Three blastomere fixation techniques were compared based on cell loss, nuclear quality, signal overlaps, and FISH errors. Technique 1 used acetic acid/methanol fixation, Technique 2 used Tween 20, and Technique 3 used a combination of Tween 20 and acetic acid/methanol. A total of 100, 106, and 114 blastomeres were fixed using each technique, respectively. Technique 2 resulted in the poorest nuclear quality with more cytoplasm, overlaps, and errors. Technique 1 produced better quality but was difficult to perform. Technique 3 showed reasonably good quality while being easier to learn and use for PGD studies.
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 document describes a multiplexed fluorescence microscopy method (MxIF) that enables quantitative analysis of multiple protein and nucleic acid targets in formalin-fixed paraffin-embedded tissue samples. The method uses a chemical solution to inactivate fluorescent dyes after each imaging round, allowing reuse of common dyes in iterative staining and imaging cycles. This overcomes limitations of standard fluorescence microscopy by permitting high levels of multiplexing. The document demonstrates applications of MxIF including analysis of 61 protein markers in colorectal cancer samples at single-cell resolution, revealing extensive tumor heterogeneity.
Jerry Angel Report for karyotype & FISH trainingJerry Angel
This document describes research conducted on detecting Edward syndrome (trisomy 18) using karyotyping and fluorescence in situ hybridization (FISH). It provides background on both techniques, limitations of karyotyping, principles and applications of FISH including for detecting structural abnormalities. Details are given on Edward syndrome including symptoms, diagnosis, genetics, tests used and epidemiology. Limitations of both techniques and differential diagnosis are also summarized.
Webinar_Exosome Isolation and Monitoring- from cell culture to clinically rel...Ketil Winther Pedersen
1. This webinar discusses methods for isolating and analyzing exosomes directly from cell culture media and urine using magnetic bead capture with Dynabeads.
2. Direct capture of exosomes with magnetic beads is recommended for monitoring exosome production during cell culture, analysis of limited sample volumes, simplified workflow, and compatibility with downstream applications like flow cytometry and western blotting.
3. Pre-enrichment methods like ultracentrifugation may be needed for samples with low exosome concentrations but yield less sample and are more complex, while direct capture is shown to work well for cell culture media and urine samples.
Multiphoton microscopy was used to study spermatogenesis in rat testes at different developmental stages. It was able to identify the stage of spermatogenesis in seminiferous tubules without labels. Tubules with and without sperm showed differences in fluorescence that could be used to distinguish them. Imaging rat testes with multiphoton microscopy resulted in minimal DNA fragmentation, indicating low risk of damage. Multiphoton microscopy has potential to help identify sperm-containing tubules during testicular sperm extraction in humans, improving outcomes and reducing risks.
The exosomes isolated from serum of patients with systemic sclerosis contained higher levels of profibrotic miRNAs and lower levels of antifibrotic miRNAs compared to exosomes from healthy individuals. When applied to normal dermal fibroblasts in culture, the scleroderma patient exosomes stimulated the expression of profibrotic genes, mimicking the phenotype of scleroderma fibroblasts. This effect was partially reduced when the exosomes were pre-treated to degrade either the RNA or protein content, indicating both components contribute to the exosomes' ability to induce a profibrotic phenotype in target cells and suggesting a potential mechanism for the transmission and progression of fibrosis in scleroderma.
This document summarizes a study that investigated the cytotoxic activity of a chloroform extract and four diterpenes isolated from Salvia ballotiflora against five cancer cell lines. The extract and isolated compounds were tested using an MTT assay to determine their IC50 values. 19-Deoxyisoicetexone had the greatest effect on HeLa cells with an IC50 of 3.2 μg/ml, while the chloroform extract showed the best cytotoxicity against A549 cells with an IC50 of 2.29 μg/ml. These effects were similar to the IC50 values of cisplatin in the respective cell lines. The study isolated and identified the active compounds from S. ballotiflora
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 document describes a multiplexed fluorescence microscopy method (MxIF) that enables quantitative analysis of multiple protein and nucleic acid targets in formalin-fixed paraffin-embedded tissue samples. The method uses a chemical solution to inactivate fluorescent dyes after each imaging round, allowing reuse of common dyes in iterative staining and imaging cycles. This overcomes limitations of standard fluorescence microscopy by permitting high levels of multiplexing. The document demonstrates applications of MxIF including analysis of 61 protein markers in colorectal cancer samples at single-cell resolution, revealing extensive tumor heterogeneity.
Jerry Angel Report for karyotype & FISH trainingJerry Angel
This document describes research conducted on detecting Edward syndrome (trisomy 18) using karyotyping and fluorescence in situ hybridization (FISH). It provides background on both techniques, limitations of karyotyping, principles and applications of FISH including for detecting structural abnormalities. Details are given on Edward syndrome including symptoms, diagnosis, genetics, tests used and epidemiology. Limitations of both techniques and differential diagnosis are also summarized.
Webinar_Exosome Isolation and Monitoring- from cell culture to clinically rel...Ketil Winther Pedersen
1. This webinar discusses methods for isolating and analyzing exosomes directly from cell culture media and urine using magnetic bead capture with Dynabeads.
2. Direct capture of exosomes with magnetic beads is recommended for monitoring exosome production during cell culture, analysis of limited sample volumes, simplified workflow, and compatibility with downstream applications like flow cytometry and western blotting.
3. Pre-enrichment methods like ultracentrifugation may be needed for samples with low exosome concentrations but yield less sample and are more complex, while direct capture is shown to work well for cell culture media and urine samples.
Multiphoton microscopy was used to study spermatogenesis in rat testes at different developmental stages. It was able to identify the stage of spermatogenesis in seminiferous tubules without labels. Tubules with and without sperm showed differences in fluorescence that could be used to distinguish them. Imaging rat testes with multiphoton microscopy resulted in minimal DNA fragmentation, indicating low risk of damage. Multiphoton microscopy has potential to help identify sperm-containing tubules during testicular sperm extraction in humans, improving outcomes and reducing risks.
The exosomes isolated from serum of patients with systemic sclerosis contained higher levels of profibrotic miRNAs and lower levels of antifibrotic miRNAs compared to exosomes from healthy individuals. When applied to normal dermal fibroblasts in culture, the scleroderma patient exosomes stimulated the expression of profibrotic genes, mimicking the phenotype of scleroderma fibroblasts. This effect was partially reduced when the exosomes were pre-treated to degrade either the RNA or protein content, indicating both components contribute to the exosomes' ability to induce a profibrotic phenotype in target cells and suggesting a potential mechanism for the transmission and progression of fibrosis in scleroderma.
This document summarizes a study that investigated the cytotoxic activity of a chloroform extract and four diterpenes isolated from Salvia ballotiflora against five cancer cell lines. The extract and isolated compounds were tested using an MTT assay to determine their IC50 values. 19-Deoxyisoicetexone had the greatest effect on HeLa cells with an IC50 of 3.2 μg/ml, while the chloroform extract showed the best cytotoxicity against A549 cells with an IC50 of 2.29 μg/ml. These effects were similar to the IC50 values of cisplatin in the respective cell lines. The study isolated and identified the active compounds from S. ballotiflora
Motion-Based Angiogenesis Analysis_A Simple Method to Quanitfy Blood Vessel G...Joe Lee
This document describes a new method called motion-based angiogenesis analysis (MBAA) to quantify blood vessel growth through the motion of blood cells. The method involves recording a video of the regenerating tissue in zebrafish fins after amputation. Image analysis software is used to analyze the video frames and highlight pixels where motion occurs, revealing all blood vessels. Basic fibroblast growth factor and vascular endothelial growth factor were used to stimulate angiogenesis and an inhibitor was used to suppress it. Both ImageJ and ENVI software produced comparable results quantifying the area of new vasculature formed. This simple, accurate, and cost-effective method provides an easy way to quantify angiogenesis without using fluorescent agents or transgenic zebrafish.
This document describes the generation of recombinant antibody-like proteins called FingRs that bind to endogenous synaptic proteins PSD-95 and Gephyrin with high affinity. FingRs were selected from a fibronectin-based library using mRNA display. The best binding FingRs were identified using a cellular localization assay in COS cells. When fused to GFP, selected PSD-95 and Gephyrin FingRs labeled endogenous proteins in cultured neurons without affecting expression or synapse number/strength. These FingRs allow visualization of excitatory and inhibitory synapses in living neurons.
1) Researchers have developed a microfluidic chip-based cell sorting system called On-chip Sort that is gentler on cells than conventional cell sorters and can be operated sterilely.
2) They propose a "Negative Cell Sorting" method to remove non-target or undifferentiated cells from cell populations more efficiently. This method increases collection speed of target cells 100-fold when non-target cells comprise 1% of the total.
3) They applied this method to remove all undifferentiated cells from neural cell populations differentiated from iPS cells, collecting 500,000 differentiated cells after sorting.
This presentation was created by Ioanna Leontiou and it is intended as a creative and flexible tool for students on Biological sciences who focus on the chromosome segregation. It is created to facilitate students performing research projects in our lab (especially during Covid restrictions), but it is suitable for every student who wants to learn more about chromosomes and the molecular mechanism controlling chromosome segregation. The presentation includes a generic overview of the cell division, illustrates the chromosome structure and provides molecular details of the spindle assembly checkpoint, an important pathway that ensures high fedility of chromosome segregation through mitosis. It also includes an introduction to some of the molecular biology techniques used in a yeast lab and incoporates some fluorescent microscopy images/videos. At the end of the presentantion there is a list of open access scientific publications for further reading on the the molecular mechanism of spindle checkpoint and some links of some very interesting sites, which include a range of videos on laboratory molecular biology techniques, research talks and guided papers. The purpose of this presentantion is to create a piece of work that students could return to when needed. Diagramms and illustrations are also encouranged to be used by scientists, science communicators and educators.
This presentation is licensed under a Creative Common Attribution-ShareAlike 4.0 (CC BY-SA 4.0), unless otherwise stated on the specific slide.
ISEV2014 - Introduction to EV biogenesis and secretion (C. Thery)andyfhill
Slides from ISEV2014 presentation. Introduction to Extracellular Vesicle biogenesis and secretion presented by Clotilde Thery.
For more information go to www.isev.org
This document discusses several microfluidic separation methods for isolating circulating tumor cells (CTCs) from blood. It describes how microfluidics can accurately manipulate flow conditions to efficiently separate CTCs from blood cells based on differences in their biophysical properties such as size and deformability. Using these microfluidic approaches, viable CTCs can be retrieved from cancer patient blood samples with high isolation efficiency and purity. Identification of CTCs aids in cancer detection, disease monitoring, and insights into metastasis. The document also discusses using magnetic nanoparticles coupled with doxorubicin chemotherapy drug and an external magnetic field to more effectively deliver the drug to breast cancer cells and increase mortality rates.
This summary provides the key details from the document in 3 sentences:
The document discusses several recent scientific studies, including the discovery of two Saturn-sized planets orbiting a star, a clinical trial showing success for a targeted skin cancer drug, and research finding that nets of DNA and proteins in blood vessels help fight infection and provide a scaffold for blood clots. It also summarizes studies on lobster behavior, vision restoration using artificial corneas, tree death in the Amazon, and more. The research highlights cover topics in astronomy, cancer biology, animal behavior, tissue engineering, ecology, and other fields.
Ossifying fibroma vs fibrous dysplasia of the jaw/rotary endodontic courses b...Indian dental academy
Indian Dental Academy: will be one of the most relevant and exciting training center with best faculty and flexible training programs for dental professionals who wish to advance in their dental practice,Offers certified courses in Dental implants,Orthodontics,Endodontics,Cosmetic Dentistry, Prosthetic Dentistry, Periodontics and General Dentistry.
This study examined the expression of endothelial nitric oxide synthase (eNOS) in testicular tissue from men with normal spermatogenesis (control group) and men with non-obstructive azoospermia (NOA group). Immunohistochemistry revealed that eNOS was present in Leydig cells, Sertoli cells, immature spermatids and abnormal germ cells in both groups. However, eNOS expression was significantly higher in Leydig cells of the control group and higher in Sertoli cells and abnormal germ cells of the NOA group. The higher eNOS expression in the NOA group suggests it may play a role in the apoptosis of abnormal germ cells and the disruption of sper
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
The study tested the effect of adding umbilical cord blood to cultures used in a clonogenic assay. Peripheral blood was collected from healthy individuals and mononuclear cells were isolated. Cultures were prepared with and without cord blood using methylcellulose media and cytokines. Cultures containing cord blood yielded significantly more hematopoietic colonies than cultures without cord blood after 14 days, as determined by a paired t-test. The addition of cord blood enhanced colony growth in the clonogenic assay.
This document summarizes a study on the cytogenetics of seven ornamental Chrysanthemum species. The key findings are:
1) The species showed varying chromosome numbers of 2n=18, 2n=36. Some diploid species like C. carinatum showed structural heterozygosity involving reciprocal translocations.
2) Meiosis in most species involved bivalents, with some like C. morifolium and C. carinatum also forming multivalents. This led to irregular meiotic divisions and reduced pollen fertility in some cases.
3) Chiasma frequency varied from 14-16.8 per cell across species. C. leucanthemum in particular
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 study examined the expression of endothelial nitric oxide synthase (eNOS) in testicular tissue from men with normal spermatogenesis (control group) and men with non-obstructive azoospermia (NOA group) using immunohistochemistry. eNOS was detected in Leydig cells, Sertoli cells, immature spermatids and abnormal germ cells in both groups. The expression of eNOS was significantly higher in Leydig cells of the control group compared to the NOA group. In contrast, eNOS expression was higher in Sertoli cells of the NOA group. Abnormal germ cells with picnotic nuclei also showed higher eNOS staining in the NOA group. This
Applications of flow cytometry to clinical microbiologyLAB IDEA
This document provides an overview of the applications of flow cytometry to clinical microbiology. It discusses how flow cytometry can be used for the direct detection of bacteria, fungi, parasites and viruses through detection of antigens or nucleic acids. It also describes how flow cytometry can be applied to serological diagnosis and antimicrobial susceptibility testing. The document outlines how flow cytometry allows for rapid analysis and monitoring of infections and antimicrobial therapy. In conclusion, it discusses the benefits flow cytometry provides for clinical microbiology and its future potential.
Morphological and molecular analysis was used to identify parasites collected from Lake Winnibigoshish in Minnesota. Parasites were stained and examined under microscopy to measure morphological characteristics, which supported identification as Cotylurus brevis, Cotylurus flabelliformis, and Apatemon gracillis based on comparisons to previous studies. Genetic sequencing of the COX1 gene was initiated but not completed. Results from staining were consistent with identification of the three species based on features such as testis shape, ovary placement, and body ratios being within reported ranges. Molecular analysis may further support identifications but has not been finished.
Basic Formal Ontology (BFO) and DiseaseBarry Smith
The document discusses different approaches to conceptualizing health, disease, and biological kinds across multiple levels of granularity. It notes that traditional biology data conceptualized entities based on observable instances, while new biology data represents entities at the molecular level through genetic sequences. It argues that linking different kinds of phenomena represented at various levels requires annotation with terms from controlled vocabularies like ontologies. Ontologies provide a structured framework for integrating data across databases and supporting logical reasoning by standardizing references to biological entities, processes, and functions.
Trypsin was more efficient than dispase at isolating keratinocytes from tonsil tissue. Keratinocytes cultured with the Rho kinase inhibitor Y27632 survived over 30 population doublings without changing phenotype, whereas those without survived less than 10. A tissue-engineered model of tonsil epithelium was created using tonsil keratinocytes, fibroblasts, and de-epithelialized dermis to form a multi-layered differentiated epithelium resembling normal tonsil surface epithelium. This model responded to S. pyogenes by increasing pro-inflammatory cytokine expression.
Novel Way to Isolate Adipose Derive Mesenchymal Stem Cells & Its Future Clini...IOSR Journals
Abstract: Adipose-derived stem cells (ADSCs), were isolated from discarded human fat tissue, obtained from csection with our recently modified methods, in Stem Cell & Regenerative Medicine Lab, VSBT. Here we develop
two methods to isolate Adipose derived mesenchymal stem cells with enzyme digestion and use of
phosphatidylcholine and deoxycholate. Surface protein expression was analyzed by flow cytometry to
characterize the cell phenotype. The multi-lineage potential of ADSCs was testified by differentiating cells with
adipogenic inducer. ADSCs can be cultured in vitro for up to one month without passage. Also, the flow
cytometry analysis showed that ADSCs expressed high levels of stem cell related surface marker CD105.
ADSCs have strong proliferation ability, maintain their phenotypes, and have stronger multi-differentiation
potential. The molecular basis of ADSC differentiation was studied using bioinformatics tools with an aim to
identify the key proteins involved in differentiation, such that they could be used as potential targets for drug
development for the treatment of obesity. The key proteins involved were found to be PPARG and C/EBPα. The
structures of the proteins were retrieved from MMDB (Molecular Modelling Database) and PDB (Protein Data
Bank) respectively. Key Words: Adipose-derived stem cells, Mesenchymal stem cells, Enzyme digestion, Phosphatidylcholine, Deoxycholate, PPARG, C/EBPα, etc.
The whitepaper discusses how procurement functions can transform into world-class strategic operations that enhance profits and mitigate risks. It outlines that procurement needs to expand its remit beyond purchasing goods and services to strategic areas like partnerships and restructuring. To achieve this, procurement requires the right skills, board support, and an end-to-end mandate. A transformed procurement function would provide commercial oversight, act as a trusted advisor, and develop collaborative supplier partnerships to maximize shareholder value through cost savings, risk management, and profitability gains. The whitepaper argues that world-class companies need world-class procurement to compete in today's challenging economic environment.
Motion-Based Angiogenesis Analysis_A Simple Method to Quanitfy Blood Vessel G...Joe Lee
This document describes a new method called motion-based angiogenesis analysis (MBAA) to quantify blood vessel growth through the motion of blood cells. The method involves recording a video of the regenerating tissue in zebrafish fins after amputation. Image analysis software is used to analyze the video frames and highlight pixels where motion occurs, revealing all blood vessels. Basic fibroblast growth factor and vascular endothelial growth factor were used to stimulate angiogenesis and an inhibitor was used to suppress it. Both ImageJ and ENVI software produced comparable results quantifying the area of new vasculature formed. This simple, accurate, and cost-effective method provides an easy way to quantify angiogenesis without using fluorescent agents or transgenic zebrafish.
This document describes the generation of recombinant antibody-like proteins called FingRs that bind to endogenous synaptic proteins PSD-95 and Gephyrin with high affinity. FingRs were selected from a fibronectin-based library using mRNA display. The best binding FingRs were identified using a cellular localization assay in COS cells. When fused to GFP, selected PSD-95 and Gephyrin FingRs labeled endogenous proteins in cultured neurons without affecting expression or synapse number/strength. These FingRs allow visualization of excitatory and inhibitory synapses in living neurons.
1) Researchers have developed a microfluidic chip-based cell sorting system called On-chip Sort that is gentler on cells than conventional cell sorters and can be operated sterilely.
2) They propose a "Negative Cell Sorting" method to remove non-target or undifferentiated cells from cell populations more efficiently. This method increases collection speed of target cells 100-fold when non-target cells comprise 1% of the total.
3) They applied this method to remove all undifferentiated cells from neural cell populations differentiated from iPS cells, collecting 500,000 differentiated cells after sorting.
This presentation was created by Ioanna Leontiou and it is intended as a creative and flexible tool for students on Biological sciences who focus on the chromosome segregation. It is created to facilitate students performing research projects in our lab (especially during Covid restrictions), but it is suitable for every student who wants to learn more about chromosomes and the molecular mechanism controlling chromosome segregation. The presentation includes a generic overview of the cell division, illustrates the chromosome structure and provides molecular details of the spindle assembly checkpoint, an important pathway that ensures high fedility of chromosome segregation through mitosis. It also includes an introduction to some of the molecular biology techniques used in a yeast lab and incoporates some fluorescent microscopy images/videos. At the end of the presentantion there is a list of open access scientific publications for further reading on the the molecular mechanism of spindle checkpoint and some links of some very interesting sites, which include a range of videos on laboratory molecular biology techniques, research talks and guided papers. The purpose of this presentantion is to create a piece of work that students could return to when needed. Diagramms and illustrations are also encouranged to be used by scientists, science communicators and educators.
This presentation is licensed under a Creative Common Attribution-ShareAlike 4.0 (CC BY-SA 4.0), unless otherwise stated on the specific slide.
ISEV2014 - Introduction to EV biogenesis and secretion (C. Thery)andyfhill
Slides from ISEV2014 presentation. Introduction to Extracellular Vesicle biogenesis and secretion presented by Clotilde Thery.
For more information go to www.isev.org
This document discusses several microfluidic separation methods for isolating circulating tumor cells (CTCs) from blood. It describes how microfluidics can accurately manipulate flow conditions to efficiently separate CTCs from blood cells based on differences in their biophysical properties such as size and deformability. Using these microfluidic approaches, viable CTCs can be retrieved from cancer patient blood samples with high isolation efficiency and purity. Identification of CTCs aids in cancer detection, disease monitoring, and insights into metastasis. The document also discusses using magnetic nanoparticles coupled with doxorubicin chemotherapy drug and an external magnetic field to more effectively deliver the drug to breast cancer cells and increase mortality rates.
This summary provides the key details from the document in 3 sentences:
The document discusses several recent scientific studies, including the discovery of two Saturn-sized planets orbiting a star, a clinical trial showing success for a targeted skin cancer drug, and research finding that nets of DNA and proteins in blood vessels help fight infection and provide a scaffold for blood clots. It also summarizes studies on lobster behavior, vision restoration using artificial corneas, tree death in the Amazon, and more. The research highlights cover topics in astronomy, cancer biology, animal behavior, tissue engineering, ecology, and other fields.
Ossifying fibroma vs fibrous dysplasia of the jaw/rotary endodontic courses b...Indian dental academy
Indian Dental Academy: will be one of the most relevant and exciting training center with best faculty and flexible training programs for dental professionals who wish to advance in their dental practice,Offers certified courses in Dental implants,Orthodontics,Endodontics,Cosmetic Dentistry, Prosthetic Dentistry, Periodontics and General Dentistry.
This study examined the expression of endothelial nitric oxide synthase (eNOS) in testicular tissue from men with normal spermatogenesis (control group) and men with non-obstructive azoospermia (NOA group). Immunohistochemistry revealed that eNOS was present in Leydig cells, Sertoli cells, immature spermatids and abnormal germ cells in both groups. However, eNOS expression was significantly higher in Leydig cells of the control group and higher in Sertoli cells and abnormal germ cells of the NOA group. The higher eNOS expression in the NOA group suggests it may play a role in the apoptosis of abnormal germ cells and the disruption of sper
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
The study tested the effect of adding umbilical cord blood to cultures used in a clonogenic assay. Peripheral blood was collected from healthy individuals and mononuclear cells were isolated. Cultures were prepared with and without cord blood using methylcellulose media and cytokines. Cultures containing cord blood yielded significantly more hematopoietic colonies than cultures without cord blood after 14 days, as determined by a paired t-test. The addition of cord blood enhanced colony growth in the clonogenic assay.
This document summarizes a study on the cytogenetics of seven ornamental Chrysanthemum species. The key findings are:
1) The species showed varying chromosome numbers of 2n=18, 2n=36. Some diploid species like C. carinatum showed structural heterozygosity involving reciprocal translocations.
2) Meiosis in most species involved bivalents, with some like C. morifolium and C. carinatum also forming multivalents. This led to irregular meiotic divisions and reduced pollen fertility in some cases.
3) Chiasma frequency varied from 14-16.8 per cell across species. C. leucanthemum in particular
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 study examined the expression of endothelial nitric oxide synthase (eNOS) in testicular tissue from men with normal spermatogenesis (control group) and men with non-obstructive azoospermia (NOA group) using immunohistochemistry. eNOS was detected in Leydig cells, Sertoli cells, immature spermatids and abnormal germ cells in both groups. The expression of eNOS was significantly higher in Leydig cells of the control group compared to the NOA group. In contrast, eNOS expression was higher in Sertoli cells of the NOA group. Abnormal germ cells with picnotic nuclei also showed higher eNOS staining in the NOA group. This
Applications of flow cytometry to clinical microbiologyLAB IDEA
This document provides an overview of the applications of flow cytometry to clinical microbiology. It discusses how flow cytometry can be used for the direct detection of bacteria, fungi, parasites and viruses through detection of antigens or nucleic acids. It also describes how flow cytometry can be applied to serological diagnosis and antimicrobial susceptibility testing. The document outlines how flow cytometry allows for rapid analysis and monitoring of infections and antimicrobial therapy. In conclusion, it discusses the benefits flow cytometry provides for clinical microbiology and its future potential.
Morphological and molecular analysis was used to identify parasites collected from Lake Winnibigoshish in Minnesota. Parasites were stained and examined under microscopy to measure morphological characteristics, which supported identification as Cotylurus brevis, Cotylurus flabelliformis, and Apatemon gracillis based on comparisons to previous studies. Genetic sequencing of the COX1 gene was initiated but not completed. Results from staining were consistent with identification of the three species based on features such as testis shape, ovary placement, and body ratios being within reported ranges. Molecular analysis may further support identifications but has not been finished.
Basic Formal Ontology (BFO) and DiseaseBarry Smith
The document discusses different approaches to conceptualizing health, disease, and biological kinds across multiple levels of granularity. It notes that traditional biology data conceptualized entities based on observable instances, while new biology data represents entities at the molecular level through genetic sequences. It argues that linking different kinds of phenomena represented at various levels requires annotation with terms from controlled vocabularies like ontologies. Ontologies provide a structured framework for integrating data across databases and supporting logical reasoning by standardizing references to biological entities, processes, and functions.
Trypsin was more efficient than dispase at isolating keratinocytes from tonsil tissue. Keratinocytes cultured with the Rho kinase inhibitor Y27632 survived over 30 population doublings without changing phenotype, whereas those without survived less than 10. A tissue-engineered model of tonsil epithelium was created using tonsil keratinocytes, fibroblasts, and de-epithelialized dermis to form a multi-layered differentiated epithelium resembling normal tonsil surface epithelium. This model responded to S. pyogenes by increasing pro-inflammatory cytokine expression.
Novel Way to Isolate Adipose Derive Mesenchymal Stem Cells & Its Future Clini...IOSR Journals
Abstract: Adipose-derived stem cells (ADSCs), were isolated from discarded human fat tissue, obtained from csection with our recently modified methods, in Stem Cell & Regenerative Medicine Lab, VSBT. Here we develop
two methods to isolate Adipose derived mesenchymal stem cells with enzyme digestion and use of
phosphatidylcholine and deoxycholate. Surface protein expression was analyzed by flow cytometry to
characterize the cell phenotype. The multi-lineage potential of ADSCs was testified by differentiating cells with
adipogenic inducer. ADSCs can be cultured in vitro for up to one month without passage. Also, the flow
cytometry analysis showed that ADSCs expressed high levels of stem cell related surface marker CD105.
ADSCs have strong proliferation ability, maintain their phenotypes, and have stronger multi-differentiation
potential. The molecular basis of ADSC differentiation was studied using bioinformatics tools with an aim to
identify the key proteins involved in differentiation, such that they could be used as potential targets for drug
development for the treatment of obesity. The key proteins involved were found to be PPARG and C/EBPα. The
structures of the proteins were retrieved from MMDB (Molecular Modelling Database) and PDB (Protein Data
Bank) respectively. Key Words: Adipose-derived stem cells, Mesenchymal stem cells, Enzyme digestion, Phosphatidylcholine, Deoxycholate, PPARG, C/EBPα, etc.
The whitepaper discusses how procurement functions can transform into world-class strategic operations that enhance profits and mitigate risks. It outlines that procurement needs to expand its remit beyond purchasing goods and services to strategic areas like partnerships and restructuring. To achieve this, procurement requires the right skills, board support, and an end-to-end mandate. A transformed procurement function would provide commercial oversight, act as a trusted advisor, and develop collaborative supplier partnerships to maximize shareholder value through cost savings, risk management, and profitability gains. The whitepaper argues that world-class companies need world-class procurement to compete in today's challenging economic environment.
Por qué los niños se aburren en las escuelasMONICA VASQUEZ
Este documento discute por qué los niños se aburren en la escuela y propone formas de mejorar la educación. Explica que las escuelas se enfocan demasiado en la memorización de datos en lugar de desarrollar la creatividad y capacidad de pensamiento crítico. También señala que las artes reciben poca atención a pesar de su importancia para fortalecer otras habilidades. Finalmente, propone que los maestros deben servir como guías que motivan a los estudiantes a aprender a través de experiencias significativas más
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.
Este documento lista diferentes tipos de basura común como botes de lubricantes, botes plásticos PETS, trapos y guantes contaminados que deben desecharse adecuadamente.
Este documento lista las direcciones, números de teléfono y fax de las embajadas de México en Alemania, Grecia, República Checa, Suiza, Portugal, Polonia, Finlandia, Irlanda, Hungría e Italia, incluyendo los nombres de los embajadores.
China's Housing Development Strategy in the New NormalSTLLab
As China has entered into a new development phase of "balanced transition" (also known as the "new normal"), the housing sector is facing tough challenges in maintaining the previous growth rate. In this turning point, the question of how to cultivate new development strategies for both the government and private sectors represents an urgent task. Dr. Shao's talk will focus on three issues:
1. How to understand the existing market situation;
2. How to evaluate housing policies; and
3. How to design a new strategy for stabilizing the real estate sector.
Role of icj in solving internation disputegagan deep
The International Court of Justice (ICJ) helps resolve international disputes through binding judgments. It is the primary judicial branch of the United Nations, composed of 15 judges elected by the UN General Assembly and Security Council. Only states can bring cases to the ICJ, and its jurisdiction is based on state consent. One example is the 1986 case of Nicaragua v. United States, where the ICJ ruled the US violated international law by supporting Contra rebels against Nicaragua's government. While the decision was binding, the US refused to participate and blocked its enforcement, showing the limited power of the ICJ without state cooperation.
International law is the set of rules that are accepted as binding by states in their relations with each other and individuals. It emerged in the 16th century from thinkers like Grotius and serves as the framework for organized international relations. There are various sources of international law, including treaties, customary practice, and general legal principles. The key subjects that international law applies to are states and non-state actors like individuals and international organizations. The 10 main principles of international law include sovereign equality of states, non-use of force, territorial integrity, and human rights.
This document discusses setting up IUI and IVF services. It covers the key components needed, including good lab design, infrastructure, equipment, and personnel. For infrastructure, it recommends building from scratch in a pollution-free area for an embryo-friendly environment. It provides details on room classification, air handling units, electricity, and gas supply. Essential and desirable equipment are outlined for IUI and IVF labs. It emphasizes the importance of a cohesive team approach. Startup costs are estimated at 1-1.5 lakhs for IUI and 40 lakhs for IVF, with an ideal setup costing around 100 lakhs plus 40-50 lakhs for infrastructure. Effective service provision focuses
Over the past 150 years, Nokia has evolved from a small paper mill in Finland to a global telecommunications leader. Nokia has disrupted into various industries before becoming a telecommunications giant. Nokia's mobile phone platforms included Symbian, MeeGo, and Meltemi. In 2011, Nokia announced a partnership with Microsoft to build a new mobile ecosystem using the Windows Phone platform. Currently, Nokia focuses on Windows Phone and Symbian 40, having decided not to continue development of MeeGo and Meltemi. Nokia's main competitors are Samsung, Apple, RIM, and HTC.
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.
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 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.
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 describes a method for rapidly detecting whether plant mutants are homozygous or heterozygous using CEL-I endonuclease. CEL-I endonuclease is a mismatch-specific endonuclease that can cleave DNA at sites of mismatches between DNA strands. The method involves isolating genomic DNA from mutant plants, amplifying a region by PCR, and treating the PCR products with CEL-I endonuclease. Digestion products are then analyzed by gel electrophoresis. For homozygous mutants, no cleavage will occur as there are no mismatches. For heterozygous mutants, cleavage will occur at mismatch sites, producing distinctive banding patterns that allow identification of heterozygotes. This method provides a low
This document describes a method for rapidly detecting whether plant mutants are homozygous or heterozygous using CEL-I endonuclease. CEL-I endonuclease is a mismatch-specific endonuclease that can cleave DNA at sites of mismatches between DNA strands. The method involves isolating genomic DNA from mutant plants, amplifying a region by PCR, and treating the PCR products with CEL-I endonuclease. Digestion products are then analyzed by gel electrophoresis. For homozygous mutants, no cleavage will occur as there are no mismatches, while heterozygous mutants will show cleavage fragments indicating mismatches. This provides a low-cost method to determine mutational status without sequencing or genetic analysis.
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 document analyzes 2,204 human oocytes and embryos from fertilization through the blastocyst stage using microarray comparative genomic hybridization to determine chromosome abnormalities. It finds that aneuploidy rates increase dramatically with female age and that complex abnormalities are common. Chromosome errors originate from failures in meiotic cell division and early mitosis. Most aneuploid embryos survive until the blastocyst stage but likely fail to implant, indicating selection against aneuploidy occurs late in preimplantation development.
This study analyzed 2,204 human oocytes, embryos, and blastocysts to investigate the origin and impact of chromosomal abnormalities during early human development. The results showed that aneuploidy rates increased dramatically with female age and many abnormalities were present until the blastocyst stage, suggesting selection against aneuploid embryos occurs late in development. Mechanisms like anaphase lag and congression failure contributed to errors in the first cell divisions. A wide variety of chromosome abnormalities were detected, with implications for understanding the sources of aneuploidy and how they influence embryo viability.
Assessment of Embryotoxicity of Compounds in Cosmetics by the Embryonic Stem ...v2zq
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This document provides background information on human chromosome analysis and describes a laboratory exercise for students to learn techniques for analyzing human chromosomes. The exercise is divided into two sessions: the first focuses on staining techniques to identify chromosomes, and the second has students practice karyotyping using a dichotomous key. The document provides extensive background on human cytogenetics, chromosome structure, banding patterns, and nomenclature to help students understand and complete the laboratory activities.
Troy University Surface of Membrane Cells Summary.pdfsdfghj21
- Mouse and human cells were fused together using Sendai virus, producing hybrid cells containing both mouse and human surface antigens.
- Within 40 minutes of fusion, over 90% of the hybrid cells showed total mixing and intermingling of the mouse and human surface antigens across the cell membrane.
- Treatments with metabolic inhibitors like lowered temperature were able to prevent the rapid mixing of surface antigens, suggesting the antigens freely diffuse across the fluid cell membrane.
Prenatal diagnosis employs techniques like amniocentesis and chorionic villus sampling to determine the health of the unborn fetus. It is helpful for managing the pregnancy, determining outcomes, and planning for complications. It allows decisions about continuing the pregnancy and identifying conditions that could affect future pregnancies. Maternal serum screening measures markers like alpha-fetoprotein to screen for neural tube defects and other abnormalities by detecting higher than normal levels that cross the placenta.
The document summarizes research on the metastatic spread of breast cancer cells in mice. Key findings include:
- Line 4T1, a metastatic breast cancer cell line, primarily spreads through hematogenous metastasis to the lungs followed later by the liver. Necropsy found lung and liver nodules.
- Line 66cl4 also metastasized to the lungs and liver but spread more through lymph nodes than 4T1.
- The non-metastatic line 67NR was unable to intravasate and spread, as no clonogenic cancer cells were found in distant organs.
The use of time lapse photography in an in vitro fertilization programme for ...鋒博 蔡
This study examined the timing of early cell divisions in human embryos using time-lapse imaging. It found that embryos producing high-quality blastocysts and resulting in pregnancies showed uniform timing of cleavage cycles and interphases. Specifically, the second interphase lasted 11±1 hours, the third interphase was 15±1 hours, and the fourth interphase was 23±1 hours. The corresponding cleavage cycles lasted 15±5 minutes, 40±10 minutes, and 55±15 minutes. In contrast, embryos with shortened or prolonged cell cycles showed poor implantation and development. The study also discovered trichotomic mitosis, where embryos cleaved into an abnormal number of cells, in 17% of cases. Only time-
This study analyzed the timing of early cell divisions in 180 human embryos using time-lapse imaging. Embryos with uniform timing of cleavages and interphases were more likely to develop into high-quality blastocysts and result in pregnancies, while abnormalities in timing predicted poor development and implantation failure. Specifically, embryos with shortened or prolonged cell cycles showed higher rates of morphological anomalies, lower blastocyst formation rates, and zero implantation, even when blastocyst formation occurred. The study demonstrates that time-lapse imaging can identify viable embryos with high specificity by analyzing cleavage uniformity and rule out non-viable embryos with 100% specificity.
The document describes a pilot study that investigated the presence of DNA in blastocyst fluids (BFs) and whether the chromosomal status predicted by analyzing this DNA corresponds to the status in trophectoderm (TE) cells and the whole embryo. The study found that:
1) DNA was detected in the BFs of 76.5% of blastocysts tested, allowing chromosomal analysis of these samples.
2) In 97.4% of cases, the ploidy condition (euploid vs. aneuploid) predicted by BF analysis matched the condition in TE cells.
3) BF analysis predicted the ploidy condition of the whole embryo with 100% accuracy
This document summarizes research comparing outcomes of fresh embryo transfers versus frozen embryo transfers (FET). Key points include:
- FET outcomes were found to equal or exceed fresh outcomes, suggesting endometrial asynchrony with fresh cycles due to ovarian stimulation effects.
- Slower developing day 6 blastocysts showed lower implantation rates than day 5 blastocysts with fresh but not FET transfers, again indicating endometrial asynchrony issues with fresh cycles.
- Studies directly comparing matched fresh and FET cycles found significantly higher pregnancy and implantation rates with FET, demonstrating cryopreservation can overcome negative endometrial effects of ovarian stimulation.
This document summarizes research comparing outcomes of fresh embryo transfers versus frozen embryo transfers (FET). It finds that FET results in better pregnancy and implantation rates than fresh transfers, likely due to ovarian stimulation negatively impacting endometrial receptivity in fresh cycles. Specifically, FET cycles have higher success rates for slower developing embryos and embryos transferred in cycles with premature progesterone elevation. FET outcomes in young patients can rival fresh donor egg cycles. Randomized trials show significantly higher pregnancy rates with FET compared to fresh transfer in normal responders.
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 study analyzed aneuploidy rates, apoptotic markers, and DNA fragmentation in sperm samples from normozoospermic men with unexplained infertility. Samples underwent density gradient centrifugation and then magnetic activated cell sorting (MACS). MACS significantly reduced the percentage of aneuploid, apoptotic, and DNA-damaged sperm. A positive correlation was found between reduced aneuploidy and lower DNA damage after MACS, but no correlation with apoptotic markers. The interactions between apoptotic markers, DNA integrity, and aneuploidy, as well as the effects of MACS on these parameters, require further investigation.
This document discusses the impact of fetal fraction, the percentage of cell-free DNA in maternal plasma that is of fetal origin, on the performance of next generation sequencing tests for detecting common fetal aneuploidies such as Down syndrome. It finds that test performance is better with higher fetal fractions. Specifically, the distribution of test results for Down syndrome pregnancies improves and separates more from normal pregnancies as fetal fraction increases. Additionally, false negative rates and rates of low fetal fractions are highest for women with high maternal weights. When a fetus has mosaicism for a trisomy, the degree of mosaicism affects the effective fetal fraction and thus impacts test performance.
This document describes a new statistical method called FetalQuant that can deduce the fractional fetal DNA concentration directly from massively parallel sequencing (MPS) data of DNA in maternal plasma, without requiring prior genotype information. FetalQuant implements a binomial mixture model to estimate the fractional fetal DNA concentration by maximum likelihood using only the allelic count data from targeted MPS. This allows improved determination of the fetal DNA fraction without additional laboratory steps. The authors believe FetalQuant can help expand the applications of MPS-based non-invasive prenatal diagnosis.
This document discusses how exome sequencing is revolutionizing the identification of genes that cause Mendelian diseases. It provides three main points:
1) Exome sequencing has identified over 30 new disease genes since 2009, improving clinical diagnosis, genotype-phenotype correlations, and understanding of rare genetic variation.
2) Our view of Mendelian diseases is changing as exome sequencing is less biased than previous methods and is identifying disease genes in cases where the genetic cause was unclear.
3) Exome sequencing is now the primary tool for studying Mendelian diseases as it can sequence hundreds of patient exomes per year more efficiently than whole genome sequencing.
1) Genome-wide gene expression analysis identified immune response and lymphangiogenesis pathways as implicated in the pathogenesis of fetal chylothorax (FC). Genes involved in immune response were universally up-regulated, while genes related to lymphangiogenesis were down-regulated in fetal pleural fluids of FC cases.
2) Expression of the ITGA9 gene, which is important for lymphangiogenesis, was concordant with trends in the lymphangiogenesis pathway. ITGA9 mutations have previously been associated with FC.
3) For one fetus (Ind) carrying an ITGA9 mutation, immune response pathways decreased after successful treatment of FC with OK-432 pleurodesis, while lymphangiogenesis pathways
- The study analyzed 22,384 maternal plasma samples to determine the effects of gestational age and maternal weight on the percentage of fetal cell-free DNA (cfDNA) in maternal plasma.
- They found that the percentage of fetal cfDNA increases with gestational age, rising 0.1% per week between 10-21 weeks and 1% per week after 21 weeks. Fetal cfDNA percentage decreases with increasing maternal weight.
- Of samples that were redrawn due to initially low fetal cfDNA, 56% of second draws had over 4% fetal cfDNA, showing that fetal percentage often improves with redraws, especially at later gestational ages.
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 describes a new noninvasive method for sequencing the entire fetal genome using cell-free DNA found in a pregnant woman's blood. The method works by counting parental haplotypes - combinations of maternal and paternal chromosomes passed to the fetus. Since a small percentage of cell-free DNA comes from the fetus, haplotypes inherited by the fetus can be identified by which have a higher count. Researchers tested this method on two pregnancies and were able to determine the fetal genomes without any invasive procedures. This noninvasive prenatal testing could allow comprehensive screening for genetic diseases.
This document describes a study that used massively parallel sequencing of cell-free DNA in maternal blood to assess zygosity and detect fetal aneuploidies in twin pregnancies. The study determined zygosity by analyzing apparent fractional fetal DNA concentrations across genomic regions. It then calculated individual fetal DNA concentrations for dizygotic twins to assess each fetus. The study detected trisomy 21 in one twin and trisomy 18 in the other twin of two pregnancies. It demonstrated that noninvasive prenatal testing for aneuploidies can be achieved for twin pregnancies using this method.
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.
This document describes a case study of preimplantation genetic diagnosis (PGD) performed on a breast cancer patient carrying a novel genomic deletion in the BRCA2 gene. Researchers first used single sperm haplotyping on the patient's carrier brother to establish linkage to the mutation. They then used BLAST analysis to locate putative hairpin structures in the genome and PCR screening to identify a 2,596 bp deletion in BRCA2 involving exons 15-16. PGD was performed using both direct mutation detection and linkage analysis to avoid misdiagnosis from recombination. This identified unaffected embryos, one of which was transferred, resulting in a live birth.
This document describes a case study of preimplantation genetic diagnosis (PGD) performed on a breast cancer patient carrying a novel genomic deletion in the BRCA2 gene. Researchers first used single sperm haplotyping on the patient's carrier brother to establish linkage to the mutation. They then used BLAST analysis to locate putative hairpin structures in the genome and PCR screening to identify a 2,596 bp deletion in BRCA2 involving exons 15-16. PGD was performed using both direct mutation detection and linkage analysis to avoid misdiagnosis from recombination. This identified unaffected embryos, one of which was transferred, resulting in a live birth.
- Dr. Chang informs 37-year-old patient Niki about declining fertility with age and recommends an AMH test to evaluate her ovarian reserve since Niki wants to have children.
- Niki's test results show very low AMH levels. Dr. Goldstein, who is covering for Dr. Chang, is upset that Dr. Chang informed Niki without considering her lack of a partner and career focus.
- The commentary argues that physicians have a responsibility to provide patients information relevant to their reproductive goals and futures to allow for informed decision making, even if the news is unexpected or unwelcome.
This study compared two microarray technologies, single nucleotide polymorphism (SNP) and comparative genomic hybridization (aCGH), for preimplantation genetic diagnosis and screening (PGD/PGS) of embryos from couples where one parent has a balanced reciprocal translocation. The study found:
1) There was no significant difference in the rates of euploid embryos without translocation imbalances, euploid embryos with imbalances, or aneuploid embryos between the SNP and aCGH technologies.
2) Clinical pregnancy rates were also equivalent for SNP (60%) and aCGH (65%) microarrays.
3) Both SNP and aCGH microarrays effectively identified unbalanced translocations
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.
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 but significantly fewer multiple pregnancies compared to double embryo transfer. Vitrification is an improved cryopreservation technique with higher post-thaw survival and pregnancy rates compared to slow freezing.
3. In-vitro maturation allows retrieval of immature eggs for fertilization, and may help avoid ovarian hyperstimulation syndrome in high-risk patients
1. RBMOnline - Vol 4. No 3. 106–115 Reproductive BioMedicine Online; www.rbmonline.com/Article/467 on web 25 February 2002
Articles
6
Blastomere fixation techniques and risk of
misdiagnosis for preimplantation genetic
diagnosis of aneuploidy
Esther Velilla began her studies in biology in 1990 at Universitat Autonoma de Barcelona
(Spain) and was awarded her Bachelor degree in 1995. In 1998 she obtained her M.Sc.
degree in Cellular Biology in the Science Faculty at the same University. She then moved
to the Veterinary Faculty to develop her Ph.D. thesis on ‘in-vitro maturation and fertilization
studies on prepubertal and adults goat oocytes’. During the same period she began her
work in human reproduction at the clinic Instituto de reproduccion CEFER (Barcelona). In
2001 she began working at The Institute for Reproductive Medicine and Science of Saint
Barnabas (Livingston, NJ, USA) in the field of preimplantation genetic diagnosis under the
direction of Santiago Munné.
Esther Velilla, Tomas Escudero, Santiago Munné1
Institute for Reproductive Medicine and Science of Saint Barnabas Medical Centre, 101 Old Short Hills Road, Suite
501, West Orange, NJ-07052, USA
1Correspondence: Tel. +1-973–3226236, Fax. +1-973–3226235, e-mail: santi.munne@embryos.net
Introduction
Preimplantation genetic diagnosis (PGD) for gender selection
(Griffin et al., 1992; Munné et al., 1993a), aneuploidy (Munné
et al., 1993b, 1995, 1999; Verlinsky et al., 1995; Gianaroli et
al., 1999), and structural abnormalities (Munné et al., 1996,
1998; Verlinsky and Evsikov, 1999) involves the biopsy of one
or both polar bodies or the biopsy of one or two blastomeres,
fixation to glass slides, followed by fluorescence in-situ
hybridization (FISH) analysis. One of the most critical steps in
this process is cell fixation, because ideally not a single cell
should be lost, and each cell should be informative in order to
have the best possible results for each embryo.
The traditional fixation method, first developed by Tarkowski
et al. (1966) and later modified in different ways by many
authors, involves the use of acetic acid/methanol (Carnoy)
solution applied to a small drop of hypotonic solution
containing the blastomere to be fixed. This process requires
considerable practice and skill, and for this reason at least, two
other cell fixation methods have been developed, one using
Tween 20 solution (Coonen et al., 1994) and the other a
combination of Tween 20 and Carnoy solution (Dozortsev and
McGinnis, 2001). These two last methods require less skill and
seem to be less prone to cell loss during fixation (Xu et al.,
1998; Dozortsev and McGinnis, 2001). However, because the
two newer methods rely on total or partial drying of the cell, a
large nuclear diameter is hard to achieve (Hliscs et al., 1997);
large diameters are desirable because they have been inversely
correlated to signal overlaps and FISH errors (Munné et al.,
1996). To date, no study has yet analysed the FISH error rate
of the two last methods; the traditional Carnoy’s method
produces about 10–15% errors (Munné and Weier, 1996;
Munné et al., 1998, 1999).
The purpose of this study was to compare the three fixation
methods based on number of cells lost after fixation, average
rate of informative cells, rate of signal overlaps and FISH errors.
Esther Velilla
Abstract
One of the most critical steps in preimplantation genetic diagnosis (PGD) studies is the fixation required to obtain good
fluorescence in-situ hybridization (FISH) nuclear quality without losing any of the cells analysed. Different fixation
techniques have been described. The aim of this study was to compare three fixation methods (1, acetic acid/methanol; 2,
Tween 20; 3, Tween 20 and acetic acid/methanol) based on number of cells lost after fixation, average rate of informative
cells, rate of signal overlaps and FISH errors. A total of 100, 106 and 114 blastomeres were fixed using techniques 1, 2 and
3 respectively. Technique 2 gave the poorest nuclear quality with higher cytoplasm, number of overlaps and FISH errors.
Although technique 1 showed better nuclear quality in terms of greater nuclear diameter, fewer overlaps and FISH errors, it
is difficult to perform correctly. However, technique 3 shows reasonably good nuclear quality and is both easier to learn and
use for PGD studies than the others.
Keywords: cell fixation, FISH, fluorescence in-situ hybridization, preimplantation genetic diagnosis, signal overlap
2. 7
Articles - Fixation techniques for PGD of aneuploidy - E Velilla et al.
Materials and methods
Source of embryos
Embryos were obtained from patients undergoing IVF at The
Institute for Reproductive Medicine and Science at Saint
Barnabas Medical Centre (Livingston, NJ, USA) in
accordance with guidelines approved by the internal review
board. Written patient consent for donated embryo research
and for PGD was provided for all embryos.
There were two sources of embryos. The first source was
supernumerary embryos with compromised morphology
and/or development not used in embryo replacement or
cryopreservation. Severely compromised development
included embryos with fewer than four cells on day 3, or with
a normal number of cells but with >35% fragmentation or
multinucleation on day 2 of development (Alikani et al., 1999,
2000). The other source was embryos that after PGD were
found to be chromosomally abnormal.
Only those embryos with three or more cells with observed
nuclei after fixation were included in this study.
Blastomere fixation and FISH
The PGD embryos had one cell biopsied on day 3 of
development (Grifo, 1992). Those embryos considered to be
chromosomally, morphologically and developmentally normal
were replaced. Many of the embryos classified as normal by
PGD were transferred to the patient on the same day as the
analysis. The non-transferred embryos, either chromosomally
normal or abnormal, had their zonae pellucida removed by
exposure to Pronase solution (3 mg/ml, Sigma), were
transferred to Ca/Mg-free media for 10 min, and were then
fixed each cell individually as described below. Infrequently,
some chromosomally and morphologically normal embryos, in
excess of the ones transferred, were disaggregated and
analysed, because cryopreservation of biopsied embryos is
considered inefficient (Magli et al., 1999).
Only one person performed the fixation for all three methods.
This person had no previous experience in fixation, and started
the study once she felt proficient in all three methods.
Embryos were randomly assigned to one of three fixation
methods: (1) acetic acid/methanol (Tarkowski, 1966, modified
by Munné et al., 1998); (2) Tween 20HCl (Coonen et al.,
1994); or (3) Tween 20-HCL and acetic acid/methanol
(Dozortev et al., 2001).
Method 1
This method was described by Tarkowski (1966) and modified
for single blastomere fixation by Munné et al. (1998). The
whole process was performed using a stereoscope (Leica
MZ9.5, Leica Wild MZ8) with a base having a mirror that
could move from vertical to horizontal position; for this
process, the mirror was at a 30° angle. The blastomere was
exposed to hypotonic solution [0.075 mol/l KCl supplemented
with 0.6% BSA (w/v)] for 2 min. Then the blastomere was
placed onto the microscope slide in a small hypotonic drop
(1–2 μl) using a 0.16 mm inner-diameter microneedle. After
that, 1 drop of methanol:acetic acid (3:1) fixative was added
over the blastomere. Often, the blastomere moved after the
first fixative drop, but was easily detectable if the slide was
clean of dust because the position of the stereoscope mirror
produced a three-dimensional effect that allowed localization
of objects protruding from the glass slide (Figure 1).
Blastomeres before cytoplasm breakdown appear refringent
and with smooth circular edges. After the blastomere settled,
but before the cytoplasm burst, a second drop of hypotonic
was added. While the second drop was drying, humidity was
added by breathing over the blastomere to facilitate cytoplasm
Table 1. Analysable cells depending on different fixation methods and studies.
Method Fixed Cells Found No Nucleated Analysable (%)
lost (%) nuclei
Xu et al. (1998)
1 121 26 (21.5)a 95 ND ND 76 (62.8)c
2 131 8 (6.1)b 123 ND ND 60 (45.8)d
Dozortsev
et al. (2001)
1 16 2 (13.0) ND ND ND 13 (81.0)
2 16 1 (16.0) ND ND ND 14 (87.0)
3 18 0 (0.0) ND ND ND 18 (100.0)
Present study
1 110 4 (3.6) 106 15 91 89 (84.0)e
2 106 3 (2.8) 103 22 81 71 (68.9)f
3 114 3 (2.6) 111 10 101 92 (82.9)
a versus b, c versus d: P < 0.001; e versus f: P < 0.025.
ND = not determined
3. Articles - Fixation techniques for PGD of aneuploidy - E Velilla et al.
Figure 1. An expanded blastomere before cytoplasm burst,
showing a three-dimensional effect.
Figure 2. Blastomeres were analysed by FISH with probes for
chromosomes 13 (red), 16 (pale blue), 18 (dark blue), 21
(green), 22 (gold). Blastomeres (nucleus diameter = 62 μm)
were fixed using method 1.
Figure 3. Blastomere (nucleus diameter = 17 μm) shows
overlaps between chromosomes 16 and 13, 16 and 21, 21 and
22, and 22 and 18. Blastomeres were analysed by FISH with
probes for chromosomes 13 (red), 16 (pale blue), 18 (dark
blue), 21 (green), 22 (gold) and fixed using method 2.
Figure 4. A binucleated blastomere (nucleus diameter = 20
and 10 μm) shows overlaps in the top nucleus between
chromosomes 13 and 18, and 21 and 22. In addition excess
cytoplasm can be seen. Blastomeres were analysed by FISH
with probes for chromosomes 13 (red), 16 (pale blue), 18
(dark blue), 21 (green), 22 (gold) and fixed using method 2.
Table 2. Number of signal overlaps and FISH errors in the present study.
Method Analysablea Diameter Nuclei with No. total FISH (%)
±SD (μm) errors (%) overlaps
1 89 58.5 ±20.7 12 (13.5)a 16d 9 (10.1)g
2 71 30.8 ±12.9 41 (57.7)b 88e 21 (29.6)h
3 92 46.0 ±18.7 36 (39.1)c 45f 16 (17.4)
a versus b, a versus c, d versus e, d versus f: P <0.001. g versus h: P < 0.005.
aSee differences between techniques in Table 1 for “Analysable”. 8
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Figure 5. Nucleus (diameter = 21 μm) with overlaps between
chromosomes 16, 18 and 21 and between 16 and 18, with
considerable cytoplasm. Blastomeres were analysed by FISH
with probes for chromosomes 13 (red), 16 (pale blue), 18 (dark
blue), 21 (green), 22 (gold) and fixed using method 2.
membrane breakdown. Once cytoplasm breakdown occurred,
the nucleus could be seen under the stereoscope for a few
seconds; but not once the fixative dried completely.
Method 2
This method was first described by Coonen et al. (1994). The
blastomere was placed (5–10 s) into hypotonic solution (1%
sodium citrate in 0.2 mg/ml BSA) and transferred onto a Petri
dish containing Tween 20 solution for 2 min. After that, it was
transferred onto a glass slide within approximately 3 μl of
Tween 20 solution. Tween 20 solution was continuously added
to the drop until cell membrane breakdown under stereoscope
observation. After membrane breakdown, the slide was
allowed to dry completely and the slide was treated with 1%
pepsin to digest the remaining cytoplasm.
Figure 6. Another binucleated blastomere (nucleus diameter =
24 and 27 μm) with overlaps between chromosomes 13, 16
and 18 and between 13 and 16 in the top nucleus, and overlaps
between chromosomes 13, 16 and 21 and between 13 and 16
in the bottom nucleus. Blastomeres were analysed by FISH
with probes for chromosomes 13 (red), 16 (pale blue), 18 (dark
blue), 21 (green), 22 (gold) and fixed using method 2.
Method 3
This method was described by Dozortsev and McGinnis
(2001). The blastomere was placed (5–10 s) in hypotonic
solution (1% sodium citrate in 0.2 mg/ml BSA) and then
washed in Tween 20 solution (1% of Tween 20 in 0.01 N HCL,
1% in 0.01 N HCL) for 40 s. After that, it was placed onto a
glass slide with 3–4 μl of Tween 20 under a stereoscope
microscope and allowed to dry completely. In order to remove
the remaining cytoplasm, several drops of methanol: acetic
(3:1) fixative were added.
For all three methods, the temperature and humidity conditions
were the same (22ºC and 30–45% respectively), based on
previous observations that nuclear diameters are a function of
temperature and humidity (Spurbeck et al., 1996).
FISH analysis was performed using probes for chromosomes
X, Y, 13, 15, 16, 17, 18, 21 and 22 following the previously
published protocol (Munné et al., 1998), except that instead of
a locus-specific probe for chromosome 14, a centromeric one
was used for chromosome 17, also labelled in Spectrum
Orange (Vysis).
Data evaluation and scoring criteria
Classification of chromosomal abnormalities in cleavage-stage
embryos, usually with 2–12 cells, requires scoring criteria
based on the analysis of as many cells as possible to
differentiate mosaicism (30% of cleavage-stage embryos;
Munné et al., 1995) from FISH errors (10% of single cells
analysed; Munné et al., 1998). In this study, the previously
described criteria distinguishing mosaics from FISH errors
were used without modification (Munné et al., 1994; Munné
and Cohen, 1998). Previous criteria were also used to
differentiate close signals from split signals when analysing
Figure 7.Anucleus with split signals for chromosomes 21 and
22. Blastomeres (nucleus diameter = 85 μm) were fixed using
method 1 and analysed by FISH with probes for chromosomes
13 (red), 16 (pale blue), 18 (dark blue), 21 (green), 22 (gold).
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Table 3. Example of 20 embryos with FISH errors.
Embryo No. cells 13 16 18 21 22 XY 15 17 Embryo diagnosis
1 1 2 2 2 2 2 xy 1a 2 Normal
2 2 2 2 2 2 xy 2 2
1 2 2 2 2 2 xy 2 1a
2 1 1a 1a 2 2 2 xy 2 2 Normal
7 2 2 2 2 2 xy 2 2
3 6 2 2 2 2 2 XY 2 2 Mosaic 2N/4N
1 3a 4 4 4 4 XXYY4 4
1 2 2 2 nr 1a XY 2 2
4 2 3 3 3 3 3 xxx 3 3 Triploid
3 3 2a 3 3 3 xxx 3 3
5 1 2 nr 2 2 2 xy 2 3a Normal
3 2 2 2 2 2 xy 2 2
6 3 2 2 3 2 2 xx nr 1 Trisomy 18, and
1 2 3a 4 2 2 xx nr 3 Aneuploid mosaic (18,17)b
1 2 2 2 2 2 xx 2 2
7 3 2 2 2 2 2 xx 2 2 Normal
1 2 nr 2 2 2 xx 1a 2
8 1 2 2 2 3 2 xy 0a 1a Trisomy 21
2 2 2 2 3 2 xy 2 2
9 1 2 2 2 2 2a xx 2 2 Trisomy 22
3 2 2 2 2 3 xx 2 2
10 3 2 2 2 2 3 xx 2 2 Trisomy 22
1 2 nr 2 2 2a xx 2 2
11 1 2 2 nr 3 2 xx 1 2 Monosomy 15, and
4 2 2 2 2 2 xx 1 2 Aneuploid mosaic (21)b
1 1a 2 2 1 2 xx 1 1a
1 2 3a 2 2 2 xx 2a 2
12 1 2 nr 2 2 2 xy 1a 2 Normal
5 2 2 2 2 2 xy 2 2
13 1 2 3a 2 2 2 xy 2 2 Normal
3 2 2 2 2 2 xy 2 2
14 1 2 1a 2 2 1 XY 2 2 Monosomy 22
3 2 2 2 2 1 XY 2 2
15 2 2 nr 2 2 2 xy 2 2 Trisomy 16
4 2 3 2 2 2 xy 1a 2
5 2 3 2 2 2 xy 2 2
1 2 3 2 2 2 xy 2 2
16 3 2 2 2 2 1 xy 3 2 Monosomy 22
1 2 2 2 2 1 xy 2a 2
1 2 1a 2 2 1 xy 3 2
17 1 4 4 4 4 4 xxyy 4 4 Mosaic 2N/4N
7 2 2 2 2 2 xy 2 2
1 3a 4 4 4 4 xxyy 4 4
18 2 2 2 2 2 2 xx 2 2 Normal
1 2 nr 2 2 nr xx 2 2
1 2 nr 2 nr 2 xx 1a 2
19 1 3a nr 2 2 2 XX 2 2 Normal
6 2 2 2 2 2 XY 2 2
1 2 2 2 2 2 XY 1a 2
20 1 1 1 1 2a 1 xx 2 2 Complex mosaic
3 2 2 2 2 nr 4x 4 4
aConsidered errors.
bAneuploid mosaic for the chromosomes in brackets.
nr = no result
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single cells in PGD cases (Munné and Weier, 1996). Embryos
were classified as normal, aneuploid, polyploid, haploid
and/or mosaic according to guidelines described elsewhere
(Munné et al., 1995; Munné and Cohen, 1998).
Parameters to be evaluated
Cells lost during fixation
These were cells that in method 1 were lost after adding the
fixative, and where the refringent protrusion was not observed.
For methods 2 and 3, the blastomere seldom moved but the cell
could burst, and these cells were also counted as lost.
Analysable cells
A cell was considered analysable when it had at least five
informative chromosomes out of the eight analysed.
Nuclear diameter
This was measured in microns under phase contrast
observation before FISH analysis.
Nuclei with overlaps
Overlaps between the two-homologue chromosomes cannot
be precisely quantified in a single cell and are one source of
misdiagnosis (see below, FISH errors). However, any other
overlap between non-homologue chromosomes can be easily
measured if these chromosomes were labelled in different
colours, as is the case here. Thus the parameter ”nuclei with
overlaps” indicated the presence of overlaps between non-homologous
chromosomes in a specific nucleus.
Total number of overlaps
This is the measure of the total number of overlaps between
non-homologous chromosomes in a specific nucleus.
FISH errors
To differentiate between FISH errors and mosaicism in this
study, previously described criteria were used (Munné et al.,
1994).
AChi-square test using the algorithm GENSTAT (1988 version)
was used to evaluate statistical differences between proportions.
The significance chosen for the test was P < 0.025.
Results
The three fixation techniques were evaluated according to the
following parameters: cells lost during fixation, nucleated
cells with no result, analysable cells, nuclear diameter, nuclei
with overlaps, and total number of overlaps, and FISH errors,
as defined in the materials and methods (Tables 1 and 2).
These parameters were also compared with other previously
published studies (Table 1), although FISH errors and
overlaps had not been previously evaluated in relation to
blastomere fixation methods.
The present results indicate that similar rates of lost cells were
observed for the three methods evaluated (Table 1), ranging
from 2.8 to 3.6%. This contrasts markedly with data published
by others reporting lost cells ranging from 0 to 21.5% (Table
1). The fraction of nucleated cells that was analysable after
FISH was significantly higher for method 1 than for method 2,
in this study as well as in that of Xu et al. (1998) (Table 1).
As far as is known, no other study has yet evaluated the
number of signal overlaps and FISH errors according to
fixation technique. The present results indicate a significant
differences in nuclear diameter after fixation, with an average
of 58 μm for method 1, 31 for method 2, and 46 for method 3
(P < 0.001) (Table 2), which translates to higher rates of
nuclei with signal overlaps (from 14% for method 1 to up to
58% for method 2, P < 0.001), total number of overlaps, and
FISH errors (from 10% for method 1 to 30% for method 2, P
< 0.005) (Table 2).
Examples of embryos with FISH errors are shown in Table 3
and examples of overlaps in Figures 3–7. The correlation
between overlaps and FISH errors and small nuclear diameter
was also observed irrespective of the type of fixation. For
instance, taking all embryos together and grouping them by
diameters, those cells of <30 μm in diameter had more
overlaps and FISH errors than those cells of >60 μm in
diameter (P < 0.005) (Table 4).
Discussion
The two most important aspects of cell fixation are to ensure
that each single cell is fixed and that the fixed nucleus is
informative. One of the steps in method 1 involves the mixture
of fixative with the drop of hypotonic solution containing the
blastomere. This act produces turbulence, during which the
cell may be lost, and the risk is about 3% in expert hands; but
could be higher for technicians using method 1 only
occasionally (Xu et al., 1998; Dozortsev and McGinnis,
2001). In contrast, methods 2 and 3 overcome the turbulence
step and are easier to learn, but they have other problems.
For instance, the presence of cytoplasm interferes with probe
binding to the nucleus especially with locus specific probes.
These probes are longer than the repetitive ones and easily
attach to the cytoplasm debris, increasing the background
signal and limiting the attachment of the probes to their target.
Moreover, cytoplasm is refringent by itself, masking the
signals. In short, cytoplasm can increase misdiagnosis or
render the nucleus non-informative (Figures 4 and 6). This is
a considerable problem in method 2, although modifications
made by Xu et al. (1998) to this method reduced the number
Table 4. Nuclear diameter in relation to overlaps and FISH
errors.
Diameter Analysable Average No. nuclei Total no. No. FISH
(μm) diameter with overlaps errors
(μm) overlaps (%) (%)
<30 55 21.63 28 (50.9)a 62 14 (25.5)c
30–60 139 42.97 45 (32.4) 71 29 (20.9)
>60 58 78.72 16(27.6)b 16 3 (5.2)d
a versus b: P < 0.025, c versus d: P < 0.005.
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of nuclei with cytoplasm to <5%. Removal of cytoplasm by
pepsin may also be detrimental because overexposure to
pepsin may degrade the DNA (Xu et al., 1998; Dozortsev and
McGinnis, 2001).
Another source of background interference found in this study
was poly-L-lysine, used in method 2 to avoid nuclear loss in
attaching the cell to the slide. This substance produces a mask
along the slide, which interferes with the analysis of the
signals. In methods 1 and 3, the cells are fixed to the slide by
methanol:acetic acid instead of poly-L-lysine. In consequence,
these fixation methods show clearer nuclei for FISH analysis
(Figure 7).
Another factor in PGD analysis is the nuclei diameter. Small
nuclei preserve their three-dimensional structure, and as a
consequence the signals lie on different focus planes, making
the analysis more prone to misdiagnosis (Figures 3 and 6). In
addition, two signals close to each other or overlapping could
be misdiagnosed as a single signal (Munné et al., 1996).
Methods 1 and 3 provide flat nuclei with all the signals in the
same focus plane, thereby reducing the frequency of overlaps.
It was observed in this study that the ideal diameter is 60 μm
and above (Figure 2). However, nuclei >80 μm show
excessively decondensed chromatin, as the signals are spread
more widely and are weaker than regular sized nuclei.
Sometimes they are almost imperceptible, which leads to
misdiagnosis of false nullisomies, monosomies or disomies. It
is important to mention that nuclear diameter is not only
dependent on the fixation technique, but also on temperature
and humidity (Spurbeck et al., 1996; Hliscs et al., 1997).
Previous observations that nuclear diameter is linked to FISH
errors (Munné et al., 1996) were confirmed.
In this respect, method 1, which produced the largest nuclei,
produced the fewest number of signal overlaps and the fewest
errors after FISH (Figure 2). In contrast, method 2 (Coonen et
al., 1994) produced the highest number of overlaps and three
times more FISH errors than method 1 (Figures 2–5).
Method 2 has been previously used in PGD for gender
determination (Harper et al., 1994, 1995; Coonen et al., 1996;
Soussis et al., 1996) and PGD diagnosis on translocation
carriers (VanAsche et al., 1999; Coonen et al., 2000; Iwarsson
et al., 2000; Scriven et al., 2001) using probes for one or two
chromosome pairs. The probes used for gender determination
bind to repetitive sequences in the satellite regions (centromere
X and 18, or most of the q arm of chromosome Y), producing
a large signal, usually even under the worst fixation
conditions. In addition, misdiagnosis of gender using FISH can
be improved because the mere presence of different colours
(irrespective of the number of signals) can identify the sex,
even though signal overlaps could still misdiagnose the ploidy.
For translocation carriers, only the chromosomes involved in
the translocation are diagnosed, usually involving only a
centromeric probe and a pair of distal probes instead of the five
probes simultaneously used for the first aneuploidy
hybridization. In addition, for PGD of translocations, either
telomeric or home-made locus-specific (LSI) probes are used,
while the LSI probes used for PGD of aneuploidy (for
chromosomes 13, 21, and 22) are different. The three fixation
methods were evaluated for their use in PGD of aneuploidy
and method 2 may still be used for gender determination based
on FISH errors or translocation PGD cases, but it is certainly
not recommended for PGD of aneuploidy.
The FISH error rate observed for method 1, 10%, was
comparable to that found in previous studies using the same
fixation method (Munné et al., 1996). Although in this study
PGD was not performed using the three techniques, if the error
rates observed here for methods 2 and 3 are maintained when
performing PGD, as for method 1, a 30% error rate for method
3 would render it useless for PGD of aneuploidy.
In conclusion, method 1 gives the best nuclear quality for PGD
analysis, even though it is the hardest to accomplish. Method
3 gives reasonably good nuclear quality, and is easy to learn.
Method 2 is also easy to learn, but under the conditions of this
study, the quality of the results was not sufficiently good for
PGD of aneuploidy.
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