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Review article

Preimplantation genetic testing

Ana Jeremić1, Dragana Vuković1, Srna Subanović1, Jovana Broćić1, Biljana Macanović1
  • The Obstetrics and Gynecology Clinic “Narodni front”, Belgrade, Serbia

ABSTRACT

The application of preimplantation genetic testing (PGT) began in the late 1980s. Preimplantation genetic testing, as the earliest possible method of prenatal diagnosis, enables the selection of embryos with a normal karyotype for embryo transfer.

The use of preimplantation genetic testing has proven to be a useful method in the following three groups of inherited diseases: monogenic disorders (single gene defects), trinucleotide repeat disorders, and chromosomal abnormalities.

The success rate of in vitro fertilization (IVF) has increased significantly since the introduction of PGT into clinical practice.

This paper presents a literature review with the aim of clearly determining the role of PGT in embryo selection before embryo transfer, as well as the role of this type of testing in increasing the success rate of IVF. One of the goals of the paper is also to review the development of molecular genetic methods that are currently, or have once been, in routine use when performing PGT.

The current literature is an indicator of the development and progress of molecular genetics techniques applied in PGT. At the same time, it provides an opportunity and an incentive for further extensive research that will lead to the improvement of preimplantation genetic testing and thus increase the success rate of in vitro fertilization.


INTRODUCTION

Preimplantation genetic testing (PGT) encompasses procedures, previously known as preimplantation genetic diagnostics (PGD) and preimplantation genetic screening (PGS). According to the PGT Consortium of the ESHRE (European Society for Human Reproduction and Embryology), PGT is defined as a test that is carried out in order to analyze the DNA of oocytes (polar bodies) or embryos (cleavage stage embryos or blastocyst stage embryos) for HLA (human leukocyte antigen) typing or for the determination of genetic abnormalities [1].

PGT, as the earliest possible form of prenatal diagnostics, is applied in couples carrying a risk of passing on a hereditary disease onto their children [2]. Until the late 1980s, when PGT was developed, invasive and noninvasive prenatal diagnostics could only ascertain that the embryo in the mother’s womb was indeed afflicted with, or would develop, a hereditary disease. Couples were then faced with one of the most difficult decisions of their lives, whether to terminate or continue with the pregnancy, despite the knowledge that their child would not be healthy. The development of PGT provided a valuable alternative [3], as the method is applied on preimplantation embryos. This provides that only healthy embryos with a normal karyotype are selected for embryo transfer [3].

Preimplantation genetic screening (PGS), previously often referred to as low risk PGD, was applied in infertile couples who were carrying a low risk of passing on a hereditary disease onto their offspring, and who were undergoing IVF, for the purpose of increasing the success rate of achieving pregnancy and producing a healthy child.

The precondition for PGT is in vitro fertilization (IVF). In stimulated cycles, its aim is to produce the maximum number of ova and embryos. This is very important for couples faced with the problem of sterility, as it offers the opportunity of selecting only healthy embryos [3]. If there are indications for the application of PGT during IVF, classic in vitro fertilization (IVF) is avoided, as PGT implies the step of DNA amplification [4]. This is why the method of intracytoplasmic sperm injection (ICSI) is employed. This is the method of choice, as its application excludes the possibility of contamination with unwanted sperm DNA [2].

PGT was first applied in clinical practice in 1990, for determining the sex of the embryo, with the aid of PCR (polymerase chain reaction), due to suspected X-linked disease [2]. Several years later, the application of fluorescence in situ hybridization (FISH) became the standard method for the selection of embryo by sex, for the purpose of detecting numerical and structural chromosome aberrations. Today, these methods are no longer in use. They have been replaced by newer, more sophisticated, more sensitive and more specific methods, such as micrroarray CGH (comparative genomic hybridization) and NGS (next generation sequencing).

Based on the data depicted in the Introduction, the goals of the present paper have been defined. These are:

1. Determining the role of PGT in the selection of healthy euploid embryos before embryo transfer;

2. Determining the role of PGT in increasing the success rate of IVF;

3. Review of genetic methods applied in preimplantation genetic testing.

PREIMPLANTATION GENETIC TESTING

According to the PGT Consortium of the ESHRE, PGT is classified into the following categories:

1. preimplantation genetic testing for aneuploidies (PGT-A)

2. preimplantation genetic testing for chromosomal structural rearrangements (PGT-SR)

3. preimplantation genetic testing for monogenic disorders (PGT-M) [1].

INDICATIONS

PGT is applied in couples at risk of having children with hereditary disease or chromosome aberrations, in the following cases: older maternal age [5], severe form of male infertility [6], repeated failure of embryo implantation after IVF [3], recurrent miscarriage [5], the occurrence of disease in a child already born to the couple or in a family member [3], when one of the partners is a carrier of a monogenic disorder (single gene defect), chromosome aberrations, and mitochondrial DNA mutations, when there is a genetic predisposition to malignancy, and in HLA typing [2].

OLDER MATERNAL AGE

It is well known that there is a clear positive correlation between numerical chromosome aberrations, most frequently aneuploidies, and the maternal age. Studies have shown that up to 70% of oocytes of women past the age of 40 have numerical chromosome aberrations [5]. One of the explanations for a decreased implantation rate, including both natural conception and IVF, is a higher percentage of embryos with aneuploidies. PGT-A of embryos has shown that aneuploidies are frequent and that their percentage significantly increases with maternal age. In such cases, it is possible to perform polar body biopsy or trophectoderm biopsy of the blastocyst [5].

HLA TYPING

PGT has found its application in HLA typing in reproductive medicine. In a couple who has a child in need of hematopoietic stem cell transplantation, PGT is applied in order to select embryos that are not carriers of the mutation related to disease and are also compatible donors for their sick brother or sister. This approach was first successfully applied in Fanconi anemia [2].

It is a known fact that predisposition towards certain types of malignancy is hereditary (breast and ovarian tumors, tumors of the gaster and colon, certain types of leukemia), which is why PGT is applied in this sphere as well. [7].

GENETIC CAUSE OF MALE INFERTILITY

The genetic cause of male infertility is very heterogenous, which is why all PGT techniques are applied. Thus far, more than 2,300 genes, whose mutations can lead to the development of male infertility, have been identified, while chromosome aberrations have been confirmed as the cause of male infertility in 20% of cases. Both types of cases are characterized by poor semen analysis parameters [6]. This part of the infertile population benefits greatly from PGT, along with additional urological procedures, or without them.

Klinefelter syndrome (47, XXY) is characterized by abnormal spermatogenesis, and azoospermia is also often present. It has been determined that, in at least 50% of cases, spermatozoa can be obtained by means of testicular sperm extraction (TESE) [6]. In men with this syndrome, there is an increased incidence of aneuploidies in their offspring, which is why the application of PGT is invaluable [6].

Owing to the application of the PGT-SR method, carriers of heterologous Robertsonian translocations may increase their chances of having healthy children. In couples with this indication, PGT-SR enables the selection of healthy embryos, of which there are only 25% [6].

PGT-SR and PGT-M are applied in the following cases: if there is a microdeletion of the AZF-c region of the Y chromosome [6], in patients with Kartagener’s syndrome [7], in patients with globozoospermia [8], in a large number of numerical and structural chromosome aberrations, and in certain monogenic disorders.

MITOCHONDRIAL DISEASES

Mitochondrial diseases are relatively common metabolic disorders, and, in 15% of cases, they are caused by maternal mitochondrial DNA (mtDNA) mutations [9]. Since they lead to severe phenotypic expression (loss of neurological function, respiratory and cardiac problems, etc.), the use of the PGT-M method in patients with this indication enables the selection of healthy embryos during IVF.

According to the PGT Consortium of the ESHRE, all pathogenic mtDNA variants, which occur in individual blastomeres, are representative of the entire embryo, which is to be expected, as mtDNA does not replicate until the cleavage stage. At the stage of the blastocyst, mtDNA replication begins, which leads to the occurrence of different new variants [1].

BIOPSY

Biopsy entails extracting cells, whose genetic material is to be analyzed, by means of one of the molecular diagnostics methods. There are different types of biopsy, namely: biopsy of the polar body at the oocyte or zygote stage; blastomere biopsy on day three of embryo development; trophectoderm biopsy of the embryo on day five or day six of development, i.e., biopsy of the blastocyst [3].

Biopsy begins with ablation on the glycocalyx coat (Lat. zona pellucida). In the past, this used to be done mechanically, and then chemically, and as of 2003, it has been done exclusively with the use of the laser. Once an opening is made in the zona pellucida, and a pathway is made, the polar body of the blastomere or trophectoderm cell, whose genetic material is analyzed, is aspirated [10].

POLAR BODY BIOPSY

Polar body biopsy was first employed in 1990 for detecting cystic fibrosis. This procedure was developed with an aim to decrease the invasiveness of blastomere biopsy. Genetic material of the polar body represents only the DNA from the oocyte, which is why polar body biopsy is especially useful for the detection of maternally inherited monogenic disorders and numerical and structural chromosome aberrations. The drawback of this method is that there is no possibility of obtaining any information on the DNA of the father or the DNA of the embryo [10]. Today, this method is mainly used to overcome ethical issues and concerns in countries where embryo biopsy is not allowed.

BLASTOMERE BIOPSY

Blastomere biopsy on day three of embryo development is performed between 66 and 72 hours after the application of the ICSI method, when the embryo has 6 – 8 blastomeres, which are still totipotent, and the boundaries between them are clearly visible [2].

The drawbacks of this method are the following: the relevance of the results obtained through the analysis of an individual cell, bearing in mind the high percentage of mosaicism that occurs in embryos; as well as the lack of information on the negative effect that the removal of a blastomere, or blastomeres, may have on the further development of the embryo [10].

Biopsy that would be performed at an earlier stage, at the level of a four-cell embryo, could damage the relation between the future inner cell mass (ICM) and the trophectoderm (TE).

Bearing in mind all of the above, the main strategy for the application of this method is day three embryo biopsy, with the embryo comprising 6 to 8 blastomeres at that moment [2]. The problem with this type of biopsy are the blastomeres which can easily lyse, which would lead to the loss of genetic material, necessitating a new blastomere for analysis.

Compaction occurring at the level between the stage of the eight-cell embryo and the morula stage additionally complicate PGT. During compaction, the cell-cell boundaries disappear, and it becomes impossible to differentiate individual cells, which is why it is difficult to extract only one blastomere [11].

Before the actual blastomere biopsy, the incubation of the embryo in mediums devoid of calcium and magnesium is necessary, in order to slow down the creation of bonds among cells and facilitate the biopsy. When the genetic material of the blastomere is sent for PCR analysis, the recommended method of oocyte fertilization is ICSI. In case the fertilization method is classic IVF, contamination and amplification of the sperm DNA instead of the embryo DNA may occur, which is why this method is not recommended.

TROPHECTODERM BIOPSY

Trophectoderm biopsy (TE) of the blastocyst can be performed on the fifth or sixth day upon fertilization. The advantage of this method is the possibility of performing a biopsy of a higher number of cells of the trophectoderm (5 to 10 cells) [10], without damaging the ICM [12]. Analysis of a greater number of cells is the preferred method in the diagnostics of monogenic disorders [2]. This number of cells can be considered representative for the entire embryo, except in the case of placental mosaicism [3], which has been observed in over 1% of pregnancies [13]. Studies have shown that blastocytes are characterized by a high level of mosaicism, which is why TE cells cannot be considered suitable for PGT analysis [3].

Blastocyst biopsy with cryopreservation has been the standard for performing some of the PGT methods for a certain period of time now [2]. Blastocyst freezing by means of the vitrification method upon biopsy provides time for all the necessary analyses [2]. The main problem of blastocyst biopsy is the fact that only a limited number of embryos reaches this stage and the appropriate quality, despite the refined cultivation mediums. The embryos that do not reach the blastocyst stage may have a high percentage of aneuploidies, which include chromosomes X, Y, 16, 18 and 21 [10].

GENETIC ANALYSES

Methods which were more frequently used in the past for the analysis of genetic material obtained through biopsy are the following: PCR, FISH (fluorescence in situ hybridization), CGH, SNP (single nucleotide polymorphism), while today these are being replaced by the NGS method [10]. The selection of the appropriate method depends on medical indications.

PCR

The PCR method was used for the detection of gene-level mutations, for the detection of the number of trinucleotide repeats, and for determining the sex of the embryo [2]. The two main problems of the PCR method in PGT-M testing are the following: sample contamination and allele dropout [10].

PCR of a single cell is a sensitive method as there is the danger of the amplification of foreign DNA (DNA of the cumulus cells or sperm DNA). In order to avoid this problem, the procedure is carried out in a special PCR room with positive air pressure, and the method of fertilization is exclusively ICSI [2]. The solution to this problem would be multiplex PCR which would identify all 4 parental alleles, which would, in turn, ensure that the amplified DNA is exclusively of embryonic origin [2].

The second problem is DNA allele dropout or preferential amplification, whereby one of the two alleles is preferentially amplified in relation to the other, which, in dominant heterozygotes, may produce a falsely negative or falsely positive result [10].

FLUORESCENCE IN SITU HYBRIDIZATION – FISH

Fluorescence in situ hybridization (FISH) was first used in PGT in 1991 for the purpose of analyzing embryonic chromosomes in order to determine the sex of the embryo and chromosome aberrations, primarily aneuploidies [2].

As opposed to the PCR method, in FISH, there is no risk of sample contamination and we are therefore not limited only to the ICSI method in oocyte fertilization. The chromosomes most commonly analyzed are X, Y, 13, 16, 18, 21, and 22 [7]. By repeating the cycles and including a higher number of analyses, the efficiency of the procedure is diminished, and the probability of false positive and false negative results is increased. As FISH can be used to analyze only certain chromosomes, and since the number of reported false positive and false negative results is high, this method is no longer used in preimplantation genetic testing [14].

COMPARATIVE GENOMIC HYBRIDIZATION AND MICROARRAY- CGH

In the meantime, new genetic methods have been developed, enabling simultaneous analysis of all chromosomes with far greater precision. One of these methods is metaphase comparative genomic hybridization - mCGH [2]. Although this molecular cytogenetic method could reliably detect aneuploids, its weakness lay in the time necessary for analysis (3 to 5 days) which is why embryo transfer could not be performed within the required time interval. Abandoning the previous freezing method (slow freezing) and employing the more sophisticated vitrification method, which is still in use nowadays (survival of blastocysts upon thawing is over 96%) [15], enabled the application of mCGH. Vitrification upon biopsy provides enough time for genetic analysis and interpretation of results and allows for embryo transfer to be performed at a time of optimal endometrial receptivity [14].

Subsequently, mCGH was substituted with the microarray-CGH method. The advantage of this technique over other methods lies in the shortening of the analysis time to one day, in increasing the number of chromosomes analyzed, as well as in a more precise detection of aneuploidies [2].

SNP and NGS

The SNP method discovers, not only aneuploidies, but also duplications and deletions, and it can also provide information on the origin of the chromosomes from the father and the mother in uniparental disomy (UPD)[2],[16].

Today, NGS is used as the standard in clinical practice. This method includes previous whole genome amplification, which enables performing multiple analyses, at the same time, on only one cell. NGS detects mutations, highly polymorphic sequences, aneuploidies, as well as the epigenetic profile [17]. Targeted NGS strategy, focused on the amplification and analysis of specific sequences, has shown a much greater power of detecting mosaicism than all the previous methods [8].

APPLICATION

Three groups of hereditary diseases can be diagnosed with the use of PGT [3]:

• monogenic disorders, i.e., single gene defects,

• trinucleotide repeat disorders,

• chromosome aberrations.

MONOGENIC DISORDERS

Monogenic disorders (single gene defects) can have autosomal dominant inheritance, autosomal recessive inheritance and X-linked inheritance.

The first autosomal dominant diseases to be diagnosed with the PGT-M method were: Marfan syndrome, familial adenomatosis, myotonic dystrophy, and brittle bone disease (lat. Osteogenesis imperfecta). Nowadays, PGT-M is used for diagnosing most autosomal dominant diseases [3].

Some of the autosomal recessive diseases that can be diagnosed with the PGT-M method are the following: cystic fibrosis, sickle cell anemia, Tay-Sachs disease, spinal muscular atrophy, beta thalassemia, adrenogenital syndrome, and hypophosphatemia.

Beta thalassemia is caused by mutation in the beta-globin gene. However, there is a large number of different mutations within the beta-globin gene, especially among different ethnic groups, which additionally complicates PGT-M [3].

Bearing in mind the fact that cystic fibrosis is the most common single gene autosomal recessive disorder in Caucasians, the predominant use of PGT-M is justified, in cases where this disease is suspected. What may impede this practically routine procedure is the existence of 800 different mutations which are linked to the development of this pathological state [3].

Thanks to the application of PGT-M, when there is suspicion of X-linked disorders, it is possible to perform the selection of embryos that are not carriers of the mutation for embryo transfer, regardless of sex – healthy male and female embryos with a normal karyotype [3].

TRINUCLEOTIDE REPEAT DISORDERS

It is possible to diagnose trinucleotide repeat disorders occurring in the presence of dynamic mutations with the application of the PGT-M method.

The number of triplet repeats increases from generation to generation, which results in a more severe clinical presentation and an earlier onset of the disease in the next generation [14].

Huntington’s disease and the fragile X syndrome were the first diseases in the group of trinucleotide repeat disorders to be diagnosed with the PGT-M method [3].

There are cut-off values defined for all trinucleotide repeat disorders. PGT-M enables the differentiation of the embryos that will develop disease caused by mutation from the healthy embryos.

CHROMOSOME ABERRATIONS

The third group of diseases diagnosed by PGT are chromosome aberrations. Chromosome aberrations can be numerical and structural.

Numerical autosomal chromosome aberrations are mostly lethal, with the exception of trisomy 13, 18, and 21, while in sex chromosomes some of the aneuploidies are compatible with life (Turner syndrome - 45, XO; Klinefelter syndrome - 47, XXY; trisomy X - 47, XXX; Jacob’s syndrome - 47, XYY), and they can all be detected with PGT methods.

Structural chromosome aberrations may be balanced and unbalanced. PGT-SR diagnostics discovers the carriers of balanced chromosomal rearrangements, of which the most common ones in the population are balanced translocations. The carriers are characterized by a normal phenotype, however, the problem of infertility, recurrent miscarriages, and birth of children with chromosomal anomalies often occur [6].

ETHICAL CONSIDERATIONS OF PGT

When speaking of PGT, it is necessary to consider the ethical and moral aspects in detail. In the process of IVF, multiple embryos may be obtained, however, embryos with a genetic load will remain unused. The question remains as to what happens with the residual embryos [18]. The current prevailing attitude within the American judiciary and health policy systems is that discarding embryos at this stadium is far more ethically acceptable than the destruction of the fetus during abortion [18].

On the other hand, there are certain oppositions to PGT, stemming from the same ethical reasons as the opposition to gene therapy and genetic engineering. Selective implantation unequivocally leads to the prevention of the existence of certain genotypes, whereby genetic diversity is jeopardized and a certain type of disability discrimination is developed. The proponents of this attitude suggest that this is a mockery of the true meaning of parenthood, depriving the parents and the children of an opportunity for personal and moral growth, which is achieved through maximal utilization of the potentials provided by nature [18].

Decreasing genetic diversity, as a problem of utmost importance, has been brought forward by representatives of persons with disability. They state, as a key argument, the fact that expensive and unnatural procedures of selecting embryos without abnormalities in the genome, send a message of discrimination towards persons with disabilities. Although the reasonableness of this claim is not to be disputed, there is no way to limit the reproductive liberties of couples who seek to reduce the risk of giving birth to a child with disability [18].

All the techniques currently in use have been developed with the aim of favoring health over disease. The possibility of abusing these techniques for the purpose of choosing the sex of the embryo, or favoring any other trait, which is not connected with health issues, is undoubtedly present. This is the very reason for concern expressed by bioethicians [18].

A justified and acceptable reason for selecting the sex of a child is the existence of a high risk of developing a disorder that is X-linked or Y-linked. Selection of the baby’s sex for the purpose of balancing the family ratio of boys and girls has not met with approval [19].

In families where there is already a child with a severe monogenic disorder or in couples at high risk of the occurrence of aneuploidies, the use of PGT, ethically speaking, is being met with approval, as it enables avoiding abortion or early death of the newborn and enables couples to have healthy children. Additionally, the use of PGT for the purpose of donating stem cells or tissue to a sick brother or sister, the so-called preimplantation tissue typing, is also being met with approval in most countries of the European Union [20].

The selection of traits linked to the development of certain talents, certain physical properties, or any other traits that are not directly linked to health, is being met with strong disapproval [19]. David King expresses concern regarding this type of offspring selection, as it would imply a determinism that would be far stronger than the genes themselves. He feels that the gene pool would thereby be jeopardized, since it would be influenced by temporary culturological concepts aimed at producing the perfect human being [19].

CONCLUSION

PGT enables a more precise selection of embryos of the best quality, with a clear moral goal – the birth of a healthy euploid child.

Bearing in mind the sensitivity and the resolution of the NGS method, which is used today as the diagnostic standard, it is clear why the application of NGS in PGT, when compared to methods employed earlier, significantly increases the success rate of conception in IVF.

The data currently available in literature provide us with clear information on the advancement of the methods applied in preimplantation genetic testing, however, they also open the possibility for the development of new, less invasive, and noninvasive methods of analyzing and assessing the quality of embryos, all for the purpose of producing healthy offspring.

The importance and benefit of developing PGT is undeniable. However, we must always bear in mind the broader picture, in order to preempt potential abuse of the methods developed so far, and not lose sight of the ethical and moral goals of PGT

LIST OF ACRONYMS AND ABBREVIATIONS

CGH – comparative genomic hybridization

DNA – deoxyribonucleic acid

ESHRE – European Society for Human Reproduction and Embryology

FISH – fluorescence in situ hybridization

HLA typing – human leukocyte antigen typing

ICM – inner cell mass ICSI – intracytoplasmic sperm injection

IVF – in vitro fertilization

mCGH – metaphase comparative genomic hybridization

mtDNA – mitochondrial

DNA NGS – next generation sequencing

PCR – polymerase chain reaction

PGD – preimplantation genetic diagnostics

PGS – preimplantation genetic screening

PGT – preimplantation genetic testing

PGT-A – preimplantation genetic testing for aneuploidies

PGT-SR – preimplantation genetic testing for chromosomal structural rearrangements

PGT-M – preimplantation genetic testing for monogenic disorders (single gene defects) S

NP – single nucleotide polymorphism T

E – trophectoderm

TESE – testicular sperm extraction

UPD – uniparental disomy

  • Conflict of interest:
    None declared.

Informations

Volume 2 No 2

Jun 2021

Pages 52-63
  • Keywords:
    preimplantation genetic testing – PGT, in vitro fertilization – IVF, embryo
  • Received:
    02 February 2021
  • Revised:
    13 May 2021
  • Accepted:
    17 May 2021
  • Online first:
    25 June 2021
  • DOI:
  • Cite this article:
    Jeremić A, Vuković D, Subanović S, Broćić J, Macanović B. Preimplantation genetic testing. Serbian Journal of the Medical Chamber. 2021;2(2):52-63. doi: 10.5937/smclk2-30790
Corresponding author

Ana Jeremić
The Obstetrics and Gynecology Clinic “Narodni front”
62 Kraljice Natalije Street, 11000 Belgrade, Serbia
E-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.


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REFERENCES

1. Carvalho F, Coonen E, Goossens V, Kokkali G, Rubio C, Meijer - Hoogeveen M, et al. ESHRE PGT Consortium Steering Commitee. Hum Reprod. 2020.[CROSSREF]

2. Traeger-Synodinos J, Staessen C. Preimplantation genetic diagnosis. In Sermon K VS. Textbook of Human Reproductive Genetics. Third Edition ed.: Cambridge University Press; 2014. p. 347-79.[CROSSREF]

3. Harper J. Preimplantation genetic diagnosis. In Elder K, Dale B. In-Vitro Fertilization. Third Edition ed.: Cambridge University Press; 2011. p. 238-51.[CROSSREF]

4. Yaron Y, Hiersch L, Gold V, Peleg-Schalka S, Malcov M. Genetic analysis of the embryo. In Gardner D, Weissman A, Howles C, Shoham Z. Textbook of Assisted Reproductive Techniques. Volume 1: Laboratory Perspectives. Fifth Edition ed.: CRC Press; 2018. p. 359-72.

5. Montag M. Polar body biopsy and its clinical application. In Gardner D, Weissman A, Howles C, Shoham Z. Textbook of Assisted Reproductive Techniques. Volume 1: Laboratory Perspectives. Fifth Edition ed.: CRC Press; 2018. p. 339-49.

6. Yatsenko S, Rajkovic A. Chromosomal causes of infertility. In Sermon K, Viville S. Textbook of Human Reproductive Genetics.: Cambridge University Press; 2014. p. 213-48.

7. Stouffs K, Lissens W, Seneca S. Severe male factor infertility. In Gardner D, Weissman A, Howles C, Shoham Z. Textbook of Assisted Reproductive Techniques. Volume 1: Laboratory Perspectives. Fifth Edition ed.: CRC Press; 2018. p. 326-38.

8. Maxwell S, Colls P, Hodes-Wertz B, McCulloh D, McCaffrey C, Wells D, et al. Why do euploid embryos miscarry? A case-control study comparing the rate of aneuploidy within presumed euploid embryos that resultet in misscarriage or live birth using next - generation sequencing. Fertility Sterility. 2016; 106(6): p. 1414-9.[CROSSREF]

9. Sallevelt S, Dreesen J, Drusedau M, Spierts S, Coonen E, van Tienen F, et al. Preimplantation genesis diagnosis in mitochondrial DNA disorders: challenge and success. Journal of Medical Genetics. 2013; 50(2): p. 125-32.[CROSSREF]

10. Kofinas J, McCaffrey C, Grifo J. Human embryo biopsy procedures. In Gardner D, Weissman A, Howles C, Shoham Z. Textbook of Assisted Reproductive Techniques. Volume 1: Laboratory Perspectives. Fifth Edition ed.: CRC Press p. 168-76.

11. Carlson B. Cleavage and Implantation. In Carlson B. Human Embryology and Developmental. Fifth Edition ed.: Saunders; 2013.

12. Carlson B. Formation of Germ Layers and Early Derivatives. In Carlson B. Human Embryology and Developmental Biology. Fifth Edition ed.: Saunders; 2013.

13. Baart E, Van Opstal D. Chromosomes in early human embryo development. In Sermon K, Viville S. Textbook of Human Reproductive Genetics.: Cambridge University Press; 2014. p. 117-51.

14. Lewin J, Wells D. Preimplantation genetic diagnosis for infertility. In Gardner D, Weissman A, Howles C, Shoham Z. Textbook of Assisted Reproductive Techniques. Volume 1: Laboratory Perspectives. Fifth Edition ed.: CRC Press; 2018. p. 350-8.

15. Maggiulli R, Giancani A, Cimadomo D, Ubaldi F, Rienzi L. Human Blastocyst Biopsy and Vitrification. J Vis. 2019.[CROSSREF]

16. Slater H, Bayle D, Ren H, Cao M, Bell K, Nasioulas S, et al. High-Resolution Identification of Chromosomal Abnormalities Using Oligonucleotide Arrays Contining 116,204SNPs. The American Jurnal of Human Genetics. 2008; vol. 77(issue 5): p. 709-26.[CROSSREF]

17. Kumar P, Zamani Esteki M, van Der Aa N, Voet T. How to analyze a single blastomere: Application of whole genome technologies: microarrays and next generation sequencing. In Sermon K VS. Textbook of Human Reproductive Genetics.: Cambridge University Press; 2014. p. 42-77.

18. Lagay F. Preimplantation Genetic Diagnosis. AMA Journal of Ethics, August 2001; Virtual Mentor. 2001;3(8).[CROSSREF]

19. King DS. Preimplantation genetic diagnosis and the “new” eugenics. J Med Ethics. 1999;25(2):176-82.[CROSSERF]

20. Asplund K. Use of in vitro fertilization – ethical issues. Upsala Journal of Medical Sciences. 2019;192-9.[CROSSREF]

1. Carvalho F, Coonen E, Goossens V, Kokkali G, Rubio C, Meijer - Hoogeveen M, et al. ESHRE PGT Consortium Steering Commitee. Hum Reprod. 2020.[CROSSREF]

2. Traeger-Synodinos J, Staessen C. Preimplantation genetic diagnosis. In Sermon K VS. Textbook of Human Reproductive Genetics. Third Edition ed.: Cambridge University Press; 2014. p. 347-79.[CROSSREF]

3. Harper J. Preimplantation genetic diagnosis. In Elder K, Dale B. In-Vitro Fertilization. Third Edition ed.: Cambridge University Press; 2011. p. 238-51.[CROSSREF]

4. Yaron Y, Hiersch L, Gold V, Peleg-Schalka S, Malcov M. Genetic analysis of the embryo. In Gardner D, Weissman A, Howles C, Shoham Z. Textbook of Assisted Reproductive Techniques. Volume 1: Laboratory Perspectives. Fifth Edition ed.: CRC Press; 2018. p. 359-72.

5. Montag M. Polar body biopsy and its clinical application. In Gardner D, Weissman A, Howles C, Shoham Z. Textbook of Assisted Reproductive Techniques. Volume 1: Laboratory Perspectives. Fifth Edition ed.: CRC Press; 2018. p. 339-49.

6. Yatsenko S, Rajkovic A. Chromosomal causes of infertility. In Sermon K, Viville S. Textbook of Human Reproductive Genetics.: Cambridge University Press; 2014. p. 213-48.

7. Stouffs K, Lissens W, Seneca S. Severe male factor infertility. In Gardner D, Weissman A, Howles C, Shoham Z. Textbook of Assisted Reproductive Techniques. Volume 1: Laboratory Perspectives. Fifth Edition ed.: CRC Press; 2018. p. 326-38.

8. Maxwell S, Colls P, Hodes-Wertz B, McCulloh D, McCaffrey C, Wells D, et al. Why do euploid embryos miscarry? A case-control study comparing the rate of aneuploidy within presumed euploid embryos that resultet in misscarriage or live birth using next - generation sequencing. Fertility Sterility. 2016; 106(6): p. 1414-9.[CROSSREF]

9. Sallevelt S, Dreesen J, Drusedau M, Spierts S, Coonen E, van Tienen F, et al. Preimplantation genesis diagnosis in mitochondrial DNA disorders: challenge and success. Journal of Medical Genetics. 2013; 50(2): p. 125-32.[CROSSREF]

10. Kofinas J, McCaffrey C, Grifo J. Human embryo biopsy procedures. In Gardner D, Weissman A, Howles C, Shoham Z. Textbook of Assisted Reproductive Techniques. Volume 1: Laboratory Perspectives. Fifth Edition ed.: CRC Press p. 168-76.

11. Carlson B. Cleavage and Implantation. In Carlson B. Human Embryology and Developmental. Fifth Edition ed.: Saunders; 2013.

12. Carlson B. Formation of Germ Layers and Early Derivatives. In Carlson B. Human Embryology and Developmental Biology. Fifth Edition ed.: Saunders; 2013.

13. Baart E, Van Opstal D. Chromosomes in early human embryo development. In Sermon K, Viville S. Textbook of Human Reproductive Genetics.: Cambridge University Press; 2014. p. 117-51.

14. Lewin J, Wells D. Preimplantation genetic diagnosis for infertility. In Gardner D, Weissman A, Howles C, Shoham Z. Textbook of Assisted Reproductive Techniques. Volume 1: Laboratory Perspectives. Fifth Edition ed.: CRC Press; 2018. p. 350-8.

15. Maggiulli R, Giancani A, Cimadomo D, Ubaldi F, Rienzi L. Human Blastocyst Biopsy and Vitrification. J Vis. 2019.[CROSSREF]

16. Slater H, Bayle D, Ren H, Cao M, Bell K, Nasioulas S, et al. High-Resolution Identification of Chromosomal Abnormalities Using Oligonucleotide Arrays Contining 116,204SNPs. The American Jurnal of Human Genetics. 2008; vol. 77(issue 5): p. 709-26.[CROSSREF]

17. Kumar P, Zamani Esteki M, van Der Aa N, Voet T. How to analyze a single blastomere: Application of whole genome technologies: microarrays and next generation sequencing. In Sermon K VS. Textbook of Human Reproductive Genetics.: Cambridge University Press; 2014. p. 42-77.

18. Lagay F. Preimplantation Genetic Diagnosis. AMA Journal of Ethics, August 2001; Virtual Mentor. 2001;3(8).[CROSSREF]

19. King DS. Preimplantation genetic diagnosis and the “new” eugenics. J Med Ethics. 1999;25(2):176-82.[CROSSERF]

20. Asplund K. Use of in vitro fertilization – ethical issues. Upsala Journal of Medical Sciences. 2019;192-9.[CROSSREF]


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