Understanding implantation window, a crucial phenomenon

Abstract

Embryo implantation is a well-defined and precise process, in which various factors come into play one after the other. There is only a specific period of time during which implantation is possible i.e. “implantation window”. Selectins were proposed to have an important role in this phase to ensure suitable rolling of the blastocyst. To prevent the blastocyst from adhering to an area with poor chances of implantation, an important role is played by the repellent activity of MUC-1, which is widely expressed throughout the endometrium and, surprisingly, even increases before implantation. In particular endometrial areas, secretion of chemokines and growth factors will attract the blastocyst to landing platforms known as pinopods. These pinopods are fully developed for only 1 or 2 days and extend over the tips of the microvilli expressing the repellent MUC-1. At this stage, adhesion molecules such as integrins and cadherins intervene to ensure adhesiveness between the embryo and the endometrium.

INTRODUCTION

Embryo implantation represents the most critical step of the reproductive process in many species. It consists of a unique biological phenomenon, by which the blastocyst becomes intimately connected to the maternal endometrial surface to form the placenta that will provide an interface between the growing fetus and the maternal circulation.[1] Successful implantation requires a receptive endometrium, a normal and functional embryo at the blastocyst developmental stage and a synchronized dialogue between maternal and embryonic tissues. Implantation occurs about 9 days after ovulation, ranging between 6 to 12 days.[2]

The implantation window

There are many conditions that must be satisfied in order for a successful implantation to take place. There is only a specific period of time during which implantation is possible;[3] this is the “implantation window”. The implantation window is started by preparations in the endometrium of the uterus, both structurally and in the composition of its secretions.

Adaptation of uterus

To enable implantation, the uterus goes through changes in order to be able to receive the embryo.

Predecidualization

The endometrium increases thickness, becomes more vascularized and its glands grow to be tortuous and boosted in their secretions. These changes reach their maximum about 7 days after ovulation.

Decidualization

Decidualization succeeds predecidualiza-tion if pregnancy occurs. This is an expansion of it, further developing the uterine glands, the zona compacta and the epithelium of decidual cells lining it. The decidual cells become filled with lipids and glycogen and take the polyhedral shape characteristic for decidual cells. It is likely that the blastocyst itself makes the main contribution to this additional growing and sustaining of the decidua. An indication of this is that decidualization occurs at a higher degree in conception cycles than in non-conception cycles. Furthermore, similar changes are observed when giving stimuli mimicking the natural invasion of the embryo.[3]

Decidua throughout pregnancy

After implantation, the decidua remains at least through the first trimester. However, its most prominent time is during the early stages of pregnancy, during implantation. Its function as a surrounding tissue is replaced by the definitive placenta. However, some elements of the decidualization remain throughout pregnancy.[3]

Pinopodes (Uterodomes)

Pinopods are bleb-like protrusions found on the apical surface of the endometrial epithelium.[4] They appear between day 19 and day 21 of gestational age.[3] This corresponds to a fertilization age of approximately 5 to 7 days, which corresponds well with the time of implantation. They only persist for 2 to 3 days.[3] The development of them is enhanced by progesterone. These structures are several micrometers wide and project into the uterine lumen above the microvilli level. They were first described in mice[5] and later in human endometrium.[6,7] The term ‘pinopod’, derived from the Greek word ‘drinking foot’. Electron microscopy is the major tool used to observe these structures.[6,7] Pinopod expression is limited to a brief period of maximum 2 days in the menstrual cycle corresponding to the putative window of implantation.[8,9] Others have detected that pinopods are present throughout the mid- to late-secretory phase, however, displaying cycle-dependent morphological changes. This suggests that morphology, rather than pinopod presence or absence, is of great significance.[4] The pinopod-regulated expression pattern throughout the menstrual cycle advocates their use as markers of implantation.

The detection of pinopods during the mid-secretory phase may be extremely useful for an assessment of endometrial receptivity to optimize implantation rates. Pinopods endocytose uterine fluid and macromolecules in it. By doing so, the volume of the uterus decreases, taking the walls closer to the embryoblast floating in it. Thus, the period of active pinocytes might also limit the implantation window.[3] Pinopods continue to absorb fluid, and removes most of it during the early stages of implantation.

Nourishment

The embryoblast spends approximately 72 hours in the uterine cavity before implanting. In that time, it cannot receive nourishment directly from the blood of the mother, and must rely on secreted nutrients into the uterine cavity, e.g. iron and fat-soluble vitamins.[3]

Growth and implantation

In addition to nourishment, the endometrium secretes several steroid-dependent proteins, important for growth and implantation. Cholesterol and steroids[3] are also secreted. Implantation is further facilitated by synthesis of matrix substances, adhesion molecules and surface receptors for the matrix substances.

Mechanism

Implantation is initiated when the blastocyst comes into contact with the uterine wall. The process of implantation may be classified into 3 stages: Apposition, adhesion and invasion.[10] During blastocyst apposition, trophoblast cells adhere to the receptive endometrial epithelium. The blastocyst will subsequently anchor to the endometrial basal lamina and stromal extracellular matrix (ECM). At this point, the achieved embryo–endometrial linkage can no longer be dislocated by uterine flushing. This is followed by an invasive blastocyst penetration through the luminal epithelium.[10]

Even though the blastocyst can implant in different human tissues, surprisingly in the endometrium, this phenomenon can only occur during a self-limited period spanning between days 20 and 24 of a regular menstrual cycle (day LH + 7 to LH + 11). Throughout this period, namely the window of implantation,[11] the human endometrium is primed for blastocyst attachment, given that it has acquired an accurate morphological and functional state initiated by ovarian steroid hormones.[12]

Zona hatching

To be able to perform implantation, the blastocyst first needs to get rid of its zona pellucida. This process can be called “hatching”. A substance probably involved is plasmin. Plasminogen, the plasmin precursor, is found in the uterine cavity, and blastocyst factors contribute to its conversion to active plasmin. This hypothesis is supported by lytic effects in vitro by plasmin.[3]

Apposition

The very first, albeit loose, connection between the blastocyst and the endometrium is called the apposition.[3]

Location On the endometrium, the apposition is usually made where there is a small crypt in it, perhaps because it increases the area of contact with the rather spherical blastocyst. On the blastocyst, on the other hand, it occurs at a location where there has been enough lysis of the zona pellucida to have created a rupture to enable direct contact between the underlying trophoblast and the decidua of the endometrium.[3]

Adhesion

Adhesion is a much stronger attachment to the endometrium than the loose apposition. The trophoblasts adhere by penetrating the endometrium, with protrusions of trophoblast cells.

Cellular adhesion molecules family

The cellular adhesion molecule (CAM) family is composed of 4 members known as integrins, cadherins, selectins, and immunoglobulins. These surface ligands, usually glycoproteins, mediate cell-to-cell adhesion. Their classical functions include maintenance of tissue integration, wound healing, morphogenic movements, cellular migrations, and tumor metastasis.

Integrins

Integrins are a family of transmembrane glycoproteins. A large variety of integrins have been described within the luminal and glandular endometrial epithelium.[13] Whereas the majority of the integrins are constitutively expressed throughout the entire menstrual cycle, others exhibit an interesting regulated pattern within the cycle.[13] Integrins whose expression is increased in the mid-luteal phase were proposed as markers for the frame of the window of implantation.[14] 3 cycle-specific integrins are co-expressed by the human endometrium defined histologically on days 20 – 24 of the human menstrual cycle: α1β1, α4β1 and αVβ3. In regard to its expression pattern along with its epithelial localization, αVβ3 has been proposed as a potential receptor for embryonic attachment.[15]

Integrins are also expressed by the human trophoblast at the time of implantation.[16] Considering expression and regulation, αVβ3 represents a promising clinical and research marker of the human implantation process.

Selectins

Selectins are glycoproteins, which also belong to the CAM family. They include P-selectin, L-selectin, and E-selectin. The human L-selectin is of importance in the implantation process.[17]

Cadherins

Cadherins constitute a group of glycoproteins responsible for the calcium-dependent cell-to-cell adhesion mechanism. They are divided into subclasses E-, P-, and N-cadherins that are distinct in immunological specificity and in tissue distribution. In regard to implantation, E-cadherin represents the most studied subclass.

Immunoglobulins

Among the CAMs family, the immunoglobulins superfamily is the most extensive. Intercellular adhesion molecule-1 (ICAM-1 or CD54) is a transmembrane glycoprotein that belongs to the immunoglobulin superfamily and is constitutively expressed on the cell surface of a variety of cell types, such as fibroblasts, leukocytes, endothelial, and epithelial cells.[18]

Although ICAM-1 was not shown to be indispensable for the early steps of blastocyst interactions with the endometrium, it could participate indirectly in this process by interacting with the immune system. A clearer picture of human endometrial pathophysiologies may be acquired by further studies of ICAM-1 expression and function.

Mucins

Mucins are high molecular weight (MW) glycoproteins. Among the 14 cloned human mucins, only Mucin-1 (MUC1) and to a lesser extent, MUC6 have been found in the human endometrium.[19] MUC1 appears to be a negative factor for an embryo implantation. Indeed, in the area where implantation takes place, MUC1 disappears. This effect was shown to be controlled in vitro mainly by the sheddase family enzymes that are modulated by blastocyst and endometrial derived factors. Because endometrial MUC1 increases at the time of implantation, it has been suspected that this factor has a crucial role to direct the embryo temporally and spatially to effective implantation.

Cytokines (T_H1, T_H2, T_regs)

Cytokines comprise a group of proteins that separately or in concert modulate a variety of cellular functions, such as cellular proliferation and differentiation. They play a major role in the reparative and inflammatory-like processes occurring every menstrual cycle in the human endometrium, but they are also implicated in critical reproductive events such as ovulation and implantation.[20]

T cells have also been a focus of research. Initially, T cells were thought to influence implantation through a T_H1/T_H2 balance.[21] Pregnancy was postulated to be a T_H2-mediated event; T_H1 cytokines such IFN-γ and TNF-α are associated with infertility and abortion, and these effects can be reversed in mice by injecting the T_H2 cytokine IL-10.[22] Tregs may play a role in implantation and are essential for the establishment of peripheral tolerance; they suppress (auto-) reactive T cells.[23] Tregs have been characterized as CD4+CD25+ cells.[24] Forkhead box p3 (Foxp3) is present exclusively in Tregs and is necessary for their development and function.[25] In the context of pregnancy, Tregs have been characterized as essential to the establishment of allotolerance. CD4+CD25+ T cells have been found in the human decidua at various stages of pregnancy. Their levels are highest in the peripheral blood during the first trimester of pregnancy.[26] Furthermore, autoimmune diseases improve during the course of pregnancy and subsequently relapse after delivery.[27] In a recent experiment, endometrial biopsies of (unexplained) infertile and proven fertile women are compared for the expression of transcription factors that determine T cell differentiation. It was found that expression of Foxp3, necessary for T_reg development and function, was significantly lower in the infertile group of women, whereas the transcription factors T-bet and GATA-3 (for T_H1 and T_H2, respectively) did not differ between both groups.[28] This supports the pivotal role of T_regs in successful human implantation. In conclusion, Tregs are of key importance to successful establishment of pregnancy. Much research is still needed to identify the precise roles of CD4+CD25+ T cells and possibly other subsets of T_regs.

Leukemia inhibiting factor

Leukemia inhibiting factor (LIF) is an IL-6 family pleiotropic cytokine, which also includes oncostatin M (OSM), ciliary neurotrophic factor (CNTF), and cardiotrophin 1. A recombinant human LIF (r-hLIF) has been investigated in preclinical and clinical trials to improve endometrial receptivity in RIF patients. In view of the important role of LIF in implantation, an administration of such r-hLIF could be valuable in future studies.

Interleukins

IL-6 is a pleiotropic cytokine, originally identified as a factor inducing immunoglobulin production in activated B cells and initially designated as IFN-β2 and B-cell differentiation factor or B-cell stimulatory factor-2. The fact that IL-6 is maximally expressed[29] during the window of implantation. IL-1 system may be an important paracrine/autocrine mediator of local intercellular interactions in the endometrial tissue.[30]

Colony stimulating factor-1

Colony stimulating factor-1 (CSF-1) expression and receptors for CSF-1 are found both in human endometrium (peaking in decidua) and in the pre-implantation embryo. Mice with aninactivating mutation in the CSF-1 gene are infertile because of low rates of implantation and fetal viability.[31]

Prostaglandins

The process of implantation can be thought of as a proinflammatory reaction,[32] given that embryo attachment and invasion into the endometrium require connection to the maternal vascular system. It has long been speculated that prostaglandins (PGs), as vasoactive factors, play an important role in ovulation, fertilization, and in late-pregnancy processes leading to the onset of labor.[33] PGs are members of the ‘eicosanoids’ family, which also comprises leukotrienes (LTs) and thromboxanes (TXa). They consist of 4 members, named PGD2, PGE2, PGF2a and prostacyclin (PGI2), which are generated from the membrane phospholipids by the consecutive action of two enzymes, cytosolic phospholipase A2(cPLA2) and cyclooxygenase (COX).

The lack of either of these enzymes leads to an absence of PG synthesis, which then results in several implantation defects.

PGs were shown to be essential for embryo implantation. Their role consists in timing the window of implantation. Delayed timing of blastocyst implantation has a ripple effect that presents in mice as embryo crowding near the cervix, abnormal placentation, and fetal resorption. PGs supplementation can partially restore a normal phenotype. Whether PGs have a similar role in human implantation should be further explored.

Proteoglycan receptors

another ligand-receptor system involved in adhesion is proteoglycan receptors, found on the surface of the decidua of the uterus. Their counterparts, the proteoglycans, are found around the trophoblast cells of the blastocyst. This ligand-receptor system also is present just at the implantation window.[3]

Invasion

Invasion is an even further establishment of the blastocyst in the endometrium.

Syncytiotrophoblasts

The protrusions of trophoblast cells that adhere into the endometrium continue to proliferate and penetrate into the endometrium. These penetrating cells differentiate to become a new type of cells, syncytiotrophoblast. The prefix syn- refers to that the boundaries between these cells disappears, forming a single mass of a multitude of cell nuclei (a syncytium). The rest of the trophoblasts, surrounding the inner cell mass, are hereafter called cytotrophoblasts.

Invasion continues with the syncytiotrophoblasts reaching the basal membrane beneath the decidual cells, penetrating it, and further invading into the uterine stroma. Finally, the whole embryo is embedded in the endometrium. Eventually, the syncytiotrophoblasts come into contact with maternal blood and form chorionic villi. This is the initiation of forming the placenta.

CONCLUSION

Embryo implantation is the result of a well-orchestrated sequence of events including cellular adhesion, invasion, and immune regulatory mechanisms, some of which are controlled through genetic processes by the ovarian hormones. It is proposed that an embryo implantation is a well-defined and precise process, in which various factors come into play one after the other, yet remaining in close collaboration.