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.
Trophectoderm dna fingerprinting by quantitative real time pcr successfully d...t7260678
This study validated a novel method of quantitative real-time PCR (qPCR) to distinguish sibling human embryos using DNA fingerprinting of trophectoderm biopsies. The study analyzed cell lines and excess DNA from trophectoderm biopsies of sibling human blastocysts using qPCR of 40 SNPs. Results showed the method accurately assigned relationship between samples, with sibling relationships averaging 57.2% similarity compared to 95.1% for self relationships. This qPCR-based DNA fingerprinting technique can unequivocally discriminate between sibling human embryos and will enable research on markers of embryonic reproductive potential.
This study evaluated the use of quantitative PCR (qPCR) to genotype single nucleotide polymorphisms (SNPs) near a mutation in the RTEL1 gene for preimplantation genetic diagnosis of Dyskeratosis Congenita, compared to the standard method using short tandem repeats (STRs). The standard STR method misdiagnosed 3 of 14 embryos due to recombination between the distant STR marker and mutation. In contrast, qPCR of closely linked SNPs identified recombination in 9 of 17 embryos and correctly diagnosed all embryos. This case demonstrates that qPCR of SNPs provides improved sensitivity over STRs for detecting recombination near telomeric mutations.
Elevated mitochondrial DNA copy number is observed in aneuploid embryos, indicating this parameter could become an additional tool for prioritizing embryo transfer. The study analyzed mitochondrial DNA copy number in 606 embryos using next generation sequencing and found higher numbers in aneuploid versus normal embryos. While most factors like embryo quality, sex, and patient age showed no correlation, implanted 5-day embryos had significantly higher mitochondrial DNA amounts than non-implanted embryos. This suggests mitochondrial DNA copy number may predict embryo viability and improve selection.
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 of single nucleotide polymorphisms (SNPs) closer to the mutation found the STR-based method misdiagnosed 3 of 14 embryos due to undetected recombinations between the STR and mutation.
4) The improved resolution of SNPs near the mutation detected recombinations and corrected misdiagnoses, demonstrating
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 and compares different methods for comprehensive chromosome screening (CCS) of human oocytes and embryos. It discusses the capabilities and limitations of various CCS methodologies, including:
1) Array comparative genomic hybridization (aCGH), single nucleotide polymorphism (SNP) microarrays, quantitative real-time PCR (qPCR), and other technologies can screen all 24 human chromosomes, unlike previous fluorescence in situ hybridization (FISH) methods.
2) CCS methods vary in terms of accuracy, minimum detectable imbalance, ability to predict monogenic diseases and origins of aneuploidy, and time required to obtain results. SNP microarrays and qPCR allow for results in time for fresh
Invicta eshre-poster-pregnancy rate after frozen blastocystINVICTA GENETICS
Assessment of pregnancy rate after comprehensive chromosome screening using next-generation sequencing-based preimplantation genetic diagnosis (NGS PGD) of trophectoderm in frozen blastocyst transfer.
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.
Trophectoderm dna fingerprinting by quantitative real time pcr successfully d...t7260678
This study validated a novel method of quantitative real-time PCR (qPCR) to distinguish sibling human embryos using DNA fingerprinting of trophectoderm biopsies. The study analyzed cell lines and excess DNA from trophectoderm biopsies of sibling human blastocysts using qPCR of 40 SNPs. Results showed the method accurately assigned relationship between samples, with sibling relationships averaging 57.2% similarity compared to 95.1% for self relationships. This qPCR-based DNA fingerprinting technique can unequivocally discriminate between sibling human embryos and will enable research on markers of embryonic reproductive potential.
This study evaluated the use of quantitative PCR (qPCR) to genotype single nucleotide polymorphisms (SNPs) near a mutation in the RTEL1 gene for preimplantation genetic diagnosis of Dyskeratosis Congenita, compared to the standard method using short tandem repeats (STRs). The standard STR method misdiagnosed 3 of 14 embryos due to recombination between the distant STR marker and mutation. In contrast, qPCR of closely linked SNPs identified recombination in 9 of 17 embryos and correctly diagnosed all embryos. This case demonstrates that qPCR of SNPs provides improved sensitivity over STRs for detecting recombination near telomeric mutations.
Elevated mitochondrial DNA copy number is observed in aneuploid embryos, indicating this parameter could become an additional tool for prioritizing embryo transfer. The study analyzed mitochondrial DNA copy number in 606 embryos using next generation sequencing and found higher numbers in aneuploid versus normal embryos. While most factors like embryo quality, sex, and patient age showed no correlation, implanted 5-day embryos had significantly higher mitochondrial DNA amounts than non-implanted embryos. This suggests mitochondrial DNA copy number may predict embryo viability and improve selection.
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 of single nucleotide polymorphisms (SNPs) closer to the mutation found the STR-based method misdiagnosed 3 of 14 embryos due to undetected recombinations between the STR and mutation.
4) The improved resolution of SNPs near the mutation detected recombinations and corrected misdiagnoses, demonstrating
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 and compares different methods for comprehensive chromosome screening (CCS) of human oocytes and embryos. It discusses the capabilities and limitations of various CCS methodologies, including:
1) Array comparative genomic hybridization (aCGH), single nucleotide polymorphism (SNP) microarrays, quantitative real-time PCR (qPCR), and other technologies can screen all 24 human chromosomes, unlike previous fluorescence in situ hybridization (FISH) methods.
2) CCS methods vary in terms of accuracy, minimum detectable imbalance, ability to predict monogenic diseases and origins of aneuploidy, and time required to obtain results. SNP microarrays and qPCR allow for results in time for fresh
Invicta eshre-poster-pregnancy rate after frozen blastocystINVICTA GENETICS
Assessment of pregnancy rate after comprehensive chromosome screening using next-generation sequencing-based preimplantation genetic diagnosis (NGS PGD) of trophectoderm in frozen blastocyst transfer.
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 summarizes the use of the nematode C. elegans as a model organism for studying human diseases. It discusses how CRISPR/Cas9 can be used to introduce disease-related mutations into C. elegans to model retinitis pigmentaria, cancer, and responses to chemotherapy. Drug screens and RNAi screens in these mutant worms have identified genetic modifiers and potential drug targets for treating human diseases. The small size, rapid life cycle, and genetic tractability of C. elegans make it a valuable pre-clinical model for validating targets and precision medicines before testing in mammalian systems.
This study compared the diagnostic performance of non-isotopic in situ hybridization (NISH) and dot-blot hybridization for detecting human papillomavirus (HPV) in cervical samples. Cervical samples from 122 patients were tested using both methods. NISH detected HPV in 33.3% of samples while dot-blot detected HPV in 12.4% of samples. NISH was more sensitive at detecting HPV even in samples with koilocytosis (abnormal cells indicative of HPV infection), detecting HPV in 90% of these samples compared to 40% for dot-blot. The study concluded that NISH is a more sensitive screening tool for detecting HPV infection in cervical samples than dot-bl
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 describes a method for performing RNA sequencing on single nuclei. Key points:
1) The authors demonstrate that double-stranded cDNA can be synthesized from single mouse neural progenitor cell nuclei and hippocampal tissue nuclei, allowing for whole transcriptome sequencing.
2) On average, sequencing of single nuclei detected over 16,000 of the approximately 24,000 mouse protein-coding genes.
3) Analysis of single nuclei avoids issues with dissociating intact cells from complex tissues, is applicable across eukaryotic species, and provides insight into nuclear gene regulation.
4) RNA sequencing of single nuclei is a powerful new method for investigating gene expression at the single cell level without disrupting cells.
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.
The document summarizes research using chick embryo models to study cancer metastasis. It describes several advantages of the chick embryo model, including its ability to support rapid growth of xenografted human tumor cells. Several chick embryo cancer models are outlined, including models for spontaneous metastasis, analyzing tumor cell interactions with vasculature, and studying invasion and extravasation. Examples are given of studies using these models to analyze tumor growth and metastasis of various cancer cell lines and effects of potential therapeutics.
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.
Generation of Clonal CRISPR/Cas9-edited Human iPSC Derived Cellular Models an...Thermo Fisher Scientific
This document describes a workflow for generating clonal CRISPR-edited human induced pluripotent stem cell (hiPSC) lines. Key aspects of the workflow include:
1) Developing a hiPSC line that stably expresses Cas9 to facilitate efficient genome editing.
2) Optimizing delivery methods for CRISPR/Cas9 editing tools and improving single cell clone survival and isolation using laminin-521 and StemFlex medium.
3) Applying the workflow to generate hiPSC lines carrying disease-relevant mutations and testing the cell models in assays, finding increased sensitivity to stress in models of Parkinson's disease.
Gene expression profiling in apoptotic mcf 7 cells infected with newcastle di...Mohamed Khalid Ali Xundhur
The document summarizes a study that examined gene expression profiling in MCF-7 breast cancer cells infected with Newcastle disease virus (NDV). The study found that NDV infection changed the expression levels of many genes involved in tumor progression, cell cycle regulation, apoptosis, and other cellular processes. Specifically, the study used a gene expression kit to measure changes in 21 genes related to these processes in MCF-7 cells infected with NDV. It found that NDV infection altered the expression of genes like PUMA, Bcl-2, ESR1, and MYBL2. The results provide insight into the gene regulation mechanisms by which NDV selectively kills cancer cells.
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 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.
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 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 summarizes preimplantation genetic diagnosis (PGD), which screens embryos for genetic disorders prior to embryo transfer during in vitro fertilization (IVF). PGD can detect single-gene disorders, chromosomal abnormalities, and HLA types. It allows transferring only unaffected embryos, avoiding termination of affected pregnancies. PGD is requested for couples at high risk of passing genetic disorders to offspring or to select euploid embryos to improve IVF success rates. The document outlines the PGD process and discusses its use for various genetic conditions like recessive, dominant, sex-linked, and chromosomal disorders. While PGD aims to prevent disease transmission, some argue its use for selecting traits could enable eugenics.
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.
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.
This document summarizes the use of the nematode C. elegans as a model organism for studying human diseases. It discusses how CRISPR/Cas9 can be used to introduce disease-related mutations into C. elegans to model retinitis pigmentaria, cancer, and responses to chemotherapy. Drug screens and RNAi screens in these mutant worms have identified genetic modifiers and potential drug targets for treating human diseases. The small size, rapid life cycle, and genetic tractability of C. elegans make it a valuable pre-clinical model for validating targets and precision medicines before testing in mammalian systems.
This study compared the diagnostic performance of non-isotopic in situ hybridization (NISH) and dot-blot hybridization for detecting human papillomavirus (HPV) in cervical samples. Cervical samples from 122 patients were tested using both methods. NISH detected HPV in 33.3% of samples while dot-blot detected HPV in 12.4% of samples. NISH was more sensitive at detecting HPV even in samples with koilocytosis (abnormal cells indicative of HPV infection), detecting HPV in 90% of these samples compared to 40% for dot-blot. The study concluded that NISH is a more sensitive screening tool for detecting HPV infection in cervical samples than dot-bl
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 describes a method for performing RNA sequencing on single nuclei. Key points:
1) The authors demonstrate that double-stranded cDNA can be synthesized from single mouse neural progenitor cell nuclei and hippocampal tissue nuclei, allowing for whole transcriptome sequencing.
2) On average, sequencing of single nuclei detected over 16,000 of the approximately 24,000 mouse protein-coding genes.
3) Analysis of single nuclei avoids issues with dissociating intact cells from complex tissues, is applicable across eukaryotic species, and provides insight into nuclear gene regulation.
4) RNA sequencing of single nuclei is a powerful new method for investigating gene expression at the single cell level without disrupting cells.
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.
The document summarizes research using chick embryo models to study cancer metastasis. It describes several advantages of the chick embryo model, including its ability to support rapid growth of xenografted human tumor cells. Several chick embryo cancer models are outlined, including models for spontaneous metastasis, analyzing tumor cell interactions with vasculature, and studying invasion and extravasation. Examples are given of studies using these models to analyze tumor growth and metastasis of various cancer cell lines and effects of potential therapeutics.
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.
Generation of Clonal CRISPR/Cas9-edited Human iPSC Derived Cellular Models an...Thermo Fisher Scientific
This document describes a workflow for generating clonal CRISPR-edited human induced pluripotent stem cell (hiPSC) lines. Key aspects of the workflow include:
1) Developing a hiPSC line that stably expresses Cas9 to facilitate efficient genome editing.
2) Optimizing delivery methods for CRISPR/Cas9 editing tools and improving single cell clone survival and isolation using laminin-521 and StemFlex medium.
3) Applying the workflow to generate hiPSC lines carrying disease-relevant mutations and testing the cell models in assays, finding increased sensitivity to stress in models of Parkinson's disease.
Gene expression profiling in apoptotic mcf 7 cells infected with newcastle di...Mohamed Khalid Ali Xundhur
The document summarizes a study that examined gene expression profiling in MCF-7 breast cancer cells infected with Newcastle disease virus (NDV). The study found that NDV infection changed the expression levels of many genes involved in tumor progression, cell cycle regulation, apoptosis, and other cellular processes. Specifically, the study used a gene expression kit to measure changes in 21 genes related to these processes in MCF-7 cells infected with NDV. It found that NDV infection altered the expression of genes like PUMA, Bcl-2, ESR1, and MYBL2. The results provide insight into the gene regulation mechanisms by which NDV selectively kills cancer cells.
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 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.
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 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 summarizes preimplantation genetic diagnosis (PGD), which screens embryos for genetic disorders prior to embryo transfer during in vitro fertilization (IVF). PGD can detect single-gene disorders, chromosomal abnormalities, and HLA types. It allows transferring only unaffected embryos, avoiding termination of affected pregnancies. PGD is requested for couples at high risk of passing genetic disorders to offspring or to select euploid embryos to improve IVF success rates. The document outlines the PGD process and discusses its use for various genetic conditions like recessive, dominant, sex-linked, and chromosomal disorders. While PGD aims to prevent disease transmission, some argue its use for selecting traits could enable eugenics.
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.
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.
The document discusses individualizing controlled ovarian stimulation (COS) regimens for in vitro fertilization (IVF) based on a woman's age, ovarian reserve tests, and risk of poor or hyper response. The number of oocytes retrieved during COS is strongly associated with live birth rates, but the relationship is non-linear and very high numbers can reduce success. Tests like antral follicle count and anti-Mullerian hormone help predict ovarian response and customize protocols to optimize oocyte yield safely for each patient.
This document discusses next generation sequencing (NGS) and its applications in preimplantation genetic diagnosis (PGD). It describes how NGS can simultaneously detect chromosome abnormalities and gene defects in single cells. Studies show NGS has the same accuracy as array comparative genomic hybridization for detecting aneuploidy, and can also detect mutations. NGS has been successfully used to analyze blastocysts and single cells to identify euploid and aneuploid embryos as well as specific gene mutations. This makes NGS useful for PGD to select embryos without chromosome issues or gene defects.
This document discusses the use of array comparative genomic hybridization (aCGH) for preimplantation genetic diagnosis (PGD) and screening (PGS). It finds that aCGH allows for high-resolution screening of chromosomal abnormalities, including aneuploidies and microdeletions. While it cannot detect balanced rearrangements, aCGH has advantages over techniques like fluorescence in situ hybridization (FISH) and can analyze more of the genome. The document also presents the author's own findings that aCGH-PGS may improve pregnancy and implantation rates compared to no PGS or FISH-PGS. Overall, aCGH is presented as a suitable approach for PGD/PGS
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 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 of a real time pcr method for rapid sexing of human preimplantati...t7260678
This document describes the development of a real-time PCR method for rapid sex determination of human preimplantation embryos as an alternative to fluorescence in-situ hybridization (FISH), the current gold standard technique. The researchers developed primer and probe sets to identify sex chromosomes in single blastomeres and buccal cells. They found that real-time PCR allowed rapid analysis of embryo sex for 60% of embryos tested, and provided sex determination for an additional 40% where FISH could not. The real-time PCR method was able to successfully determine embryo sex from single cells and may provide a faster and more accurate alternative to FISH for preimplantation genetic diagnosis.
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 culture, and vitrification for improved cryopreservation.
2. A randomized controlled trial in Brussels found that preimplantation genetic screening (PGS) did not clearly increase implantation or decrease abortion rates compared to controls.
3. Vitrification through the Cryotop method has improved oocyte and embryo survival rates after freezing and thawing compared to slow cooling methods.
1) The likelihood of abnormal embryos and non-chromosomal implantation failure increases with maternal age, ranging from 54% of embryos being abnormal for women under 35 to 82% for women 41-42.
2) A study found chromosomally abnormal embryos detected by array CGH had delayed blastulation compared to normal embryos.
3) Studies have found both positive and no effects of preimplantation genetic screening using day 3 embryo biopsy and FISH, with some finding a negative effect. A recent study found day 3 biopsy reduced implantation rates compared to blastocyst biopsy or no biopsy.
4) Data suggests preimplantation genetic testing for aneuploidies by methods like array C
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.
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 various methods for embryo selection to improve IVF outcomes, including embryo morphology, genomics/PGS, and embryo metabolism techniques.
2) Embryo morphology assessment is currently the most common method but has limitations like variability between labs and observers and inability to assess genetic content. New techniques like time-lapse may help standardize morphology analysis.
3) Genomic analysis through PGS shows promise but has technical limitations and some studies found it may be detrimental. Improved methods like blastocyst biopsy are being studied.
4) Metabolism analysis of amino acid and glucose uptake and techniques like metabolomics and respirometry aim to provide a non-invasive assessment of embryo viability
Similar to Development and validation of an accurate quantitative real time polymerase chain reaction-based assay for human blastocyst comprehensive chromosoma
Comprehensive Chromosome Screening (CCS) methodologies have been developed to analyze all 24 human chromosomes in oocytes and embryos, including methods such as array comparative genomic hybridization (aCGH), single nucleotide polymorphism (SNP) microarrays, and quantitative real-time PCR (qPCR). These methods vary in terms of accuracy, minimum detectable imbalance, analysis time, and ability to detect other genetic abnormalities beyond aneuploidy. While aCGH and SNP microarrays can both detect smaller deletions and duplications, the level of resolution differs between platforms. CCS now allows for comprehensive screening to be completed in time for fresh embryo transfers using techniques like trophectoderm biopsy followed by qPCR
Trophectoderm dna fingerprinting by quantitative real time pcr successfully d...t7260678
This study validated a novel method of DNA fingerprinting using quantitative real-time PCR (qPCR) to distinguish sibling human embryos. qPCR was performed on 40 single nucleotide polymorphisms (SNPs) from cell line samples modeling limited biopsy material (2-5 cells) as well as excess DNA from trophectoderm biopsies of human blastocysts. The method accurately assigned relationships between self, sibling, and unrelated samples with 100% accuracy, demonstrating its ability to unequivocally discriminate between sibling embryos. This inexpensive and practical qPCR-based DNA fingerprinting technique will support research to identify new markers of embryonic reproductive potential and evaluate interventions that may influence development.
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 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
This study used SNP microarray analysis to reanalyze 50 blastocysts that had previously been diagnosed as aneuploid by FISH at the cleavage stage. 58% of blastocysts were found to be euploid in all sections analyzed despite an aneuploid FISH result. Aneuploid blastocysts showed no evidence of preferential segregation of abnormalities to the trophectoderm. Additionally, mechanisms of self-correction like chromosome extrusion or duplication were not observed. These findings support the conclusion that cleavage-stage FISH has poor predictive value for aneuploidy in morphologically normal blastocysts.
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.
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.
This study evaluated the use of targeted next-generation sequencing (NGS) for preimplantation genetic diagnosis (PGD) of single-gene disorders. The study compared NGS results from embryo biopsies to results from two established PGD methods. NGS provided 100% consistency with the established methods in diagnosing point mutations and small insertions/deletions in six couples at risk of transmitting single-gene disorders. Additionally, NGS allowed for parallel single-gene disorder screening and comprehensive chromosome screening from the same biopsy sample. The study demonstrates NGS can provide accurate and consistent PGD results and could serve as a model for further development of this emerging technology in PGD.
This study evaluated the use of targeted next-generation sequencing (NGS) for preimplantation genetic diagnosis (PGD) of single-gene disorders. The study compared NGS results from embryo biopsies to results from two established PGD methods. NGS provided 100% consistency with the established methods in diagnosing point mutations and small insertions/deletions in six couples at risk of transmitting single-gene disorders. Additionally, NGS allowed for parallel single-gene disorder screening and comprehensive chromosome screening from the same biopsy sample. The study demonstrates NGS can provide accurate and consistent PGD results and could serve as a model for further development of this emerging technology in PGD.
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
Development and validation of an accurate quantitative real timeMoa-ren Hong
This study developed and validated a quantitative real-time polymerase chain reaction (qPCR)-based assay for comprehensive chromosomal aneuploidy screening of human blastocysts. The assay was tested in two phases using cell lines and blastocyst biopsies. Results demonstrated 97.6% reliability in diagnosis and 100% consistency in chromosome copy number assignments for cell lines. For blastocysts, the assay showed 99.94% chromosome-specific consistency and 98.6% consistency in 24-chromosome diagnosis. Comparison with single-nucleotide polymorphism microarrays showed high consistency. The qPCR-based method allows same-day biopsy and aneuploidy screening with potential for fresh blastocyst transfer.
This study aimed to develop an unbiased RNA profiling approach for the early detection of colorectal cancer (CRC) and advanced adenomas (AA) using blood samples. The researchers combined a literature review with microarray analysis of circulating RNA purified from plasma to identify RNA biomarker panels. They tested the panels on two cohorts, detecting CRC with 75% sensitivity and 93% specificity using an 8-gene panel, and detecting AA with 60% sensitivity and 87% specificity using a 2-gene panel. The study demonstrates the feasibility of unbiased molecular diagnosis of CRC and AA from blood and introduces circulating RNA profiling as a potential non-invasive screening approach.
An integrated approach to analyzing breast cancer at the proteomic and genomic level is presented using a cytometric readout. The approach analyzes fine needle aspirates to assess proteins, mRNA expression of HER2, and genomic integrity. Feasibility testing used a model system of mixed cell lines and analyzed 40 breast tumors and 10 normal tissues fixed in two solutions. The clinical performance relates to the model system and the cell-based assay could apply to xenograft models and circulating tumor cells.
Clinical investigational studies for validation of a next-generation sequenci...Frank Ong, MD, CPI
Clinical investigational studies were conducted to demonstrate the accuracy and reproducibility of the Illumina MiSeqDx CF System, a next-generation sequencing (NGS) in vitro diagnostic device for cystic fibrosis testing. Two assays were evaluated in both an Accuracy Study and a Reproducibility Study, with comparison to reference methods. The studies found that both assays achieved high positive agreement, negative agreement, and overall agreement compared to reference methods. Sample pass rates were also high. This is the first systematic evaluation of a NGS platform for broad clinical use as an in vitro diagnostic for cystic fibrosis testing.
Identifying novel and druggable targets in a triple negative breast cancer ce...Thermo Fisher Scientific
In this study, we developed a CRISPR/Cas9-based high throughput loss-of-function screen for identifying target genes responsible for the tumor proliferation and growth in TNBC. Our initial focus was to identify essential kinases in MDA-MB-231 cell line using the Invitrogen™ LentiArray™ Human Kinase CRISPR Library, which targets 840 kinases with up to 4 different gRNAs per protein kinase for complete gene knockout. This functional screen identified over 90 protein kinases that are essential for cell viability and cell proliferation. Ten of these hits (CDK1, CDK2, CDK8, CDK10, CDK11A, CDK19, CDK19, CDC7, EPHA2 and WEE1) are well-known targets validated in the literature. Currently, we are in the process validating the novel hits through target gene sequencing, western blotting and target specific small molecule kinase inhibitors.
Applications of Next generation sequencing in Drug Discoveryvjain38
This presentation gives an overview of the Next generation Sequencing (NGS) technology and what are is current applications in the drug discovery process.
1) The study found that circ-LDLRAD3, STAT3 expression were upregulated while miR-876-3p expression was downregulated in pancreatic cancer tissues and cell lines compared to normal tissues/cells.
2) Knockdown of circ-LDLRAD3 suppressed pancreatic cancer cell proliferation, migration, and invasion.
3) Mechanistically, circ-LDLRAD3 was found to directly regulate miR-876-3p expression, and miR-876-3p targets STAT3. Downregulation of circ-LDLRAD3 inhibited cell proliferation, invasion and migration by regulating the miR-876-3p/STAT3 axis.
This study aimed to optimize the CRISPR/Cas9 genome editing protocol for efficient homozygous gene knock-in in human induced pluripotent stem cells (iPSCs). The researchers targeted the CD90 locus for replacement with the mouse ortholog Cd90 and tested various experimental conditions. After optimization, CRISPR efficiency increased from 0.28% to 11.8% homozygous knock-in as determined by flow cytometry. Key conditions implicated in higher efficiency included plasmid concentrations and quality, Cas9 delivery method, nucleofection device, recovery conditions, and cell concentration during nucleofection.
Similar to Development and validation of an accurate quantitative real time polymerase chain reaction-based assay for human blastocyst comprehensive chromosoma (20)
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.
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.
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.
2. SEMINAL CONTRIBUTION
bodies from the oocyte and zygote, blastomeres from the
cleavage-stage embryo, and trophectoderm from the blasto-cyst
(3). Although there are advantages and disadvantages
to each of these components of aneuploidy screening, the
amount of time required to perform comprehensive chromo-some
screening (CCS) by any of these methodologies typically
exceeds 12 hours. Although this is less critical when applied to
polar bodies or blastomeres, analysis of aneuploidy in the tro-phectoderm
would likely require cryopreservation of the blas-tocyst
to provide sufficient time for analysis while also
preserving the appropriate synchrony between embryo and
endometrial development (4). Although methods of blastocyst
cryopreservation have improved, some risk remains, and
many patients prefer to avoid the additional expense and
time associated with a frozen embryo transfer cycle. To rou-tinely
perform comprehensive aneuploidy screening of the
blastocyst without cryopreservation, more rapid methodolo-gies
need to be developed.
Indeed, a more rapid method of preimplantation-stage
DNA analysis, polymerase chain reaction (PCR), has been in
clinical practice to manage patients with risk of transmitting
monogenic disorders for20 years (5). However, the ability to
reliably diagnose aneuploidy of all 24 chromosomes with the
use of PCR has not been established. Because of the initial
success with PCR-based preimplantation genetic diagnosis
(PGD), many improvements in the application of PCR have
been made, including the incorporation of multiplexing, nest-ing,
and fluorescence detection (6). Particularly noteworthy is
the development of quantitative real-time PCR (qPCR) (7). Al-though
qPCR has typically been applied to gene expression
studies where relative transcript quantities are determined,
there is also potential to evaluate the quantity of DNA in
a given sample (8). Nevertheless, the ability of qPCR to quan-tify
chromosomes in limited numbers of cells has yet to be
demonstrated. The present study characterizes the accuracy
of a novel method of 24 chromosome quantificatation in lim-ited
starting material as a preclinical step toward the applica-tion
of a rapid CCS method in the diagnosis of human
embryonic aneuploidy at the blastocyst stage of development.
MATERIALS AND METHODS
Experimental Design
This study was conducted in two phases with emphasis on eval-uating
the technical variation of qPCR-based 24-chromosome
copy number assignments by avoiding analysis of samples
with potential biologic variation. In phase I, only cell lines
with little to no evidence of mosaicism (biologic variation)
for the previously well characterized whole-chromosome aneu-ploidies
were used. In a similar attempt to avoid the impact of
mosaicism in embryos in phase 2, only blastocysts with two
consistent SNP microarray–based diagnoses were reevaluated
by qPCR. Randomized and blinded evaluation of consistency
of qPCR with cell line karyotypes and embryo SNP microarray
diagnoses were used as a measure of accuracy.
Phase 1: Cell Lines
Nine established and stable cell lines (fibroblasts and lympho-cytes)
were purchased from the Coriell Cell Repository (Cam-den,
NJ) and cultured as recommended by the supplier.
Included were GM09286 (47,XY,þ9), GM02948 (47,XY,þ13),
GM04610 (47,XX,þ8[75]/46,XX,þ8,dic(14;21)(14qter 14p
13::21p13 21qter)[25]), GM04435 (48,XY,þ16,þ21[45]/
47,XY,þ21[5]), GM00323 (46,XY), AG16777 (47,XX,þ21
[21]/47,XX,þ21,t(21;22)(q22;q13)[29]), AG16778 (46,XX),
AG16782 (46,XY), and GM01454 (47,XY,þ12[48]/47,XY,
þ12,add(13)(q34)[52]). Earlier studies have indicated that the
typical trophectoderm biopsy contains about five cells (9). To
model this in evaluating cell lines, 5-cell samples were prepared
by placement offive individual cells into a PCRtube under a dis-secting
microscope, as previously described (10). Lymphocyte
lines were prepared directly and fibroblast lines after trypsin
EDTA treatment. Seven 5-cell samples from cell line
GM00323 were used to serve as a reference dataset to interpret
results from 42 randomized and blinded 5-cell test samples
(GM00323, n ¼ 10; GM09286, n ¼ 4; AG16777, n ¼ 5;
AG16778, n ¼ 3; AG16782; n ¼ 3; GM01454, n ¼ 5;
GM02948, n ¼ 5; GM04610, n ¼ 5; and GM04435, n ¼ 2), as
described subsequently. Randomization was performed using
Microsoft Excel to avoid potential bias fromsequential analysis
ofmultiple samples fromthe same cell line. The identification of
the origins of each samplewas blinded by using decoded sample
names created in Microsoft Excel. The amount of time to com-plete
the procedure was recorded for each sample.
Phase 2: Embryos
Seventy-one embryos were included in this study. All em-bryos
had two consistent SNP microarray–based aneuploidy
screening results of trophectoderm biopsies (from days 5
and 6), as previously described (9, 11, 12). Thirty-seven of
the 71 embryos included in this study had arrested by day 6
and were subsequently found to be euploid by SNP microar-ray
analysis. The remaining 34, despite developmental nor-malcy
on day 6, were found to possess aneuploidy by SNP
microarray analysis. A third biopsy of each of the 71 day 6
embryos was randomized, blinded, and evaluated by qPCR
with the same seven 5-cell reference sample set used in the
cell line study described above. Again, randomization and
blinding was performed in Microsoft Excel to avoid interpre-tation
bias. The amount of time to complete the procedure was
recorded for each embryo.
qPCR
Cell line 5-cell samples and embryo biopsies were processed by
alkaline lysis as previously described (13). Multiplex amplifi-cation
of 96 loci (four for each chromosome, as previously de-scribed
[14]) was performed with the use of TaqMan Copy
Number Assays and TaqMan Preamplification Master Mix as
recommended by the supplier (Applied Biosystems), and in
a 50-mL reaction volume for 18 cycles using an Applied Bio-systems
2720 thermocycler. Real-time PCR was performed in
quadruplicate for each of the individual 96 loci using TaqMan
Gene Expression Master Mix (Applied Biosystems), a 5-mL
reaction volume, a 384-well plate, and a 7900 HT sequence
detection system, as recommended by the supplier (Applied
Biosystems). A unique method of the standard delta delta
threshold cycle (ΔΔCt) method of relative quantitation (15)
820 VOL. 97 NO. 4 / APRIL 2012
3. was applied. First, a chromosome-specific ΔCt was calculated
from the average Ct of the 16 reactions targeting a specific
chromosome (four replicates of four loci) minus the average
Ct of all of the 336 reactions targeting all of the remaining au-tosomes
(four replicates of four loci of 21 remaining auto-somes).
The same process was used to individually determine
the ΔCt for each of the 24 chromosomes in the test sample.
Each chromosome-specific ΔCT was then normalized to the
average chromosome specific ΔCt values derived from the
same evaluation of seven normal male (GM00323) 5-cell sam-ples
(reference set). The resulting chromosome-specific ΔΔCt
values were used to calculate fold change by considering the
ΔΔCt values as the negative exponent of 2, as previously de-scribed
(15). All autosome fold changes were then multiplied
by 2, whereas the sex chromosome fold changes were used
as is, to determine the 24-chromosome copy number in each
sample. This methodology was designed to specifically iden-tify
whole-chromosome but not segmental aneuploidy.
Statistics
Sample specific concurrence. To evaluate the utility of
a previously established strategy for identifying poor-quality
data independent of knowing its accuracy (12), the
overall concurrence was calculated for each sample. In this
analysis, it is first assumed that the qPCR assay can assess
only whole-chromosome aneuploidy, such that the four
copy number assignments within each chromosome should
always agree. Therefore, the standard deviation of the four
measurements of copy number for each chromosome was cal-culated.
The standard deviations of each of the 24 chromo-somes
were then averaged for each sample. Outliers
(nonconcurrent samples) were defined as samples found out-side
an interquartile range of 1.5 from the overall distribution
of average sample-specific standard deviations for each sam-ple
type as determined with the use of Analyse-It software for
Microsoft Excel. Means and variations of the rates of concur-rence
in cell lines and embryos were evaluated for signifi-cance
with a Student t test and an F test, respectively.
Consistency of diagnosis. Consistency of the cell line 5-cell
samples’ qPCR-based 24-chromosome copy number predic-tions
with the cell lines’ karyotype (previously established
by the Coriell Cell Repository by conventional karyotyping)
was evaluated at the level of individual chromosome copy
numbers and for the entire 24 chromosomes of each sample
tested. Consistency of embryo qPCR-based 24-chromosome
copy number assignments with previously established SNP
microarray–based diagnoses was also evaluated at the level
of individual chromosome copy numbers for the entire 24
chromosomes of each sample tested and for the overall diag-nosis
of aneuploidy or euploidy. Results were evaluated with
and without the application of a threshold of concurrence as
described above.
RESULTS
Phase 1: Cell Lines
Forty-two randomized blinded samples were evaluated for
24-chromosome copy number and compared for consistency
FIGURE 1
Fertility and Sterility®
Examples of qPCR-based 24-chromosome copy number results from
5-cell samples derived from nine cell lines with previously well
characterized karyotypes.
Treff. 4-hour qPCR-based 24-chromosome CCS. Fertil Steril 2012.
with the cell lines’ karyotype previously determined by con-ventional
g-banding at the commercial provider’s laboratory.
Examples of qPCR results for 5-cell samples from the cell lines
are shown in Figure 1. One of the samples (GM00323; 46,XY)
produced a false positive trisomy 18, giving an overall consis-tency
of chromosome copy number assignment of 99.90%
(1,007/1,008) and an overall 24-chromosome diagnosis con-sistency
of 97.6% (41/42). There were no false negative
VOL. 97 NO. 4 / APRIL 2012 821
4. SEMINAL CONTRIBUTION
diagnoses for aneuploid chromosomes or inaccurate predic-tions
of gender. Analysis of concurrence identified the only
discordant cell line sample as the only outlier (i.e., nonconcur-rent;
Fig. 2). Therefore, by applying a threshold of concur-rence,
the cell line study resulted in 97.6% reliability of
obtaining a diagnosis and a 100% level of consistency of
chromosome-specific (n ¼ 984) and 24-chromosome copy
number (n ¼ 41) assignments. The amount of time taken to
complete the procedure for each sample was 4 hours.
Phase 2: Embryos
Seventy-one embryos with consistent SNP microarray–based
24 chromosome aneuploidy screening results from 2 biopsies
were rebiopsied, randomized, and blinded for analysis of con-sistency
of qPCR-based diagnoses. These were selected to re-duce
the risk of mosaicism. Examples of embryo biopsy qPCR
results are shown in Figure 3, and the details of karyotype pre-dictions
are included in Supplemental Table 1 (available on-line
at www.fertstert.org). In one embryo, consistently
diagnosed as 45,XY,13,14,þ18 by SNP microarray analy-sis
of two biopsies, qPCR failed to detect monosomy 14. All of
the remaining chromosomes for all of the remaining samples
were consistent between qPCR and microarray, giving an
overall chromosome-specific consistency of 99.94% (1,703/
1,704) and an overall 24-chromosome diagnosis consistency
of 98.6% (70/71). There were no false positive aneuploid chro-mosomes
observed or inaccurate predictions of sex. The over-all
rate of concurrence in cell lines from phase 1 was
equivalent to the rate of concurrence in embryos in phase 2
(P¼.96). The variation in concurrence rates within cell lines
and embryos was also equivalent (P¼.34). Analysis of con-currence
identified only one embryo sample as an outlier
(i.e., nonconcurrent; Fig. 2). However, this was not the sample
with the false negative monosomy 14, and therefore the
consistency of the embryo results was the same with or with-out
applying a threshold for concurrence. Because the only
false negative aneuploidy diagnosis occurred in an embryo
with other consistently diagnosed aneuploidies (monosomy
13 and trisomy 18), the overall qPCR-based diagnosis of
aneuploidy or euploidy was 100% consistent with SNP
FIGURE 2
Box-whisker plots representing the distribution of average 24-
chromosome four-loci copy number standard deviations for each of
the 42 cell line samples and 71 blastocyst biopsies. For each sample
type, one outlier was identified, including the only cell line sample
with an inconsistent qPCR diagnosis.
Treff. 4-hour qPCR-based 24-chromosome CCS. Fertil Steril 2012.
FIGURE 3
Examples of (gray) single-nucleotide polymorphism microarray– and
(white) qPCR-based 24-chromosome copy number results from
blastocyst-stage embryo biopsies.
Treff. 4-hour qPCR-based 24-chromosome CCS. Fertil Steril 2012.
822 VOL. 97 NO. 4 / APRIL 2012
5. microarray based predictions. The amount of time taken to
complete the procedure for each sample was 4 hours.
DISCUSSION
Results of the present study have demonstrated the validity of
a new 4-hour method for CCS in human blastocysts. The tech-nical
accuracy was measured in two phases. The first phase
involved the use of cell lines with previously well character-ized
karyotypes. Although it is possible for biologic variation
of cell line karyotypes to exist as a result of extended culture
(16, 17) or from unidentified low-level mosaicism in the orig-inal
sample used to create the cell line, the potential impact of
these biologic artifacts can be avoided by the use of early pas-sages
of cell lines that show little to no evidence of mosaicism
by conventional karyotyping. With this strategy we demon-strated
a consistency of qPCR-based CCS of concurrent
5-cell samples of 100%.
To evaluate a more relevant tissue type, the second phase
of the study involved the evaluation of discarded human em-bryos.
Because the presence of mosaicism in embryos as a re-sult
of postzygotic mitotic aneuploidy development
represents a well documented phenomenon that could con-tribute
to biologic variation in blastocysts (11, 18), we
selected embryos which specifically demonstrated
consistent SNP microarray diagnoses from 2 biopsies. This
approach may help reduce the impact of mosaicism and
biologic variation on evaluating the technical accuracy of
new methods such as qPCR. Indeed, analysis of these well
controlled blastocysts by qPCR demonstrated 98.6% 24-
chromosome consistency with the highly validated method
of SNP microarray–based aneuploidy screening (12). Impor-tantly,
all SNP microarray–based euploid embryos were diag-nosed
as euploid and all SNP microarray–based aneuploid
embryos as aneuploid by qPCR (100% laboratory diagnostic
consistency). Furthermore, because trophectoderm biopsies
may not all possess five cells as modeled in phase I, the results
of evaluating actual trophectoderm biopsies in phase II pro-vides
additional evidence of validity to samples with variable
and potentially fewer numbers of cells.
Another important observation regarding the perfor-mance
of this qPCR methodology was the equivalent levels
of concurrence measured in cell lines and embryos (Fig. 2).
It has been suggested that PGD-based assays typically per-form
differently on different cell types (i.e., lymphocytes, fi-broblasts,
and embryonic cells) (19). Given the high degree
of similarity in performance between cell lines and embryos
in the present study, qPCR-based aneuploidy screening ap-pears
to be a robust methodology independent of the cell
type. This may be in part due to the use of locus-specific mul-tiplex
PCR rather than whole-genome amplification for the
initial processing of the sample. It is also possible that the
use of trophectoderm biopsies, which may possess more
than five cells, provided an advantage compared with the
use of five lymphocytes or fibroblasts for providing consistent
copy number assignments across each chromosome. The same
advantage might be expected when comparing concurrence
of trophectoderm with either blastomeres or polar bodies
where less template DNA is present. Although this method
Fertility and Sterility®
was not applied to blastomeres or polar bodies (single cells),
it is theoretically possible. In addition, this methodology
could also be applicable to evaluating segmental aneuploidies
associated with inheritance of unbalanced translocations by
simply adding specific assays targeting positions on either
side of the breakpoints of the chromosomes involved. Finally,
one important challenge that should be considered is the need
to process multiple embryos in parallel. Although this cer-tainly
involves an additional expense (a limitation on its
own), the procurement of multiple thermal cyclers and the
use of standard laboratory automation solutions can be used
to completely circumvent this challenge.
In conclusion, with these measures of accuracy in place
and the fact that this protocol can be accomplished within 4
hours of receiving a biopsy, this qPCR-based methodology
provides the first opportunity for same-day trophectoderm bi-opsy
24-chromosome aneuploidy screening and fresh blasto-cyst
transfer. Given the level of consistency with an
established method of aneuploidy screening that has also
demonstrated excellent predictive value for clinical outcome
(20), this qPCR method can now be justifiably evaluated for
clinical efficacy in a randomized controlled trial (RCT). In-deed,
preliminary RCT results of 24-chromosome aneuploidy
screening with qPCR on trophectoderm biopsies and subse-quent
fresh euploid blastocyst transfer indicate a significant
increase in the success of IVF (21).
REFERENCES
1. Harper JC, Harton G. The use of arrays in preimplantation genetic diagnosis
and screening. Fertil Steril 2010;94:1173–7.
2. Treff NR, Su J, Tao X, Northrop LE, Scott RT Jr. Single-cell whole-genome am-plification
technique impacts the accuracy of SNP microarray-based geno-typing
and copy number analyses. Mol Hum Reprod 2011;17:335–43.
3. Delhanty JD. Is the polar body approach best for pre-implantation genetic
screening? Placenta 2011;32(Suppl 3):S268–70.
4. van Voorhis BJ, Dokras A. Delayed blastocyst transfer: is the window shut-ting?
Fertil Steril 2008;89:31–2.
5. Handyside AH, Kontogianni EH, Hardy K, Winston RM. Pregnancies from bi-opsied
human preimplantation embryos sexed by Y-specific DNA amplifica-tion.
Nature 1990;344:768–70.
6. Harton GL, De Rycke M, Fiorentino F, Moutou C, SenGupta S, Traeger-
Synodinos J, et al. ESHRE PGD consortium best practice guidelines for
amplification-based PGD. Hum Reprod 2010;26:33–40.
7. Higuchi R, Fockler C, Dollinger G, Watson R. Kinetic PCR analysis: real-time
monitoring of DNA amplification reactions. Biotechnology 1993;11:1026–30.
8. D’Haene B, Vandesompele J, Hellemans J. Accurate and objective copy num-ber
profiling using real-time quantitative PCR. Methods 2010;50:262–70.
9. Schoolcraft WB, Treff NR, Stevens JM, Ferry K, Katz-Jaffe M, Scott RT Jr. Live
birth outcome with trophectoderm biopsy, blastocyst vitrification, and sin-gle-
nucleotide polymorphism microarray-based comprehensive chromo-some
screening in infertile patients. Fertil Steril 2011;96:638–40.
10. Treff NR, Su J, Tao X, Miller KA, Levy B, Scott RT Jr. A novel single-cell DNA
fingerprinting method successfully distinguishes sibling human embryos.
Fertil Steril 2009;94:477–84.
11. Northrop LE, Treff NR, Levy B, Scott RT Jr. SNP microarray-based 24 chromo-some
aneuploidy screening demonstrates that cleavage-stage FISH poorly
predicts aneuploidy in embryos that develop to morphologically normal blas-tocysts.
Mol Hum Reprod 2010;16:590–600.
12. Treff NR, Su J, Tao X, Levy B, Scott RT Jr. Accurate single cell 24 chromosome
aneuploidy screening using whole genome amplification and single nucleo-tide
polymorphism microarrays. Fertil Steril 2010;94:2017–21.
13. Cui XF, Li HH,Goradia TM, Lange K, Kasasian HH Jr,Galas D, et al. Single-sperm
typing: determination of genetic distance between the G gamma-globin and
VOL. 97 NO. 4 / APRIL 2012 823
6. SEMINAL CONTRIBUTION
parathyroid hormone loci by using the polymerase chain reaction and allele-specific
oligomers. Proc Natl Acad Sci U S A 1989;86:9389–93.
14. Treff NR, Tao X, Su J, Lonczak A, Northrop LE, Ruiz A, et al. Tracking embryo
implantation using cell-free fetal DNA enriched from maternal circulation at
9 weeks gestation. Mol Hum Reprod 2011;17:434–8.
15. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative
CT method. Nat Protoc 2008;3:1101–8.
16. Maitra A, Arking DE, Shivapurkar N, Ikeda M, Stastny V, Kassauei K, et al.
Genomic alterations in cultured human embryonic stem cells. Nat Genet
2005;37:1099–103.
17. Draper JS, Smith K, Gokhale P, Moore HD, Maltby E, Johnson J, et al. Recur-rent
gain of chromosomes 17q and 12 in cultured human embryonic stem
cells. Nat Biotechnol 2004;22:53–4.
18. Fragouli E, Wells D. Aneuploidy in the human blastocyst. Cytogenet Ge-nome
Res 2011.
19. Glentis S, SenGupta S, Thornhill A, Wang R, Craft I, Harper JC. Molecular
comparison of single cell MDA products derived from different cell types. Re-prod
Biomed online 2009;19:89–98.
20. Scott RT Jr, Ferry K, Su J, Tao X, Scott K, Treff NR. Comprehensive chromo-some
screening is highly predictive of the reproductive potential of human
embryos: a prospective, blinded, nonselection study. Fertil Steril 2012. doi:
10.1016/j.fertnstert.2012.01.104.
21. Scott RT Jr, Tao X, Taylor D, Ferry K, Treff N. A prospective randomized con-trolled
trial demonstrating significantly increased clinical pregnancy rates
following 24 chromosome aneuploidy screening: biopsy and analysis on
day 5 with fresh transfer. Fertil Steril 2010;94:S2.
824 VOL. 97 NO. 4 / APRIL 2012