 and 20, all chromosomes were
affected by aneuploidy at least once in this set of samples, with
chromosome 22 displaying the greatest number of errors.
Monosomies and trisomies occurred at similar frequencies.
A second biopsy was taken from each embryo, coded and
blindly assessed using a well-established microarray-comparative
genomic hybridisation (CGH) approach. After decoding, the
diagnostic results from the two analyses were compared. In all
cases, the tests were in agreement concerning the embryos that
were chromosomally normal and those that were aneuploid
(figure 1). The karyotypes predicted by microarray comparative
genomic hybridisation (aCGH) and NGS were entirely identical
for all but two embryos. The embryos with discrepant results
(numbers 34 and 37 in table 1) were considered highly abnor-mal
using both methods, each affected by several aneuploidies
(>3 abnormal chromosomes each). Considering the 1296 chro-mosomes
assessed in the 54 samples used to evaluate the accur-acy
of NGS, the concordance rate per chromosome was 99.7%.
Chromosomal analysis of embryos using NGS can be carried
out at a speed, throughput and cost appropriate for use in
conjunction with standard embryo biopsy and transfer
protocols
Given the quantity of sequence data produced from each
sequencing chip, it seemed likely that a large number of samples
could be simultaneously tested, significantly reducing costs per
sample and greatly increasing throughput. To verify this, 32
samples derived from cells biopsied from human embryos were
multiplexed together. Different adapters of unique DNA
sequence (ie, ‘barcodes’) were ligated to the DNA fragments
obtained from each embryo and samples were pooled together
and then sequenced simultaneously. Once the process was com-plete,
the sequence of the barcode at the beginning of each
DNA fragment was examined allowing the fragment to be
assigned to an individual embryo (see online supplementary
figure S2). The cytogenetic diagnoses obtained were found to be
identical regardless of whether cells were analysed individually
or multiplexed together. The robust results obtained for the sim-ultaneous
analysis of 32 samples suggest that even higher levels
of multiplexing are likely to be possible.
Figure 1 Aneuploidy analysis of cells biopsied from a human blastocyst. (A) Analysis of embryo #41 using next-generation sequencing (NGS)
predicting a female trisomic for chromosome 21; (B) Microarray-CGH analysis of a second embryo biopsy sample from embryo #41, confirming the
NGS result.
556 Wells D, et al. J Med Genet 2014;51:553–562. doi:10.1136/jmedgenet-2014-102497
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Chromosomal analysis of cells from embryos could be com-pleted
within 15 h using the optimised NGS protocol. However,
even faster testing was shown to be possible using the new Ion
Isothermal Amplification Chemistry (Life Technologies), which
streamlines the sequencing procedure, eliminating the need for
emulsion PCR. Using this approach, chromosome screening
data could be obtained within 8 h (see online supplementary
table S3). A total of 17 embryo biopsy specimens were simultan-eously
tested using this new approach and chromosomal copy
number assessments were shown to be concordant with aCGH
in all cases (see online supplementary table S4).
NGS allows the potential for simultaneous chromosomal
analysis and diagnosis of gene mutations in single cells
In the current study, low-pass sequencing of WGA products
yielded <0.1% coverage of the genome, so the chances of a
given locus being represented were extremely low. For this
reason, attempts to provide data on specific mutations required
a semitargeted approach, enriching the genomic sequences rele-vant
for diagnosis (ie, the mutation sites and/or informative
linked polymorphisms) before carrying out NGS. As a proof of
concept, single cells were isolated from a cell line homozygous
for the common CFTR ▵F508 mutation, associated with cystic
fibrosis. After cell lysis and WGA, an aliquot of the amplified
product was subjected to PCR, amplifying a 95 bp DNA frag-ment
encompassing the ▵F508 mutation site. Sequencing librar-ies
were generated from the original WGA product and the
locus-specific PCR product. The two libraries were then com-bined
together and simultaneously sequenced. This approach
provided sufficient DNA sequence information across all chro-mosomes
to allow detection of aneuploidy, while also sequen-cing
the region of CFTR containing ▵F508 multiple times
(sequenced to a coverage depth of approximately 30×). All of
the CFTR sequence reads obtained confirmed the
homozygous-affected genotype of the tested cells (see online
supplementary figure S3).
The NGS method provides quantitative data on mtDNA copy
number and mutation load
Not only did the optimised NGS protocol allow accurate detec-tion
of aneuploidy, it also succeeded in sequencing the entirety
of the mitochondrial genome in single cells to a coverage depth
of 20–60×. Interestingly, when the ratio of mitochondrial DNA
(mtDNA) sequences to nuclear DNA sequences was considered,
significant differences were seen between embryos derived from
the same couple. In most cases, the embryo with the largest
quantity of mtDNA from a single couple had three to sixfold
more than the embryo with the least. However, more extreme
variations were also detected. In one instance, a 97-fold differ-ence
in mtDNA content was observed between two embryos
derived from the same parents. It seems highly likely that such
dramatic differences in a key organelle would have functional
consequences for the affected cells. Indeed, a potentially
important observation was that embryos with high levels of
mtDNA had an elevated risk of aneuploidy (p<0.05) (figure 2).
The use of real-time PCR to quantify a fragment of the mito-chondrial
genome in trophectoderm samples from human blas-tocysts
verified the results of NGS-based mtDNA quantification
(see online supplementary figure S4).
In the current study, a blinded analysis was carried out on
individual cells isolated from a cell line affected with an mtDNA
disorder. Analysis of DNA from the cell line using conventional
methods had previously shown that ∼70% of the mitochondrial
genomes were affected by a clinically significant 7438 bp
Methods
deletion. The single cell NGS method was capable of detecting
the mutation, correctly defining the breakpoints of the deletion,
and provided an accurate estimation of the proportion of mito-chondria
affected (see online supplementary figure S5).
Additionally, the chromosomal status of the cell tested (euploid)
was simultaneously confirmed using the same method discussed
above. Other research supports the suggestion that mtDNA can
be accurately sequenced in small numbers of cells derived from
human preimplantation embryos.11
Clinical application of NGS to screen human embryos for
aneuploidy, resulting in pregnancies and births
After confirmation of the accuracy of aneuploidy detection in
the blinded study outlined above, the NGS method was used to
help guide the selection of embryos produced by two infertile
couples (women aged 35 and 37 years). A total of five blastocyst
stage embryos suitable for biopsy were produced in one IVF
cycle and two in the other. A trophectoderm sample (composed
of ∼5 cells) was taken from each embryo and tested for aneu-ploidy
using the NGS method. Two embryos were found to
have abnormalities, predicted to lead to failure of implantation
or early pregnancy loss and were excluded (table 1, samples 57
and 58). The remaining embryos were shown to be euploid.
One chromosomally normal embryo, containing typical quan-tities
of mtDNA, was transferred in each instance and resulted
in the birth of a healthy baby in the summer of 2013 in both
cases. The sex and chromosomal status of the two children were
confirmed to be identical to that predicted by NGS.
DISCUSSION
This study describes the design, optimisation, validation and
clinical application of an aneuploidy detection method applic-able
to single cells. The method, based on NGS, was shown to
be rapid and highly accurate. Compatibility with utilisation in a
Figure 2 Relationship between blastocyst aneuploidy and relative
mitochondrial DNA (mtDNA) quantity in human blastocyst stage
embryos. The amount of mtDNA relative to nuclear DNA is significantly
lower in biopsies of trophectoderm cells from euploid embryos
(p<0.05, two-tailed t test). Red spots indicate the mtDNA content of
the two chromosomally normal embryos that were transferred to
patients and produced viable pregnancies.
Wells D, et al. J Med Genet 2014;51:553–562. doi:10.1136/jmedgenet-2014-102497 557
6. Methods
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clinical setting was demonstrated by the testing of embryos pro-duced
using IVF technology, assisting in the identification and
transfer to the uterus of chromosomally normal embryos. This
resulted in viable pregnancies and the birth of healthy children.
Although alternative methods for the detection of aneuploidy in
human preimplantation embryos already exist, NGS offers sig-nificant
advantages in terms of the breadth and scope of the
information provided and the cost of testing.
The number of patients requesting preimplantation genetic
testing of their embryos is growing significantly. In our own
laboratory, we have seen referrals for preimplantation genetic
diagnosis (PGD) and preimplantation genetic screening (PGS)
rise by over 50% in the last 2 years and many other clinics have
witnessed similar increases. The upsurge in the number of cases
has intensified the need to develop methods capable of deliver-ing
accurate diagnosis of embryos at reduced cost, thereby redu-cing
the financial burden on patients and healthcare systems.
The results of the current research confirm that NGS provides
highly accurate aneuploidy detection at substantially lower cost
than existing methods used for PGS and that testing can be per-formed
within a fresh IVF cycle, avoiding the complication of
cryopreservation. A small number of previous studies had sug-gested
that the combination of WGA and NGS might be
capable of producing useful genomic data from single cells.12–15
However, previous attempts to harness massively-parallel
sequencing methods for the detection of aneuploidy in single
cells required several days to complete, meaning that preimplan-tation
genetic testing could only be carried out if embryos were
cryopreserved after biopsy. The addition of embryo cryopreser-vation
represents a deviation from the most widely used strat-egies
for embryo analysis, which increases the cost of the
procedure and carries a risk of harm to the embryo (most fertil-ity
clinics report that 5%–10% of embryos do not survive freez-ing
and thawing).
The need for rapid methods for the genetic testing of preim-plantation
embryos stems from the fact that the window of time
during which implantation can occur is narrow. If embryo
biopsy is undertaken at the blastocyst stage (5 days after fertilisa-tion
of the oocyte)—increasingly the preferred strategy for PGD
and PGS—less than 24 h are available for genetic analysis. In
the current study, a protocol requiring under 15 h was used suc-cessfully
in clinical cycles, a turnaround time similar to speeds
achieved using the most rapid microarray-CGH methods cur-rently
available and considerably quicker than alternative PGD
and PGS methods based on the use of SNP microarrays (see
online supplementary table S2). Subsequent work, carried out
after the initial clinical cases, demonstrated that reliable aneu-ploidy
detection in single cells biopsied from human embryos is
possible in even shorter timeframes (less than 8 h) when using
NGS.
As well as the requirement for rapid results, methods used for
the analysis of preimplantation embryos must also permit simul-taneous
testing of multiple samples. Individual patients undergo-ing
IVF/PGD treatment typically produce several embryos, each
of which needs to be examined. The fact that each patient is
associated with multiple tests is problematic for some PGD and
PGS methods: technical bottlenecks mean that equipment used
for analysis must be duplicated in order to accommodate all of
the samples. NGS has the advantage in that it allows large
numbers of samples to be processed concurrently. Up to 32
samples were processed simultaneously during this study and
the data obtained suggest that two or three times that number
could potentially be multiplexed together without any signifi-cant
loss of accuracy. Multiplex analysis also has implications in
terms of the price of the test. Although, NGS is becoming less
expensive every year, at present each run is still associated with
an appreciable cost. However, consumable expenses can be
shared across several samples tested during the same run using
molecular ‘barcoding’ (see online supplementary figure S2). In
the current study, this approach allowed NGS to reduce the per-embryo
cost by more than a third compared with the most
widely used microarray-based approaches (based on 32-plex
analysis and manufacturer’s list prices). Such a reduction has
substantial implications for embryo chromosome screening,
strengthening the health-economics argument for clinical utilisa-tion
and potentially making it accessible to much greater
numbers of infertile couples.
The data presented here provide reassurance concerning the
accuracy of aneuploidy detection in cells from preimplantation
embryos using NGS. Near complete concordance was achieved
in comparison with results obtained using a highly validated
aCGH method (see online supplementary tables S5 and S6).4 16
A total of 54 samples were tested during the initial validation
phase of this study and the diagnosis (normal or abnormal)
using the two methods was identical in all cases. Agreement was
seen for 99.7% of the 1296 chromosomes evaluated. The only
two embryos with any discrepant findings (numbers 34 and 37
in table 1) were each highly abnormal, containing multiple
aneuploidies (>3 abnormal chromosomes each). Embryos
affected by several chromosome abnormalities frequently display
chromosomal mosaicism and consequently a degree of cytogen-etic
divergence between the different biopsy specimens is
expected. In these two cases, most of the aneuploidies were
present in both biopsies taken from the same embryo and may
have been a consequence of meiotic errors. However, additional
aneuploidies were present in one of the two samples. The dis-crepant
findings could be explained by mitotic errors occurring
after fertilisation, but this cannot be proven conclusively on this
occasion as no further embryo material was available for ana-lysis.
The spectrum of abnormalities detected in the embryos
analysed during this study are in agreement with the findings of
numerous previous investigations, using a variety of cytogenetic
techniques, and confirm that a wide range of abnormalities,
including types never seen in established pregnancies or miscar-riages
and presumably lethal during early development, are
common prior to implantation.
Diagnosis of mutations responsible for single gene disorders
in embryos produced using IVF technology (ie, PGD) is increas-ingly
requested by couples at high-risk of transmitting an inher-ited
disorder to their children.17 In most cases, this involves
biopsy of single cells at the cleavage stage, genetic testing and
transfer of unaffected embryos to the mother’s uterus. Any
pregnancies that result following this process should be free of
the familial disorder and therefore the issue of selected preg-nancy
termination is avoided. However, on some occasions the
embryos transferred miscarry due to a spontaneously arising
chromosome abnormality. This is a particular issue for couples
requesting PGD who carry a single gene mutation and are at
increased risk of aneuploid conception due to female age.
Although the research reported in this paper was principally
focused on the use of NGS for preimplantation aneuploidy
detection, a pilot experiment was undertaken to assess the feasi-bility
of testing for alterations of DNA sequence in parallel with
chromosome screening. Historically, the combination of DNA
sequence analysis and chromosome copy number testing in
single cells has been challenging. Of the hundreds of protocols
for the PGD of gene mutations published in the literature, only
a handful are compatible with comprehensive chromosome
558 Wells D, et al. J Med Genet 2014;51:553–562. doi:10.1136/jmedgenet-2014-102497
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analysis.18–21 The data presented here are preliminary, but indi-cate
that NGS has the potential to deliver accurate mutation
detection as well as aneuploidy screening, overcoming a limita-tion
of traditional PGD tests. It is important to note, however,
that rates of allele dropout (failure to amplify one of the two
alleles in a heterozygous cell) are appreciable at the single cell
level and consequently a significant error rate would be
expected for diagnostics based on analysis of the mutation site
alone. For this reason, analysis of several flanking polymorph-isms
remains advisable, supplementing direct mutation detection
with redundant diagnoses based on linkage analysis. Strategies
using multiple linked markers are commonly used for PGD of
single gene disorders.22
While there is accumulating evidence to suggest that aneu-ploidy
is the single most important factor affecting the ability of
an embryo to implant and form a viable pregnancy,6–10 23 24 it
remains the case that even the transfer of a chromosomally
normal, morphologically perfect embryo to the uterus cannot
guarantee a pregnancy. Clearly there are other, less well-defined
aspects of embryo biology that are also critical for successful
development. A prime candidate in this regard is mitochondrial
copy number, which previous studies have linked to various
aspects of oocyte and embryo competence.25 26 Recent research
in our laboratory has demonstrated that an unusually high level
of mtDNA in human preimplantation embryos is associated
with failure of implantation in the uterus.27 Unlike all other
methods of embryo aneuploidy screening currently in use, NGS
is capable of simultaneously providing data on mtDNA copy
number as well as chromosome imbalance. While further work
is needed to replicate our observations, the evidence thus far
suggests that quantification of mtDNA may provide an extra
dimension to embryo assessment, enhancing our ability to rec-ognise
viable embryos and thereby improving the likelihood of
successful IVF treatment.27
In the current study, the embryos from clinical cycles that pro-duced
babies had typical mtDNA levels, but too few embryos
were transferred to allow any conclusions to be drawn concern-ing
the relevance of mtDNA in terms of IVF success. However,
an observation of biological and clinical importance was that
embryos with high levels of mtDNA had an elevated risk of
aneuploidy (p<0.05) (figure 2). The link between chromosomal
abnormality and mtDNA content, revealed by NGS, was subse-quently
confirmed in our laboratory using qPCR and is con-cordant
with a previous study that employed real-time PCR
methods.28 It is likely that the detection of greater quantities of
mtDNA is indicative of an increased number of mitochondria
present in the cells tested, although this remains to be conclu-sively
proven. Raised quantities of mitochondria could lead to
abnormally elevated metabolism, a factor that previous studies
have speculated might be associated with reduced embryo viabil-ity,
the so-called ‘Quiet Embryo’ hypothesis.29 Alternatively,
expansion of the mitochondrial population could represent a
compensatory mechanism, symptomatic of the presence of
defective organelles, perhaps a consequence of mtDNA
mutation.
The ability of the NGS method described to detect and quan-tify
mtDNA mutation was also demonstrated during this study.
mtDNA disorders are responsible for a number of devastating
conditions for which there is no cure and few treatment
options. In most cases, affected individuals are heteroplasmic,
having a mixture of normal and mutant mitochondria in their
cells. The severity of the symptoms is determined by the propor-tion
of organelles that are defective. As many as one person in
400 is affected by an mtDNA disorder30 and the phenomenon
Methods
of the mitochondrial bottleneck means that the phenotype can
swing from mild to severe in a single generation. One possibility
for the avoidance of mtDNA disorders is to perform PGD,
testing cells biopsied from preimplantation embryos and trans-ferring
to the uterus only those with entirely normal mitochon-dria
or with low (subclinical) mutation loads.31–37 However,
accurate quantification of mtDNA mutations has been technic-ally
difficult at the single cell level, requiring extensive protocol
design and optimisation for each mutation tested. It seems likely
that NGS will provide a single robust method for the detection
and quantification of any mtDNA mutation, streamlining the
PGD process.
CONCLUSIONS
Worldwide, approximately a million assisted reproductive treat-ment
cycles end in failure each year, emphasising the urgent
need for improvements to existing techniques. There is mount-ing
evidence from randomised clinical trials that many infertile
couples can achieve increased IVF success rates if the embryos
chosen for transfer to the uterus have been shown to be
euploid, yet only a small fraction of IVF cycles currently include
chromosome screening. The wider application of genetic ana-lysis
is restrained, in part, by the high costs associated with
testing. The technique detailed in this paper has the capacity to
cut the cost of analysis significantly and should therefore
increase patient access to embryo aneuploidy screening. Unlike
previous studies applying NGS to single cells, this method is
compatible with the most widely used strategies for embryo
testing, which require diagnosis to be completed within ∼24 h
of embryo biopsy. The clinical applicability of the test was
demonstrated in IVF cycles, resulting in the birth of healthy
children. These represent the first births achieved following
NGS-based screening of human embryos in the Western hemi-sphere.
The children produced are healthy and developing nor-mally,
at 1 year of age.
In theory, NGS methods such as those described here could
be extended to allow whole genome sequencing of embryos.
While our data and that of other research groups suggest that
this is technically feasible, sequencing of the entire genome
would greatly increases costs and the time required for analysis,
eliminating two of the chief benefits of the strategy described in
this paper. Furthermore, knowledge of the genome sequence of
a human embryo prior to transfer to the uterus has the potential
to reveal unexpected genetic findings that may be difficult to
interpret (ie, incidental findings of unknown clinical signifi-cance),
not to mention the ethical concerns that may be raised
by the acquisition of such detailed genetic information and its
potential use in embryo selection. The test outlined here deliber-ately
avoided sequencing the whole genome (less than 0.1% was
sequenced from each embryo), focusing on low cost, rapid ana-lysis
and detection of serious genetic abnormalities that have a
well-defined impact on health or embryo viability.
METHODS
Samples analysed during preliminary optimisation and
validation
Samples were derived from cytogenetically characterised cell
lines (Coriell Cell Repositories) or from human embryos previ-ously
shown to be aneuploid during routine preimplantation
genetic screening (Reprogenetics). Initial analyses began in July
2011, with clinical cases initiated in April 2012. Clinical IVF
cycles using NGS took place at Main Line Fertility and
New York University Fertility Center. Genetic analyses were
carried out at Reprogenetics UK and the University of Oxford.
Wells D, et al. J Med Genet 2014;51:553–562. doi:10.1136/jmedgenet-2014-102497 559
8. Methods
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Single cell lysis
An amount of 2.5 μL of the alkaline lysis buffer (0.75 μL
PCR-grade water (Promega, Madison, USA); 1.25 μL dithio-threitol
(DTT) (0.1 M) (Sigma, Gillingham, UK); 0.5 μL NaOH
(1.0 M) (Sigma)) was added to a 0.2 mL microcentrifuge tube
containing a single cell or trophectoderm biopsy (5–10 cells)
suspended in 0.5–2.0 μL of phosphate buffered saline. The
samples were then incubated for 10 min at 65°C in a thermal
cycler.
Whole genome amplification
After lysis, whole genome amplification reagents were then
added to each tube (44 μL per sample): 12.5 μL of PCR-grade
water (Promega); 2.5 μL of tricine (0.4 M) (Sigma); 29 μL of
Repli-g Midi Reaction buffer (Qiagen, Crawley, UK); and 1 μL
of Repli-g Midi DNA polymerase (Qiagen). Samples were incu-bated
at 30°C for 120 min and 65°C for a further 5 min.
Optimised NGS protocol
WGA product was quantified using a Qubit (HS dsDNA) and
100 ng fragmented using NEBNext DNA fragmentation enzyme
for 17.5 min for Ion Torrent 200 bp chemistry.
Electrophoresis was performed and the sample fraction at
300 bp was excised using E-Gel SizeSelect Gels (Life
Technologies).
A minimum of five cycles of adaptor mediated amplification
was required to generate quantifiable sequence-ready libraries.
The samples were evaluated using a 2100 Bioanalyser High
Sensitivity Chip (Agilent Technologies). All samples were diluted
to a 24 pmol concentration and a 5 mL aliquot of the sample
was used as a template for clonal amplification using the Ion
OneTouch system (Life Technologies). Sample enrichment was
performed on the Ion OneTouch ES module and Ion 316 chips
were used for sequencing. The chip was prepared for sequen-cing
by the use of a modified protocol whereby 30 mL of
sample was added between five rounds of 2 min centrifugation
steps, resulting in high levels of data generated with each
sequencing run.
Data were analysed using the coverageAnalysis (V.2.2.3)
plugin in the Torrent Suite V.2.2 (Life Technologies), providing
the percentage of DNA sequence reads mapped to each chromo-some.
The percentage of the reads derived from a given
chromosome for an embryo (test) sample was divided by the ref-erence
value for the same chromosome as described in the
Results section (see also online supplementary table S1).
Chromosomal gains were associated with ratios >1.2 and losses
with ratios <0.7. For each sample, the number of mtDNA reads
was divided by the number of reads attributable to the nuclear
genome. This provided an indication of the amount of mtDNA
per cell.
Up to 32 samples were multiplexed together and sequenced on
the same chip. In order to achieve this, prior to sequencing, ampli-fied
DNAs from each sample were fragmented and different adap-ters
of unique DNA sequence (ie, barcodes) from the Ion Xpress
Barcode Adapters 1–16 and 17–32 kits were ligated, allowing for
postsequencing demultiplexing. The samples were diluted to a con-centration
of 24 pmol and pooled prior to clonal amplification on
the Ion OneTouch system. Semiconductor sequencing of 100 bp
was performed on the Ion PGM Sequencer using 200 bp chemistry
on a single Ion 316 chip. The fragments sequenced were assigned
to specific samples based on their unique barcode.
Procedures for blinded analysis of aneuploidy
A second biopsy was taken from each embryo and coded by a
technician who had no other involvement in the study. Analysis
of the coded samples was undertaken using a well-established
microarray-CGH approach, carried out by scientists in a differ-ent
laboratory from those performing NGS. The results of both
tests (ie, aneuploidy diagnosis using NGS and aCGH) were
communicated to an independent adjudicator, samples decoded
and diagnostic results compared.
Confirmation of NGS-based mtDNA quantification using
real-time PCR
Whole genome amplification products from cells biopsied from
blastocyst stage embryos were analysed using real-time PCR. A
custom-designed TaqMan Assay (Life Technologies, UK) was used
to target and amplify the mitochondrial 16 s ribosomal RNA
sequence (probe sequence: AATTTAACTGTTAGTCCAAAGAG).
Normalisation of input DNA took place with the reference to a
second TaqMan Assay amplifying the multicopy Alu sequence
(YB8-ALU-S68) (probe sequence: AGCTACTCGGGAGGCTG
AAGGCAGGA). This normalisation was employed to avoid issues
such as variation in whole genome amplification efficiency and dif-ferences
in the number of cells contained in the biopsy specimen.
Additionally, a reference DNA sample consisting of amplified 46,
XY DNA was included each time real-time PCR was undertaken,
allowing variation between experiments to be controlled for. A
negative control (nuclease free H2O and PCR master-mix) was also
included in all experiments. Alu and mtDNA sequences were amp-lified
in triplicate from each PicoPlex product. Reactions contained
1 μL of whole genome amplified embryonic DNA, 8 μL of
nuclease-free H2O, 10 μL of TaqMan Universal Master-mix II
(2X)/no UNG (Life Technologies, UK) and 1 μL of the 20×
TaqMan mtDNA or Alu assay (Life Technologies, UK), for a total
volume of 20 μL. The thermal cycler used was a StepOne
Real-Time PCR System (Life Technologies, UK), and the following
conditions were employed: incubation at 50°C for 2 min, incuba-tion
at 95°C for 10 min and then 30 cycles of 95°C for 15 s and
60°C for 1 min.
PCR amplification of the CFTR gene ▵F508 mutation site
After cell lysis and WGA, a 0.5 μL aliquot was removed from the
MDA product and added to 14.5 μL of PCR mixture. The PCR
mixture was composed of the following: 1.45 μL 10× Enzyme
Buffer (5 Prime, Hamberg, Germany); 0.15 μL of primer mixture
(Forward GTTTTCCTGGATTATGCCTGGCAC; Reverse
GTTGGCATGCTTTGATGACGCTTC; each at 10 μM); 0.2 mM
dNTPs (Sigma); 0.45 units of Hot Master Taq enzyme (5 Prime);
and PCR-grade water (Promega). The PCR programme was: 96°C
for 1 min; 30 cycles of 94°C for 15 s, 59°C for 15 s, 65°C for 45 s;
65°C for 2 min. This yielded a DNA fragment encompassing the
▵F508 mutation site (95 bp in length if normal, 92 bp if ▵F508
was present). Sequencing libraries were generated from the original
WGA product and the resulting PCR product. For clonal amplifica-tion
and based on template dilution factors calculated according to
manufacturer protocol, 1.3 mL of the library from the PCR
product (diluted 1:500) was added to 3.7 mL of a 1:16 dilution of
the library from the original WGA. The WGA-PCR library mixture
was then analysed using NGS as described above.
Microarray-CGH
Microarray-CGH analysis was undertaken according to our pre-viously
validated protocol using 24Sure Cytochip (Illumina,
Cambridge, UK) (see online supplementary tables S5 and S6).
560 Wells D, et al. J Med Genet 2014;51:553–562. doi:10.1136/jmedgenet-2014-102497
9. Downloaded from jmg.bmj.com on October 10, 2014 - Published by group.bmj.com
Lysis and whole genome amplification of single cells biopsied
from embryos were achieved using the SurePlex kit (Illumina).
The entirety of this procedure took place according to the man-ufacturer’s
instructions. The fluorescence labelling system
(Illumina) was used for the labelling of the amplified ethylene-diaminetetraacetic
acid-Tris (TE) samples and also for labelling a
commercially available reference DNA (Illumina). Test TE
samples were labelled with Cy3 while the reference 46,XY
DNA was labelled with Cy5. Test and reference DNAs’
co-precipitation, their denaturation, array hybridisation and the
posthybridisation washes all took place according to protocols
provided by the manufacturer. The hybridisation time was 16 h.
A laser scanner (InnoScan 710, Innopsys, Carbonne, France)
was used to analyse the microarrays after washing and drying.
The resulting images were stored in TIFF format file and exam-ined
by the BlueFuse Multi analysis software (Illumina).
Chromosome profiles were examined for gain or loss with the
use of a 3× SD assessment.
Clinical application of NGS for preimplantation aneuploidy
detection
Two infertile couples (women aged 35 and 37) gave consent for
NGS analysis of their embryos following counselling. The pro-tocols
used for ovarian stimulation, IVF and embryo culture did
not differ from methods considered to be standard. At the
blastocyst stage, 5 days after oocyte fertilisation, the zona pellu-cida
encapsulating the embryo was breached with a laser and ∼5
trophectoderm cells were carefully removed using a micromani-pulator.
The cells were washed in three 5 μL droplets of PBS
+0.1% polyvinyl alcohol and then transferred to a 0.2 mL
microcentrifuge tube in a total volume of 1.5 μL. Cell lysis,
MDA and NGS were carried out as described above. One
euploid embryo was transferred in each case, while additional
euploid embryos remained cryopreserved (vitrified).
Preliminary evaluation of an ultra-rapid method of
NGS-based aneuploidy screening
Subsequent to the validation and clinical application of NGS for
the purposes of aneuploidy detection, an even more rapid evo-lution
of the original protocol was evaluated. This involved ana-lysis
of 17 samples (∼5 cells each) derived from human
blastocysts. In some cases, embryos were biopsied twice, allow-ing
independent testing of cells derived from the inner cell mass
and trophectoderm. For all samples, cell lysis and whole
genome amplification (MDA) were carried out as described
above. This was followed by creation of barcoded libraries from
100 ng of MDA samples using the Ion Xpress Plus protocol
described previously with six cycles of amplification. The ampli-fied
libraries were run on an EGel SizeSelect 2% agarose gel
(Invitrogen) and the fraction corresponding to a 350 bp insert
was purified with 0.5× of AMPure beads (Agencourt).
Concentrations were estimated by qPCR using the Ion Library
Quantitation Kit (Life Technologies) and 1.7 mL of a 50 pM
dilution was used as an input for Ion Isothermal Amplification
Chemistry (Life Technologies). This generated templated
spheres in 30 min. Spheres were then enriched, loaded on an
Ion 316 chip (10 samples multiplexed at a time) and sequenced
on the Ion PGM for 260 flows using the Ion PGM 200 V.2
Sequencing kit (Life Technologies). Analysis of the DNA
sequence reads produced was carried out as described above.
Acknowledgement This work was supported by the Oxford NIHR Biomedical
Research Centre Programme.
Methods
Contributors All authors had essential roles in this study and each participated in
the writing and/or review of the manuscript. DW—study concept, laboratory work,
manuscript preparation; KK—laboratory work; JG and MG—patient counselling and
treatment (IVF); JCT, EF, SM—genetics support, data analysis.
Competing interests None.
Ethics approval NRES Committee South Central.
Provenance and peer review Not commissioned; externally peer reviewed.
Open Access This is an Open Access article distributed in accordance with the
Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which
permits others to distribute, remix, adapt, build upon this work non-commercially,
and license their derivative works on different terms, provided the original work is
properly cited and the use is non-commercial. See: http://creativecommons.org/
licenses/by-nc/4.0/
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Clinical utilisation of a rapid low-pass whole
genome sequencing technique for the
diagnosis of aneuploidy in human embryos
prior to implantation
Dagan Wells, Kulvinder Kaur, Jamie Grifo, et al.
J Med Genet 2014 51: 553-562
doi: 10.1136/jmedgenet-2014-102497
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References
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