Spectacular progress in the therapeutic management of patients with severe male infertility was obtained with the implementation of intracytoplasmic sperm injection (ICSI) and has opened avenues to treating previously untreatable cases of infertility. However, it must be recognized that in the vast majority of ICSI cycles, the selection of the spermatozoon followed by the intracytoplasmic injection is done very quickly at low magnification and without detection possibilities for potential pathological morphological anomalies of spermatozoa. Strong correlations between sperm morphological characteristics and male fertility have been demonstrated. Thus, it seems reasonable to implement optimized non-invasive sperm selection techniques in ART based on morphological ’normalcy’ of spermatozoa. Almost 20 years ago, ’motile-sperm organelle morphology examination’ (MSOME) was introduced by Bartoov et al. Using Nomarski differential interference contrast optics (DIC) an improved three-dimensional view of the head and midpiece became available. This chapter will describe different aspects of sperm selection by morphology.

Intracytoplasmic sperm injection (ICSI) was first described in 1992 as the ultimate tool for treating couples plagued by male factor infertility. Since then, it has become the most prevalent assisted reproductive technology (ART) treatment in the world, due to its incredible versatility and expanding cohort of indications for use. ICSI requires both a dedicated laboratory setup and knowledgeable and skilled embryologists in order to competently treat infertile couples. In this chapter, we will briefly describe the procedure, indications, required equipment and proper protocol in order to achieve reliable ICSI results and maintain consistent clinical outcome.

Piezo-ICSI is one of the intracytoplasmic sperm injection (ICSI) techniques using a piezo-actuator. This technique requires the use of a drive unit, a micropipette with a blunt-ended tip and an operation liquid. Piezo-ICSI can be applied for sperm immobilization, opening of the zona pellucida without oocyte deformation, and to semi-automatically break the oolemma membrane without the need for aspiration of cytoplasm into the injection pipette. The piezo-ICSI process is stable and easy and can contribute to standardizing the ICSI technique. In this chapter, we describe the set-up and protocol of piezo-ICSI for human oocytes.

Selection of the best embryo to transfer, that is, the one that will have the highest potential to result in a live birth, is one of the most important decisions in an in vitro fertilization (IVF) laboratory. A zygote forms with the amalgamation of the female and male gametes, and much information about the viability of an embryo can be gained within 16 to 20 hours after insemination. Interaction of the oocyte and sperm should first be examined with an accurate assessment of fertilization.

Since the advent of in vitro fertilization (IVF), conventional insemination has remained essentially unchanged and for most IVF laboratories involves overnight co-incubation of sperm with oocytes. Optimization of insemination methods to better mimic in vivo conditions may achieve better embryo quality and clinical outcomes. A short insemination protocol is one method that addresses this divergence. Studies on a short insemination protocol (1–4 hours instead of 15–20 hours) demonstrate similar fertilization rate, embryo cleavage rates and pregnancy rates. Since human sperm capacitation requires only 45 minutes of contact between oocytes and spermatozoa, these observations are not unexpected. These findings and the information presented in this chapter illustrate that short insemination is an effective method for IVF that, further- more, provides benefits for programmes using time-lapse (TL) imaging. While historically only intracytoplasmic sperm injection (ICSI) oocytes can be observed for fertilization with TL imaging, short co-incubation insemination facilitates early TL culture for all IVF cycles, making early-morning fertilization checks obsolete and freeing staff to perform other duties. Furthermore, TL imaging affords the advantage of identifying oocytes that show normal fertilization outside the standard window of time used for fertilization assessment.

The implementation of intracytoplasmic sperm injection (ICSI) in the field of assisted reproduction has allowed fertilization in couples with previous fertilization failure. However, complete fertilization failure after ICSI shows a prevalence of up to 3%. Moreover, low or moderate fertilization (<30%) can be observed for some patients. In such cycles of bad prognosis, the underlying problem is most likely related to a failure of oocyte activation. In vivo, a sperm-derived enzyme mediates the physiological stimulus of the complex process of oocyte activation. In vitro, a complete fertilization failure can eventually be compensated by several artificial oocyte activation (AOA) techniques, all of which aim for an increase in internal Ca²⁺. AOA is only applied with the proper indication and not as a routine method. The lack of standardized AOA methodologies led to the development and launch of a ready-to-use AOA solution and the number of publications dealing with AOA is constantly increasing. This chapter describes a proven protocol for AOA that has been used in clinical practice for more than two decades.

Appropriate procedures must be documented that describe all the steps performed during an insemination of oocytes for in vitro fertilization (IVF). These procedures must be designed bothe to optimize the fertilization of oocytes and to prevent circumstances that increase the risk of introduction, transmission or spread of communicable diseases through the collection, preparation, culture and/or transfer of patients’ gametes and embryos.

For most people, the most obvious thought or image that sexual reproduction brings to mind is that of sexual intercourse, a mating between two individuals of opposite sexes, which will result in the birth of their common offspring. While biparental reproduction is certainly the most common mode of sexual reproduction among all eukaryotes, it is not the only one, and the way it is carried out can depart substantially, in many different ways, from the ‘canonical’ description above. What is common to all these modes is that two distinct sexually compatible individuals (parents) undertake a sexual exchange that leads to the generation of new individuals with a genetic constitution obtained from the association and/or the reassortment of those parents’ genomes. The key event in this mode of reproduction, technically called amphigony, is the fusion of two gametes or two nuclei functioning as gametes (syngamy), each produced by one parent, to form a zygote. While in species with anisogamy (i.e. with distinct male and female gametes; Chapter 4), only gametes of opposite sex are compatible, the two individuals that produce them are not necessarily a male and a female.

We are all familiar with the changes in an organism during development, followed by its reproduction, which are repeated generation after generation. Biologists describe this development–reproduction sequence as the life cycle: the series of transformations and reproductive events that, from a given stage of life of an organism, leads to the corresponding stage in a subsequent generation. We can describe a biological cycle as going from zygote to zygote, but also from adult to adult, or from embryo to embryo: in a cyclical process, the choice of the ‘initial phase’ is arbitrary or conventional, as the notorious ‘the chicken or the egg’ dilemma beautifully illustrates.

In the course of their lives, organisms spend time and energy on a number of activities and functions, of which reproduction is only one – think of growth, defence against predators and pests, and others. How many resources are used for reproduction, how much time is devoted to it and how this time is distributed over the course of life are all elements that characterize the different reproductive strategies. From an even wider perspective, in those organisms that at certain times in their lives can opt for one or another reproductive mode (e.g. sexual or asexual reproduction, as in many plants and many marine invertebrates), a reproductive strategy includes also this reproductive policy.

On February 1997 the birth was announced of a sheep named Dolly, the first mammal to be cloned from an adult cell of a mother individual. The event attracted enormous media attention. Dolly, born on 5 July 1996, actually had three ‘mothers’: one provided the egg (whose nucleus was removed), another the nucleus with the DNA picked out from a somatic cell (i.e. a cell of the body not specialized for reproduction), while the third mother carried the cloned embryo in her womb until parturition.

Ever since living beings arose from non-living organic compounds on a primordial planet, more than 3.5 billion years ago, a multitude of organisms has unceasingly flourished by means of the reproduction of pre-existing organisms. Through reproduction, living beings generate other material systems that to some extent are of the same kind as themselves. The succession of generations through reproduction is an essential element of the continuity of life. Not surprisingly, the ability to reproduce is acknowledged as one of the most important properties to characterize living systems. But let’s step back and put reproduction in a wider context, the endurance of material systems.

Acquiring the traits specific to a given sex, during early development or at another point during the life of an organism, is usually a complex process. Although the sex condition of an individual is conventionally defined based on the type of gametes it is able to produce (Chapter 4), the sex-specific phenotype is generally not limited to the organs of reproduction. Each of these characters can maintain a certain degree of independence from other sexual traits in the same organism, be subject to different developmental control, and show different degrees of sensitivity to the environment. Therefore, sexual differentiation extends to the development of the secondary sexual characters, which can be morphological, physiological, behavioural, or combinations of these. An exploration of this fascinating subject requires some preliminary clarification about systems and mechanisms of sex determination and sex differentiation.

In Chapter 1 we defined sexual reproduction as a form of reproduction that generates new individuals carrying a genome obtained by the association and/or the reassortment of genetic material from more than one source. In the most familiar form of sexual reproduction, the new genome is formed by the union of (partial) copies of the genomes of two parents through the fusion of two special cells produced for that purpose, the gametes, into a single cell, the zygote. This is the way most multicellular eukaryotes, ourselves included, reproduce sexually.

A zygote does not necessarily derive from the fusion of gametes or gametic nuclei produced by different individuals. Both egg and sperm may instead be produced by the same individual, a sufficient simultaneous hermaphrodite (Chapter 4). In this case, the offspring has only one parent. However, the gametes that merge are the products of independent processes of meiosis undergone by different germ cells, although in the same individual: this distinguishes self-fertilization (or selfing) from some forms of parthenogenesis where there is the fusion of two of the four nuclei deriving from the same meiosis, as we will see in the next sections (Figure 6.1).