2015 Group Project 6
|2015 Student Projects|
|2015 Projects: Three Person Embryos | Ovarian Hyper-stimulation Syndrome | Polycystic Ovarian Syndrome | Male Infertility | Oncofertility | Preimplantation Genetic Diagnosis | Students|
|2015 Group Project Topic - Assisted Reproductive Technology|
|This page is an undergraduate science embryology student and may contain inaccuracies in either description or acknowledgements.|
- 1 Preimplantation Genetic Diagnosis and Preimplantation Genetic Screening
- 1.1 Introduction
- 1.2 History
- 1.3 Preimplantation Genetic Diagnosis
- 1.4 Preimplantation Genetic Screening
- 1.5 Biopsy Methods
- 1.6 Genetic Techniques
- 1.7 Diagnosis
- 1.8 Laws & Legal status
- 1.9 Future/Current Research
- 1.10 Glossary
- 1.11 References
- 1.12 Glossary Links
Preimplantation Genetic Diagnosis and Preimplantation Genetic Screening
Preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS) are reproductive options for couples with known family histories of genetic disease or couples undergoing IVF procedures due to infertility issues. PGD can diagnose many genetic disorders caused by known chromosomal abnormalities (number and/or structure) or single gene mutations, and, thus decrease the risk of termination of pregnancy or miscarriages and enable such couples to have an unaffected child. Through major improvements in PGD/S and general laboratory technology, the testing for abnormalities in fetuses has shifted from prenatal diagnosis during the first 2 trimesters , testing for overall fetal growth, complications of pregnancy, and birth defects, to an embryonic focus especially in advancements in Artificial Reproductive Technologies (ART). In 1990 PGD for a recessive X-linked disease resulted in the first live birth and has since been incorporated in clinical routine and applied for a variety of genetic diseases, such as sickle cell anemia, thalassemia, or cystic fibrosis.
|Preimplantation Genetic Diagnosis|
The advances in reproductive technology during the second half of the 20th century, led to PGD's first clinical application in 1990 .
|1967||First PGD on rabbit blastocysts (Gardner& Edwards) |
|1986||First Cleavage Biopsy (Wilton & Trounson)|
|1987||First Blastocyst Biopsy (Muggleton-Harris, Monk, Rawlings, & Whittingham)|
|1988||First Polar Body Biopsy (Yury Verlinksy)|
|1990||First Clinical PGD using PCR testing for X-linked (Handyside, Kontogianni, Hardy, & Winston)|
Preimplantation Genetic Diagnosis
PGD is used to test the genetic makeup of embryos to detect single gene disorders, chromosomal abnormalities and mitochondrial disorders. It also has applications in gender selection for diseases with unequal gender distributions . Some diseases commonly involved with PGD include cystic fibrosis, spinal muscular atrophy and beta – thalassaemia  . It was first used in the United Kingdom in the 1980s, primarily focusing on sex- linked disorders . PGD is now capable of detecting single cell defects (molecular) and chromosomal disorders resulting from the inversion, translocation or deletion of chromosomes (cytogenic)  . PGD can be applied to the embryo at different stages. That is on polar bodies, blastomeres or blastocyst . Depending on the type of genetic disorder, PGD utilises different methods of genetic testing. These include Fluorescence in situ hybridisation (FISH) which is used for sex – linked disorders and detects chromosomal rearrangements  and Embryo halotyping which allows the identification of chromosomes causing the inherited disorder through knowledge of the pattern of closely linked markers . Polymerase chain reaction (PCR) is also widely used to detect molecular abnormalities . PGD is tightly regulated and supported by large organisations namely The American Society for Reproductive Medicine, The European Society for Human Reproduction and Embryology (ESHRE), The European Society of Human Genetics and the Preimplantation Genetic Diagnosis International Society .
The determination of a disease as gender specific usually correlates with the presence or absence of specific genes such as SRY on the Y chromosome. It is known that females have two X chromosomes and males have an X and a Y chromosome where abnormalities are more prevalent on the X chromosome. PGD can be used for sex selection where only male embryos are transferred to reduce the chance of inheriting X-linked disorders. However, this does not completely eradicate the problem as male embryos remain susceptible to inheriting an affected X chromosome. Sex determination is only used when the specific mutation is unknown and has yet to be discovered .
Single gene defects
Single gene defects can be dominant, recessive, autosomal or X-linked. They are commonly diagnosed using PCR and although the PCR available today is complex and capable of combatting a large range of disease the development of new protocols has been proven to be difficult due to the small DNA sample available .
Mitochondrial disorders also known as oxidative phosphorylation disorders arise from mutations in the nuclear DNA or mitochondrial DNA . They pose as a problem because they are unrecognisable until the mutations in the cell reach a detrimental level . Mitochondrial disorders cause miscarriages and stillbirths as well as death in children and young adults. The effects can either be contained in a single organ or more commonly involve multiple organ failure where organs with high energy demands such as the brain, liver muscle and heart and heavily influenced. They are usually occur spontaneously or result from inheritance from the mother. Since mitochondria are solely inherited from the mother oocyte donations have been used as a solution to combat mitochondrial disorders. Additionally, there has been an increasing use in PGD where embryos that stay under the given threshold of 18% gene-mutations are allowed to be transferred and result in normal development. New technology in the areas of nuclear gene transfer and genome editing are also being experimented with .
Chromosomal disorders can be reciprocal, Robertsonian translocations, inversions, deletions and insertions . Data from ESHRE collected between 2010 and 2011 has shown that the most common chromosomal abnormality confronted in PGD are reciprocal chromosomal abnormalities . PGD has also been used successfully for Robertsonian translocations (RT), a type of structural translocation. Children who carry RT are phenotypically normal, however in their later years it is found that they will suffer from infertility and repeated miscarriages due to the high frequency of abnormal embryos . PCR and FISH are the two main techniques used for chromosomal disorders. To be able to conduct the examinations the cells are required to be at the metaphase stage .
Preimplantation Genetic Screening
PGS involves an array of methods or ideas that aim to segregate embryos that have genetic flaws and those that are healthy . The occurrence of aneuploidy is high around the stages of early embryonic development and they are the most common cause if miscarriages and congenital birth defects . They have little effect on the morphology of the embryo making them difficult to identify thus identification heavily relies on genetic testing . Genetic sampling is most commonly conducted using Microarray Comparative Genomic Hybridisation (aCGH) as well as FISH, Quantitative PCR and Single Nucleotide Polymorphism (SNP) . These methods collectively aim to assess numeral and structural chromosomal errors . Studies have also introduced Next- Generation Sequencing (NGS)  and Whole Genome Amplification used to screen imbalances in the complete 24-chromosomes . Statistics from ESHRE have shown that the most common indications for PGS is advanced age, followed by repeated implantation failure or recurrent miscarriage and male infertility .
A low percentage of structural abnormalities in chromosomes are responsible for the cause of miscarriages. Despite this, they are the most prevalent type of chromosomal abnormality accounted for in PGD . The presence of specific gene cycles, initiation of embryonic protein synthesis and evident physiological development are all indicative of a successful in vitro fertilisation procedure . To eliminate further errors from occurring during the PGD procedure it is recommended to undertake further prenatal testing such as amniocentesis in the later stages .
Advanced maternal age
Data provided by the ESHRE has shown that the mean age of women undergoing PGS is 39 years . Women of advanced age have been shown to have a lower rate of pregnancies reaching childbirth . The highlighted concern revolves around the increased occurrence of aneuploidy following maternal age. The optimal age range for the lowest aneuploidy incidence was found to be between 27 to 37 years of age (6%) then progressively higher in women aged up to 42 (33%) and most common in those 44 and above (53%) .
Recurrent pregnancy loss / IVF failure
Recurrent pregnancy loss is defined as three or more IVF failures after cumulative transfer of more than 10 good-quality embryos. These are primarily caused by two main factors, reduced endometrium receptivity or embryonic defects. Endometrium receptivity can be negatively influences by instances including uterine pathologies such as thin endometrium, altered expression of adhesive molecules and immunological factors. Additionally, embryonic defects may be due to genetic abnormalities, embryonic aneuploidy or zona hardening. Endometriosis and hydrosalpinx has been known to effect both the endometrium and embryo .
Human leukocyte antigen matching
First used in 2001, HLA matching is an option given to parents to save a child with haematological or immunological disease through conceiving another child who would potentially be able to donate cord blood or haematopoietic stem cells from the bone marrow for transplantation. The process namely PGD-HLA has shown to improve haematopoietic stem cell transplant (HSCT). PGD-HLA can only be performed when HSCT is not needed urgently due to the time needed to conceive and delivery the baby . It is most commonly applied to children suffering from relapsed leukaemia . In a case study PGD-HLA was proven to successfully cure 10 diseases including Fanconi anaemia, Diamond-Blackfan anaemia and beta thalassemia .
Biopsy, the removal of genetic materials, from oocytes or embryos in the preimplantation stage is the primary step in PGD. For the past two decades these biopsies have been performed at three stages, the polar body, blastomere, and blastocyst, and the methodologies optimized to ensure the embryo’s viability. The most common approach involves biopsies at the cleavage stage. However, polar body and blastocyst biopsies are increasingly more often tested and applied. Approached for opening the zona pellucida involve next to the traditional mechanical and chemical means, novel approaches such as noncontact lasers. Their application may simplify and secure the procedure significantly. The most challenging question about PGD procedures remains at what stage biopsies should be taken. Much controversy has developed around this topic, highlighting the varying disadvantages and advantages of temporal biopsies .
|Time of Biopsy||Prevalence||Advantages||Disadvantages|
|Polar Body||Day 1||~16%||Little to no harm is caused to the oocyte and both PBs can be extracted (more genetic material).||Only the maternal DNA is tested, often PB biopsies need to be coupled to other biopsies, and difficulties arise in distinguishing between the first and second PB. Lower reliability of results compared to other biopsy methods have been reported.|
|Blastomere||Day 3||~80%||Biopsies are safe for good quality embryos and it is performed relatively early, so fresh transfer is possible, yet, it includes both paternal and maternal genetic contributions.||Relatively large decrease in implantation rates for low quality embryos have been reported, embryo mosaicism can influence genetic analysis, and only one to two cells can be safely removed.|
|Trophectoderm||Day 5 and 6||~ 2%||Little harm to the embryo and large amount of genetic material can be extracted, which allows for more accurate genetic analysis and lessen effects of mosaicism.||The biopsy takes place relatively late and, thus, the time window for procedure is small and embryos often need to be cryopreserved.|
Polar Body Analysis
Polar body (PB) biopsy offers a promising alternative to biopsies performed at the blastomere stage for PGD/S indications on legal and practical grounds. The ESHRE calculated the proportion of PB biopsies to be about 16.3%. Embryo development does not necessitate the presence of the first and second PB and their removal may not be crucial. The idea behind PB biopsies is that each abnormality found in the PB corresponds to an error in the oocyte. On the other hand, in women with known single gene mutations, it is assumed that if the PB contains the mutated allele ,the oocyte will have the normal allele, thus, resulting in a healthy embryo. PB biopsy requires precise timing. Keeping track of the meiotic cell cycle is necessary to perform a successful biopsy and, therefore, PB biopsy usually is applied in combination with intracytoplasmic sperm injection (ICSI). During the maturation from the germinal vesicle stage to the metaphase-II stage the first PB is formed. A cytoplasmic bridge containing spindle remnants, that are still in contact with the cellular genetic material, links this PB to the oolemma for about 90 minutes after extrusion. It is possible to visualize these remnants by polarization microscopy and it is crucial to not perform the biopsy until the first PB is no longer firmly attached to the oolemma as this indicates an immature embryo.The oocyte tolerates mechanical zona dissection best during hours four until six after ICSI as the oolemma has stabilized by that time because of the cortical granule reaction. Over time the first PB degenerates stressing a temporal biopsy and its optimal extraction time window is four to 12 hours after ICSI. The second PB forms around two to four hours after ICSI. Its optimal time window for biopsy is set to be eight to 16 hours after ICSI due to the second PB being attached to the oolemma with spindle remnants until six hours after ICSI. Biopsy at this point may cause enucleation of the oocyte. Studies have shown that the amplification efficiency of second PB’s DNA is worse if the PB is extracted prior to eight hours after ICSI. Thus, it is possible to perform sequential and simultaneous biopsies for the first and second PB. If performed, sequentially the first PB may be removed four to 12 hours and the second PB eight to 16 hours after ICSI. The optimal time window for a biopsy for both the first and second PB simultaneously is eight to 12 hours after ICSI. It is highly preferred to analyze both PBs due to potential aneuploidies in either PB and crossing overs during meiosis .
Chemical opening achieved with for example acidic tyrode’s solution of the zona pellucida is not tolerated by the oocyte and may have detrimental effects on the embryo’s development. Therefore, the access to the perivitelline space of the oocyte is provided by mechanical zona dissection or by laser. Both techniques work well if performed by experienced embryologist. However, laser-assisted biopsy is less time consuming when compared to manual dissection. It is critical to consider the size of the introduced opening as it will remain permanent. If it is too large the blastomere may be lost during embryo development and if it is too small it may interfere with hatching of the embryo during blastocyst stage.
|Laser-assisted polar body biopsy|
Advantages & Disadvantages
PB biopsies can only investigate the maternal contributions to the embryo as they are of maternal origin. Thus, this procedure is appropriate for PGD solely for monogenetic diseases from the maternal side. Recessive diseases may be evaluated with PB biopsies on the basis that the embryo’s outcome will be determined by the paternal contribution. It may still be applied for PGS as it is a fairly safe biopsy option and most aneuploidies either arise during meiosis or origin from the maternal genome. However, diagnosis error has been reported due to the lack of considering paternal contributions. About 10% of PB biopsies appear to be wrongfully diagnosed with aneuploidies. Because PB biopsies are performed very early it is not yet known whether the oocyte will develop into a viable embryo. Thus, PBs are commonly frozen or fixed after biopsy and depending on the state of the embryo only chosen PBs will be tested. This is primarily an economic issue as many genetic testing procedures are very cost-intensive. Generally the sustained implantation predictive value of screening of PBs is significantly lower than of, for example, biopsies of the blastocyst stage. Moreover, it is often difficult to distinguish between the first and the second PB. As the first one degenerates quicker, this may influence diagnostic procedures.
Blastomere biopsy has been the prevalent method for PGD and PGS in the last two decades. In 2013 the ESHRE reported 79.8% of biopsies to be performed at the cleavage stage. At least one, but up to two, blastomeres are biopsied on day three of the cleavage stage embryo. The right number of blastomeres removed is a controversial topic as two cells allow for more genetic material and more accurate results. However, removing two cells might be too invasive and damaging to the embryo. As PGS tries to improve implantation rates, whereas PGD sets out to avoid known genetic disorders, for the former only one cell is removed, while for the latter often two need to be removed, to ensure results as correct as possible.
Initially a hole was drilled into the zona pellucida using acid Tyrode's solution or by mechanical means. Nowadays the zona pellucida is largely opened using a laser and calcium and magnesium free media have been introduced to decrease junctions between blastomeres, which facilitates the biopsy. This if followed by the consequent aspiration of blastomeres with a pipette.. The blastomeres can also be removed by applying pressure on the outside of the zona.
|Laser-assisted blastomere biopsy|
Advantages & Disadvantages
Blastomere biopsy is limited by the presence of embryo mosaicism, which can severely influence the interpretation of the genetic analysis. Approximately 15%-80% of all embryos display mosaicism on day three. Thus, any results might be a misrepresentation of the embryo as a whole. However, if good quality embryos are selected, the procedure overall is safe and does not negatively influence the embryos transition to the blastocyst stage. In a typical IVF cycle, however, not all embryos are of good quality, particularly if they have undergone cryopreservation. Studies have found that in these embryos biopsies reduce implantation rates by 12.5%-25%. Furthermore, unlike PB biopsy, the cells at the blastomere stage contain both paternal and maternal contribution, giving the genetic analysis a fuller perspective on the embryo's genetic make up. Since blastomere biopsy is performed at the third day after fertilization, it is possible complete fresh embryo transfer. Thus, no storage procedures, such as cryopreservation, are necessary.
Day 5 and 6
The improvement of embryo culture media allowed the in vitro development of human embryos until the blastocyst stage and opened up the possibility to take blastocyst biopsies. While currently according to ESHRE datasets only about 2.3% of biopsies are performed at the blastomere stage. During day three to day five the haploid maternal and paternal genomes come together for the first time to form the genome of the embryo. The maternal epigenetic control lessens significantly while preparing for implantation with precisely arranged events happening. The first event includes a rapid increase in the number of embryonic cells which are active in mitotic divisions and apoptosis of aberrant cells. This is followed by the formation of blastocoel (cavitation) which results from the flattening of cell located on the outside of the blastomere. Now the blastocyst will expand until the embryo hatches by rupturing the zona pellucida. The cells of the blastocyst will differentiate to form two distinct cell lineages, the outer trophectoderm and the inner cell mass.
Trophectoderm biopsy is usually performed in Hepes buffered biopsy medium and opening approaches include needle cutting which has been replaced by lasers in past years. Different research groups report different timings of the opening to be the most successful. Some open the blastocyst on day three or four by creating a 25 µm which causes the trophectoderm to herniate through this hole and is, thus, accessible for biopsy. Others create this hole about four hours before biopsy which allows sufficient herniation of trophectoderm cells. It is also possible to open the blastocyst immediately before biopsy. This avoids an extra step and the inner cell mass usually is easy to locate. Blastocyst biopsies involve the removal of trophectoderm cells and the ideal time for the procedure is day five. Successful biopsies on day six have been performed, while little is known about the results of biopsies on day seven. However, since the window of implantation in humans is from day eight to ten after ovulation, day seven biopsies appear to be possible.
|Laser-assisted blastocyst biopsy (at 00:50 min)|
Advantages & Disadvantages
Blastocyst biopsies are believed to be less damaging to the embryo and appear to be unrelated to implantation rates. In addition, this biopsy method allows for a larger extraction of cells for genetic testing. However, since they are performed late in in vitro development, the time window for genetic testing is relatively small. Fast and accurate genetic testing methods are needed to ensure a successful and safe result from this biopsy. Thus, blastocyst biopsies may gain more popularity in the future when such methods have been developed or improved. Furthermore, the extraction of multiple cells may lessen the effects of mosaicism and problems during PCR, such as ADO. Studies comparing the implantation rate and screening accuracy have found that blastocysts are significantly safer. Blastocyst biopsies decrease implantation rates significantly, while biopsies at day five or six do not seem to influence implantation and delivery rates.
Polymerase Chain Reaction
PCR amplifies DNA specific to genetic sequence of interest . PCR was developed by Kay Mullis in the 1980's, for which he was awarded the Nobel prize for chemistry in 1993 . This technique enables clinicians to monitor and diagnose diseases using minute samples such as embryonic cells . PCR is used to detect genetic disorders, as a part of PGD, in conjunction with IVF. It is used to detect molecular abnormalities, such as single gene disorders including Tay Sach, Cycstic fibrosis, Duchenne Muscular Dystrophy, Thalassemia, Huntington disease, Spinal muscular atrophy and many more. Molecular and genetic analysis require a significant amount of DNA, which can be delivered by PCR. It revolutionized the study of DNA, replacing all previous recombinant DNA technology and is a significant component of PGD procedures.
|Fast and inexpensive way of copying a target sequence of DNA. |
|Particularly useful for the diagnosis of single cell defects.|
|Rapid generation of results (within hours).]|
|Highly sensitive, a single molecule of DNA is sufficient for reaction.|
|Generates DNA copies exponentially.|
|This enables DNA amplification required for molecular and genetic diagnostic analysis .|
|Only during the exponential DNA replication phase, the starting quantity sequence contained in the original sample (template DNA strand) can be determined.|
|PCR reaction is limited to by presence of inhibitors in the sample.|
|Self annealing due to the accumulation of the product and the stopping exponential amplification for the target sequence and reaching of a plateau
quantification of the end point of reaction of PCR products make real time quantitative RT-PCR necessary.
|Not a diagnostic method on its own, but requires further analysis.|
|Low-quantity DNA template may result in amplification failure.|
|Allele drop-out (ADO) in heterozygous loci is possible.|
Samples are obtained from the the blastocyst, a polar body biopsy or the blastomeres stages of the embryo.
- Stage 1 Denaturing: separating the target strands of DNA
The obtained sample is heated to roughly 90 degrees celcius, this heat breaks the relatively weak bonds between nucleotides that form DNA. The double stranded DNA is split into two single strands of DNA that are used as templates.
- Stage 2 Annealing: Binding the Primers to the target DNA sequence 
PCR will only copy the target sequence of DNA specified by specific PCR primer. These synthesized primers oligonucleotides are small artificial pieces of DNA. TAQ polymerase enzyme synthesize two new strands of DNA duplicate to the single sample DNA stand template indicated by these primers. During this stage the reaction is cooled to a temperature between 40-60 degrees Celsius.
- Stage 3- Extension- making copies
Each of these two copies are then used again as templates generating two further replications This cycle can occur as many 30 -40 times within a couple hours leading to billions of extra copies of the original DNA segment. Generally the optimal temperature for the further replication is roughly 72 degrees Celsius, although, this may vary according to the analysis machines used.  This process mediated by a thermocycler machine that is programmed to alter the temperature of the reaction every couple of minutes, perpetuating the cycle of DNA denaturing and synthesis. This process, generates exponentially exact copies of the original template DNA sequence, as visible in the expandable table below. 
|PCR Cycle||Target Copies|
Fluorescent In Situ Hybridisation
FISH is the one of the most effective and rapid  method as part of PGD. This technique locates a specific DNA sequences within a chromosome. FISH facilitates the clinical diagnosis of chromosomal abnormalities indicated by sequential duplications, deletions and rearrangements of chromosome, that are usually missed with microscopic analysis. This technique is especially relevant for female embryos with X-linked diseases. As part of PGD, it enabled the screening for aneuploidies and increased live birth rates in women with advanced age . FISH is 99% effective when used in conjunction with competitive Genomic Hybridisation (CGH) to diagnose chromosomal abnormalities.
|FISH is 99% effective when used in conjunction with CGH to diagnose chromosomal abnormalities.|
|Results are rapidly generated. |
|Very small translocations and aneuploidies that occur within chromosomes, that would usually be missed under microscopic analysis, can be identified.|
|The most common chromosomal abnormalities, such as down syndrome chromosomes 13,16,18,21 and 22, and inheritable X-linked disease or sex chromosome anomalies such as Duchenne's Muscular Dystrophy, hemophilia, ectodermal dysplasia can be identified.|
|FISH destroys all cells tested .|
|It does not fully access all chromosomes (only ~ 12).|
|The analysis of results is dependent upon the dot quality impacted by hybridisation efficiency or the camera sensitivity.|
|Some studies indicate that using FISH on a day-3 embryo biopsy decreases the rate of live births.|
Samples collected from the embryo are collected, processed, and its DNA strands are heated and denatured causing their the individual DNA strands to break apart. Then, probes of single complimentary stands of DNA a that have been tagged with small chemical agents that glow brightly in the presence of a specific region on a chromosome are added. These specific probes then hybridize and join to their complementary DNA strand. The fluorescent tags enable the correct identification of the presence or lack thereof and location of specific chromosomes that are tested for. The number and the relative location of the fluorescent dots generated by the FISH images is analysed and gives rise to diagnosis. The probe will not fully hybridise if there has been a duplication or a deletion of the DNA - indicating chromosomal and sex chromosomal anomalies like trisomies and aneuploidies. . Different probes are used for different purposes :
- Locus specific tags detect very small imbalances, and locate isolated small portions
of genes within a chromosome
- Alphoid/Centomeric Repeat probes are developed from the repetitive sequence located in the middle region of each chromosome, useful in determining the number of chromosomes , detecting rearrangement and when used in conjunction with locus probes to determine the absence of genetic material on a chromosome.
- Paint Probes are collections of smaller probes with their own stains that bind to a different section on the chromosome - allowing the full chromosome too be labeled a unique colour, this "full colour map" can be used to know the spectral karyotype- full chromosomal mapping is useful in examining chromosomal abnormalities.
Array Comparative Genomic Hybridisation (aCGH)
aCGH also known as Microarray analysis efficiently scans the entire genome for chromosomal imbalances. CGH was initially developed to detect the number of changes in a solid tumor mass. It uses 2 genomes comparing the sample to the control, with each labeled in a different fluorescent dye. Earlier CGH techniques were limited by the resolution of the imaging , these initial limitations were overcome by using Microarrays in conjunction with CGh to improve the resolution of the imaging, Array Comparative Genomic Hybridisation (aCGH). This method compares sample and control microarrayed slides containing small segments of DNA (probes).  the Probes used will vary according from the small (25-85 base pairs) oligonucleotides manufactured to highlight different target sequences, to the very large genomic clones (80,000- 200,00 base pairs), and as these are significantly smaller than the traditional metaphase chromosomes used for CGH, generating a higher resolution of image. .aCGH is used as a diagnostic tool for prenatal detection f chromosomal abnormalities .
|Multiple applications: prenatal genetic diagnosis, cancer diagnosis,  genetic screening for developmental delay (learning disabilities) . or congenital anomalies that are suspected to be genetic in origin |
|trace represents all chromosomes present in the human genome chromosomes 1-22 and the X & Y chromosomes, and therefore is the most accurate method for testing whole embryo aneuploidy|
|aCGH has been extensively tested, and Validated, and is now used world wide.|
|Detects submicroscopic alterations|
|Detects deletions and additions and rearrangements as well as amplification, of the WHOLE genome, simultaneously.|
|Used as a diagnostic tool for prenatal detection of chromosomal abnormalities |
|Provides high resolution genomewide screening of segmental genomic copy number variations|
|Very accurate when used in conjunction with FISH|
|Alone is more reliable and in detecting chromosomal abnormalities, with a higher implantation success rate, than FISH |
|Allows an in depth research focus upon specific types of rearrangements within selected chromosomal regions, a recent particular area of interest is subtelomeric and pericentromeric rearrangements|
|Reduces the risk of failed implantation and miscarriage, improving the chance of a healthy baby >|
|Translocations and inversions of DNA are not detected|
|Limited ability diagnosing specific polyploidies such as triploidy |
|Will not detect mosaicism detection <20% (cultures where >1 of 5 cells are trisomy 12)|
|Will not detect balanced chromosomal rearrangement|
|Will not detect duplications or deletions <80kb|
|Will not detect point mutations within genes |
|will not detect chromosomal position of genomic gains|
|Will not detect loos of heterozygosity (LOH) or Absence of heterozygosity (AOH) |
As a part of PGD fetal cell samples are collected from; the fertilized egg polar bodies, the blastomere (day 3 embryo) or the blastocyst/tropoectoderm stage (day 5 embryo). Sample DNA is labeled with one fluorescent dye, and the control DNA is labeled with a different colored fluorescent dye. the control DNA is used as the base point of reference. heated and denatured single DNA strands then hybridize to their complementary single strand probes, which are then combined and applied to a microarray and the results are run through a computer program and a digital imaging system is used to quantify the results( fluorescent intensities of the labeled probes). The fluorescent ratio and the hybridization signal at different locations on the genome of the control DNA are used to identify any variances present in the sample DNA. aCGH facilitates the clinical diagnosis of submicroscopic chromosomal duplication, deletion and rearrangements indicative of chromosomal disorders such as trisomies 1-22 and specific sex linked disorders. Duplication in the DNA are displayed by the computer program as spikes/ peaks over an established threshold and deletions in DNA are displayed by the computer program as spikes/ toughs beneath this threshold.
Next Generation Sequencing
The development and advances within ART in the past 20 years, as well as the increasing popularity of IVF, has lead to an influx of new technologies developed to screen embryos for chromosomal anomalies, which are covered by the umbrella term of Next generation Sequencing (NGS). NGS is a general term used to describe all of the new and emerging screening techniques currently being introduced and used as part of PGD for IVF. NGS screens for single gene disorders as well as conducting extensive and very comprehensive chromosome diagnosis by sequencing, counting, and accurately assembling millions of DNA reads, simultaneously. NGS is expected to replace the other limited and outdated testing techniques and be used as the standard test in the future.
|Cost effective  |
|Accurately tests the whole genome |
|Tests for the presence of monogenic diseases of known genetic background.|
|Reduces the number of biopsies required for diagnosis.|
|Higher detection rate of small translocations is possible.|
|Testing for compound point mutations, chromosomal duplication, deletions and insertions is highly accurate.|
|It accurately detects chromosomal aneuploidy and unbalanced rearrangement.|
|NGS single gene disorder screenings can be conducted in conjunction with PCR comprehensive chromosomal screening.|
|Human error is reduced.|
|It detects the presence of mosaicism better.|
|Work well in conjunction with CGH and aCGH as part of PGD, improving the chances of IVF. |
|There are limited information available to clinical applications of NGS.|
|More research and progress needed to establish a clinical manifestation.|
Samples are collected from either blastomere or the blastocyst/tropoectoderm and processed for analysis by a computer system. The methodology of each process is unique to the technique being used. The popularity of the new and emerging techniques is due to the cost effective nature of their testing, their speed and the accuracy of their results. Please see the table above for advantages of NGS.
There are a large amount of diseases that PGD can apply to, below are descriptions of the diseases that PGD are more commonly used for. Upon completion of the genetic testing for the genes of concern the cells the embryos are discarded and priority is given to those that are healthy. 
Cystic fibrosis is a single gene disorder. It is autosomal recessive and involves mutations in the cystic fibrosis transmembrane conductance regulator (CTFR) gene. The CTFR gene is normally responsible for the decrease in chloride and the transport of bicarbonate in epithelial cells thus playing a major physiological role. In PGD procedures, identification of the gene is assisted by microsatellite markers that have similar composition to the CTFR gene itself. Biopsy of two cells at the blastocyst stage is recommended if markers are not available. There are many variations of cystic fibrosis the most common being P.Phe508del .
Autosomal Recessive Meckel-Gruber Syndrome
Autosomal recessive genetic defect caused by the TMEM67 gene. It results in cystic dysplasia of the kidneys with fibrotic change in the liber and occipital encephalocele. Other malformations could also be present in the central nervous system. In PGD whole genome amplification of single blastomeres are taken to identify the gene, this is also coupled with PCR techniques. Maternal plasma can also be extracted for PGS. 
β – Thalassemia
β – Thalassemia is known as the most common type of autosomal recessive inherited disorder among haemoglobinopathies. It involves the adult β-globin gene and is associated with the absent or decreased expression of the gene. This is commonly caused by a single nucleotide change in the gene. PGD has been used successfully worldwide to identify the β-globin gene where its application is usually performed on a single blastomere or polar body .
In the collapsible table is a list of diseases and their corresponding genes. These genes are tested for in PGD for the identification of a specific disease. Note that not all the diseases applicable to PGD are listed. Some of the genes listed also have a link attached to them which will bring you to the Online Mendelian Inheritance in Man website which provides extensive information about that particular gene. This web site can be accessed when you click here if you would like to find out more about any of the other genes.
|Applicable diseases for PGD   |
|5 Alpha Reductase Deficiency (5ARD)||SRD5A2 |
|Acute Intermittent Porphyria||ALAD  , ALAS2 , CPOX , FECH , HMBS , PPOX , UROD , or UROS |
|Adrenoleukodystrophy (Adrenomyeloneuropathy)||ABCD1 |
|Agammaglobulinaemia (x-linked)||BTK |
|Agammaglobulinemia Bruton Tyrosine Kinase (BTK)||BTK |
|Aicardi Goutieres Syndrome 1 (AGS1)||TREX1  , RNASEH2A  , RNASEH2B  , RNASEH2C , SAMHD1 |
|Alagille Syndrome||JAG1  or NOTCH2|
|Alpers-Huttenlocher Syndrome||POLG |
|Alpha-1-antitrypsin deficiency||SERPINA1 |
|Alpha Thalassemia||HBA1or HBA2 |
|Alports Syndrome||COL4A3  , COL4A4  , COL4A5 |
|Alzheimer's Disease - early onset (Type 3 and 4)||APP  , PSEN1 , or PSEN2 |
|Amyotrophic Lateral Sclerosis 1 (ALS1)||C9orf72 , SOD1 , TARDBP , FUS , ANG  , ALS2 , SETX , VAPB |
|Argininosuccinic Aciduria||ASL |
|Arrhythmogenic Right Ventricular Cardiomyopathy/ Dysplasia (ARVC/D)||DSG2 ; DSP  ; PKP2 |
|Ataxia Telangiectasia||ATM |
|Autosomal Recessive Meckel-Gruber Syndrome||TMEM67 |
|Bardet-Biedl Syndrome (BBS)||BBS1 ; BBS10 |
|Barth Syndrome||TAZ |
|Beta Thalassaemia||HBB |
|Breast Ovarian Cancer Familial Susceptibility (BRCA2)||BRCA1; BRCA2|
|Carnitine-Acylcarnitine Translocase Deficiency||SLC25A20|
|Cerebral Arteriopathy with Subcortical Infarcts & Leukoencephalopathy (CADASIL)||NOTCH3|
|Cerebral Cavernous Malformation||CCM1|
|Charcot-Marie-Tooth Disease||GJB1; MPZ; NEFL; PMP22|
|Chronic Granulomatous Disease||CYBB; NCF1|
|Congenital Adrenal Hyperplasia||CYP21A2|
|Congenital Cataracts||GJA8; VSX2|
|Congenital Diarrhea, Syndromic||SPINT2|
|Congenital Disorders of Glycosylation (CDG)||ALG1; ALG6; CDG1C; DOLK; PMM2|
|Cornelia de Lange Syndrome||NIPBL|
|Cysteinyl Leukotriene Receptor 1 Deficiency||CYSLTR1|
|Diamond –Blackfan Anemia||RPS19|
|Duchenne Muscular Dystrophy||DMD|
|Dyskeratosis congenita (Male embryos only)||DKC1|
|Ectodermal dysplasia (Hypohidrotic)||EDA; EDA1; GJB6; IKBKG|
|Familial Adenomatous polyposis coli (FAP)||APC|
|Fanconi Anemia||FANCA; FANCC; FANCD2; FANCF; FANCG; FANCJ|
|Fragile X Syndrome (FRAX)||FMR1|
|Glutaric Acidemia (aciduria)||GCDH|
|Glycogen Storage Disease||G6PC; GAA; SLC37A4|
|Hereditary Nonpolyposis Colorectal Cancer: Lynch Syndrome||MLH1; MSH2; MSH6|
|Holt Oram Syndrome||TBX5|
|Incontinentia Pigmenti (IP)||NEMO|
|Joubert Syndrome 5||INPP5E|
|Leber Congenital Amaurosis (LCA)||CEP290; GUCY2D|
|Leigh Syndrome (Infantile Subacute Necrotising Encephalopathy)||LRPPRC|
|Lesch Nyan Syndrome||HPRT1|
|Leukocyte Adhesion Deficiency (Type I)||ITGB2|
|Macular Dystrophy Retinal||VMD2|
|Maple Syrup Urine Disorder (MSUD)||BCKDHB|
|Mitochondrial DNA Depletion Syndrom||POLG; RRM2B; SUCLA2; TK2|
|Mucolipidosis type II||GNPTAB|
|Multiple Endocrine Neoplasia||MEN1; MEN2A; MEN2B|
|Multiple Exostoses||EXT1; EXT2|
|Neurofibromatosis Type 1||NF1|
|Neurofibromatosis Type 2||NF2|
|Noonan Syndrome||KRAS, PTPN11; SOS1|
|Oculocutaneous Albinism||OCA2; TYR|
|Ornithine Transcarbamylase Deficiency||OTC|
|Osteogenesis imperfeca||COL1A1; COL1A2|
|Osteopetrosis||CLCN7; OSTM1; TCIRG1|
|Pachyonychia Congenita||KRT16; KRT6A|
Laws & Legal status
|Australia||PGD is currently used to detect serious genetic conditions to improve the outcome of ART. In very rare cases it may be used to select an embryo with compatible tissue for a sibling who has a life-threatening disease where other means of treatment is unavailable. This is with the conditions that the use of PGD will not affect the welfare and interests of the child to be born. The parents must also receive adequate counselling and have full understanding of the procedures of PGD. 
The use of PGD for sex-selection is prohibited with the exception of reducing the risk of the transmission of serious sex-linked genetic conditions. 
|Brazil||The laws regarding PGD in Brazil are not strictly regulated. They are vague and there is limited information surround the activities of assisted reproduction in general in the country .|
|Czech Republic||The laws in the Czech Republic allow those with a “defined indication in order to exclude risk of serous genetically conditioned disease and defects with embryos before they are implanted into the cavity of the uterus”. Couples that undergo any assisted reproductive treatment including PGD have to be married. Sex-selection is illegal except in regards to serious sex-linked genetic diseases .|
|Greece||In Greece, ART is open to those up to the age of 50 with written consent. It can only be used if a medical necessity is present such as the susceptibility of serious hereditary diseases. Sex selection is banned apart from cases of sex-linked diseases and sexually transmitted diseases .|
|India||In India there is a cultural preference for boys thus the Prenatal Diagnostic Techniques (Regulation and Prevention of Misuse) Act was introduced in 1994. This law limits the use of PGD for sex determination except in cases of specific congenital diseases. These laws however are not strictly enforced. |
|Portugal||ART is only available to those who are married or have a similar type of relationship for at least two years. The couples cannot be of the same sex and must be at least 18 years of age. Sex selection is illegal except in cases of sex-linked diseases. The use of PGD is illegal if the predictive value of genetic tests are very low .|
|Spain||PGD can be applied to “serious hereditary diseases not amenable to postnatal curative treatment” and “detection of other abnormalities which may compromise the viability of the pre-embryo”. If PGD is to be used for any other purpose authorisation must be acquired .|
|Sweden||The use of PGD is strictly regulated and couples must achieve authorisation of the National Board of Health and Welfare. It can only be used it can only be used if the child is at risk to inheriting a serious chromosomal or monogenetic disease. It cannot be used for selection of specific characteristics .|
|United Kingdom||Those involved with carrying out the IVF treatment must have a license from the Human Fertilisation and Embryology Authority (HFEA). Only embryos that are approved by the HFEA can be implanted where the embryonic nuclear or mitochondrial DNA cannot be altered with the exception of preventing mitochondrial diseases .|
In addition to research efforts for improving overall performance of established PGD/S techniques, such as finding the ideal zona pellucida insection site in an fully automatic manner, many research efforts have been spent to find alternative, non-invasive techniques to test for diseases and abnormalities.
Non-invasive Preimplantation Genetic Testing without Embryo Biopsy
Different parameters of gametes, zygotes, embryos (“vacuoles in sperm heads, spindle position in mature oocytes, cleavage intervals of zygote, and embryo developmental dynamics”) may correlate with aneuploidy rates. This knowledge may be applied in potential noninvasive preimplantation diagnostic methods. Several methods have been proposed and are currently further researched.
Intracytoplasmic morphologically selected sperm injection (IMSI) is common procedure in IVF treatment to improve fertilization rates in patients with poor semen quality. In addition, studies have found that IMSI improves embryo development and that spermatozoa with large vacuoles in their heads correlate with increased aneuploidy rates and disturbed chromosomal structures. Thus, selecting spermatozoa based on morphological hallmarks may decrease aneuploidy rates in the fertilized embryos. As with polar body biopsies, however, this approach will solely be applicable if evidence for severe male detrimental contribution is given.
Blastocoel Fluid Extraction
In addition, a less invasive retrieval of material for diagnosis could include the extraction of the blastocoel fluid. The cavity of the blastocyst, lined by the trophectoderm, is filled with this blastocoel fluid, which contains metabolites of both trophectoderm and inner cell mass origin. The retrieval does not require a biopsy but merely a small opening to extract the fluid and, thus, causes less harm to the embryo. Multiple studies have applied this method   and it may in the future become of clinical relevance.
Next to metabolites, the blastocoel fluid can contain DNA. Studies have shown that genomic DNA is present in about 90% of the blastocoel fluid samples tested. Typically the fluid is removed with an ICSI pipette from a day five blastocyst through its mural trophectoderm until the blastocyst fully collapses around the embryo. Several concerns regarding this method in a clinical setting have been raised. The collected DNA may be contaminated by the culture media that contains DNA fractions. The DNA may also be least representative of the actual embryos genome as the DNA may originate from abnormal or degenerated cells. Even though labelled noninvasive, the blastocyst still undergoes manipulation to some degree which may affect its viability. Thus, more research is required until this method can evolve from research to clinical use.
Proteomics and Medium Based PGD
The media in which embryos are kept during the early IVF procedures, gives rise to potential non-invasive techniques. For instance, the protein secretome of blastocysts may be representative of its chromosome constitution Recent studies have found biomarkers such as lipocalin-1, interleukin-10, tumor necrosis factor, stem cell factor, and chemokine ligand 13 to be differently secreted by aneuploid blastocyst than by euploid ones. The most significant biomarker appears to be interleukin-10. Paired with novel proteomic technologies and mass spectrometry this knowledge when extended may contribute to a new invasive PGD method. In addition, medium based non-invasive PGD has been researched for the diagnose of human α-thalassemia-SEA. Genomic DNA was collected from the media and the study surprisingly resulted in increased diagnosis efficacy compared to biopsy-based methods.
Using time lapse imaging the embryo’s morphology can be closely observed and potential aneuploidy characteristics detected. Such characteristics may include the time of division to five cells, the time between the division from three to four cells, and the duration of the division from one to two and subsequently to three. In addition, the morphological quality of ICM an TE has been positively associated with aneuploidy or euploidy prognosis. These may give rise to an embryo quality screening prior to implantation.
Allele: One of two or more versions of a gene
Aneuploidy: Presence of an abnormal number of chromosomes in a cell
Biopsy: Sample of tissue taken for examination
Blastocyst: Stage of embryo at approximately day 5 consisting of an outer (trophoblast) layer and inner (embryoblast) cell mass.
Blastomere: Cell type formed through cleavage of the zygote after fertilization
Chromosome Thread-like structure, which is made up of protein and DNA, within the nucleus of a cell
CTFR: Cystic Fibrosis Transmembrane Conductance Regulator, responsible for transport of chloride across the cell membrane
Denaturing: Proteins or nucleic acids lose their quaternary, tertiary, and secondary structure
DNA: DeoxyriboNucleic Acid, hereditary material
Endometriosis: Condition in which the endometrium, the tissue lining the uterus, grows outside of it
Enucleation: Removal of the nucleus
Epigenetic: Phenotypic trait variations due to external or environmental factors that influence gene expression
ESHRE: European Society of Human Reproduction and Embryology
FISH: Fluorescent In situ Hybridisation, technique used to locate specific gene sequences using fluorescence tags
Heterozygote: Diploid organism that contains two different alleles of one gene
Hydrosalphinx: Fluid filled fallopian tube
Intracytoplasmic Morphologically Selected Sperm Injection (IMSI): IVF technique involving morphological selection of sperm under the microscope to be injected to oocyte
Intracytoplasmic Sperm Injection (ICSI): IVF technique used to treat male infertility and involves direct injection of one sperm into an oocyte
IVF: In Vitro Fertilisation
Leukocyte: White blood cell, involved in immune system
Leukaemia: Cancer of the bone marrow, increased numbers of abnormal or premature leukocytes are formed by bone marrow and other organs
NGS: Next Generation Sequencing, term used to describe the collection of recent findings regarding embryo screening
Oolemma: Plasma membrane of the oocyte
PB: Polar Body, cell formed during the meiotic stages of the oocyte containing extra genetic material
PCR: Polymerase Chain Reaction, technique used to amplify DNA to study a specific genetic sequence
Polyploidy: Having more than two sets of homologous chromosomes
PGD: Preimplantation Genetic Diagnosis, genetic testing conducted to identify abnormalities in an embryo before implantation in parents with genetic disease history
PGS: Preimplantation Genetic Screening, similar to PGS but in couples seeking IVF due to infertility issues to improve implantation rates
Perivitelline Space: Space between the oolemma and the zona pellucida
RT: Robertsonian Translocations, a type of structural chromosomal translocation
Trisomies: Presence of three copies of a chromosome instead of two
Trophoectoderm: Outer layer of the mammalian blastocyst
Zona Pellucida: Thick membrane surrounding the mammalian oocyte
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- 2015 Course: Week 2 Lecture 1 Lecture 2 Lab 1 | Week 3 Lecture 3 Lecture 4 Lab 2 | Week 4 Lecture 5 Lecture 6 Lab 3 | Week 5 Lecture 7 Lecture 8 Lab 4 | Week 6 Lecture 9 Lecture 10 Lab 5 | Week 7 Lecture 11 Lecture 12 Lab 6 | Week 8 Lecture 13 Lecture 14 Lab 7 | Week 9 Lecture 15 Lecture 16 Lab 8 | Week 10 Lecture 17 Lecture 18 Lab 9 | Week 11 Lecture 19 Lecture 20 Lab 10 | Week 12 Lecture 21 Lecture 22 Lab 11 | Week 13 Lecture 23 Lecture 24 Lab 12 | 2015 Projects: Three Person Embryos | Ovarian Hyper-stimulation Syndrome | Polycystic Ovarian Syndrome | Male Infertility | Oncofertility | Preimplantation Genetic Diagnosis | Students | Student Designed Quiz Questions | Moodle page
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Cite this page: Hill, M.A. (2019, September 21) Embryology 2015 Group Project 6. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/2015_Group_Project_6
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