Progesterone is essential for the transformation of an oestrogen-primed endometrium in preparation for implantation. With the advent of cDNA array technology, several researchers have applied this methodology to identify the various classes of genes that are upregulated during the window of implantation, both in animal models and human endometrium. The relative paracrine signalling of the individual prostanoids acting via their receptors to facilitate reproductive processes, such as implantation, has been largely unexplored. During adhesion of the implanting blastocyst to the endometrium, locally regulated uterine apoptosis is necessary to promote tissue remodelling and invasion of the epithelium. This process of programmed cell death is seen in rodents, where a progressive, continuous wave of induction of apoptosis is observed in the maternal tissues surrounding the conceptus in the rat. Over the past few years a great deal of progress has been made towards identifying the endocrine and local factors involved in implantation.

Reproductive immunologists have always followed mainstream immunology and tried to fit the immunology of the maternal-fetal relationship into the paradigms of the day. Attachment of the blastocyst to the uterine lining is thought to trigger the differentiation of the trophectoderm into two layers: an inner cytotrophoblast layer and an outer syncytium of syncytiotrophoblast. Adults homozygous for a highly truncated 'null' allele of human leucocyte antigen G (HLA-G) have been identified. The birth of these individuals suggests that the expression of full-length, membrane-associated HLA-G in trophoblast is not an obligate requirement for pregnancy. The various trophoblast populations differ with respect to both their expression of major histocompatibility complex (MHC) and which maternal immune cells they encounter. In humans, uterine natural killer (uNK) cells are present before implantation and in nonpregnant cycles where the number of uNK cells changes during the course of the menstrual cycle.

This chapter focuses on the role of progestin-induced decidualisation in regulating endometrial haemostasis and vascular stability. Observations made by in situ hybridisation and immunohistochemistry of endometrial sections, together with in vitro studies of human endometrial stromal cells (HESCs), indicate that progest-ininduced decidualisation creates a pro-haemostatic, vascular-stabilising milieu that resists bleeding. During decidualisation of human endometrium, the interstitial-type extracellular matrix (ECM) of the follicular phase, which is enriched in fibronectin and collagen types I, III,V and VI, is converted to a mixture of residual interstitial proteins and new peridecidual basal laminar-type components. The resulting peridecidual cell ECM plays an integral role in implantation by modulating migration of the invading trophoblast and in counteracting the threat of local haemorrhage during endovascular trophoblast invasion by serving as a vascular support and stabilising scaffolding structure. Uterine natural killer (NK) cells have been implicated in maintaining decidualisation and in limiting trophoblast invasion.

Experimental studies of human implantation in which embryos hatch and attach to endometrial epithelial cells in monolayer culture have shown that Mucin (MUC1) disappears from a small area of cells that surrounds the attached embryo. The term 'marker' is used advisedly because, while the inclusion criteria include the ability to function as an adhesion molecule, as well as presence at the implantation site, in vivo functional (IVF) evidence is so far mostly lacking. Heparin-binding EGF-like growth factor (HB-EGF) has been suggested to play two different roles: in addition to accelerating embryo development, it has a membrane-bound variant that can mediate intercellular attachment by binding to ErbB4 and heparan sulphate proteoglycan, both of which are present on the blastocyst. Critically, the biochemical dialogue between the embryo and the endometrium that determines endometrial receptivity at the attachment site cannot emerge from an analysis of either one alone.

The generation of an appropriate environment for embryonic development requires the development of a receptive endometrium. Examination of routinely fixed sections of endometrial tissue reveals a striking feature of the endometrial vasculature, namely the presence of the so-called spiral arterioles. The presence of such vascular smooth muscles enables these vessels to regulate blood flow within the endometrium. The profound changes that occur in the endometrial thickness and structure during the menstrual cycle, one would expect that endothelial growth might show similar changes over the menstrual cycle. Endothelial cells from different tissues differ in a variety of ways and, the vasculature within the endometrium shows considerable variation at different anatomical sites and it also varies during the menstrual cycle. The vascular endothelial growth factor (VEGF) system interacts with other signalling families to cooperatively regulate vessel growth and adaptation.

This chapter deals with the regulation of matrix metalloproteinase-9 (MMP9) transcription in tissue remodelling at the fetal-maternal interface. MMPs, also called matrixins, are a family of at least 20 human zinc-dependent endopeptidases collectively capable of degrading all components of the endometrial extracellular matrix (ECM) essentially. Gelatinase B (MMP9) is considered to be instrumental in trophoblast invasion since in vitro, it mediates cytotrophoblastic cells (CTB) invasion into a Matrigel. The MMP9 gene contains 13 exons and 12 introns for a total size of 7.7 kbp. The regulatory region of this gene has been described in two studies showing multiple cis-regulatory elements. These cis-regulatory elements are specific DNA sequences that bind trans-activators or trans-repressors (transcription factors), which are thus proteins encoded by other genes. As is the case with many other genes, MMP9 can be regulated by co-repressors or coactivators.

This chapter presents published data on the embryonic regulation of endometrial epithelial molecules such as chemokine receptors, the leptin system and the relaxin receptor LGR7. Chemokines and their receptors are divided into two families based on structural and genetic considerations. A specific molecular cross-talk between embryo and endometrium has been reported to take place during human implantation process. In human and rodents, two major forms of leptin receptors (OB-R) are expressed: the short form (OB-RS) and the long form (OB-RL). Relaxin is a peptide hormone produced by the corpus luteum during luteal phase of the menstrual cycle and during first trimester of pregnancy. It has been associated with a wide range of functions related to pregnancy, such as parturition and lactation. Advances in gene expression profiling facilitated by the development of DNA microarrays represent major progress in global gene expression analysis.

Failure of the embryo to implant successfully into the endometrium is one of the major causes of infertility. Many causes of infertility, such as sperm dysfunction and certain disorders of the fallopian tube or ovary, can be identified and either treated or bypassed by in vitro fertilisation (IVF). There are few models of human embryo implantation that have been exploited to dissect the implantation process and from which information about the function of specific molecules involved has been obtained. Solid-phase assays have been used previously to determine the function and signalling properties of extracellular matrix (ECM) components in the adhesion and trophoblast outgrowth of mouse blastocysts. The interaction of the blastocyst with various cellular components of the endometrium can be investigated in co-cultures of isolated cells and peri-implantation blastocysts. Model systems for the study of complex processes such as implantation of the human embryo can involve varying degrees of complexity.

In vivo, only a species-specific number of ovarian follicles are selected to release their oocytes for fertilisation during each reproductive cycle. While many factors play a role in selecting the follicles for ovulation, the overriding factor is availability and follicular response to the circulating gonadotrophins, folliclestimulating hormone (FSH) and luteinising hormone (LH). Initially, oocyte growth is commensurate with follicle growth. In most species, oocytes are generally thought to have completed their growth phase by the early antral stage of follicular development. While gonadotrophins and steroids have been the focus of much research into follicular development, numerous other growth factors and cytokines have been identified as also having a role in oocyte developmental competence. Some of these factors affect the selection of follicles that proceed to the ovulatory stages while others directly enhance oocyte maturation. Another aspect of oocyte maturation is the epigenetic phenomenon of genomic imprinting.

This chapter discusses the four important measurable aspects of sperm function that define a good sperm: sperm transport, sperm-epithelial contact, sperm-egg recognition and fertilisation, and DNA quality. One of the important aspects of sperm transport to emerge over the past ten years is the role of sperm-epithelial contact in regulating sperm physiology during the preovulatory period. It has been shown that co-culture of sperm with epithelium from the human endosalpinx prolongs sperm viability, increases motility and delays the process of sperm capacitation. Whether fertilisation occurs in vitro or in vivo, it is almost certain that the cellular and molecular processes of sperm-egg recognition and fertilisation are identical. Studies of sperm DNA quality have shown that DNA integrity is directly related to the probability of conception, both in the general population and in couples attending for assisted conception treatment.

The cell lineage heterogeneity is an essential component of early morphogenesis and is associated with the maintenance of developmental potential for the embryo. The mammalian embryo has long been considered to exhibit remarkable regulative capacity, presumably reflecting a plasticity in developmental potential to combat different environmental conditions. Cell contact patterns between blastomeres have a major influence on this dynamic state. While compaction and epithelial cell polarisation at the 8-cell stage is mediated by cell adhesion, contact patterns remain effective in steering developmental pathways throughout the cleavage period. The capacity to recavitate and re-form trophectoderm-like outer cells is progressively lost when inner cell masses (ICMs) are isolated from older, more expanded blastocysts which then form predominantly primary endoderm-like structures. The cell contact pattern, either asymmetric or symmetric, maintaining trophectoderm and ICM lineage pathways respectively, acts upstream of signal transduction mechanisms regulating phenotypic status.

This chapter reviews epigenetic reprogramming during early development, describes the background to the technique of nuclear transfer (NT), and discusses the present knowledge on reprogramming in cloned embryos. The chapter focuses on nuclear modifications related to somatic cell nuclear transfer (SCNT) and the strategies that have been developed to improve the efficiency of nuclear reprogramming. An analysis of nuclear structure remodelling has been made by the analysis of the structure of the nuclear envelop. Components of the oocyte cytoplasm are capable of inducing changes in nuclear architecture as well as modifications of the DNA and DNA-associated molecules, rendering the somatic chromatin amenable for reprogramming its gene expression. The ability of the egg to reprogramme the genome of a somatic cell, as occurs in animal cloning, is helpful to define the role of epigenetic control during embryonic development and cellular differentiation.

Many of the phenotypes associated with the large offspring syndrome (LOS) have features reminiscent of some naturally occurring human overgrowth syndromes, such as the Beckwith-Wiedemann syndrome (BWS), that are associated with errors in an imprinted cluster of genes on human chromosome. The major obstetric challenges facing those involved with assisted reproductive technologies (ART) continue to be preterm deliveries, low-birthweight babies and additional complications associated with multiple births. The alterations in genomic imprinting correlate with a varied range of aberrant fetal phenotypes that depend on the species in question, the type of insult during oocyte and/or preimplantation development, the gene(s) affected and the nature of the epigenetic modification. The process is known to involve active demethylation of paternal chromatin prior to syngamy in some but not all species, and may go some way to explain interspecies variation in imprinted gene expression following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).

Pre-implantation genetic testing (PGT) can be classified as either pre-implantation genetic diagnosis (PGD) or pre-implantation genetic screening (PGS). PGS aims to improve the outcome of assisted reproductive technology (ART) treatment for the subfertile by testing for a number of the more frequent chromosome aneuploidies in an attempt to improve implantation and reduce the incidence of miscarriage. The first successful clinical application of PGD, to avoid the X-linked condition adrenoleukodystrophy, used polymerase chain reaction (PCR) to amplify a specific repeat on the Y chromosome in order to sex embryos. Patients performing embryo biopsy must be approved and licensed by the Human Fertilisation and Embryology Authority (HFEA) after inspection. The use of PGD for the prevention of transmission of serious genetic disease has largely been overtaken in volume by use of embryo biopsy to improve in vitro fertilisation (IVF) outcome for subfertile couples.

This chapter deals with the role of leukaemia inhibitory factor (LIF) at the time of embryo implantation. LIF is involved in endometrial function in humans and domestic species. The lack of LIF signalling in a pregnant female prevents implantation, showing that the action of LIF in the uterus is essential for this process. The absence of LIF has recently been shown to result in low levels of a number of progesterone-responsive genes in the uterus In LIF-null mice, pinopods do not develop over the apical cell membranes, which remain microvillous up to day 7 of pregnancy. Transcript analysis of expression at the implantation site in LIF-null mice suggests molecular abnormalities associated with the lack of decidualisation. One of the most distinctive aspects of the LIF-null phenotype is the complete absence at the implantation site of expression of oncostatin M (OSM), a member of the interleukin 6 (IL6) family.

This chapter reviews how microarray studies to date have advanced understanding of critical aspects of the biology of implantation and discusses potential applications in the future. All gene arrays for expression profiling depend on hybridisation of complementary strands of DNA or RNA. Progesterone receptor (PR) has long been known to be essential for the preparation of a receptive endometrium, until now relatively little has been known about the molecular pathways regulated by progesterone in the endometrium. One of the novel genes upregulated by progesterone is the lipid-metabolising enzyme leucocyte 12/15-lipoxygenase. In women, the action of the progesterone receptor is also essential for the development of receptivity, which occurs between day luteinising hormone (LH)+5 and day LH+10. The ability of microarrays to analyse thousands of mRNAs simultaneously provides a powerful new approach to the identification of markers of receptivity.

Sporadic early pregnancy loss occurs in approximately 15% of clinically recognised pregnancies. One of the most remarkable aspects of the first-trimester miscarriage of pregnancy is the fact that the majority of karyotypically abnormal pregnancies miscarry in the first trimester and the majority of karyotypically normal pregnancies continue. Comparative genomic hybridisation (CGH) can be used to analyse the cytogenetics of formalin-fixed, paraffin-embedded miscarriage specimens. The newly introduced medical therapeutic abortion methodology has allowed detailed examination of undamaged first-trimester pregnancies. The traditional 'gold standard' management for miscarriage was dilatation and surgical curettage of the uterus. This was initiated to prevent blood loss and infection. An alternative to surgical management with drugs has been developed since the early 1990s. Obtaining an accurate aetiology should be within the capabilities of all hospital clinics and is indispensable in informing and supporting the management of miscarriage.

This chapter develops the hypothesis that some women with a history of recurrent miscarriage (RM) are in a prothrombotic state outside of pregnancy. The causes of RM have been grouped into six main categories: genetic, anatomical, infective, endocrinological, immunological and unexplained. The thrombophilic disorders play a part in the aetiology of recurrent pregnancy loss at various gestations. Haemostasis in vivo is a balancing act between the coagulation and fibrinolytic pathways, and plays a vital role in the establishment and maintenance of pregnancy. Prospective studies have shown an increased prevalence of acquired thrombophilic disorders in women with a history of RM. Women with a history of RM are at greater risk of later pregnancy complications such as pre-eclampsia, fetal growth restriction and intrauterine death. Revised criteria for the diagnosis of Primary antiphospholipid syndrome (PAPS) recognise the obstetric manifestations of antiphospholipid antibodies (aPL), such as a history of pre-eclampsia and preterm labour.

This chapter reviews the epidemiological evidence in support of a pathological link and discusses the implications for clinical management. Several epidemiological studies have investigated pregnancy outcome in singleton pregnancy after in vitro fertilisation/intracytoplasmic sperm injection (IVF/ICSI). The lack of consensus diagnostic criteria and the heterogeneous nature of contemporary infertility investigations make it difficult to define the risk of adverse pregnancy outcome in relation to specific reproductive disorders, such as endometriosis or polycystic ovary syndrome (PCOS). In humans, the formation of a functional placenta requires invasion of fetal trophoblast not only into the maternal decidua and inner myometrium (interstitial invasion) but also into the maternal spiral arteries. Progesterone support improves pregnancy outcome after IVF treatment and two recent randomised trials have reported that progesterone also markedly reduces the incidence of preterm labour in at-risk women.

Among all women who had ever been pregnant, 21% reported ever having had a miscarriage. The results of the National Women's Health Study confirmed some well-established risk factors, including increased maternal age and previous history of miscarriage and infertility, and also the protective effect of nausea. The response rate for the study was 73% for the more targeted Stage 2 questionnaire. Six hundred and three cases and 6116 controls were included in the case-control analysis of risk factors for first-trimester miscarriage. The results obtained from this study, after adjusting for year of conception, maternal age, previous miscarriage and previous livebirth, are listed in this chapter. The reduced risks associated with taking vitamins, consumption of fresh fruit and vegetables and feeling happy and relaxed during pregnancy are perhaps not surprising, but further work is needed to establish causal pathways and whether these results can be explained by selection or reporting biases.

This chapter presents suggested options for the elimination of multiple pregnancies including selective fetal reduction, single blastocyst transfer and elective single-embryo transfer (eSET). Selective fetal reduction carries a risk of miscarriage and poses serious ethical and legal questions. Three trials comparing eSET with elective double embryo transfer (DET) were identified. All involved women who had undergone embryo transfer in a fresh in vitro fertilisation (IVF)/intracytoplasmic sperm injection (ICSI) cycle. It is clear that in a selected population the cumulative outcome of eSET is comparable with that of DET while virtually eliminating multiple pregnancies. The outcome of an eSET policy can be substantially enhanced in conjunction with an efficient and reliable embryo cryopreservation programme. Twin pregnancies resulting from IVF treatment are associated with higher maternal and perinatal morbidity. More data from pragmatic trials are to demonstrate whether a policy of eSET is effective, acceptable and financially viable in other clinical settings.