Apposition: The Embryo/Endometrial Dialogue

The embryo enters the uterus 3 or 4 days after ovulation, and hatches from the zona pellucida as an expanded blastocyst on Day 5–6, so that the hatched blastocyst is ‘apposed’ to the uterine endometrium. Apposition is facilitated by the transient appearance of specialized epithelial cellular membrane protrusions or ‘pinopodes,’ which surround the pits in endometrial glands and actively reabsorb the uterine secretory phase fluid. Their appearance is progesterone-dependent, they are present for only 2–3 days (between Days 5 and 7 post ovulation), and they are a morphological marker of the opening of the implantation window (Nikas et al., 1995).

The uterine epithelium is coated with a network of glycoproteins (glycocalyx), which acts as a physical barrier to cell–cell interaction. The uterus is not receptive at this stage: the thick mucin coat, long microvilli and negative charge on the cellular surface membranes prevent blastocyst attachment. The embryo must first overcome this ‘glycocalyx barrier,’ which contains mucins expressed by endometrial cells, via localized (i.e., embryo driven) enzymatic cleavage of the extracellular glycoproteins, or via binding of receptors expressed by the embryo and subsequent cleavage of the mucins. During the receptive stage, the microvilli on the apical surface of the uterine epithelial cells shorten and MUC1 expression decreases in the immediate vicinity of the blastocyst. In response to estrogen, expression of maternal factors such as LIF is increased, which promotes endometrial receptivity to blastocyst attachment. Under these conditions, the progesterone-primed uterine wall increases expression of epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF). EGF and HB-EGF bind EGF receptors and heparin sulfate proteoglycans on the trophoblast surface, which induces receptor phosphorylation and a second messenger cascade by the trophoblast; the blastocyst then hatches from the zona pellucida. When apposition is achieved, the adhesion molecules have access to each other, and the embryonic cytotrophoblast cells of the trophectoderm become attached to the endometrial cells.

Adhesion

The embryo is thought to attach (adhere) to the endometrium on Day 6–7 post ovulation, mediated by the polar trophectoderm, the trophoblast cells immediately next to the inner cell mass. Once the embryo and endometrial adhesion molecules have free access to each other, the embryonic trophoblast cells attach to the endometrial epithelial cells: an erosive syncytium of trophoblast cells (syncytiotrophoblast) spreads from the polar trophectoderm into the basal decidua (Figure 6.2a). Several families of adhesion molecules are thought to be involved to a greater or lesser degree in human embryo attachment, including the integrins, tastin/trophinin complexes, heparan sulfate proteoglycans, cadherins, lectin/glycan interactions and glycan/glycan interactions. The integrin family is one of the key adhesion factors involved in human implantation. Experimental evidence has shown that hCG upregulates the integrin avb3, and interleukins IL-1α and IL-1β from the embryo are also involved. Human oocytes, early embryos and blastocysts express integrins, and they are also widely expressed by the glandular epithelium. Some are synthesized at a constant rate (α2, α3, α6, β1, β4, β5), whereas others vary during stages of the cell cycle (α1, α9, αv, β3, β6). The αv family of integrins are most likely to be involved in attachment; they bind fibronectin, osteopontin, vitronectin and other components of the extracellular matrix.

Figure 6.2

(a) Preimplantation and peri-implantation blastocyst, showing both polar and mural trophectoderm, and the emergence of the primitive syncytiotrophoblast from the polar trophectoderm. (b and c) Postimplantation trophoblast development. Note the development of lacunae in the syncytiotrophoblast and the emergence of the primary villi (b).

(d) Anatomy of the definitive placenta, showing both the villous trophoblast organized into chorionic villi and the extravillous cytotrophoblast cells organized into trophoblast cell columns.

Adapted with permission from Baines & Renaud (2017), with thanks to Alejandra Ontiveros.

Invasion

Pre-villous trophoblast starts to differentiate between Days 7 and 12, and endometrial transformation (stromal decidualization) is underway by Day 12 post ovulation (Duc-Goiran et al., 1999). Cytotrophoblasts of the embryonic trophectoderm are mononuclear undifferentiated stem cells that are precursors of all trophoblast forms. These differentiate into subsets via a complex process of gene repression to prevent their self-renewal and induction of genes that promote new biochemical functions (see Baines & Renaud, 2017 for review).

1. 
   

   1. Junctional trophoblast cells from the polar trophectoderm mediate the attachment of the placenta to the uterus.

   
   
2. 
   

   2. Invasive intermediate trophoblasts migrate into the uterine tissue.

   
   
3. 
   

   3. Villous syncytiotrophoblast makes the majority of the placental hormones, including hCG, and regulates nutrient exchange between maternal and fetal blood.

Mononuclear trophoblast cells fuse to form a mitotically inactive syncytiotrophoblast with small chambers (lacunae) that connect together and fill with maternal blood from eroded decidual blood vessels. This invasion of the uterine decidua establishes an interface between fetus and mother, so that nutrients and waste products can be transferred. It also fulfills a mechanical function by stabilizing the placental tissue within the uterus. Cytotrophoblast cells proliferate rapidly to form large finger-like villous projections that penetrate the syncytiotrophoblast (VCT). At their tips, the villi continue as columns of cytotrophoblast cells that make contact with the endometrium and extend laterally, fusing with neighbors to form the cytotrophoblast shell. The shell represents the maternal–fetal interface, anchoring the conceptus to the endometrium. A subpopulation of trophoblast, the extravillous trophoblast (EVT), arises from the outer surface. These cells migrate into the wall of the uterus and play a critical role in remodeling the uterine spiral arteries that ultimately supply the placenta. These villi initially radiate out from the entire conceptus, and then regress gradually, apart from those adjacent to the decidua basalis, where the placenta will develop (Figure 6.2b).

EVTs invade the endometrium and upper layers of the myometrium and remodel the extracellular matrix in order to selectively permeate uterine spiral arteries. The colonized blood vessels are then modified to yield widened, low-resistance channels that can carry an increased maternal blood flow to the placenta. The mother must protect herself from these invasive trophoblasts migrating toward the uterine spiral arteries, and the endometrial stroma transforms itself into a dense cellular matrix known as the decidua, which will form the maternal part of the placenta. During decidualization, the spindle-shaped stromal fibroblasts enlarge and differentiate into plump secretory decidual cells, creating a tough extracellular matrix that is rich in fibronectin and laminin. This transformation occurs under the influence of progesterone.

The reaction is initially localized to cells surrounding the spiral arteries, but subsequently spreads to neighboring cells. The majority of cells of the decidua express leukocyte antigens. The largest single population of white blood cells in the endometrium are natural killer large granular lymphocytes (or natural killer [NK] cells), with smaller numbers of macrophages and T cells also present. The abundance of NK cells increases dramatically between ovulation and implantation, influenced by steroid hormones (King et al., 1998). The decidua forms a physical barrier to invasive cell penetration and also generates a local cytokine milieu that promotes trophoblast attachment.

The first signs of the decidualization reaction can be seen as early as Day 23 of the normal menstrual cycle (10 days after the peak of the luteinizing hormone surge), when the spiral arteries of the endometrium first become prominent. Over the next few days, the effect of progesterone causes the stromal cells surrounding the spiral arteries to transform and differentiate into predecidual cells. This progressive decidualization of the endometrial stroma prepares the uterine lining for the presence of the invasive trophoblasts, but simultaneously closes the door to implantation. At this stage the embryo first becomes visible to the maternal immune system.

The invasion stage requires a very delicate balancing of conflicting biological needs between the early fetus and the mother, with a complex regulation of adhesion molecule expression, coordinated in time and in space. The invading cells use collagenases, and also express plasminogen activator inhibitor type 1, suggesting that the plasminogen activator system may also be involved. They lose integrins associated with basement membrane interactions (possibly laminin), and gain integrins that can interact with fibronectin and type I collagen. The outer layer trophoblast cells fuse to form a multinucleated syncytiotrophoblast layer that covers the columns of invading cells. This layer proliferates rapidly and forms numerous processes, the chorionic villi (the chorion is the layer that surrounds the embryo and extraembryonic membranes). Cyclic AMP and its analogs, and more recently hCG itself, have been shown to direct cytotrophoblast differentiation toward a syncytiotrophoblast phenotype that actively secretes the placental hormones.

At the point where chorionic villi make contact with external extracellular matrix (decidual stromal, ECM), another population of trophoblasts proliferates from the cytotrophoblast layer to form the junctional trophoblast. The junctional trophoblasts make a unique fibronectin, trophouteronectin (TUN), which appears to mediate the attachment of the placenta to the uterus. Transforming growth factor-β (TGF-β) and, more recently, leukemia inhibitory factor (LIF) have been shown to downregulate hCG synthesis and upregulate TUN secretion. These cells also make urokinase-type plasminogen activator and type 1 plasminogen activator inhibitor (PAI-1). Experiments using in-vitro model systems showed that phorbol esters increase trophoblast invasiveness and upregulate PAI-1 in cultured trophoblasts. Uterine prostaglandins (PGF2 and PGE2) are regulated by steroids and are involved in regulating the formation of the decidua.

The Implantation Window

The existence of a transient implantation window has been well documented for rodent species. In these species, the window is maternally directed, and the receptive state is sustained for less than 24 hours. In the human, the window appears to be approximately 5 days long (Day 6 to Day 10 post ovulation in the normalized 28-day menstrual cycle), and the opening of the receptive phase is not as clearly defined as its termination. In the mouse, hatched blastocysts readily attach and spread in an integrin-dependent process on any surface that includes ligands found in the stroma during pregnancy (fibronectin, collagen, laminins, vitronectin, thrombospondin, etc.) as well as on artificial substrates like Matrigel. However, during human embryo implantation, trophoblast is penetrated through the epithelium, and the process is likely to be more strongly regulated by complex and multifactorial interactions between embryo and endometrium.

Summary

Successful implantation requires both a synchronous development and a synchronized interaction between blastocyst and endometrium. Direct signaling from the embryo to the endometrium upregulates molecules such as the integrins, and this signaling promotes blastocyst adhesion. The blastocyst initially derives nourishment from uterine secretions, but in order to continue growing the conceptus must develop its own vascular system and, as a first step, induces a highly specialized reaction in the uterine stroma that initiates sprouting and growth of capillaries – the primary decidual reaction. The decidualized stromal cells make pericellular fibronectin. There is a dramatic transformation of endometrial stromal cells and a massive leukocyte infiltration by NK cells and macrophages. Maternal hormones influence the communication between the embryo and the endometrium, via effects on cytokines, adhesion molecules, prostaglandins, metalloproteases and their inhibitors, and angiogenic growth factors.

The molecular mechanisms behind this complex and sophisticated process have been studied using animal models, and knockout (KO) mouse studies have positively identified genes for receptivity (LIF, HMX3), responses to embryo (Cox2) and decidualization (IL-11R). Other factors identified as having a role include immune response gene (IRG1), progesterone receptor knock-out (PRKO), estrogen receptor knock-out (ERKO), homeobox protein A10 (Hoxa10), IHH (Indian Hedgehog gene) and immune regulating hormone 1 (IIRH1). Estrogen and progesterone receptors and the signaling pathways that interact with them are clearly important, including the IGF-I and EGF family and prostaglandins. Research in humans continues, now using endometrial organoid culture and microarray technology to look at gene expression in order to identify those factors that determine a receptive endometrium, with the hope of elucidating mechanisms that may enhance successful implantation after ART (Sherwin et al., 2007; Simon et al., 2009; Turco et al., 2017).