Intracellular signaling in the developing blastocyst as a consequence of the maternal-embryonic dialogue

The success of blastocyst implantation is dependent on signaling between the embryo and the receptive endometrium. Intercellular signaling molecules, which include hormones, growth factors, and cytokines, have been identified that participate in the maternal-embryonic dialogue. These biologically active molecules may target uterine and/or embryonic tissues in a biochemical cascade that coordinates the two developmental programs during implantation. Two notable uterine products are calcitonin and heparin-binding epidermal growth factor-like growth factor, which are both expressed during the receptive phase of the endometrium in humans and in rodent models. We review data that demonstrate the ability of these molecules to accelerate blastocyst differentiation and delineate the respective intracellular signaling pathways that advance the embryonic developmental program. An understanding of the mediators regulating embryonic development in utero and their biochemical mechanisms of the action may provide insights for improvement of embryo culture in vitro prior to blastocyst transfer.     

Network of cytokines, integrins and hormones in human trophoblast cells.

Trophoblast cells of the developing embryo are unique not only for transporting oxygen and nutrients from the mother to the fetus but also for their array of other functions throughout the pregnancy beginning from the attachment of the blastocyst to the endometrium during the process of implantation, its well regulated invasion in the uterine tissue, proliferation, differentiation and immuno-endocrine functions which subsequently maintain the pregnancy. Using human trophoblast cells in culture, we have tried to understand the molecular mechanisms which regulate such a variety of functions for trophoblast cells. Our RT-PCR studies show that trophoblast cells express the laminin and collagen receptors: integrins alpha1 and alpha2, which are both stimulated by IL-1 and IL-6. These two cytokines, also synthesised by the trophoblast cells themselves, act in an autocrine/paracrine manner to induce their own expression. In addition, IL-1 expression seems to be modulated by a large variety of cytokines and growth factors usually present in the uterine milieu whereas IL-6 expression appears to be significantly stimulated by growth factors like EGF and bFGF only. Hormones like estrogen, progesterone and hCG exhibit a general negative modulation in the expression of IL-1 and IL-6. Since, both IL-1 and IL-6 are known to be involved in the proliferation, invasion and differentiation of trophoblast cells, they might be the key factors involved in trophoblast functions.     

Human implantation: the last barrier in assisted reproduction technologies?

Implantation processes are highly complex involving the actions of numerous hormones, immunoglobulins, cytokines and other factors in the endometrium. They are also essential matters for the success of assisted reproduction. The nature of early embryonic development is of equal significance. It involves ovarian follicle growth, ovulation, fertilization and preimplantation growth. These processes are affected by imbalanced chromosomal constitutions or slow developmental periods. Post-implantation death is also a significant factor in cases of placental insufficiency or recurrent abortion. Clearly, many of these matters can significantly affect birth rates. This review is concerned primarily with the oocyte, the early embryo and its chromosomal anomalies, and the nature of factors involved in implantation. These are clearly among the most important features in determining successful embryonic and fetal growth. Successive sections cover the endocrine stimulation of follicle growth in mice and humans, growth of human embryos in vitro, their apposition and attachment to the uterus, factors involved in embryo attachment to uterine epithelium and later stages of implantation, and understanding the gene control of polarities and other aspects of preimplantation embryo differentiation. New aspects of knowledge include the use of human oocyte maturation in vitro as an approach to simpler forms of IVF, and new concepts in developmental genetics.     

The 2 differentiation pathways of the human trophoblast

The trophoblast is the major component of the human placenta. It is directly involved in blastocyst implantation and in feto-placental growth and development. Human trophoblast follows two major pathways of differentiation: the villous trophoblast, bathing in maternal blood of intervillous spaces and involved in matemo-fetal exchanges and in placental endocrine functions; the extra-villous trophoblast involved in uterine spiral arteries remodeling and in the placental anchorage into the uterine wall. It is essential to understand the cellular and molecular mechanisms involved in human trophoblast differentiation: cellular proliferation, migration, invasion and differentiation by cell-cell fusion. Abnormal trophoblast differentiation is implicated in the major pathologies of human pregnancy such as pre-eclampsia and intrauterine growth retardation.     

Molecular and cellular events involved in the completion of blastocyst implantation

Blastocyst implantation is an interactive process between the embryo and the uterus. The synchronization of embryonic development with uterine differentiation to a receptive state is essential for a successful pregnancy. The period of uterine receptivity for implantation is limited. Although implantation involves the interaction of numerous signaling molecules, our understanding of the hierarchical mechanisms that coordinate with the embryo–uterine dialogue is not yet sufficient to prevent infertility caused by implantation failure. This review highlights our knowledge on uterine receptivity and hormonal regulation of blastocyst implantation in mice. We also discuss the adhesion molecules, cross-linker proteins, extracellular proteins, and matricellular proteins involved in blastocyst implantation. Furthermore, our recent study reveals that selective proteolysis in an activated blastocyst is associated with the completion of blastocyst implantation after embryo transfer. A better understanding of uterine and blastocyst biology during the peri-implantation period would facilitate further development of reproductive technology.     

Biomarkers of Endometrial Receptivity—A Review

Implantation is a phenomenon that involves an interaction between the embryo and maternal endometrium. There is, in the menstrual cycle, a short and precise period of time in which the maternal-embryonic interaction is optimal and culminates with adhesion and invasion of the blastocyst into the progesterone-induced secretory endometrium. This period is called nidation or implantation window. In the implantation window changes occur in endometrial epithelial morphology, characterized by the appearance of membrane projections called pinopodes. Pinopodes are progesterone-dependent organelles, that look like apical cellular protrusions appearing between days 20 and 21 of the natural menstrual cycle. There are many factors that regulate the changes typical of the implantation window and the appearance of the pinopodes. The embryonic and maternal expression of growth factors and cytokines, calcitonin, HOX genes and cell adhesion molecules might all play a major role in the phenomenon of implantation. The cytokines function as chemical messengers and can serve as biomarkers of uterine receptivity. Understanding the function of these biomarkers and their role in determining the implantation window in women, will help us to diagnose and treat infertile couples more efficiently.     

Embryo-maternal interactions at the implantation site: a delicate equilibrium

Blastocyst implantation and successful establishment of pregnancy require delicate interactions between the embryo and the maternal environment. During preimplantation, maternal/embryo communication is mediated by the trophectoderm. In the late luteal phase, physiological changes occur in the endometrium to allow blastocyst implantation. The "window of implantation" represents the period of maximum uterine receptivity for implantation. In response to signals from the embryo, pregnancy-specific proteins are released in maternal serum and a series of morphological, biochemical and immunological changes occur in the uterine environment. These systemic and local modifications can be considered to constitute "the maternal recognition of pregnancy". The human hemochorial placenta arises primarily through proliferation, migration and invasion of the endometrium and its vasculature by the embryonic trophoblast. The complex invasive processes accompanying implantation of the embryo are controlled at the embryo-maternal interface by factors from decidualized endometrium and the trophoblast itself. An inflammatory reaction and a proper maternal immune response allow survival and development of the feto-placental unit. In this review, we focus on interactions between trophoblast and uterine tissues and on cellular mechanisms and molecular signals involved in the closely regulated process of implantation.     

Compaction and lineage divergence during mouse preimplantation embryo development

The preimplantation embryo development leads to the formation of a blastocyst made of two cell lineages: an outer layer of epithelial cells, the trophectoderm, that will give rise to some embryonic annexes, and a mass of undifferentiated cells, the inner cell mass, that will form the foetus and the remaining embryonic annexes. The trophectoderm encloses the inner cell mass and protects it from the external medium. Moreover, after hatching, the trophectoderm invades the uterine tissue, a crucial step for the implantation of the embryo. Therefore, the divergence between these two lineages is of crucial importance for the emergence of the foetus itself and for the postimplantation development to take place correctly. The setting up of cell polarity during compaction at the eight-cell stage allows asymmetric divisions to take place, thereby leading to lineage divergence. Phenotypic properties of these two cell populations are progressively reinforced through cell-cell interactions, outer cells undergoing epithelial differentiation while inner cells remain undifferentiated. Although cellular mechanisms controlling the divergence of the first two lineages are quite well known, important efforts have been carried out this last decade to identify the molecular machinery involved in this process and will be presented in this review.     

The role of Wnt signaling members in the uterus and embryo during pre-implantation and implantation

Wnt family members are best known for their roles in cell fate determination, differentiation, proliferation and apoptosis during embryonic development. Wnt signaling becomes effective during these cellular processes through the proper interaction between its ligands, receptors, effectors and inhibitors. Here we review Wnt signaling in terms of embryonic development to the blastocyst stage implantation with emphasis on endometrial changes that are critical for receptivity in the uterus. The relationship between Wnt signaling and implantation clearly reveals that, Wnt family members are critical for both early embryonic development and changing of the endometrium before implantation. Specific Wnt signaling pathway members are demonstrated to be critical for endometrial events such as decidualization and endometrial gland formation in addition to cyclic changes in the endometrium controlled by reproductive hormones. In conclusion, specific roles of Wnt members and associated factors for both uterine function and embryonic development should be further investigated with respect to the efficiency of human ARTs.     

The Chemokine Connection: Hormonal and Embryonic Regulation at the Human Maternal-Embryonic Interface—A Review

Chemokines are small polypeptides that attract specific leukocyte subsets by binding to cell-surface receptors. In reproductive biology, they have been implicated in ovulation, menstruation, and embryo implantation, and pathological processes such as preterm delivery, HIV infection, and endometriosis.It is known that successful implantation requires a functionally normal embryo at the blastocyst stage and a receptive endometrium that is adequately communicated through the implantation process. This crosstalk is highly regulated, with numerous molecules taking part. Accumulated evidence suggests that chemokines produced and received by the endometrial epithelium and the human blastocyst are implicated in this molecular network. Here, we present updated information on the presence and hormonal and embryonic regulation of chemokines and their receptors during human implantation.     

An integrated view of L-selectin and trophinin function in human embryo implantation

Determining molecular mechanisms of human embryo implantation is an extremely challenging task due to the limitation of materials and significant differences underlying this process among mammalian species. Recently, L-selectin and its ligand carbohydrate have been proposed as a system that mediates initial adhesion of human blastocysts to the uterine epithelia. We have also identified trophinin as a unique apical cell adhesion molecule potentially involved in the initial adhesion of trophectoderm of the human blastocyst to endometrial surface epithelia. In the mouse, the binding between ErbB4 on the blastocyst and heparin-binding epidermal growth factor-like growth factor on the endometrial surface enables the initial step of the blastocyst implantation. The evidence suggests that L-selectin and trophinin are included in human embryo implantation. This review summarizes findings relevant to the functions of L-selectin and trophinin in human embryo implantation, and proposes a model that reconciles these cell adhesion mechanisms.     

Comparative implantation and placentation

In all placental mammals, the establishment of an intimate contact between the embryo and the mother follows a succession of common critical steps whose chronology and timing may considerably vary from species to species. These processes present a great diversity based on the anatomy and the histology of the uterus, the developmental stage of the embryo at the time of implantation, and the endocrine and molecular interactions between the uterine and the embryonic tissues. This paper provides an overview of the general mechanical, endocrinological and cellular aspects involved in implantation in mammals with an emphasis on domestic species.     

Paracrine regulators of implantation.

Classical and contemporary studies have shown that endocrine regulation exerted by ovarian hormones priming the endometrium is essential for embryo implantation. Increasing evidence indicates that steroid-induced molecules acting as paracrine modulators are necessary for embryo-uterine interactions. That is the case for calcitonin, heparin-binding epidermal growth factor (EGF)-like growth factor, leukaemia inhibitory factor and other molecules. Furthermore, when the blastocyst enters the uterine cavity, it starts the complex signals that will drive embryo adhesion. The paracrinology of this process is based on the local interplay of molecules, such as the secretion of cytokines that may facilitate the acquisition of endometrial receptivity by controlling the expression of adhesion and anti-adhesion proteins. Finally, during the embryonic invasive phase, uterine stromal-trophoblast dialogue may modulate this self-controlled proteolytic and immunological process to avoid damage to both the embryo and maternal environment.     

Histamine receptors in the female reproductive system. Part I. Role of the mast cells and histamine in female reproductive system

Histamine isolated from many different tissues, acts via three types of histamine receptors: H1, H2 and H3. In peripheral tissues histamine is mainly stored in mast cells (MC). Presence of mast cells was proved also in mammals' uteri. In human uterus the majority of mast cells are located close to smooth muscle cells. It might indicate that MC plays a role in tissue remodelling during the menstrual cycle. The quantity and activity of mast cells is in connection with hormonal status of the organism. Although there are some differences, human uterine mast cells are similar to the mast cells isolated from other tissues. It is suggested that histamine is important for normal ovulation, blastocyst implantation, placental blood flow regulation, lactation and contractile activity of uterus. Histamine may also play a role in pathological processes such as pre-eclampsia or preterm delivery. The participation of mast cells and histamine in blastocyst implantation is very controversial. In W/Wv mice (without mast cells) normal implantation was observed. It denies the main role of mast cells in this process but dos not exclude histamine action. In mice the major source of histamine are uterine epithelial cells during early pregnancy. The influence of cytokines on blastocyst implantation and the role of histamine in cytokines release from the uterine mast cells are also very unclear.     

Mouse embryo toxicity of IL-6 in peritoneal fluids from women with or without endometriosis

BACKGROUND: To determine whether there is a factor (or factors) in the peritoneal fluid of endometriosis patients that impairs embryo growth and embryo implantation. METHODS: Growth and development of two-cell mouse embryos which were cultured in media with peritoneal fluid from women with or without endometriosis and interleukin-1-beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) levels in conditioned media were measured. RESULTS: The blastocyst rate in the non-endometriosis group was 46.4 +/- 31.1%, and that of the endometriosis group was 54.6 +/- 28.7%. Logistic regression analysis using the criteria of blastocyst development in 454 embryos, showed that the peritoneal fluid from endometriosis could promote (p=0.015) but IL-6 could arrest embryo growth to blastocyst (p=0.025). IL-1beta and TNF-alpha levels had no significant effect on blastocyst formation. CONCLUSION: Peritoneal fluid from women with endometriosis was not toxic to mouse embryo development. However, IL-6 in the peritoneal fluid deteriorated the growth and development of mouse embryos.     

The science of implantation emerges blinking into the light

Although embryo implantation is essential for human survival, it remains an enigmatic biological phenomenon. Following fertilization, the resulting blastocyst must signal its presence to the mother, attach to the luminal epithelium of the endometrium and embed into the decidualising stroma. Failure to do so results in infertility, which affects around 9% of women. Subsequent placental development requires remodelling of maternal blood vessels by trophoblast cells from the placenta, that invade deep into the decidua. Failure in these very early stages can compromise fetal development, resulting in diseases of pregnancy such as intrauterine growth restriction or pre-eclampsia which can also impact on health in adulthood. Abnormal implantation therefore constitutes a significant disease burden in humans. Although we have known for many years that successful implantation requires an embryo that is competent to implant and an endometrium that is receptive, the molecular basis of these processes remains poorly understood. Our inability to identify implantation-competent embryos or to diagnose/treat the non-receptive endometrium therefore limits our ability to intervene through assisted reproduction techniques. This Implantation Symposium aims to review recent exciting developments in our understanding of the biology of early implantation and to highlight the rapid progress being made to translate these into improved diagnosis and treatment.Although embryo implantation is essential for human survival, it remains an enigmatic biological phenomenon. Following fertilization, the resulting blastocyst must signal its presence to the mother, attach to the luminal epithelium of the endometrium and embed into the decidualising stroma. Failure to do so results in infertility, which affects around 9% of women. Subsequent placental development requires remodelling of maternal blood vessels by trophoblast cells that invade deep into the decidua. Failure in these very early stages can compromise fetal development, resulting in diseases of pregnancy such as intrauterine growth restriction or pre-eclampsia which can also impact on health in adulthood. Abnormal implantation therefore constitutes a significant disease burden in humans. Although we have known for many years that successful implantation requires an embryo that is competent to implant and an endometrium that is receptive, the molecular basis of these processes remains poorly understood. Our inability to identify implantation-competent embryos or to diagnose/treat the non-receptive endometrium therefore limits our ability to intervene through assisted reproduction techniques. This Implantation Symposium aims to review recent exciting developments in our understanding of the biology of early implantation and to highlight the rapid progress being made to translate these into improved diagnosis and treatment.     

Cytokines in implantation

The endometrium of most species is now recognized as an important site of production of cytokines and their receptors. The cellular origin of the cytokines varies but many predominate in the uterine glandular or luminal epithelium or in the decidualized stromal cells. From studies in genetically modified mice it is clear that implantation of the blastocyst can proceed in the absence of most individual cytokines, although leukemia inhibitory factor and interleukin-11 have indisputable roles in this process. In other cases, such as CSF-1, GM-CSF, IL-1, and IL-6, the numbers of implantation sites or litter sizes are reduced when the cytokine is absent. The same cytokines that are implicated in implantation in mice are generally maximally expressed in human endometrium with maximal production in the secretory phase, particularly during the "window of implantation," but functional studies of their role in implantation in women and other primates are still required.     

Molecules in blastocyst implantation. Role of matrix metalloproteinases,cytokines and growth factors

Initiation of implantation is due not to passive growth pressure but to an active biochemical process that requires a blastocyst to interact with a carefully prepared endometrium. This versatile and dynamic process requires a variety of different molecules secreted by human trophoblast as well as endometrial cells that play a unique role. Several molecules have been shown to regulate, by an autocrine and paracrine manner, the cross-talk between the implanting blastocyst and the endometrial epithelium. Particularly, the molecular dialogue involves either cell-to-cell or cell-to-extracellular matrix interactions, mediated by matrix metalloproteinases, cytokines and growth factors. The present overview of the literature reports on the most significant molecules involved in the implantation process and describes the mechanisms of interaction and control. Since impaired blastocyst implantation is a significant cause of natural and in vitro fertilization pregnancy failure, a better understanding of the aforementioned molecular dynamics would be useful in improving the chance of viable pregnancy.