The enzymes that are used in gene manipulation are described in Chapter 5. The discovery of restriction endonucleases, and the use of type II restriction enzymes in generating DNA fragments is outlined. The three types of fragment ends (blunt or flush-ended, 5’ protruding and 3’ protruding) enable DNA from different sources to be joined together, using the enzyme DNA ligase. DNA modifying enzymes include polymerases (DNA and RNA polymerases and reverse transcriptase), nucleases that act on the ends of molecules (exonucleases) or within a DNA strand (endonucleases), transferases, kinases and phosphatases. The enzymes make up the ‘toolkit’ that allows DNA to be manipulated in the test tube (in vitro) to generate recombinant molecules.

Bioinformatics is discussed in Chapter 11. The complex nature of the subject and its interaction with other disciplines are outlined, and the inter-dependence of bioinformatics, the development of computer hardware and the internet is stressed. The nature and range of biological databases are outlined, from the inception of nucleic acid databases in the 1970s to the present breadth of primary and secondary databases that are repositories for information on nucleic acid and protein sequences, interactions between cellular components, biochemical pathways, pharmacological targets and many other data sets derived from existing information. Genome sequence databases are used to illustrate the tools needed to assemble, collate, annotate and interrogate the data, and the impact of bioinformatics in enabling experiments and protocols to to be conducted in silico is discussed.

To introduce the subject, the history of genetics since Mendel’s work which was rediscovered in 1900 is outlined. The discovery of the structure of DNA in 1953 marked the start of the molecular genetics era. When restriction enzymes and DNA ligase were discovered, DNA fragments could be cut and joined, with the first recombinant DNA molecules generated in 1972. Rapid methods for sequencing DNA were developed in the late 1970s and eventually were improved to the level needed to enable the Human Genome Project to be undertaken. The completion of this in 2003 marked the start of the ‘post-genomic era’ that led to further development of the technology and a reduction in time and cost of genome sequencing. We are now firmly in the post-genomic era, where DNA technology is having a major impact in areas such as transgenic plants and animals, genome editing, diagnosis and treatment of disease, forensic analysis and personalised medicine.

In Chapter 13, what is needed to analyse cloned genes, and how this can be achieved are considered. The ultimate structural information is the sequence of the gene, and thus DNA sequencing has become a standard part of any cloning experiment. Although genome sequencing has led to a greater emphasis on bioinformatics-based analysis, methods such as restriction mapping, gel mobility shift assays, DNA footprinting and the various blotting techniques are still needed to confirm and link structure and function. The yeast hybrid systems have become important for analysing DNA∼protein, RNA∼protein and protein∼protein interactions, and DNA microarray technology and its extensions have changed the way in which gene expression is investigated. High-throughput analysis at genome and transcriptome levels is now routine and cost-effective. Genome projects have now generated vast amounts of sequence data, and the fields of comparative genomics and structural genomics are well established. Sequencing large numbers of genomes has now become possible and is leading to new discoveries and therapeutic interventions based on genome analysis.

Medical and forensic applications of recombinant DNA are described in Chapter 15. The range of genetically based diseases is outlined, and potential therapies discussed, covering diagnosis of infection, comparative genomics, development of vaccines, therapeutic antibodies and xenotransplantation. Treatment using gene therapy approaches is described, and the relatively limited success of gene therapy is considered in the context of its initial promise and the expectations that emerged from this. RNA-based therapies are covered by discussing RNA interference and antisense oligonucleotides, and the medical applications of genome editing are considered. The CCR5 controversy, known as the ‘CRISPR babies scandal’, is mentioned as an example of how the overall system can fail to prevent unethical practices when these are driven by determined scientists and clinicians. DNA profiling for analysis of DNA is described, and its use in forensic, legal and other applications is outlined.

Chapter 4 describes how living systems are organised at the molecular level, beginning with the chemistry of carbon-based systems and the concept of emergent properties. The genetic code and the flow of information are introduced as a key central theme, and the structure of DNA and RNA is presented. An outline of gene structure and organisation in prokaryotes and eukaryotes is followed by the description of transcription and translation as the mechanisms by which genes are expressed. A broader look at how genomes are organised leads to an outline of the transcriptome and proteome as two important concepts that are key to understanding how the genome functions in adaptive and developmental contexts.

Meiosis comprises fundamental processes that permit sexual reproduction and species evolution. In addition to producing haploid gametes, it provides for a stochastic distribution of maternally and paternally inherited chromosomes, which undergo allelic recombination thereby increasing genetic variability in the next generation. Thus it provides for diversity within a population, and is essential for the formation of euploid germ cells that will contribute to a euploid healthy embryo after fertilization. Meiosis is therefore the basis for maintaining genomic integrity, high developmental potential, and health of the embryo and offspring, and normal fertility in males and females [1,2]. Furthermore, it is the basis of changes in the genome that are important for adaptation and evolution of species.

There are two types of genomic information accurately inherited or maintained following DNA replication: the entire genomic DNA sequence and epigenetic information in the form of patterns of CpG methylation on a subset of the genome. These DNA methylation patterns are crucially important for mammalian development, primarily because they regulate gene transcription. There are cyclical declines and increases in DNA methylation during gametogenesis and embryogenesis, and the oscillations in methylation occurring across generations must depend on de novo methylation and demethylation processes to rearrange the existing methylation patterns. Within any single reproductive cycle, changes in genomic methylation are the outcome of a linked sequence of active and passive processes that rearrange genomic methylation to generate epigenetic milestones, each with a defined future role. For example, genomic imprints are established during gametogenesis by de novo methylation to generate mature gametes with complete complements of paternal methylation imprints in sperm and maternal methylation imprints in oocytes. The establishment of a collective set of imprints within a sperm and an oocyte is an epigenetic milestone, whose purpose after sperm–oocyte fusion is to ensure monoallelic expression of imprinted genes during fetal development. We postulate that another essential epigenetic milestone is found in the blastocyst-stage embryo; this milestone is achieved at the generation, through the poorly understood process of epigenetic reprogramming, of pluripotent embryo stem cells, whose role is to contribute to the development of the conceptus. Primordial germ cells (PGCs), largely devoid of genomic methylation and poised to differentiate into gametes with sex-specific imprints, and adult stem cells, poised to differentiate into organs, would be other possible epigenetic milestones. The developmental locations of three fundamental milestones (gametes, blastocyst, and PGCs), and the epigenetic processes by which they are crafted, are depicted in Figure 5.1.

The release of the human genome sequence has revealed that our genes represent only a minor fraction, with exons making up less than 2% of our DNA. In contrast, transposable elements represent some 45% of the genomic mass. Contrary to other species such as Drosophila or plants, these elements are not clustered in specific chromosomal regions in mammals, but are rather scattered throughout the genome, residing between but also inside genes. The transposon landscape of our genome reflects an evolutionary tug-of-war between integration and propagation events orchestrated by these elements, and counteracting defense mechanisms exerted by the host.

The genetic causes of male infertility are highly heterogeneous, and a large portion of these causes remains unexplained. More than 2300 testes-specific genes may contribute to male infertility [1]. Primary testicular disorders affecting spermatogenesis are commonly associated with abnormal semen parameters, including sperm concentration (oligozoospermia or azoospermia), morphology, motility, and vitality. Studies in infertile men have demonstrated that up to 20% carry constitutional chromosome aberrations [2–5]. Genomic aberrations found in these patients include numerical abnormalities, such as Klinefelter syndrome and its variants; XYY karyotype; testicular disorders of sex development, such as XX males; structural chromosome rearrangements, including Robertsonian translocations, balanced reciprocal translocations and inversions; as well as submicroscopic DNA copy number alterations (microdeletions and microduplications) encompassing genes associated with spermatogenesis or gonadal development.

This chapter on the ethics of human reproductive genetics focuses on current debates in three corners of the field of medically assisted reproduction (MAR). First, we discuss preimplantation genetic testing (PGT). Until recently, ethical debates concentrated on PGT “on indication,” formerly termed preimplantation genetic diagnosis (PGD), namely testing of embryos on behalf of couples mostly at high risk of transmitting a specific genetic disorder. As important, however, is the ethics of (the offer of) routine testing of in vitro fertilization (IVF) embryos, formerly termed preimplantation genetic screening (PGS). Although it is still unclear what the precise value of such screening may turn out be, the scenario of next generation sequencing (NGS)-based “comprehensive PGS” raises a number of challenging ethical issues. Secondly, we discuss carrier screening. In MAR, until recently this type of screening targeted gamete donors. Presently, however, a growing number of clinics have started offering carrier screening to applicants of MAR as well. As discussed in Chapter 10, new NGS technologies are expected to make broad-scope testing for many genetic conditions feasible and affordable. Accordingly, what should we test gamete donors and applicants of MAR for and why? Finally, we add a brief ethical reflection on what may become a (or the) real revolution in human reproduction: germline genome editing.

Reproduction in humans is considered to be a relatively inefficient process, as the chance of achieving a spontaneous pregnancy after timed intercourse is only approximately 30%. This is much lower than the 70–90% estimated for other species, such as the rhesus monkey, the captive baboon, or rodents and rabbits. The inefficiency of human reproduction is mainly explained by the high incidence of preclinical losses, an estimated 40–60% of all conceptions. Early pregnancy loss is mainly explained due to the occurrence of chromosome abnormalities, which have been identified in most spontaneous abortion samples investigated.

Preimplantation genetic testing (PGT), until recently known as preimplantation genetic diagnosis (PGD), is an early form of prenatal testing for couples at high risk of transmitting a genetic condition to their offspring, either for monogenic disorders (PGT-M) or chromosomal structural rearrangements (PGT-SR). The goal is to test for the specific genetic status in cells biopsied from oocytes/zygotes or embryos obtained in vitro through assisted reproductive technology (ART) and, following analysis, to transfer to the uterus only those embryos identified as genetically suitable relative to the condition under consideration. The selective transfer of unaffected embryos to the uterus for implantation means that PGT minimizes the need to consider the termination of affected pregnancies. This advantage of PGT means that it has become a widely acceptable alternative to conventional prenatal diagnosis. Of course, PGT is not applied without some ethical concerns. In patients with high risk for transmitting specific genetic conditions to their offspring, including serious single gene disorders and chromosomal rearrangements, there is usually little ethical debate. However, the increased availability and emergence of new uses, such as PGT for autosomal dominant late-onset disorders, cancer predisposition syndromes, PGT for histocompatibility (HLA) typing, and mitochondrial DNA (mtDNA) mutations, are associated with greater ethical controversy and important ethical questions (see Chapter 15).

Mitochondria are typically described as the powerhouse of the cell, because they are the cytoplasmic organelles responsible for the production of ATP through oxidative phosphorylation. Over the years, it has become clear that their function within the cell is more complex as they are also involved in numerous other processes, including lipid and carbohydrate metabolism, heme biosynthesis, apoptosis, and calcium homeostasis [1]. Human cells contain multiple mitochondria, with the exception of red blood cells that have none. The numbers, mass, morphology, and distribution vary greatly across different cell types, generally depending on the energy demands of the tissues. For instance, sperm contain 20–75 mitochondria in their midpiece, while hepatocytes and muscle cells contain thousands.

In the past decade the development of new technologies and innovations have revolutionized genetic testing. The ability to diagnose and prevent genetic disorders before an existing pregnancy and to detect embryonic and fetal genetic errors has dramatically increased, and has substantially changed the care for couples wanting a child.