Assessment of endometrial dating

The most often used proliferation markers are the DNA S-phase Ki Mib-1 molecule , the anti-apoptotic bcl-2, and the tumor suppressor gene p Both the gland and stromal cell nuclei stain strongly and diffusely for Ki, so do occasional endothelial cells of endometrial capillaries x Cytoplasmic membrane staining for bcl-2 is seen in endometrial gland cells and occasional stromal cells x Immunostaining for p53 is mainly confined to nuclei of stromal cells x Increased proliferation leads to a considerable thickening of the endometrial mucosa.

It is interesting to note that the endometrium demonstrates geographic variations in its response to hormonal stimuli. Maximum DNA synthesis is observed in the fundus and body of the uterus, whereas the isthmic and cornual regions contain comparatively lower values.

Also, nuclear DNA activity is higher in the upper third than in the lower two thirds of the functional layer. This zonal variation in sensitivity of endometrial tissue to hormonal influence may be related to different physiologic functions: Whether differences in hormonal responses are due to dissimilar vascular supply of the upper and lower layers or to the intrinsic, heterogeneous nature of the endometrial tissue in terms of receptor content, or to both, remains to be determined.

Maximum DNA synthesis during the midproliferative phase of the cycle i. Increased nuclear DNA synthesis and mitotic activity in gland cells correlate with high levels of nucleolar organizer regions. According to animal studies, DNA synthesis decreases rather than increases after 2 days of estrogen administration. Inhibition of nucleic acid synthesis is apparently not related to loss of estradiol receptors or nuclear translocation of estradiol receptors, but rather, presumably, to accumulation of the chalone-like inhibitors of DNA synthesis.

This hypothesis is attractive, but it remains to be tested.

My approach to the interpretation of endometrial biopsies and curettings

In addition to tissue proliferation, estradiol promotes the development of free and bound ribosomes, mitochondria, golgiosomes and primary lysosomes in gland cells and presumably in stromal cells. Biochemically, these organelles each provide for protein matrix, energy, and synthesis of various enzymes. Some of these enzymes, including glucosephosphatase, hexokinase, pyruvate kinase, and lactate dehydrogenase, are involved in carbohydrate metabolism. Concentrations of estradiol receptors and progesterone receptors increase in both the blood and the endometrium during the proliferative phase of the cycle see Fig.

Another characteristic feature of proliferative-surface and gland-lining cells is an increase in the number of cilia and microvilli.

These decrease considerably during the secretory phase, suggesting that endometrial ciliogenesis and microvillogenesis are estrogen dependent. Ciliated cells are especially numerous around gland openings. It has been suggested that this peculiar distribution and strong-forward and slow-recovery ciliary beat pattern facilitate mobilization and distribution of endometrial secretions during the luteal phase of the cycle. Intracytoplasmic filaments serve as a cytoplasmic 'skeleton' in gland and stromal cells. Gland cells have cytokeratin- and vimentin-positive intracytoplasmic filaments, whereas endometrial stromal cells stain strongly for vimentin, smooth muscle-related antigens and CD Fig.

Proliferative endometrium, cycle day Interglandular stromal cells strongly and diffusely immunostain for CD x Lymphoid aggregates resembling follicles may be seen in the endometrial stroma, particularly in the basal layer and during the proliferative phase of the cycle. They are unlikely to play a significant role, if any, in the local secretory immune system. Indeed, endometrial epithelial cells synthesize negligible amounts of immunoproteins, 2 and IgG-containing plasma cells are absent in normal endometrium.

The observations are consistent with the sterile nature of normal endometrium. The two main mechanisms of endometrial vessel formation have been suggested to occur via intussusception and sprouting. In the latter, endothelial cell activation, degradation of basement membrane, migration and proliferation lead to formation of tubules, stabilization of pericytes, and extracellular matrix formation. Pericytes may, in fact, play a significant role in angiogenesis and regulation of blood flow.

The cycling endometrium requires repeated, rapid, short-term proliferation and rapid arrest of neovascularisation. Angiogenesis in the endometrium is regulated and controlled by a multitude of promoters and inhibitors, all of which are believed to be under the influence of estradiol and progesterone during the menstrual cycle.

The most important promoter of endothelial growth via mitosis is the vascular endothelial growth factor VEGF. During the postovulatory, or secretory, phase the estradiol-primed endometrium is under progestagenic stimulation and undergoes secretory differentiation.

My approach to the interpretation of endometrial biopsies and curettings

Because similar changes may be produced by estrogens alone in the absence of ovulation, variably sized subnuclear vacuoles in a mitotically active endometrium are not considered specific to ovulation. The most reliable histologic alterations that are considered specific to ovulation are seen on the POD 3 or 17 th day of the cycle. Both phenomena involve every cell in a given gland Fig. Day 17 POD 3 secretory endometrial glands with S-shaped configuration, subnuclear vacuolization, and palisading of nuclei in middle of lining epithelium x At the transmission electron microscopic level, ovulation may be recognized by the appearance of giant mitochondria and the so-called nucleolar channel system in gland cells.

They are presumably produced by the infolding of the nuclear membranes under progesterone stimulation. During the first four postovulatory days, both the glandular and stromal cells are engaged in DNA synthesis and mitotic activity although to a much lower degree than is seen in the preovulatory and proliferative endometrium Fig.

Similarly, other proliferative markers such as bcl-2 and tumor suppressor gene p16 are only rarely seen, if at all Fig. Postovulatory endometrium, POD 3 cycle day Immunostaining for Ki in glands and stromal cells is reduced from that seen in the preovulatory endometrium X Immunostaining for p16 is absent from cells lining an 'S'-shaped gland x These findings indicate reduced proliferative activity and an increase in accumulation of intracytoplasmic glycogen.

The glands are engaged in intracellular but not yet in active extracellular secretion of glycoproteins. On POD 5 and 6 cycle days 19 and 20 , the intracellular secretory products are extruded into the glandular space by apocrine-type secretion. This is characterized by protrusions and eventual detachment of the apical portion of cells containing glycoproteins. Transudation of plasma from circulating blood in the endometrial mucosa also contributes to uterine secretory fluids. The peak of intraglandular secretions on POD 7 cycle day 21 coincides with the time of implantation of the free blastocyst if fertilization has taken place in this cycle.

Nucleic acid synthesis by gland cells ceases as apocrine secretory activity is initiated by POD 5 cycle day 19 Fig. Postovulatory day 11 cycle day Immunostaining for the proliferation marker Ki is confined to predecidual stromal cells as DNA synthesis is shut off within secretory-type endometrial gland cells x Immunostaining for ER is present in both gland and stromal predecidual cells but of less intensity than in proliferative endometrim x Immunostaining for PR is confined exclusively to predecidual stromal cells indicating arrest in E2-dependent glandular growth x This correlates with total lack of mitoses in the glands during the mid and late periods of the secretory phase.

It has been suggested that in mice, progesterone induces the epithelial cells to enter a nondividing G0 stage of the cell cycle during decidualization. For accurate dating purposes from cycle day 21 POD 7 onwards, the pathologist relies on changes in the stroma, rather than in the glands. These include edema, coiling of spiral arterioles, and predecidualization of the stroma.

Endometrial vascular proliferation at the implantation site is related to the blastocyst rather than to histamine or PGE2. The blastocyst has a unique biologic property that is shared only with tumor cells producing the so-called angiogenesis factor, a substance capable of inducing growth of new capillaries. Receptors for human chorionic gonadotropin and luteinizing hormone are present in vascular smooth muscle cells and endothelium, suggesting their possible role in regulating blood flow.

Although decidualized cells have receptors for estradiol, its role in this process, if any, is not clear. This change consists of cytonuclear enlargement with tetraploid nuclei resulting in plump, liver-like epithelioid cells. Predecidualization not pseudodecidualization is accompanied by an increase in nuclear DNA synthesis, mitotic activity, and the formation of a pericellular laminin substance.

Endometrial Biopsy

Consequently, predecidual cells are relatively independent of the growth-inhibitory effect of progesterone. Predecidual cells are of stromal origin Fig. They have mesenchymal-related e. CD, vimentin, desmin , not epithelial-related e. They represent precursor forms of gestational decidual cells decidua vera. Because they develop after implantation, they are not involved in the implantation process per se. The cells have several metabolic functions related either to pregnancy or, if conception has not occurred, to menstrual breakdown of the endometrium.

For example, prolactin is produced by decidual cells and is related to osmoregulation of amniotic fluid. POD 11 cycle day The predecidual cells of the compacta layer of the functionalis below the surface epithelium display a strong and diffuse immunostaining reaction for CD x Decidual tissue during early gestation is rich in lymphocytes, natural killer cells and macrophages. The latter are observed near nidation sites, are of the major histocompatibility complex MHC class II type, and have the potential to present fetal antigens to T-lymphocytes.

In addition, they secrete monokines and have immunosuppressive activity. In the nongestational endometrium, predecidual cells are engaged in phagocytosis and digestion of extracellular collagen matrix. These cellular activities may contribute to menstrual breakdown of the endometrium. The presence of progesterone, however, appears to be a prerequisite for decidualization, for without it, no such reaction is observed in vitro.

Predecidual transformation of stromal cells begins around the arterioles on POD 9 cycle day 23 and later involves the stroma under the surface epithelium, producing the compacta layer by POD By POD 12 cycle day 26 , the predecidual stromal reaction under the surface epithelium coalesces with predecidual cells around the arterioles forming large sheets of predecidua. One day later, this confluence may involve the upper two thirds of the functional layer.

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They are members of the large granular lymphocyte series. They are particularly numerous during the first trimester of pregnancy and may play a role in placentation. By POD 14 cycle day 28 , the spiral arterioles become dilated, and fissures appear in the compacta layer.

These fissures contain edematous fluid, red blood cells, and acute inflammatory exudate. The cyclic endometrium, as with many other tissue systems in the body, is subject to two fundamental types of cell death: Apoptosis, or programmed, single cell death plays a complementary but opposite role to mitosis in normal tissue homeostasis.

It is considered to be the major process responsible for cell death in various physiologic cell-turnover and differentiation events in both the embryo and the adult.

The Endometrial Cycle

These alterations were traditionally called 'polydust' before the true origin of this nuclear debris was recognized. These apoptotic bodies are subsequently engulfed by adjacent cells. An inflammatory reaction composed of leukocytes is generally absent in association with apoptotic bodies. Apoptosis is seen in gland cells and in predecidual cells, both in vivo and in vitro.

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The extent to which apoptosis contributes to endometrial degeneration and gestational decidual regression is not clear. Also unclear is whether apoptosis is more or less important than tissue necrosis. Indeed, the endometrium demonstrates morphologic evidence of coagulative tissue necrosis in response to vasoconstriction of basal arteries and ischemia. The necrotic tissue with increased hydrolytic enzyme activity is characterized by collapse of the upper two thirds of the functional layer. It contains fragmented tissue, with swelling of the cells' nuclei and membrane disruption.

The final event is heterolysis of dead cells due to the action of inflammatory cells invading the necrotic tissue. Primary lysosomes are present in the epithelial, stromal, and endothelial cells of the functional layer of the endometrium. Their release is presumably inhibited by the membrane-stabilizer effect of progesterone. The sudden decrease in estradiol and progesterone levels causes a failure in the membrane integrity of acid phosphatase-containing lysosomes.

As a result, the lysosomal enzyme is released into enzyme-free autophagic bodies filled with sequestered intracellular elements. The destructive action of the acid hydrolases leads to digestion of the incorporated cytoplasmic elements, producing empty vacuoles. Thus, portions of the cytoplasmic substance are removed by lysosomal autodigestion. It also has been suggested that the gradual increase in lysosome-membrane permeability may result in direct intracellular and intercellular diffusion of the lytic enzymes, including type II collagenase found in predecidual cells.

Relaxin, which is found in gland and decidualized cells, stimulates collagenase and plasminogen activators, contributing to tissue breakdown. It destroys the glandular and stromal cells as well as the vascular endothelium. Also, matrix metalloproteins cause breakdown of integrin-mediated adherence on the endothelium to its basement membrane and the exocellular matrix, and play an important role in the menstrual breakdown of the endometrium including its vascular system.