The Implantation Phase
Significant changes occur within the endometrium from the 7th to the 13th day postovulation (days 21-27 of the cycle). At the onset of this period, the distended tortuous secretory glands have been most prominent with little intervening stroma. By 13 days postovulation, the endometrium has differentiated into three distinct zones. Something less than one-fourth of the tissue is the unchanged basalis fed by its straight vessels and surrounded by indifferent spindle-shaped stroma. The midportion of the endometrium (approximately 50% of the total) is the lace-like stratum spongiosum, composed of loose edematous stroma with tightly coiled but ubiquitous spiral vessels and exhausted dilated glandular ribbons. Overlying the spongiosum is the superficial layer of the endometrium (about 25% of the height) called the stratum compactum. Here the prominent histologic feature is the stromal cell, which has become large and polyhedral. In its cytoplasmic expansion one cell abuts the other, forming a compact, structurally sturdy layer. The necks of the glands traversing this segment are compressed and less prominent. The subepithelial capillaries and spiral vessels are engorged.
At the time of implantation, on days 21-22 of the cycle, the predominant morphologic feature is edema of the endometrial stroma. This change may be secondary to the estrogenand progesterone-mediated increase in prostaglandin and vascular endothelial growth factor (VEGF) production by the endometrium that cause an increase in capillary permeability. Receptors for the sex steroids are present in the muscular walls of the endometrial blood vessels, and the enzyme system for prostaglandin synthesis is present in both the muscular walls and the endothelium of the endometrial arterioles. Mitoses are first seen in endothelial cells on cycle day 22. Vascular proliferation leads to the coiling of the spiral vessels, a response to the sex steroids, the prostaglandins, and the autocrine and paracrine factors produced in response to estrogen and progesterone.
During the secretory phase, so-called K (Körnchenzellen) cells appear, reaching a peak concentration in the first trimester of pregnancy. These are granulocytes that have an immunoprotective role in implantation and placentation. They are located perivascularly and are believed to be derived from the blood. By day 26-27, the endometrial stroma is infiltrated by extravasated polymorphonuclear leukocytes. The majority of the leukocytes are killer cells and macrophages, believed to be involved in the process of endometrial breakdown and menstruation. The appearance and function of these cells are regulated by the complex array of peptides and cytokines in the endometrium in response to hormonal signaling.
The gene expression pattern in the endometrium throughout the menstrual cycle is being established, with a focus on the implantation window.23,24 and 25 As expected, microarray analyses reveal a changing pattern of gene expression that correlates with each hormonal and morphological stage in the endometrial menstrual cycle.26 Ultimately this will yield a comprehensive picture, with the gene signature of each event in the estrogen and progesterone regulation of the endometrium. The regulating growth factors, cytokines, and peptide hormones that are essential for implantation will be identified.
The stromal cells of the endometrium respond to hormonal signals, synthesize prostaglandins, and, when transformed into decidual cells, produce an impressive array of substances, some of which are prolactin, relaxin, renin, insulin-like growth factors (IGFs), and insulinlike growth factor binding proteins (IGFBPs). The endometrial stromal cells, the progenitors of decidual cells, were originally believed to be derived from the bone marrow (from cells invading the endometrium), but they are now considered to emanate from the primitive uterine mesenchymal stem cells.27
The decidualization process begins in the luteal phase under the influence of progesterone and mediated by autocrine and paracrine factors. On cycle days 22-23, predecidual cells can be identified, initially surrounding blood vessels, characterized by cytonuclear enlargement, increased mitotic activity, and the formation of a basement membrane. The decidua, derived from stromal cells, becomes an important structural and biochemical tissue of pregnancy. Decidual cells control the invasive nature of the trophoblast, and the products of the decidua play important autocrine and paracrine roles in fetal and maternal tissues.
Lockwood and his colleagues assign a key role to decidual cells in both the process of endometrial bleeding (menstruation) and the process of endometrial hemostasis (implantation and placentation).28,29 and 30 Implantation requires endometrial hemostasis and the maternal uterus requires resistance to invasion. Inhibition of endometrial hemorrhage can be attributed, to a significant degree, to appropriate changes in critical factors as a consequence of decidualization; e.g., lower plasminogen activator levels, reduced expression of the enzymes that degrade the stromal extracellular matrix (such as the metalloproteinases), and increased levels of plasminogen activator inhibitor-1. Withdrawal of estrogen and progesterone support, however, leads to changes in the opposite directions, consistent with endometrial breakdown.
The Phase of Endometrial Breakdown
Predecidual transformation has formed the “compacta” layer in the upper part of the functionalis layer by day 25 (3 days before menstruation). In the absence of fertilization, implantation, and the consequent lack of sustaining quantities of human chorionic gonadotropin from the trophoblast, the otherwise fixed lifespan of the corpus luteum is completed, and estrogen and progesterone levels wane.
The withdrawal of estrogen and progesterone initiates important endometrial events: vasomotor reactions, the process of apoptosis, tissue loss, and, finally, menstruation. The most prominent immediate effect of this hormone withdrawal is a modest shrinking of the tissue height and remarkable spiral arteriole vasomotor responses. The classic concept of the vascular sequence was constructed from direct observations of Rhesus endometrium transplanted to the anterior chamber of the eye.7,8 With shrinkage of height, blood flow within the spiral vessels diminished, venous drainage was decreased, and vasodilation ensued. Thereafter, the spiral arterioles underwent rhythmic vasoconstriction and relaxation. Each successive spasm was more prolonged and profound, leading eventually to endometrial blanching. Thus these reactions were proposed to lead to menstruation because of endometrial ischemia and stasis caused by vasoconstriction of the spiral arterioles. A new model of menstruation, as discussed in Chapter 15, emphasizes enzymatic autodigestion of the functional layer of the endometrium and its capillary plexus.
In the first half of the secretory phase, acid phosphatase and potent lytic enzymes are confined to lysosomes. Their release is inhibited by progesterone stabilization of the lysosomal membranes. With the waning of estrogen and progesterone levels, the lysosomal membranes are not maintained, and the enzymes are released into the cytoplasm of epithelial, stromal, and endothelial cells and eventually into the intercellular space. These active enzymes will digest their cellular constraints, leading to the release of prostaglandins, extravasation of red blood cells, tissue necrosis, and vascular thrombosis. This process is one of apoptosis, (programmed cell death, characterized by a specific morphologic pattern that involves cell shrinkage and chromatin condensation culminating in cell fragmentation) mediated by cytokines.31 An important step in this breakdown is the dissolution of cell-tocell adhesion by key proteins. Binding of endometrial epithelial cells utilizes transmembrane proteins, cadherins, that link intercellularly with each other and intracellularly with catenins that are bound to actin filaments.
Endometrial tissue breakdown also involves a family of enzymes, matrix metalloproteinases, that degrade components (including collagens, gelatins, fibronectin, and laminin) of the extracellular matrix and basement membrane.33,34 The metalloproteinases include collagenases that degrade interstitial and basement membrane collagens; gelatinases that further degrade collagens; and stromelysins that degrade fibronectin, laminin, and glycoproteins. The expression of metalloproteinases in human endometrial stromal cells follows a pattern correlated with the menstrual cycle, indicating a sex steroid response as part of the growth and remodeling of the endometrium with a marked increase in late secretory and early menstrual endometrium.35 Progesterone withdrawal from endometrial cells increases VEGF production and induces matrix metalloproteinase secretion, probably from both endometrial stromal cells and leukocytes, which is followed by the irreversible breakdown of cellular membranes and the dissolution of extracellular matrix.36,37 and 38 Appropriately, this enzyme expression increases in the decidualized endometrium of the late secretory phase, during the time of declining progesterone levels. With the continuing progesterone secretion of early pregnancy, the decidua is maintained and metalloproteinase expression is suppressed, in a mechanism mediated by transforming growth factor-beta (TGF-&bgr😉.39 In a nonpregnant cycle, metalloproteinase expression is suppressed after menses, presumably by increasing estrogen levels.
Metalloproteinase activity is restrained by specific tissue inhibitors designated as TIMP.40 The balance of metalloproteinase and TIMP activity is an important event in successful implantation. Thus, progesterone withdrawal can lead to endometrial breakdown through a mechanism that is independent of vascular events (specifically ischemia), a mechanism that involves cytokines.31 During bleeding, both normal and abnormal, there is evidence indicating that specific genes are activated in the endometrium; one such gene has the structural features of the TGF-&bgr; family.41
There is considerable evidence to support a major role for a cytokine, tumor necrosis factor-a (TNF-a), in menstruation.31 TNF-a is a transmembrane protein whose receptor belongs to the nerve growth factor/TNF family for inducing apoptotic signals. The key change is an increase in secretion because TNF-a secretion by endometrial cells reaches a peak at menstruation, but there is no cycle change in receptor content. TNF-a inhibits endometrial proliferation and induces apoptosis; this cytokine causes a loss of adhesion proteins (the cadherin-catenin-actin complex) and induces cell-to-cell dissolution. In addition to endometrial cells, TNF-a also causes damage to vascular endothelium.
Progesterone withdrawal is also associated with an increase in vascular endothelial growth factor receptor concentrations in the stromal cells of the layers of endometrium destined to be sloughed.42 Although the vascular endothelial growth factor system is usually involved with angiogenesis, in this case these factors are involved in the preparation for menstrual bleeding, perhaps influencing the expression of matrix metalloproteinases (MMPs). Endometrial genes without classic steroid response elements can respond to the sex steroids by utilizing a family of proteins (the Sp family) that mediate steroid activity at the level of transcription (acting in a fashion similar to the steroid receptors). These proteins, induced by progesterone in stromal (decidual) and epithelial cells, can activate tissue factor, plasminogen activator inhibitor-1, IGF binding protein-1, uteroglobin, and uteroferrin. Tissue factor is involved in the clotting mechanism to sustain hemostasis. Uteroglobin is a small protein expressed in endometrial epithelial cells.43 The physiologic function of uteroglobin is uncertain. Uteroglobin, with high affinity, binds progestins and may play a role in immunosuppression. Uteroglobin gene expression is stimulated by estrogen, and this response is enhanced by progesterone. Human endometrium can secrete &bgr;-endorphin, yet another candidate for involvement in endometrial immunologic events, and its release is inhibited by both estrogens and glucocorticoids.
Eventually, considerable leakage occurs as a result of diapedesis, and finally, interstitial hemorrhage occurs due to breaks in superficial arterioles and capillaries. White cells migrate through capillary walls, at first remaining adjacent to vessels but then extending
throughout the stroma. The leukocytes add important regulatory cytokines, chemokines, and enzymes that are involved in the degradation of the extracellular matrix. During arteriolar vasomotor changes, red blood cells escape into the interstitial space. Thrombinplatelet plugs also appear in superficial vessels. The prostaglandin content (PGF2a and PGE2) in the secretory endometrium reaches its highest levels at the time of menstruation. The vasoconstriction and myometrial contractions associated with the menstrual events are believed to be significantly mediated by prostaglandins from perivascular cells and the potent vasoconstrictor endothelin-1, derived from stromal decidual cells.
As ischemia and weakening progress, the continuous binding membrane is fragmented, and intercellular blood is extruded into the endometrial cavity. New thrombin-platelet plugs form intravascularly upstream at the shedding surface, limiting blood loss. Increased blood loss is a consequence of reduced platelet numbers and inadequate hemostatic plug formation. Menstrual bleeding is influenced by activation of clotting and fibrinolysis. Fibrinolysis is principally the consequence of the potent enzyme plasmin, formed from its inactive precursor plasminogen. Endometrial stromal cell tissue factor (TF) and plasminogen activators and inhibitors are involved in achieving a balance in this process. TF stimulates coagulation, initially binding to factor VII. TF and plasminogen activator inhibitor-1 (PAI-1) expression accompanies decidualization, and the levels of these factors may govern
the amount of bleeding.30,45 PAI-1, in particular, exerts an important restraining action on fibrinolysis and proteolytic activity.46 Blood loss is also controlled by constriction of the spiral arteries, mediated by the perivascular cells, myofibroblasts that surround the spiral arteries.47 These cells respond to progesterone withdrawal by expressing prostaglandins, cytokines, and MMPs, causing not only cycling vasoconstriction and vasodilation but also modulating leukocyte entry (an important additional source of metalloproteinases) into the endometrium. Disordered growth and function of the perivascular cells are likely contributing factors in menstrual bleeding problems.
High Progesterone Levels
Perivascular Growth and Decidualization
Prostaglandin, Cytokine, and VEGF Expression
Suppression of Prostaglandin, Cytokine, and MMP Expression
Vasoconstriction, Vasodilation, Leukocyte Infiltration, and Increase in MMPs