Preimplantation Genetic Diagnosis—An Overview

Preimplantation genetic diagnosis (PGD) is gradually widely used in prevention of gene diseases and chromosomal abnormalities. Much improvement has been achieved in biopsy technique and molecular diagnosis. Blastocyst biopsy can increase diagnostic accuracy and reduce allele dropout. It is cost-effective and currently plays an important role. Whole genome amplification permits subsequent individual detection of multiple gene loci and screening all 23 pairs of chromosomes. For PGD of chromosomal translocation, fluorescence in-situ hybridization (FISH) is traditionally used, but with technical difficulty. Array comparative genomic hybridization (CGH) can detect translocation and 23 pairs of chromosomes that may replace FISH. Single nucleotide polymorphisms array with haplotyping can further distinguish between normal chromosomes and balanced translocation. PGD may shorten time to conceive and reduce miscarriage for patients with chromosomal translocation. PGD has a potential value for mitochondrial diseases. Preimplantation genetic haplotyping has been applied for unknown mutation sites of single gene disease. Preimplantation genetic screening (PGS) using limited FISH probes in the cleavage-stage embryo did not increase live birth rates for patients with advanced maternal age, unexplained recurrent abortions, and repeated implantation failure. Polar body and blastocyst biopsy may circumvent the problem of mosaicism. PGS using blastocyst biopsy and array CGH is encouraging and merit further studies. Cryopreservation of biopsied blastocysts instead of fresh transfer permits sufficient time for transportation and genetic analysis. Cryopreservation of embryos may avoid ovarian hyperstimulation syndrome and possible suboptimal endometrium.

Deep impact: sequencing embryo biopsy specimens at increasing depth

Preimplantation genetic screening (PGS) for aneuploidy in embryos after in vitro fertilization (IVF) does not improve reproductive outcome in women over 35: a prospective controlled randomized study

Today, preimplantation genetic diagnosis (PGD) is offered in over 40 centres worldwide for an expanded range of genetic defects causing disease. This very early form of prenatal diagnosis involves the detection of affected embryos by fluorescent in situ hybridization (FISH) (sex determination or chromosomal defects) or by polymerase chain reaction (PCR) (monogenic diseases) prior to implantation. Genetic analysis of the embryos involves the removal of some cellular mass from the embryos (one or two blastomeres at cleavage-stage or some extra-embryonic trophectoderm cells at the blastocyst stage) by means of an embryo biopsy procedure. Genetic analysis can also be performed preconceptionally by removal of the first polar body. However, additional information is then often gained by removal of the second polar body and/or a blastomere from the embryo. Removal of polar bodies or cellular material from embryos requires an opening in the zona pellucida, which can be created in a mechanical way (partial zona dissection) or chemical way (acidic Tyrode's solution). However, the more recent introduction of laser technology has facilitated this step enormously. Different biopsy procedures at different preimplantation stages are reviewed here, including their pros and cons and their clinical applications. The following aspects will also be discussed: safety of zona drilling by laser, use of Ca2+/Mg2+-free medium for decompaction, and removal of one or two cells from cleavage-stage embryos.

Evaluation of: Mir P, Rodrigo L, Mateu E et al. Improving FISH diagnosis for preimplantation genetic aneuploidy screening. Hum. Reprod. 25(7), 1812–1817 (2010).Mir et al. have evaluated the impact of additional hybridization rounds on FISH accuracy in preimplantation genetic screening (PGS) of aneuploidy. In a retrospective analysis of 1000 PGS cycles, embryo biopsy was performed on day 3. A greater number of embryos were diagnosed as chromosomally normal and were available for transfer after discarding ‘false monosomies’ and decreasing the number of noninformative embryos. The error rate for the FISH technique was 17.3% without and 5% with additional probes. A randomized controlled trial is needed to demonstrate improved clinical outcome. However, major obstacles in cleavage-stage PGS are chromosomal mosaicism and incomplete analysis. Therefore, polar body and/or trophectoderm analysis using 24 chromosome microarrays might be better alternatives.

Research towards preimplantation diagnosis of genetic disease was initiated in the UK in the mid 1980s with the aim of helping those couples who would prefer selection to occur at this stage rather than during pregnancy. Following in vitro fertilisation, (IVF), biopsy and removal of 1 or 2 of the totipotent cells from the cleavage stage 3 day old embryo provides the material for molecular genetic diagnosis without interfering with development. Earliest applications were in the avoidance of X-linked disease by sexing embryos and selecting females for transfer to the mother. Initially, polymerase chain reaction (PCR) amplification of DNA from the biopsied blastomeres was performed using primers specific for sequences derived from the Y chromosome and this led to the birth of several normal girls. To reduce the risk of misdiagnosis due to amplification failure, PCR based methods for sexing the embryo now employ both X and Y specific sequences, but the preferred method is currently considered to be fluorescent in situ hybridisation (FISH) with fluorochrome labelled DNA probes to the embryonic nuclei that have been fixed and spread on slides. Dual FISH with probes from X and Y chromosomes allows unequivocal diagnosis of sex and determination of chromosome copy number, avoiding transfer of embryos with abnormal numbers of sex chromosomes, including those with only the maternal X that would be at 50% risk for the X-linked disease. The application of FISH for preimplantation diagnosis has also led to the realisation that chromosomal mosaicism is common at the cleavage stage of development, a finding that has important implications for diagnosis of both dominant single gene disorders and trisomies, as well as for our understanding of early human development. Cloning and sequencing of the relevant genes has enabled the development of methods for the diagnosis of certain recessive single gene disorders in cleavage stage embryos. PCR based methods have to be developed for each condition, sometimes for each family if there is heterogeneity. Preimplantation diagnosis has been successful so far for cystic fibrosis, Tay Sachs disease, and Lesch-Nyhan syndrome. Worldwide, 32 pregnancies have been established following all types of preimplantation diagnosis and with 29 babies born, there is no evidence for any adverse effect on development.

Next-generation sequencing: challenges in reproductive genetics

The widespread use of in vitro fertilization (IVF) throughout the world provides the opportunity to study human development at the very earliest stages before implantation. Nonetheless, the study of human embryos poses a series of unique ethical and moral implications. The unique totipotent nature of a human embryo and its potential to develop into a child necessitates a level of restriction and regulatory control that is not present when studying other cell types. Although some governments outlaw any experimental procedure on human embryonic material, others allow it under appropriate control. In the latter case (e.g., in the UK), experimentation can be justified on the basis of development of a diagnostic test and/or the goal of improving patient care. A further challenge to effective study is the paucity of material available. Much of the work reported in this chapter arises from the study of only single nuclei. For these reasons, research on interphase cytogenetics in human preimplantation embryos is less advanced than in other cell types. Despite this, a fundamental insight into chromosome copy number and nuclear organization can be gleaned from this material through collaboration with an appropriate clinical program. As attested by other chapters in this book, fluorescent in situ hybridization (FISH) was first adopted for research, but clinical applications rapidly followed. Prenatal and cancer diagnostics are the best examples of this,, but the increasing use of assisted reproductive technologies, namely IVF, precipitated the use of FISH in the field of preimplantation genetic diagnosis (PGD). PGD is defined as the diagnosis of genetic disorders in human preimplantation embryos. The purpose is selective implantation of unaffected embryos in the hope of establishing genetically normal ongoing pregnancies. PGD by interphase cytogenetics was first applied for sexing (to screen for sex-linked disorders), then for chromosome translocations, and later for chromosome copy number. In the latter case, termed preimplantation genetic screening (PGS), families at risk of adverse obstetrical outcomes (referral categories include advanced maternal age and recurrent miscarriage) are targeted, rather than families at risk of transmitting inherited disorders in a classical Mendelian fashion. Clinical application of interphase cytogenetics in the IVF world has allowed the subsequent study of chromosome copy number and nuclear organization. This chapter provides an overview of interphase cytogenetics in human embryos, highlighting the progress and the sometimes contentious pitfalls that it has encountered.

Gender does not impact the developmental rate of in vitro fertilization-derived human embryos as confirmed by day 3 blastomere biopsy and preimplantation genetic diagnosis (PGD)

Three-parent in vitro fertilization: gene replacement for the prevention of inherited mitochondrial diseases

Non-invasive preimplantationsexingusingRT-PCRon the spent embryo culture media

Preimplantation Genetic Diagnosis (PGD) is currently an alternative for couples with high risk of pregnancies with genetic anomalies; it offers the possibility of avoiding the need to terminate affected pregnancies, since it allows the selection of unaffected embryos for transfer. PGD for inherited disorders has become extremely accurate (99.5%), and may currently be performed for any single gene disorders in which mutation is identified. PGD has been performed for more than 100 different conditions resulting in the birth of at least 1000 healthy children free of genetic disorder. PGD is presently also used together with preimplantation HLA typing for treatment of affected sibling with genetic and acquired disorders requiring HLA matched stem cell transplantation. This is not only to allow couples to have an unaffected child but also to select a potential donor progeny for stem cell transplantation. In Turkey, thalassemia is the most commonly seen genetic disorder the rate of thalassemia carriers is about 3–4% in Turkey. The majority of our PGD cases are thalassemia carriers. They do not only require thalassemia mutation analysis but also HLA typing for their affected child. In this study PGD results of 236 Turkish couples with or without HLA typing will be presented and discussed. A full diagnosis was achieved in 91.0% of the biopsied samples. In Group I, 17.8% of the analyzed embryos were found to be HLA compatible. HLA compatible and disease free embryos were 12.9% of all diagnosed embryos. In group II, 17.2% of embryos were found to be HLA matched and 71.4% HLA non-matched. The majority of our HLA typing combined with PGD cases were β-Thalassemia carriers (87.9%). The mutations analyzed have high heterogeneity, the most frequent mutation was IVS-I-110 G-A and comprised 46.2% of all mutations. To date, 70 healthy and HLA compatible children have been born. Twenty-five sick children have already been cured with cord blood cell and/or bone marrow transplantation. Twenty-one children are waiting for their newborn siblings to gain sufficient weight and maturity for the donation of stem cells. The successful transplantations have been performed for the following indications: β- Thalassemia (n = 19), Wiskott Aldrich syndrome (n = 2), Glanzmann Disease (n = 1), X-Adrenoleukodystrophy (n=1) and acute myeloid leukemia (n = 1) and Diamond Blackfan anemia (n = 1). This data presents one of the world’s largest experiences on preimplantation HLA typing, and the outcome of stem cell transplantation is the largest number available from one center. Our results indicate HLA typing with or without mutation analysis is a promising and effective therapeutic tool for curation of an affected sibling.

Telomere DNA is significantly reduced in aneuploid human oocytes and embryos

Preimplantation Genetic Testing for Aneuploidy (PGT-A) and Preimplantation Genetic Screening (PGS) have become indispensable tools in In Vitro Fertilization (IVF) to increase pregnancy rates and enhance embryo choice. Analyzing its application in spotting chromosomal abnormalities to reduce implantation failure, miscarriage rates, and the danger of genetic illnesses, this extensive research looks at the technological, clinical, and ethical sides of PGS. Screening techniques have developed to significantly improve accuracy and reliability from early Fluorescent In Situ Hybridization (FISH) to sophisticated Next-Generation Sequencing (NGS). Notwithstanding its benefits, PGS raises some ethical issues and therapeutic limitations, including the debate on genetic determinism, the influence of embryo biopsy on viability, and the likely misclassification of mosaics arising from mosaicism. Moreover, remaining challenges for many patients include PGS's cost burden and availability. As industry advances, non-invasive genetic testing, artificial intelligence-driven embryo selection, and ethical concerns are addressed, and CRIS pen-based genetic therapeutics might help improve IVF success be improved. This paper provides a reasonable evaluation of PGS's prospects, efficiency, and constraints, thereby directing its use in modern reproductive biology. This study aims to contribute to the ongoing discussion on improving IVF outcomes and ensuring ethical integrity in genetic screening by incorporating ethical problems and the most current studies.

Ornithine transcarbamylase (OTC) deficiency is an X-linked co-dominant disorder. A couple, with a previous history of a neonatal death and a therapeutical termination due to OTC deficiency, was referred to our center for preimplantation genetic diagnosis (PGD). The female partner has a nonsense mutation in the exon 9 of the OTC gene (R320X). We carried out nested polymerase chain reaction (PCR) for R320X mutation and fluorescence in situ hybridization (FISH) for aneuploidy screening. Among a total of 11 embryos, two blastomeres per embryo from 9 embryos were biopsied and analyzed by duplex-nested PCR and FISH, and one blastomere per embryo from 2 embryos by only duplex-nested PCR. As a result of PCR and restriction fragment length polymorphism analysis, four embryos were diagnosed as unaffected embryos having the normal OTC gene. Among these embryos, only one embryo was confirmed as euploidy for chromosome X, Y and 18 by FISH analysis. A single normal embryo was transferred to the mother, yielding an unaffected pregnancy and birth of a healthy boy. Based on our results, PCR for mutation loci and FISH for aneuploidy screening with two blastomeres from an embryo could provide higher accuracy for the selection of genetically and chromosomally normal embryos in the PGD for single gene defects.