Endometrial histopathology-Basics

Endometrium in surgical pathology

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Embryology

• The endometrium and the myometrium are of mesodermal origin and are formed secondary to fusion of the mullerian (paramesonephric) ducts between the 8th and 9th postovulatory weeks.

• Until the 20th week of gestation

– Endometrium consists of a single layer of columnar epithelium

– Supported by a thick layer of fibroblastic stroma.

• After the 20th week

– Surface epithelium invaginates into the underlying stroma

– Glandular structures formed that extend toward the underlying myometrium. aj

• At birth

– Endometrium measures less than 0.5mm in thickness

– The surface and glands are lined by a low columnar to cuboidal epithelium

– Epithelium is inactive.

• During the prepubertal years, the endometrium remains inactive(similar to postmenopausal age).

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Layers of endometrium• In the reproductive years, there are two regions

– Superficial functionalis (stratum spongiosum)

– Basalis (stratum basale).

• The functionalis has greatest degree of hormonal responsiveness, while basalis is less responsive, the morphology not varying greatly during the menstrual cycle

• A biopsy consisting entirely of basalis is not adequate for dating of the menstrual cycle.

• Usually, the endometrial glands are regularly spaced and have a perpendicular arrangement from the basalis to the surface.

• The basalis abuts the myometrium and regenerates the functionalis following its shedding during menstruation. The basalis is composed of inactive appearing glands, cellular stroma, and spiral arteries that have thicker muscular walls than those in the functionalis.aj

• The endometrium during the reproductive period undergoes cyclical morphologic changes, which are particularly evident in the superficial two thirds, the so-called functionalislayer.

• Morphologic alterations are minimal in the deeper one third, the so-called basalis layer. Functional layer develops from it

• A=functionalis layer• B=basal layer• C=myometrium

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Vascular anatomy• The endometrial vascular supply

originates from the radial arteries of the underlying myometrium.

• These arteries penetrate the endometrium at regular intervals and give rise to the basal arteries, which in turn divide into – Horizontal providing the blood supply to

the endometrial basalis– Vertical branches supplying the overlying

functionalis layer.

• The endometrial vessels in the functionalis layer are referred to as spiral arteries.

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• A differentiating feature between the endometrial and myometrial arteries is the absence of subendothelial elastic tissue in the endometrial arteries, except for those in the basal layer.

• Uterine lymphatics drain from subserosaluterine plexuses to the pelvic and para-aortic lymph nodes.

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Epithelium of endometrium

• Epithelium of the endometrium, which is tall and columnar in form. Some of the cells bear cilia, the remainder having surface microvilli.

• Stromal cells have plump, spindle-shaped nuclei and scanty cytoplasm.

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• Histology for pathologists, 4th ed, pg 1089

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• During adult reproductive age group the endometrium is undergoing constant cyclical changes under effects of hormones which is termed as menstrual cycle.

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Sampling methods

Technique Feature

Endometrial curettage (cervical dilationand endometrial curettage—D and C)

Removal of most of the uterine mucosa by scraping with a sharp curette.Considered the “gold standard”, as tissue amount is good

Endometrial biopsy (EMB) Limited sample received and performed with a smaller curette. Single strips of endometrium are usually taken from both the anterior and the posterior fundal surfaces.Does not require cervical dilation.Potential to miss focal lesions

Hysteroscopy in combination with endometrial sampling

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• Low power view of a Pipelle sample showing what appears to be much tissue, but is actually mostly blood.

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• Diagrammatic representation of the endometrial glands and arterioles throughout the menstrual cycle.

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Endometrium in pathology

• Basic questions

– Why endometrial sampling?

– Why interpretation important and difficult?

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Why do endometrial sampling?

• There are four main indications for endometrial sampling:

1. Determination of the cause of abnormal uterine bleeding.

2. Evaluation of the status of the endometrium in infertile patients, including histological dating.

3. Evacuation of products of conception, either spontaneous abortions or termination of pregnancy.

4. Assessment of the response of the endometrium to hormonal therapy, especially estrogen replacement in perimenopausal and postmenopausal women and Tamoxifen therapy for breast cancer.

5. Multiple overlapping reasonsaj

First a few terms

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Causes of AUB

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Endometrial dating

• Interpreting the cycle based on histomorphology of endometrium.

• First by Noyes et al

• Current modifications of his chart are in use for benign endometrium.

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Interobserver variability• High

• Histologic endometrial dating does not have the accuracy or the precision necessary to provide a valid method for the diagnosis of luteal phase deficiency or to otherwise guide the clinical management of women with reproductive failure.

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Accurate dating not possible

• Polyps

• Endometritis

• IUD

• Other pathological lesions

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Data required with biopsy

• Age

• Clinical presentation

• Obstetric history- LMP, details of cycle

• Gynecological history

• Drug history- hormone therapy?

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• Hypothalamus, pituitary, ovarian cortex, and endometrium.

• Two major phases

– follicular (proliferative)

– luteal (secretory) phase

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Endometrial cycle

• Typical=28 days• Range= 24-35 days• Variation due to changes in duration of PP, SP

being constant 14 days from ovulation to menstruation.

• 20% of women at some time experience an irregular cycle.

• Durations of flow may range from 2 to 7 days• Blood loss should be less than 30 ml.• Anovulatory cycles are most common before age

20 and after age 40.aj

Cyclic endometrium phases based on morphology

1. Menstrual phase

2. Early proliferative phase

3. Mid- to late proliferative phase

4. 16-day endometrium

5. Vacuole phase of early secretory endometrium

6. Secretory exhausted phase (mid)

7. Predecidua phase (late)

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Reasons for assigning a precise date to an endometrial biopsy

1. The need to establish that ovulation has taken place or not.

2. To determine if the assigned date falls within the expected range based on the patient’s last menstrual period. This requires cooperation of both the clinician and the pathologist in determining whether a disturbance in the luteal phase has occurred.

3. To determine if a morphologically visible maturation disturbance exists, such as dyssynchronous maturation of endometrial glands in relation to the stroma.

4. To become accustomed to small differences in morphology of benign endometrial changes. An appreciation of such nuances may facilitate the recognition of subtle neoplastic processes.

5. To determine if sufficient endometrial luteal maturation has occurred in a preparatory cycle for potential recipients of egg donation or frozen embryo. aj

Menstrual phase

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Menstrual phase

• Absence of implantation of a fertilised ovum, degeneration of the corpus luteum results in cessation of oestrogen and progesterone secretion.

• In turn, this initiates spasmodic constriction in the spiral arterioles

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Menstrual phase

• Morphologically it is defined by collapse of the stroma and shedding of the functionalis.

• Usually 4 days

• Condensation of predecidual cells into discrete aggregates or cordlike arrangements admixed with blood and inflammatory cells.

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Menstrual

• Diffuse pattern of stromal breakdown

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• degeneration of the superficial layers of the endometrium and leakage of blood into the stroma

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• Stroma is condensed and collapsed, and the stromal cells aggregate into tightly packed balls (stromal blue balls) and separate from the glands.

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• Apoptotic bodies are identified within both the glands and the stroma

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• Subsequently, a pattern of “scheduled breakdown” begins, with coordinate collapse of the functionalis, sheets of neutrophils, and the characteristic plump aggregates of necrotic predecidua.

• The debris contains exhausted secretory glands that range from large, irregularly shaped glands with slightly stratified nuclei (a consequence of epithelial collapse and nuclear condensation) to single-cell layered epithelium with a thin or delicate appearance

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• The exhausted glands contain delicate single-layered epithelium and cordlike aggregates of necrotic predecidualized stroma

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• Predecidual stromal aggregates or exhausted glands typifies ovulatory breakdown and, when encountered, this pattern of scheduled (diffuse) breakdown with acute inflammatory debris most commonly signifies that ovulation has occurred.

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Day 3 menses

• At day 3 of the menses, the diagnosis of ovulation can still be made provided sufficient secretory exhausted glands are still present.

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Day 3 menses

• Features of ovulation can still be appreciated due to presence of glands

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• Following this, the remainder of the breakdown occurs in the lower functionalisand the diagnosis of ovulation cannot be confirmed on histologic grounds.

• The diagnosis rendered for these changes is menstrual (ovulatory) endometrium.

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Variations in menstrual endometrium

• Irregular shedding

• In this, fragments of intact predecidualizedendometrium coexist with early proliferative-pattern endometrium.

• The explanation for this picture is persistent corpus luteum, leading to a greater range of findings in which features of early breakdown (predecidualizefragments) coexist with later regenerative (tubular glands) components

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Irregular shedding

• Early breakdown of predecidualizedendometrium

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Irregular shedding

• Early proliferative pattern with star-shaped glands

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Variations in menstrual endometrium

• Membranous dysmenorrhea

• Consisting of cohesive sheets of predecidualizedendometrium with scarce glands, alternating with conventional disaggregated endometrium.

• This pattern was anecdotally associated with dysmenorrhea in some instances, hence the term.

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Membranous dysmenorrhoea

• Intact sheets of necrotic predecidua(lower).

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Papillary syncytial metaplasia• An inaccurate term as it is not a metaplasia but rather a

regenerative or degenerative process secondary to tissue breakdown.

• Mitotic figures may be present within the papillary proliferations.

• It is also seen following surface breakdown associated with nonmenstrual conditions.

• On occasions, the micropapillary architecture is particularly striking, and, if associated with mitotic activity, this raises the possibility of a serous carcinoma

• Differentiated by – background endometrium shows features of breakdown

– p53 staining which is negative here.aj

• Florid papillary syncytial metaplasia

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Papillary syncytialmetaplasia

• Following breakdown, the endometrial glands regenerate with a surface micropapillaryarchitecture

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• Diagnostic Gynecologic and Obstetric Pathology, pp413aj

Microphotographs • Menstrual endometrium

is characterized by an

– influx of inflammatory cells into the endometrium,

– thrombi in stromalvessels

– Apoptosis

– gland–stromaldissociation

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Day 1

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Day 2

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• The dissociation of the glands from the stroma leads to stromal aggregates, which appear darker and these are called “blue balls”.aj

• Menstrual endometrium.With stromal hemorrhage, the predecidual cells collapse and they lose their abundant cytoplasm

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Day 3 and 4

• Menstrual endometrium, cycle days 3 and 4.

• Note dense stromalcell aggregates beneath the regenerative surface epithelium (arrow)

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Proliferative phase

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Proliferative phase

• During the proliferative phase, the endometrium grows from about 0.5 mm up to 4.0 to 5.0 mm in thickness, so by the late proliferative phase, a biopsy obtains a moderate amount of tissue.

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Proliferative phase

• The proliferative phase usually lasts 14 days, but under physiologic conditions may fluctuate between 10 and 20 days.

• It is therefore impossible to distinguish each day during this phase.

• Consequently, it is subdivided only into the early, middle, and late proliferative phases.

• Dating of proliferative endometrium is not utilized clinically, and so a diagnosis of proliferative endometrium is sufficient.

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Early proliferative

• The histologic features of endometrial breakdown described above may overlap and persist into the early proliferative phase.

• Early proliferative endometrium is composed of straight, relatively uncoiled glands, and confi guration may best be appreciated on a hysterectomy specimen.

• On biopsy or curettage, the glands are most often seen in cross section, and look like “little blue donuts”, small circular glands.

• The epithelium is pseudostratified

– while nuclei may be seen at various levels of the epithelium, all epithelial cells are in contact with the basement membrane of the gland, hence not truly stratified.

• Mitotic activity may be seen in the glands and stroma over the proliferative period, increasing along the cycle

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• Pseudostratifiedepithelium contains prominent nuclei with mitoses

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Mid proliferative phase

• There is tortuosity of glands due to their increase in length, which exceeds the growth in height of the endometrium.

• The glandular epithelial cells are tall columnar cells and contain slightly enlarged oval-shaped, chromatin-rich nuclei in a dense, sparse cytoplasm rich in RNA.

• Nucleoli are prominent, and mitoses frequent. • The spindle-shaped stromal cells are still poorly

differentiated, but rich in DNA and RNA with occasional mitoses and separated by interstitial edema (Harkin 1956).

• The surface epithelium has increased in height and is now low columnar.

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Late proliferative phase• Marked tortuosity of glands lined by tall columnar epithelial

cells piled up against one another with their nuclei at different levels, giving a pseudostratified appearance.

• Nuclei -enlarged, more elongated or oval shaped.

• Nucleoli -quite prominent, mitoses are frequent.

• Cytoplasm is sparse and poorly differentiated.

• Stromal cells are enlarged. Stromal mitoses are frequent. The cells are still of uniform size and without signs of differentiation. The stromal edema has subsided.

• Spiral arterioles are still absent.

• The general height may be slightly lower than that of the midproliferative phase due to decrease of edema. The surface epithelium is distinctly columnar.

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• Diagnosis of endometrial biopsies and curretings 2005, pg 11aj

• The orientation and outline of proliferative phase glands and their relationship to intact stroma are important features for recognizing this normal pattern, as hyperplastic glands or glands in a polyp can have cytologic features identical to those of glands in the proliferative phase.

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Pitfalls in dating of proliferative phase

• Variable appearance of the stroma

• Include a prominent spindled appearance. This may sometimes be misinterpreted as predecidua or progestin effect.

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• The second pitfall is intraluminal material misinterpreted as secretions.

• “Secretions” can be found in the lumen of any endometrial gland irrespective of cycle day and should not be used to assign a cycle date.

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• A third pitfall is a technical one where gland tracts are susceptible to intussusception, also termed “telescoping” or “gland in gland” artifact.

• This may be misinterpreted as hyperplasia (EIN)

• The diagnosis for this phase is simply “proliferative endometrium.”

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• Gland in gland (telescoping) artifact

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• Assessing gland orientation can be complicated, by biopsy-induced fragmentation, an especially common artifact in early to mid-proliferative phase biopsies when the mucosa is still thin.

• Detached and disrupted glands may appear abnormally crowded or irregular. To separate fragmentation artifact from true abnormalities, it is important to assess the integrity of the stroma as well as the glands and to use surface epithelium to help orient the tissue fragments.

• Detached and poorly oriented glands that show pseudostratified nuclei and mitotic activity usually represent proliferative endometrium unless better-oriented tissue suggests otherwise.

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• Proliferative phase

• Constituent cells are pseudostratified, and the characteristically elongated glandular nuclei have dense chromatin.

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• Proliferative phase glands with ciliated cells in the gland at the right. The round cell with clear cytoplasm at the 3:00 o’clock position has the characteristic appearance of ciliated cells before they have extruded their cilia into the glandular lumen. The other ciliated cells have a pyramidal shape aj

Secretory phase

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Day 15-16 (POD 2); Interval endometrium

• Ovulation as defined occurs on day 14 of the cycle.

• Both 15- and 16-day endometrium are technically synonymous with the first and second days following ovulation.

• The morphologic changes of the secretory phase begin 36 to 48 hours after ovulation.

• There is an interval phase of 36 to 48 hours between ovulation and the first recognizable histologic changes of the endometrium attributable to ovulation.

• The glands become more tortuous and begin to show subnuclear vacuoles

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• Interval endometrium has features of both proliferative and secretory endometrium.

• Features of interval endometrium can be seen whether or not ovulation has occurred, and hence the pathologist cannot confirm that ovulation has occurred on the basis of this morphology

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• Day 15 endometrium contains rare vacuoles

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• 16-day endometrium contains subnuclearvacuoles and regular mitoses.

• The extent of cytoplasmicvacuolization is not sufficient to be certain if ovulation has occurred.

• This pattern may be associated with ovulation but is not specific

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Vacuolation in proliferative phase??Must be interval!

• Focal subnuclear vacuolization may occur in the proliferative phase, at least 50% of the glands should contain vacuoles to confirm ovulation.

• In addition, at least 50% of the cells in a gland should contain vacuoles.

• If the 50% rule is not fully met, but the clinical history and morphology suggest recent ovulation, the endometrium may be in the interval phase.

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Vacuole Phase of Secretory Endometrium(17 to 19 days; POD-4 to -6)

• The first phase of the postovulatory period.

• Low power shows glands that are larger and slightly undulating and that appear more eosinophilic because of the absence of pseudostratified nuclei combined with vacuoles and intracellular secretions

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• 17-day SE characterized by uniform subnuclearvacuoles and virtually no mitotic activity.

• Piano key appearanceaj

• 18-day SE, the vacuoles are staggered toward the lumen and are accompanied by supranuclear discharge, seen as a ragged surface change.

• The vacuoles may or may not be conspicuous; however, the nuclei are prominently situated in the center of each cell, forming a uniform monolayer amidst eosinophilic cytoplasm.aj

• Day 19 SE, the nuclei have nearly migrated back to the base and are accompanied by scattered residual subnuclearvacuoles and the appearance of early secretory exhaustion; mitotic activity typically is not present.

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• The vacuoles have entirely migrated to the luminal surface by day 19

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Diagnostic pitfall in vacuole phase

• Irregular random pattern of gland enlargement leading to foci of gland crowding.

• This is sometimes erroneously interpreted as a “secretory hyperplasia” (EIN)aj

Exhaustive Phase of Secretory Endometrium(20 to 22 Days; POD-6 to -8)

• Conclusion of the vacuole phase is characterized by secretory exhaustion.

• Peak secretion is seen on day 20. The apical portions of the glands may have “snouts”.

• Rest of the secretory phase occurs in the stroma, as the glands become exhausted, and take on a sawtooth appearance.

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• SE day 20 demonstrates peak intraluminal secretions accompanied by occasional residual subnuclear vacuoles combined with slight gland enlargement and a compact subsurface stroma.

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• peak secretion is seen at day 20

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• SE day 21 exhibits an increase in stromal edema, highlighting the spiral arterioles

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• SE day 22 is characterized by a peak in stromal edema and exhibits the first hint of perivascular cuffing, but conspicuous predecidual changes are lacking

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Predecidual Phase of Secretory Endometrium(23 to 28 days; POD-9 to -14)

• This phase is initiated by the condensation of predecidua around spiral arterioles.

• Predecidua is not actually decidua because it is not the result of pregnancy; nevertheless, the term predecidua is used because the cells bear some morphologic resemblance to pregnancy-related decidual change.

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• Predecidua characteristics

– spiral aggregates of spindled cells around vessels

– slightly basophilic cytoplasm, with distinct cell borders(dark cytoplasm)

– nuclear changes with a reduction in basophiliawith finer chromatin texture.(lighter nuclei)

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• Predecidua cuffing around spiral arterioles begins at day 23.

• Cytoplasmic basophilia and nuclear hypochromasia produce a loss of cell contrast aj

Predecidua vs mimics

• This combination of abundant darker cytoplasm mixed with paler nuclei reduces the nuclear–cytoplasmic contrast, making the cells less distinct and lending a slightly “smudged” or “out of focus” appearance to the aggregates. This relative “softness” amid the higher contrast of the lower functionalisdistinguishes predecidua from its mimics

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Diagnostic pitfall day 23 SE

• Interpretation of larger arteries as spiral arterioles and confusing the two to three layers of investing adventitia around these larger arteries as predecidua.

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• Aggregates bridging multiple vessels are day 24 SE

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• day 25, predecidua is also under the surface epithelium

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• Predecidua around spiral arterioles

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• Late secretory endometrium. Stromal cells around spiral arteries show predecidual change with increased cytoplasm.

• The gland shows secretory exhaustion with patchy cytoplasmic vacuolization aj

• Day 25 SE

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• Expanding predecidua as linear sheets beneath the surface is seen on day 26

• Some granulocytes aj

• Day 27 SE entire stroma predecidulaized.

• Continuous subsurface with expansion of predeciduadownward and between the folds of the functionalisaj

• Extension of predecidua between glands by day 27 SE

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• Peak lymphocytic infiltration by day 28 SE.

• Gland–stromal dissociation begins with hemorrhageaj

• Saw toothed glands

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Diagnostic pitfall in late SE• Edematous predecidua, which may be confused with day 21.

Here predecidual cell characteristics are checked.

• Interpretation of stromal granulocytes as inflammation or chronic endometritis. – No plasma cells are present and the setting of late secretory

endometrium.

• Glands may be closely packed (more by tangential sectioning), and this can superficially resemble hyperplastic endometrium however, other features of a hyperplastic endometrium, such as mitotic activity, are absent.

• Hypersecretory endometrium vs Arias-Stella reaction

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• A, Edematous predecidua

• B, Stromal changes in a 21-day secretory endometrium may mimic predecidua

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• Endometritis

– Plasma cells

– Spindled stromaaj

• Glands may be closely packed superficially resembling a hyperplastic endometrium

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• Late-secretory endometrium. The glands exhibit the Arias- Stella reaction

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IHC of normal endometrium

• Normal– ER and PR positive

• Glands during proliferative and late secretory – diffusely positive for bcl-2

• Glands– CK 7 positive– CK 20 negative

• Stroma– CD 10 and bcl-2 negative– CD 34 positive– Active positive– Desmin negative

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ER positive glands and stroma CD 10 positive stroma

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• Article to consider-

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Some pathology for clinicians

• All clinicians may not have a sense of how pathology works or sufficient understanding of how important a history is in making a determination.

• Rarely do specimens look exactly like what is in the textbooks, and the rendering of diagnosis is often a process of developing a differential diagnosis.

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• Lack of clinical information impedes this, and may lead to a long descriptive diagnosis rather than a concise one.

• As an example, the diagnoses below are both from the same specimen.

• In the first case, no history of exogenous progesterone therapy was provided, and in the second it was:

– Diagnosis 1 (in the absence of history): endometrial tissue irregularly developed. The glands are small and inactive. The stroma is hypercellular and focally decidualized. This may represent exogenous progestational effect. Clinicalcorrelation suggested.

– Diagnosis 2 (with history): benign endometrium, with glandular and stromal features consistent with progestin effect.

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Endometrial histopathology-Basics

Health & Medicine

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