Preimplantation development

Understanding the spatial and temporal developmental processes that take place within an embryo as it develops from a single cell into a recognizable human is the challenge of embryology. The control of these processes resides within the genome; fundamental questions remain concerning the genes and interactions involved in development.

Staging of Embryos

For the purposes of embryological study, prenatal life is divided into an embryonic period and a fetal period. The embryonic period covers the first 8 weeks of development (weeks following ovulation and fertilization resulting in pregnancy). The ages and stages of early human embryos were initially estimated by comparing their development with that of other embryos or with those of monkey embryos of known postovulatory ages. Because the external and internal development of embryos at any specified time may be different, a classification of human embryo development into 23 stages based on the internal and external features of embryos was developed by , and continued by , from detailed histological study of approximately 600 sectioned embryos in the Carnegie collection. On the basis of correlating particular maternal menstrual histories and the known developmental ages of monkey embryos, growth tables were constructed so that the size of an embryo (specifically, the greatest length) could be used to predict its presumed age in postovulatory days (synonymous to postfertilizational days). emphasize that the Carnegie embryonic stages are based on external and internal morphological criteria and are not founded on length or age. Ultrasonic examination of embryos in vivo has necessitated the revision of some of the ages related to stages, and embryos of stages 6–16 are now thought to be up to 3–5 days older than the embryological estimates used previously ( ). Within this staging system, embryonic life commences with fertilization at stage 1; stage 2 encompasses embryos from two cells, through compaction and early segregation, to the appearance of the blastocoele. The developmental processes occurring during the first 10 stages of embryonic life are shown in Fig. 8.1 .

Fig. 8.1, Developmental processes occurring during the first 10 stages of development. In the early stages, a series of binary choices determine the cell lineages. Generally, the earliest stages are concerned with formation of the extraembryonic tissues, whereas the later stages are concerned with the formation of embryonic tissues. The approximate age in days is taken from O’Rahilly and Müller 2010 .

Knowledge of the early molecular processes of development is derived from experimental studies on amniote embryos, particularly the chick, mouse and rat. Figure. 8.2 shows the comparative timescales of development of these species and human development up to stage 12 and illustrates the differences in heterochrony between the species ( Ch. 11 ). The length and age, in post fertilization days, of human development during stages 10–23 is given in Fig. 8.3 , the external appearance of embryos over this time is shown in Fig. 23.1 and stage is correlated to days and weeks in Fig. 23.3 .

Fig. 8.2, Within developmental biology, evidence concerning the nature of developmental processes has come mainly from studies in vertebrate embryos, most commonly amniote embryos of the chick, mouse and rat. This chart illustrates the comparative timescale of development in these animals and in humans.

Information on developmental age after stage 23 (postfertilization days 53–58) is shown in Fig. 23.4 , where the developmental staging used throughout this book is juxtaposed with the clinical obstetric estimation of pregnancy based on maternal last menstrual period. Great care should be taken when reading developmental papers to clarify the staging system used to confer age in days on any embryos. All developmental publications using the Carnegie collection prior to 2010 use earlier estimations of age. Many of the publications since 2010, when O'Rahilly and Müller revised their correlation of age in days to stage, are still using the older unrevised ages. Publications noting changes correlated to Carnegie stage (not age) still match Streeter and O'Rahilly and Müller publications. All embryonic stages in this book are corrected to postfertilization days based on .

Fertilization

The central feature of reproduction is the fusion of the two gamete pronuclei at fertilization. In humans, the male gametes are spermatozoa, which are produced from puberty onwards. Female gametes are released as secondary oocytes in the second meiotic metaphase, usually singly, in a cyclical fashion. The signal for the completion of the second meiotic division is fertilization, which stimulates the cell division cycle to resume, completing meiosis and extruding the second polar body (the second set of redundant meiotic chromosomes).

Fertilization normally occurs in the ampullary region of the uterine tube, probably within 24 hours of ovulation. Very few spermatozoa reach the ampulla to achieve fertilization. They must undergo capacitation, a process that is still incompletely understood, and that may involve modifications of membrane sterols or surface proteins. They traverse the cumulus oophorus and corona radiata, then bind to specific glycoprotein receptors on the zona pellucida, ZP3 and ZP2. Interaction of ZP3 with the sperm head induces the acrosome reaction, in which fusion of membranes on the sperm head releases enzymes, such as acrosin, that help to digest the zona around the sperm head, allowing the sperm to reach the perivitelline space. In the perivitelline space, the spermatozoon fuses with the oocyte microvilli, possibly via two disintegrin peptides in the sperm head and integrin in the oolemma ( Figs 8.4 , 8.5A ).

Fig. 8.4, The fertilization pathway: a succession of steps. After a sperm binds to the zona pellucida, the acrosome reaction takes place (see detail at top). The outer acrosomal membrane (blue), an enzyme-rich organelle in the anterior of the sperm head, fuses at many points with the plasma membrane surrounding the sperm head. Then those fused membranes form vesicles, which are eventually sloughed off from the head, exposing the acrosomal enzymes (red). The enzymes digest a path through the zona pellucida, enabling the sperm to advance. Eventually, the sperm meets and fuses with the secondary oocyte plasma membrane and this triggers cortical and zona reactions. First, enzyme-rich cortical granules in the oocyte cytoplasm release their contents (yellow) into the zona pellucida, starting at the point of fusion and progressing right and left. Next, in the zona reaction, the enzymes modify the zona pellucida, transforming it into an impenetrable barrier to sperm as a guard against polyspermy (multiple fertilization).

Fusion of the sperm with the oolemma causes a weak membrane depolarization and leads to a calcium wave, triggered by the sperm at the site of fusion, that crosses the egg within 5–20 seconds. The calcium wave amplifies the local signal at the site of sperm–oocyte interaction and distributes it throughout the oocyte cytoplasm. The increase in calcium concentration is the signal that causes the oocyte to resume cell division, initiating the completion of meiosis II and setting off the developmental programme that leads to embryogenesis. The pulses of intracellular calcium that occur every few minutes for the first few hours of development also trigger the fusion of cortical granules with the oolemma. The cortical secretory granules release an enzyme that hydrolyses the ZP3 receptor on the zona pellucida and so prevents other sperm from binding and undergoing the acrosome reaction, thus establishing the block to polyspermy. The same cortical granule secretion may also modify the vitelline layer and oolemma, making them less susceptible to sperm–oocyte fusion and providing a further level of polyspermy block.

The sperm head undergoes its protamine to histone transition as the second polar body is extruded. The two pronuclei form, the female one cortically near the site of emission of the second polar body. They move together and are juxtaposed prior to pronuclear membrane breakdown/syngamy and cleavage after approximately 24 hours after sperm and oocyte association ( ) ( Fig. 8.5B ). Pronuclear fusion as such does not occur; no true zygote containing a membrane-bound nucleus forms. The two pronuclear envelopes disappear and the two chromosome groups move together to assume positions on the first cleavage spindle. Nucleolar ribosomal ribonucleic acid (rRNA), and perhaps some messenger RNA (mRNA), are synthesized in pronuclei. A succeeding series of cleavage divisions produces eight even-sized blastomeres by approximately 55 hours, when embryonic mRNA is transcribed.

Fig. 8.5, A , An unfertilized human secondary oocyte surrounded by the zona pellucida; the first polar body can be seen. Spermatozoa are visible outside the zona pellucida. B , A fertilized human ootid before fusion of the pronuclei. Two polar bodies can be seen beneath the zona pellucida.

The presence of the pronuclei from both parental origins is crucial for spatial organization and the controlled growth of cells, tissues and organs. In the mouse, embryos in which the paternal pronucleus has been removed and replaced with a second maternal pronucleus develop to a relatively advanced state (25 somites), but with limited development of the trophoblast and extraembryonic tissues. In contrast, embryos in which the maternal pronucleus has been replaced by a second paternal pronucleus develop very poorly, forming embryos of only six to eight somites, but with extensive trophoblast. Thus, it seems that the maternal genome is relatively more important for the development of the embryo, whereas the paternal genome is essential for the development of the extraembryonic tissues that would lead to placental formation.

This functional inequivalence of homologous parental chromosomes is called parental imprinting. It occurs during the maturational phase of production of the haploid gametes in mammals and silences one allele by DNA methylation without changing the DNA sequence ( ). Imprinted genes are heritable but in the next generation may be unmethylated during gametogenesis and re-established in the new germline. Between 50 and 90 imprinted genes expressed in the placenta and embryo have been identified, many concerned with embryogenesis and growth ( ). Although imprinted genes represent a small proportion of the genome, they are particularly important in blastocyst implantation, placentation, developmental growth rates and the embryonic nervous system. Some epigenetic modifications can lead to congenital anomalies and syndromes ( ). For example, changes to the 15q11-13 domain if paternally imprinted lead to Prader–Willi syndrome (a disorder of growth) and if maternally imprinted cause Angelman syndrome (a neurological disorder); changes to the domain 11p15 affect IGF2 if paternally expressed and H19 if maternally expressed; both of these imprinted conditions affect growth regulation and trajectory in early development, Beckwith-Wiedemann syndrome results if the region is maternally imprinted ( ). Generally, paternally expressed genes enhance growth and maternally expressed genes suppress growth ( ).

In vitro fertilization

In vitro fertilization (IVF) of human gametes is a successful way of overcoming most forms of infertility ( ). Controlled stimulation of the ovaries (e.g. pituitary downregulation using gonadotrophin-releasing hormone superactive analogues, followed by stimulation with purified, or recombinant, follicle stimulating hormone or urinary menopausal gonadotrophins) enables many preovulatory oocytes (often 10 or more) to be recruited and matured, and then aspirated transvaginally using ultrasound guidance, 34–38 hours after injection of human chorionic gonadotrophin (which is given to mimic the luteinizing hormone surge). These oocytes are then incubated overnight with motile spermatozoa in a specially formulated culture medium, in an attempt to achieve successful in vitro fertilization. In cases of severe male-factor infertility, in which there are insufficient normal spermatozoa to achieve conventional in vitro fertilization, individual spermatozoa can be directly injected into the oocyte in a process known as intracytoplasmic sperm injection (ICSI), which is as successful as routine IVF. In cases in which there are no spermatozoa in the ejaculate, suitable material can sometimes be directly aspirated from the epididymis or surgically retrieved from the testes, and the extracted sperm are then used for intracytoplasmic injection.

It is also now sometimes possible to assess embryos by preimplantation genetic testing (PGT) for the presence of monogenic/single gene diseases (PGT-M) or for structural chromosomal rearrangements (PGT-SR). A sample (biopsy) is removed from either the oocyte (polar bodies), the early cleavage stage embryo (a blastomere) or the blastocyst (small piece of trophectoderm), and subjected to specific genetic testing. Oocytes and embryos can also be biopsied and screened for errors in chromosome copy numbers using preimplantation genetic testing for aneuploidy (PGT-A). Unaffected embryos can then be identified for transfer to the recipient. Although increasingly utilized across all ages of IVF patients, there is debate over the efficacy of PGT-A. It is considered most clinically appropriate for women over 37 years of age due to increased aneuploidy levels in their oocytes. Embryos that are surplus to immediate therapeutic requirements can also be cryopreserved in liquid nitrogen for later use. Propanediol or dimethylsulphoxide are commonly used as cryoprotectants for human embryos at all stages. Conception rates per cycle using ovarian stimulation, IVF and successive transfers of fresh and cryopreserved embryos exceed those obtained during non-assisted, or natural, conception.

In 2014, some 40 years after the first IVF infant was born, more than 200,000 assisted reproductive technologies (ART) cycles were performed in the United States leading to nearly 60,000 live births. World-wide the prevalence of ART is now extensive. For details of ART the reader should consult .

Data collected on the longer-term effects of ART have indicated an associated increased risk of imprinting syndromes ( ). The complexities of ART, i.e. the processes involved in collection of oocytes and sperm, in vitro fertilization and blastocyst culture, and the manipulation of the maternal endometrium for successful implantation, challenge many developmental conceptus–maternal interactions. It is now appreciated that the temporal conversations between oocyte/zygote/blastocyst and the mother are informed by dynamic exchange of environmental information some days before the close juxtaposition of the tissues seen in implantation, and that their effects extend into fetal growth and postnatal life ( Ch. 23 ). These periconceptual influences are now being investigated ( ).