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

Prenatal Genetic Diagnosis was historically conducted post-implantation in the first 2 trimesters [1], testing for overall fetal growth, complications of pregnancy, birth defects as well as chromosomal or genetic abnormalities[2]. Due to advancements in Artificial Reproductive Technologies ( ART) in the past 2 decades , these genetic tests are now conducted as a part of Preimplantation Genetic Diagnosis (PGD)[3]. In the late 1980 Preimplantation genetic testing was being developed in the UK and was first used on humans in 1990, to diagnose recessive X-linked diseases in the embryos of pregnant women undergoing IVF. [4]


Presently Preimplantation genetic diagnosis is used as an alternative to prenatal diagnosis in detecting genetic disorders, in at risk couples. [5]

PGD improves the accuracy and effectiveness of IVf, and reduces the although post natal testing can be used to confirm original PGD diagnosis.




Indications

Preimplantation Genetic Diagnosis

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 [6]. Some diseases commonly involved with PGD include cystic fibrosis, spinal muscular atrophy and beta – thalassaemia [7] [8]. It was first used in the United Kingdom in the 1980s of arenoleucodystrophy and primarily focusing on sex- linked disorders [8][9]. PGD is now capable of detecting single cell defects (molecular) and chromosomal disorders resulting from the inversion, translocation or deletion of chromosomes (cytogenic) [7],[10]. PGD can be applied to the embryo at different stages. That is on polar bodies, blastomeres or blastocyst [7].

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 [10][11] and Embryo halotyping which allows the identification of chromosomes causing the inherited disorder through knowledge of the pattern of closely linked markers [7]. Polymerase chain reaction (PCR) is also widely used to detect molecular abnormalities [10].

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 [12].

Sex-linked disorders

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 [13].

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 [14].

Mitochondrial disorders

Mitochondrial disorders also known as oxidative phosphorylation disorders arise from mutations in the nuclear DNA or mitochondrial DNA [15]. They pose as a problem because they are unrecognisable until the mutations in the cell reach a detrimental level [6]. 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 [15].

Chromosomal disorders

Chromosomal disorders can be reciprocal, Robertsonian translocations, inversions, deletions and insertions [14]. . Data from ESHRE collected between 2010 and 2011 has shown that the most common chromosomal abnormality confronted in PGD are reciprocal chromosomal abnormalities [16]. 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 [17]. 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 [14].

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 [7]. 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 [18]. They have little effect on the morphology of the embryo making them difficult to identify thus identification heavily relies on genetic testing [7].

Genetic sampling is most commonly conducted using Microarray Comparative Genomic Hybridisation (aCGH) as well as FISH, Quantitative PCR and Single Nucleotide Polymorphism (SNP) [7]. These methods collectively aim to assess numeral and structural chromosomal errors [18]. Studies have also introduced Next- Generation Sequencing (NGS) [18] and Whole Genome Amplification used to screen imbalances in the complete 24-chromosomes [19]

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 [20].

Indications

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 [6]. 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 [21]. 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 [6].

Advanced maternal age

Data provided by the ESHRE has shown that the mean age of women undergoing PGS is 39 years [20]. Women of advanced age have been shown to have a lower rate of pregnancies reaching childbirth [22]. 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%) [23].

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 [24].

Male Infertility
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 [12]. It is most commonly applied to children suffering from relapsed leukaemia [25]. In a case study PGD-HLA was proven to successfully cure 10 diseases including Fanconi anaemia, Diamond-Blackfan anaemia and beta thalassemia [26].

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. [27]

Advantages Disadvantages
Polar Body ...
Blastomere
Trophectoderm ... ...
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[27].

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[27]

The presence of a faint but clearly identifiable strand connecting PB2 to the oolemma. The biopsy was performed ∼9 h after ICSI.

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[27].

The sustained implantation predictive value (with 95 % confidence interval) of a euploid screening result obtained from the first polar body (PB1), PB1 and the second polar body (PB2), or a direct embryo biopsy for each stage of embryo transfer (cleavage-stage and blastocyst stage)

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 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[27].

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

-Cryopreservation

(A) Some trophectoderm cells in a blastocyst started to hatch and (B) the trophectoderm cells were biopsied with assisted laser cutting. Images in C–D show the blastocysts after vitrification and warming. Blastocysts had been cultured for 2–4 hrs after warming, showing good (ICM and trophectoderm cells) hatched (C) and hatching (D) blastocysts. The hatching blastocyst in (E) has good ICM but fair trophectoderm while the blastocyst in (F) has both fair ICM and trophectoderm. Arrows indicate ICMs and arrow heads indicate trophectoderm cells. Bar = 40 µm.


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[27]. PMID 23993663 PMID 20966459 PMID 22723007 PMID 26168107 PMID 25654908

Genetic Techniques

Fluorescent In Situ Hybridisation (FISH)

Description

Fluorescence in situ hybridization (FISH) the one of the most effective and rapid [28] 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,[29] that have no other mutation specific tests [4] 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 [30]. FISH is 99% effective when used in conjunction with competitive Genomic Hybridisation (CGH) to diagnose chromosomal abnormalities.[31]


Procedure

Samples are collected from either the blastocyst, a polar body biopsy or the blastomeres stages of the embryo [32]. DNA strands are heated and denatured causing their the individual DNA strands to break apart Probes are 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. These specific probes then hybridize and join to their complementary DNA strand. 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. [33] Researcher analyse the results by identifying the number and the relative location of the fluorescent dots generated by the FISH images.[34] 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. [35]


Different probes are used for different purposes: [36]

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.


Fluorescent In Situ Hybridisation (FISH).jpg


Advantages and Disadvantages:

BENEFITS


  • FISH is 99% effective when used in conjunction with Competitive Genomic Hybridisation (CGH) to diagnose chromosomal abnormalities.[37]
  • rapid generation of results
  • enables the number and size of specific chromosomes to be known
  • identifies very small translocations and aneuploidies that occur within chromosomes, that would usually be missed under microscopic analysis
  • indicates whether the accessed chromosomes are normal [38]
  • tests for the most common chromosomal abnormalities - down syndrome chromosomes 13,16,18,21 and 22 [39]
  • can be used to identify and remove inheritable X-linked disease or sex chromasome anomalies such as Duchennes Muscular Dystrophy, hemophilia, ectodermal dysplasia [40]
  • it may be used to compare the chromosomal gene arrangements in related species


DISADVANTAGES

  • FISH destroys all cells tested and
  • it does not fully access all chromosomes ( only ~ 12),
  • there is a 40 % chance that chromosomal aneuploidy is occuring and not being targeted by this test.
  • analysis of results is dependent upon the dot quality impacted by hybridisation efficiency or the camera sensitivity [41]





PMID 17970921



PCR

Description

Procedure

Advantages & Disadvantages

Array Comparative Genomic Hybridisation (aCGH)

Description

Procedure

Advantages & Disadvantages

PMID 26100406 PMID 24771116



Single Nucleotide Polymorphism

Description

Procedure

Advantages & Disadvantages

Next Generation Sequencing

Description

Procedure

Advantages & Disadvantages

Diagnosis

Cystic Fibrosis

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 [42].

Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy is an X-linked recessive disease. It involves the Xp21 gene where majority of the mutations are chromosomal deletions with a smaller percentage resulting from duplications [43].

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 [44].

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. [45]

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. [45]

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[46].

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[46].

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[47] [48] 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[46].

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[46].

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[49]. These may give rise to an embryo quality screening prior to implantation[46].

Ethics

References

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