2015 Group Project 6

From Embryology
Revision as of 17:46, 29 September 2015 by Z5088434 (talk | contribs)
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.


Prenatal Genetic Diagnosis for ART

Introduction

History

Historically prenatal genetic diagnosis were conducted post-implantation in the first 2 trimesters, testing for overall fetal growth, complications of pregnancy, birth defects and chromosomal or genetic abnormalities, although, currently these genetic tests are conducted as a part of PreImplantation Genetic Diagnosis (PGD) made possible by the recent advances in Assisted Reproductive Technologies (ART).

PMID 20638568 (includes some history)

Indications

Preimplantation Genetic Diagnosis

--Z5088434 (talk) if you can't find enough information, you could also include the different biopsies and their application for different indications. So polar body biopsies wouldn't be applicable for Y-linked diseases (which is very obvious, but there are more indications that are specific for biopsies at the different development stages)

Preimplantation genetic diagnosis (PGD) is used to test the genetic makeup of embryos to prevent the transmission of inherited diseases with detrimental effects such as cystic fibrosis, spinal muscular atrophy and beta – thalassaemia [1] [2]. It was first used in the United Kingdom in the 1980s of arenoleucodystrophy and primarily focusing on sex- linked disorders [2][3]. PGD is now capable of detecting single cell defects (molecular) and chromosomal disorders resulting from the inversion, translocation or deletion of chromosomes (cytogenic) [1],[4]. PGD can be applied to the embryo at different stages. That is on polar bodies, blastomeres or blastocyst [1].

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 [4][5] and Embryo halotyping which allows the identification of chromosomes causing the inherited disorder through knowledge of the pattern of closely linked markers [1]. Polymerase chain reaction (PCR) is also widely used to detect molecular abnormalities [4].

Inheritance patterns

Sex-linked disorders
Single gene defects
Chromosomal disorders

Preimplantation Genetic Screening

Prenatal Genetic Screening (PGS) involves an array of methods or ideas that aim to segregate embryos that have genetic flaws and those that are healthy [1]. 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 [6]. They have little effect on the morphology of the embryo making them difficult to identify thus identification heavily relies on genetic testing [1].

Genetic sampling is most commonly conducted using Microarray Comparative Genomic Hybridisation (aCGH) as well as FISH, Quantitative PCR and Single Nucleotide Polymorphism (SNP) [1]. These methods collectively aim to assess numeral and structural chromosomal errors [6]. Studies have also introduced Next- Generation Sequencing (NGS) [6] and Whole Genome Amplification used to screen imbalances in the compete 24-chromosomes [7]

Indications

Advanced maternal age
Recurrent pregnancy loss
IVF failure
Male Infertility
Sex selection
Human leukocyte antigen matching

Biopsy Methods

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. [8]

Polar Body, Blastomere, and Trophectoderm Biopsy.jpeg

Polar body (A), blastomere (B) and trophectoderm (C) biopsies.


PMID 24305177

Polar Body Analysis

Day 1 PMID 22723007 PMID 26096028 PMID 26024488 PMID 25639967 PMID 25106935 PMID 23993663 PMID 20966459 PMID 26168107 PMID 25654908 PMID 25064409

Description

Polar body (PB) biopsy, which was introduced in 1990, offers a promising alternative to biopsies performed at the blastomere stage for PGD/S indications on legal and practical grounds. Description: Consequent embryo development does not necessitate the presence of the first and second PB and their removal may not be crucial. 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. However, its optimal time window for biopsy is set to be eight to 16 hours after ICSI. This is 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. In addition, 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[8].

Procedure

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[8].

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 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. 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[8].

Blastomere biopsy

Day 3 PMID 23993663 PMID 20966459 PMID 22723007 PMID 26168107 PMID 25654908 PMID 25816038 PMID 25516085 PMID 25624194 PMID 24581980 PMID 24301057

Description

Procedure

Aspiration of a Blastomere.jpeg

Aspiration of a Blastomere into the biopsy pipette

Advantages & Disadvantages

Trophectoderm biopsy

Day 5 and 6

Description

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. During day three to day five the haploid maternal and paternal genomes work 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[8].

Procedure

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[8]. -collapse of trophoblasts -possible: biopsy of hatched blastocyst, holding pipette careful mild suction force to hold embryo near ICM -Cryopreservation

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 whn such methods have been developed or improved[8]. PMID 23993663 PMID 20966459 PMID 22723007 PMID 26168107 PMID 25654908

Advantages Disadvantages
Polar Body ...
Blastomere
Trophectoderm ... ...

Genetic Techniques

main indicators of the presence single gene disorders (PCR) and inherited chromosome abnormalities(FISH) PMID 25154779 (might be useful?) PMID 11325751 PMID:11325751

Fluorescent In Situ Hybridisation (FISH)

Fluorescence in situ hybridization (FISH) the one of the most effective and rapid [9] Preimplantation Genetic Diagnosis (PGD). This techniques used to locate 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,[10] that have no other mutation specific tests [2] As part of PGD, samples are collected from various stages of the embyro,and they are able to conducted tests on the blastocyst, a polar body biopsy and the blastomeres [11]. FISH is 99% effective when used in conjunction with competitive Genomic Hybridisation (CGH) to diagnose chromosomal abnormalities.[12]

Fluorescent In Situ Hybridisation (FISH).jpg

Fluorescent In Situ Hybridisation (FISH) [13]

[[3]]


How does it work ?

Probes are short single strands of DNA that have been tagged with fluorescent labels to complement specific parts of each chromosome. These probes are usually target specific or used in a specific combination to test for specific loci. These fluorescent labels are small chemical agents that glow brightly in the presence of a specific region on a chromosome. Heating the DNA sample denatures it, causing the individual DNA strands to break apart, allowing the probe to hybridise (join with) the complementary strand of DNA. the fluorescent tags enable the researchers to correctly identify the presence or lack thereof and location of the specific chromosomes that they are testing for. [14] The probe will not hybridise is the there has been a duplication or a deletion of the DNA - indicating chromosomal abnormalities such as trisomies 13,18 and 21




PMID 26338801 PMID 20809319 PMID 11325751 PMID 11325751 PMC1 120169 PMID 17970921 PMID 9793305


Unique, Rare Chromosome Disorder Support Group (2013) Fluorescence in situ hybridisation (FISH) Rarechromo.org retrieved from [4]

NIS (2015) Flourescence in Situ Hydridization (FISH) National Human Genome Research Institute retrieved from [5]

PCR

Array Comparative Genomic Hybridisation (aCGH)

PMID 26100406 PMID 24771116

Single Nucleotide Polymorphism

Next Generation Sequencing

Diagnosis

Applicable diseases for PGD
Disease Involved Genes
5 Alpha Reductase Deficiency (5ARD) SRD5A2
Achondroplasia FGFR3
Acute Intermittent Porphyria ALAD, ALAS2, CPOX, FECH, HMBS, PPOX, UROD, or UROS
Acute Recurrent Autosomal Recessive Rhabdomyolysis (ARARRM)
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-Mannosidosis MAN2B1
Alpha Thalassemia HBA1 or 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
Anauxetic Dysplasia
Anderson Fabry Disease
Androgen Insensitivity Syndrome
Angelman Syndrome (UBE3A gene only)
Aniridia
Aplastic anaemia - severe
Argininosuccinic Aciduria
Arrhythmogenic Right Ventricular Cardiomyopathy/ Dysplasia (ARVC/D)
Arthrogryposis renal dysfunction and cholestasis type 1 and type 2
Ataxia Telangiectasia
Autosomal Dominant Acute Necrotizing Encephalopathy
Autosomal dominant familial exudative vitreoretinopathy types 1, 5 and 4,
Autosomal Dominant Polycystic Kidney Disease (ADPKD)
Autosomal Dominant Retinitis Pigmentosa
Autosomal Recessive Dopa Responsive Dystonia
Autosomal Recessive Severe Combined Immunodeficiency with Bilateral Sensorineural Deafness (A RSCIDBSD)
Bardet-Biedl Syndrome (BBS)
Barth Syndrome
Battens Disease (infantile)
Beta Hydroxyisobutyryl CoA Hydrolase Deficiency (Methacrylic Aciduria)
Beta Thalassaemia
Bethlem Myopathy
Bilateral Frontoparietal Polymicrogyria
Birt-Hogg-Dubé Syndrome
Branchio-Oto-Renal Syndrome (BOR)
BRCA 1 (increased susceptibility to breast cancer)
Breast Ovarian Cancer Familial Susceptibility (BRCA2)
Canavan Disease ASPA
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
CHARGE Syndrome CHD7
Cherubism SH3BP2
Choroideremia CHM
Chronic Granulomatous Disease CYBB; NCF1
Ciliary Dyskinesia DNAH5
Citrullinemia ASS1
Cleidocranial Dysplasia RUNX2
Cockayne Syndrome ERCC6
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
Craniosynostosis TWIST1
Crouzon Syndrome FGFR2
Cysteinyl Leukotriene Receptor 1 Deficiency CYSLTR1
Cystic Fibrosis CFTR

Note: This table is not complete yet --Z5017878 (talk) 17:44, 17 September 2015 (AEST)

Utilization of Diseased Cell Lines

Laws & Legal status

Australia

PGD is currently used to detect serious genetic conditions to improve the outcome of Assisted Reproductive Technologies (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. [15]

Sex-selection: 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. [15]

Future/Current Research

Noninvasive 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[16].

Sperm Selection

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[16].

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[17] [18] and it may in the future become of clinical relevance.

Aspiration of the Blastocoel Fluid.jpeg

Aspiration of the Blastocoel Fluid using a ICSI pipette


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[16].

Proteomics

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 preimplantation genetic diagnosis method[16].

Embryo Morphology

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[19]. These may give rise to an embryo quality screening prior to implantation[16].

Ethics

References

  1. 1.0 1.1 1.2 1.3 1.4 1.5 1.6 Coward, K. & Wells, D. (2013). Textbook of Clinical Embryology New York: Cambridge University Press
  2. 2.0 2.1 <pubmed>17823145</pubmed>
  3. <pubmed>23150080</pubmed>
  4. 4.0 4.1 4.2 <pubmed>11325751</pubmed>
  5. <pubmed>17876073</pubmed>
  6. 6.0 6.1 6.2 <pubmed>26085841</pubmed>
  7. <pubmed>25953353</pubmed>
  8. 8.0 8.1 8.2 8.3 8.4 8.5 8.6 <pubmed>22723007</pubmed>
  9. <pubmed>26338801</pubmed>
  10. <pubmed>20809319</pubmed>
  11. <pubmed>21748341</pubmed>
  12. <pubmed>26338801</pubmed>
  13. [1]
  14. <pubmed>17876073</pubmed>
  15. 15.0 15.1 National Health and Medical Research Council (2007). Retrieved September 17, 2015, from https://www.nhmrc.gov.au/health-ethics/ethical-issues/assisted-reproductive-technology-art
  16. 16.0 16.1 16.2 16.3 16.4 <pubmed>24783200</pubmed>
  17. <pubmed>22020776</pubmed>
  18. <pubmed>23148560</pubmed>
  19. <pubmed>26246880</pubmed>


Please do not use your real name on this website, use only your student number.

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

Glossary Links

Glossary: A | B | C | D | E | F | G | H | I | J | K | L | M | N | O | P | Q | R | S | T | U | V | W | X | Y | Z | Numbers | Symbols | Term Link