2015 Group Project 6

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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

PMID 20638568 (includes some history)

Indications

Inheritance patterns

Preimplantation Genetic Diagnosis

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].

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]

Cell Extraction 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]


PMID 24305177

Polar Body Analysis

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

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

Description

Procedure

Advantages & Disadvantages

Trophectoderm biopsy

Description

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

Advantages & Disadvantages

PMID 23993663 PMID 20966459 PMID 22723007 PMID 26168107 PMID 25654908

Genetic Techniques

PMID 25154779 (might be useful?)

Fluorescent In Situ Hybridisation (FISH)

PCR

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. [9]

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. [9]

Future/Current Research

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 <pubmed>22723007</pubmed>
  9. 9.0 9.1 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


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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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