Normal Endometrium and Infertility Evaluation

2 Normal Endometrium and Infertility Evaluation

General Considerations in Histologic biopsy or curettage is part of a comprehensive Evaluation ...... 8 workup of the patient in the operating room Histologic Dating of the Normal, that includes laparoscopy, hysteroscopy, or Cycling Endometrium ...... 10 hysterosalpingography to assess the presence Proliferative Phase Endometrium . . . . . 11 or absence of uterine or tubal lesions that con- Secretory Phase Endometrium ...... 12 tribute to infertility. In these cases, the endome- Menstrual Endometrium ...... 19 trial sampling may not be timed as precisely for Pitfalls in Dating ...... 21 the mid- to late luteal phase. Nonetheless, his- Artifacts and Contaminants ...... 23 tologic evaluation provides the gynecologist Luteal Phase Defect and Abnormal with information regarding the response of the Secretory Phase Patterns ...... 26 Clinical Queries and Reporting ...... 29 endometrium to hormonal stimulation, including indirect evidence of ovulatory function. The secretory phase is constant in the normal cycle, lasting 14 days from the time of ovula- The histologic features of what constitutes tion to the onset of menstruation.1 Variations in “normal” endometrium change with a woman’s cycle length occur because the proliferative age, through the premenarchal, reproductive, phase of the cycle varies, both between cycles perimenopausal, and postmenopausal years.1–3 and between women. Accordingly, the gynecol- During the reproductive years, the cyclical hor- ogist correlates the cycle date by histology with monal changes of the menstrual cycle provide the woman’s cycle date based on the time of a continuously changing morphologic pheno- onset of the upcoming menstrual period, not type that is “normal.” In biopsy specimens, the the last menstrual period. Ovulation with se- combination of these cyclical changes along cretory endometrial changes ceases in most with artifacts and limited sampling can make women by age 53, although rarely ovulation normal patterns difficult to interpret. During with secretory endometrium and a confirmed the reproductive years, deviations from normal, corpus luteum of the ovary has been seen up to either in histologic pattern or in temporal rela- a least age 56 (personal observation). tionship to ovulation, often indicate underlying The biopsy findings help confirm that ovula- abnormalities that may cause female infertility. tion occurred, and indicate whether there was The endometrial biopsy is an important part sufficient secretory effect, mediated by proges- of the evaluation of the woman with infertil- terone, during the luteal phase. To utilize fully ity.4;5 Biopsies for the evaluation of infertility the morphologic interpretation, the gynecolo- often are performed in the office using a small gist compares the histologic date to the clinical curette or a Pipelle aspirator and therefore data, including the date of the rise in the basal the specimens tend to be small.6 Occasionally, body temperature, the time of the serum

7 8 2. Normal Endometrium and Infertility Evaluation luteinizing hormone (LH) surge, transvaginal The subsurface endometrium is divided into ultrasound evaluation of follicular or corpus two regions, the functionalis (stratum spongio- luteum development, serum progesterone sum) and the basalis (stratum basale) (Fig. 2.1). level, or subtraction of 14 days from the onset The functionalis, situated between the surface of menses.4;7–9 Consequently, the biopsy typi- epithelium and the basalis, is important to cally is timed to coincide with the luteal (secre- evaluate because it shows the greatest degree tory) phase of the cycle. In addition to defining of hormonal responsiveness. The size and the precise histologic date, an endometrial distribution of glands as well as the cytologic biopsy is part of the infertility workup to features of the glandular epithelial cells exclude other organic uterine abnormalities. are important features in the histologic evalua- This chapter reviews the morphologic variation. Under normal conditions, the glands tions caused by ovarian hormonal stimulation should be regularly spaced and have a per- that provide a background for the interpreta- pendicular arrangement from the basalis to tion of endometrial biopsies in infertility the surface epithelium. In the secretory phase, patients. These patterns include changes result- the endometrium also shows a stratum com- ing from normal hormonal fluctuations during pactum, a thin region beneath the surface the menstrual cycle and variations in normal epithelium. In the stratum compactum the development that are caused by abnormalities stroma is dense and the glands are straight in the endogenous ovarian hormonal levels and narrow, even when the glands in the during the reproductive years. The latter repre- functionalis are tortuous. The basalis adjoins sent the so-called dysfunctional abnormali- the myometrium, serving to regenerate the ties that are, for the most part, due to abnor- functionalis and surface epithelium following malities in ovarian follicular development or in shedding during menses. The endometrium of hormone production by the corpus luteum. the basalis is less responsive to steroid hor- Ovarian dysfunction also can result in abnormal mones, and typically shows irregularly shaped, bleeding, and Chapter 5 reviews dysfunctional inactive appearing glands, dense stroma, and uterine bleeding caused by ovulatory abnor- aggregates of spiral arteries. The spiral arteries malities. During gestation the endometrium of the basalis (basal arteries) have thicker mus- undergoes other “normal,” that is, physiologic, cular walls than those in the functionalis. In alterations as discussed in Chapter 3. biopsies, tissue fragments that contain basalis often do not have surface epithelium. The glands and stroma of the basalis cannot be dated, as they are unresponsive to steroid General Considerations in hormones. A specimen consisting solely of Histologic Evaluation endometrium from the basalis is therefore inadequate for dating. Histologic evaluation begins with identification Tissue from the lower uterine segment or of surface epithelium, a prerequisite for orient- isthmus is another region of the endometrium ing the underlying glands and stroma. The sur- that is less responsive to steroid hormones. In face epithelium is less responsive to sex steroid the lower uterine segment the endometrium hormones than the underlying glands, but it has shorter, poorly developed, inactive glands often shows alteration in pathologic conditions, dispersed in a distinctive stroma (Fig. 2.2). The especially when the abnormalities are subtle or columnar cells lining the glands resemble those focal. For example, during the proliferative of the corpus. Some glands near the junction phase, estrogenic stimulation induces develop- with the endocervix show a transition to muci- ment of ciliated cells along the surface.10 In con- nous endocervical-type epithelium.The stromal trast, ciliated surface epithelial cells are far cells in the lower uterine segment are elongate more frequent in pathologic conditions, par- and resemble fibroblasts with more abundant ticularly those associated with unopposed eosinophilic cytoplasm, in contrast to the oval estrogen stimulation, such as hyperplasia and to rounded stromal cells with minimum cyto- metaplasia.2;3;11–13 plasm seen in the corpus. General Considerations in Histologic Evaluation 9

Figure 2.1. Normal secretory phase endometrium. The basalis in the lower portion of the illustration Surface epithelium orients the tissue.The midportion consists of irregular, closely spaced glands, dense of the tissue consists of functionalis where glands, stroma, and aggregates of arteries. The stratum com- stroma, and blood vessels demonstrate the typical pactum is composed of the surface epithelium and a patterns of maturation through the menstrual cycle. subjacent thin layer of dense stroma.

The tangential orientation of the functionalis this instance, gland development can be difﬁ- in biopsies and the tortuosity of the glands, par- cult to assess. Furthermore, not all fragments of ticularly in the late proliferative phase, often tissue in a biopsy or curettage include surface lead to irregular cross sections of the tissue. In epithelium, which helps to orient the glands. 10 2. Normal Endometrium and Infertility Evaluation

Figure 2.2. Lower uterine segment. Small, poorly developed glands are seen in nonreactive stroma that is composed of widely spaced spindle cells. Tissue from the lower uterine segment cannot be dated.

Nonetheless, at least focally, portions of better- of the corpus luteum.4 The regular sequence of oriented glands usually can be traced through morphologic changes determined by the fluctu- the functionalis to the surface epithelium, and ating levels of ovarian steroid hormones forms these foci are critical for assessing appropriate the basis for histologic dating. glandular and stromal development. Dating uses an arbitrarily defined “normal” cycle of 28 days, with day 1 the first day of menstrual bleeding.1 Histologic dating is most Histologic Dating of the Normal, precise in the postovulatory secretory phase, Cycling Endometrium as the follicular phase can be highly variable in length. Furthermore, proliferative phase In the ovulatory patient, normal endometrium changes are not as discrete as those in the secre- has two phases.The first is the proliferative (fol- tory phase. The date of the secretory phase is licular or preovulatory) phase characterized by expressed either as the specific day of the 28- growth of glands, stroma, and vessels that is day menstrual cycle, assuming ovulation occurs influenced by estradiol produced mainly by on day 14, or is stated as the postovulatory day granulosa cells in the ovarian follicles. Fol- (e.g., secretory day 21 or postovulatory [P.O.] lowing ovulation, the secretory (luteal or day 7). Local custom often determines the pre- postovulatory) phase reflects the effect of ferred method of stating the histologic date. the combined production of progesterone and There are nine histologic features of the estradiol by luteinized granulosa and theca cells glands and stroma that determine the phase of Histologic Dating of the Normal, Cycling Endometrium 11

Table 2.1. Morphologic features used in endome- 2.4). Glands and stroma show brisk mitotic trial dating. activity. In early proliferative phase endo- Glandular changes metrium, the functionalis contains small, 1. Tortuosity tubular glands. The glands progressively elon- 2. Mitoses gate and become tortuous from the mid- to a 3. Orientation of nuclei (pseudostratified or basal) the late proliferative phase because the gland 4. Subnuclear cytoplasmic vacuolesa 5. Secretory exhaustion (luminal secretions)a growth is disproportionate to the stromal growth. Despite the tortuosity, the glands main- Stromal changes tain a relatively regular spacing between each 1. Edemaa 2. Mitoses other. Throughout the proliferative phase, the 3. Predeciduaa epithelium lining the glands has pseudostrati- 4. Granular lymphocyte infiltratea fied, oval nuclei with small nucleoli and dense basophilic cytoplasm. The pseudostratified a Salient features used in dating the secretory phase. nuclei remain oriented to the basement membrane, but some nuclei are raised above the the cycle and the histologic date (Table 2.1).1 basement membrane, giving a two-dimensional Five of these features affect glands: (1) tortu- layering of the nuclei. The pseudostratification osity, (2) gland mitoses, (3) orientation of nuclei of the nuclei and the presence of mitotic activ- (pseudostratified versus basal), (4) basal sub- ity in the glands and stroma are two constant nuclear cytoplasmic vacuoles, and (5) luminal features of the proliferative phase. secretions with secretory exhaustion. Four In the proliferative phase, the stromal cells features relate to the stromal: (6) edema, (7) are widely separated in the functionalis (Fig. mitoses, (8) predecidual change, and (9) infil- 2.4).They are small and oval, with dense nuclei, tration of granular lymphocytes. Practically, the scant wisps of cytoplasm, and ill-defined cell most important glandular features are orienta- borders. Some stromal edema is normal at mid- tion of nuclei, subnuclear cytoplasmic vacuoles, proliferative phase. A few lymphocytes also are and luminal secretions with secretory exhaus- scattered throughout the stroma, being most tion (3, 4, and 5), and the most important prominent around the vessels. Small spiral stromal features are edema, predecidual arteries and thin-walled venules are present. change, and granular lymphocytic infiltration The orientation and outline of proliferative (6, 8, and 9). These salient features are usually phase glands and their relationship to intact readily apparent when present, allowing the stroma are important features for recognizing pathologist to assign a histologic date.

Proliferative Phase Endometrium Table 2.2. Proliferative phase changes.a During the proliferative phase, the endo- Early (4–7 days) metrium grows from about 0.5mm up to 4.0 to Thin regenerating epithelium Short narrow glands with epithelial mitoses 5.0mm in thickness, so by the late proliferative Stroma compact with mitoses (cells stellate or spindle phase, a biopsy obtains a moderate amount of shaped) tissue. Proliferative endometrium has three 2 Mid (8–10 days) stages: early, mid, and late (Table 2.2). These Long, curving glands divisions are seldom used in dating biopsies, Columnar surface epithelium however. Usually the diagnosis of proliferative Stroma variably edematous, mitoses frequent phase alone is sufﬁcient, indicating that the Late (11–14 days) endometrium is growing, shows a normal glan- Tortuous glands dular distribution, and evidence of ovulation is Pseudostratiﬁed nuclei not present. Moderately dense, actively growing stroma Growth of endometrium is the main charac- a These changes are subtle. They are rarely used for actual teristic of the proliferative phase (Figs. 2.3 and dating. 12 2. Normal Endometrium and Infertility Evaluation

Figure 2.3. Proliferative endometrium. Glands are mitotic activity. Focal hemorrhage beneath the tubular and regularly spaced in abundant stroma. surface epithelium is a result of the biopsy and does The stroma contains small vessels with thin walls. not represent a pathologic change. Both the glandular and the stromal cells show this normal pattern, as hyperplastic glands or diagnosis. Also, proliferative phase glands fre- glands in a polyp can have cytologic features quently show the telescoping artifact (see identical to those of glands in the proliferative later). phase. The regular spacing and uniform shape of the glands are characteristics of normal proliferative endometrium. Assessing gland Secretory Phase Endometrium orientation can be complicated, however, by biopsy-induced fragmentation, an especially In the secretory phase, the glands and stroma common artifact in early to mid-proliferative develop in an orderly sequence and display spe- phase biopsies when the mucosa is still thin. cific histologic features of secretory activity Detached and disrupted glands may appear from histologic day 16 through day 28. The abnormally crowded or irregular. To separate endometrium attains a thickness of up to 7.0 to fragmentation artifact from true abnormalities, 8.0mm. Unlike in the proliferative phase, the it is important to assess the integrity of the changes in the glands and stroma are relatively stroma as well as the glands and to use surface discrete, varying sharply from one day to the epithelium to help orient the tissue fragments. next, thus permitting accurate dating. Dating Detached and poorly oriented glands that show of the first half of the secretory phase is based pseudostratified nuclei and mitotic activity primarily on glandular changes whereas dating usually represent proliferative endometrium of the second half is based mainly on stromal unless better-oriented tissue suggests another alterations (Table 2.3). Histologic Dating of the Normal, Cycling Endometrium 13

Figure 2.4. Proliferative endometrium. The prolif- nuclei and indistinct cytoplasm. Scattered lympho- erative phase gland shows pseudostratiﬁed nuclei cytes are normally present. with mitotic activity. The stromal cells have oval

Table 2.3. Endometrial dating, secretory phase. Interval phase, 14–15d.a No datable changes for 36–48 Mid- to late secretory phase, 21–27d. Stromal changes hours after ovulation predominate, variable secretory exhaustion 21d. Marked stromal edema Early secretory phase, 16–20d. Glandular changes 22d. Peak of stromal edema—cells have “naked predominate nuclei” 16d. Subnuclear vacuoles (Note: Scattered small 23d. Periarteriolar predecidual change irregular vacuoles can be caused by Spiral arteries prominent estrogen alone.) 24d. More prominent predecidual change 17d. Regular vacuolation—nuclei lined up with Stromal mitoses recur. subnuclear vacuoles 25d. Predecidual differentiation begins under 18d. Vacuoles decreased in size surface epithelium. Early secretions in lumen Increased numbers of granular lymphocytes Nucleus approaches base of cell. 26d. Predecidua starts to become confluent. 19d. Few vacuoles remain. 27d. Granular lymphocytes more numerous Intraluminal secretion Confluent sheets of predecidua No pseudostratification, no mitoses Focal necrosis 20d. Peak of intraluminal secretions 24–27d. Secretory exhaustion of glands—tortuous with intraluminal tufts (saw-toothed), ragged luminal borders, variable cytoplasmic vacuolization, and lumenal secretions a d. = day of ideal 28-day menstrual cycle. To state as postovulatory day, subtract 14 14 2. Normal Endometrium and Infertility Evaluation

The morphologic changes of the secretory phase. Special stains for glycogen add little to phase begin 36 to 48 hours after ovulation. routine histologic evaluation for establishing There is an interval phase of 36 to 48 hours the presence of secretory changes. between ovulation and the first recognizable Subnuclear vacuoles are abundant by day 17, histologic changes of the endometrium attrib- and by day 18 the vacuoles begin to move from utable to ovulation. During the interval phase, the basal to the supranuclear cytoplasm (Fig. the glands become more tortuous and begin to 2.7). Concurrently, the nuclei become basally show subnuclear vacuoles (Fig. 2.5). The first oriented and line up in a single layer perpen- diagnostic evidence of ovulation, however, is dicular to the basement membrane. The cyto- the presence of abundant subnuclear glycogen plasmic contents then form mucin that is vacuoles in the undulating, tortuous glands expelled into the gland lumen. Luminal secre- (Fig. 2.6). At this time the stroma is indistin- tions peak at day 20 (Fig. 2.8). guishable from that of the late proliferative After day 20, the stromal changes are more phase. Because focal subnuclear vacuolization important for dating than the glandular may occur in the proliferative phase, at least changes. Nonetheless, the glands continue to 50% of the glands should contain vacuoles to show increasing tortuosity, and variable confirm ovulation. In addition, at least 50% of amounts of luminal secretions persist until just the cells in a gland should contain vacuoles. If before menses. From days 20 to 22 the glands the 50% rule is not fully met, but the clinical in the functionalis begin to show secretory history and morphology suggest recent ovula- exhaustion, a change that becomes more promi- tion, the endometrium may be in the interval nent by days 24 to 25 (Fig. 2.9). Secretory

Figure 2.5. Interval endometrium.The glands main- plasmic vacuolization is not sufﬁcient to be certain tain proliferative phase characteristics and show ovulation has occurred. scattered subnuclear vacuoles. The extent of cyto- Figure 2.6. Early secretory endometrium, days compared to the late proliferative phase. Inset: Every 16–17. Postovulatory changes are clearly present gland cell contains a vacuole, resulting in a uniform with a regular distribution of subnuclear vacuoles in alignment of nuclei away from the basement mem- the serpiginous glands.The stroma shows no changes brane.

Figure 2.7. Early secretory endometrium, days 17–18. Glandular cell vacuoles remain prominent but begin to migrate to the supranuclear cytoplasm. A portion of the stroma shows mild edema. Figure 2.8. Mid-secretory endometrium, days 20–21. Glands are distended with secretions.The stroma shows edema and there is no predecidual change.

Figure 2.9. Late secretory endometrium, days 23–24. Predecidual stromal change is evident around spiral arteries with intervening zones showing edema. The glands are tortuous and show secretory exhaustion. Histologic Dating of the Normal, Cycling Endometrium 17 exhaustion is characterized by the presence of glands in the stratum compactum immediately a single layer of cells that lie in disarray with beneath the surface epithelium remain small loss of orientation. The cytoplasmic border and tubular despite their increasing tortuosity along the luminal surface becomes ragged, and in the functionalis. luminal secretions are usually, although not As the glandular cells develop cytoplasmic invariably, present. By days 24 to 25 the glands vacuoles and produce luminal secretions, often develop a serrated, “saw-toothed” edema, the ﬁrst stromal change, begins and luminal border (Fig. 2.10). The glandular cells peaks quickly at days 21 to 22 (Table 2.3). Once may continue to show a variable degree of vac- stromal changes begin, the glandular changes uolization throughout the remainder of the are less important for dating. Because of the secretory phase. Cytoplasmic vacuolization is a edema, the stromal cells take on the so-called physiologic change as long as the glands other- naked nucleus appearance at days 21 to 22. wise have appropriate tortuosity; the cytoplas- With this change the stromal cells are widely mic changes from vacuolization to complete dispersed and have small nuclei with scant, secretory exhaustion with no vacuoles repre- imperceptible cytoplasm (Fig. 2.8). This phase sent a continuum of normal development. By of pure stromal edema is brief, and the subse- day 27, cellular necrosis (apoptosis) becomes quent predecidual transformation of the stroma evident with accumulation of nuclear debris in becomes the main feature in dating the late the basal cytoplasm of the glandular epithelial secretory phase. Although stromal edema is cells. Throughout the secretory phase, the maximal at days 21 to 22, edema begins in a

Figure 2.10. Late secretory endometrium. Stromal cells around spiral arteries show predecidual change with increased cytoplasm. The gland shows secretory exhaustion with patchy cytoplasmic vacuolization. 18 2. Normal Endometrium and Infertility Evaluation patchy distribution in the early secretory phase and leading to prominence of the vessels (Fig. at day 17 to 18. Therefore, some edema in the 2.9). In the predecidua there is a resurgence of earlier portion of the secretory phase does not stromal mitotic activity at day 24, while the represent an irregularity of maturation. glandular epithelium lacks mitoses. Predecidual Predecidual change characterizes the late change expands, extending to the subsurface secretory phase (days 23 to 28). With the stroma on day 25. The predecidual change appearance of predecidua (not “pseudode- around vessels and beneath the surface epithe- cidua”), the cells gain identifiable cytoplasm lium becomes confluent, forming larger sheets (Fig. 2.9). These cells become oval to polygonal by day 26 (Fig. 2.12). Predecidua is easy to rec- shaped in the functionalis and show a moder- ognize when advanced, but this change can be ate amount of eosinophilic to amphophilic subtle when it is early and not confluent. Inter- cytoplasm (Figs. 2.10 and 2.11). Just below the vening stroma often shows some edema, and surface epithelium they can be spindle shaped. dating remains based on the most advanced Cell borders of predecidual cells often are changes. By day 27, predecidual change is indistinct in formalin-fixed specimens. Prede- extensive. cidual transformation begins on day 23 around With predecidual transformation, the stroma spiral arteries, making the walls appear thicker shows a gradually increasing number of smaller

Figure 2.11. Predecidua and granular lymphocytes plasm. At this time in the secretory phase, mitotic in late secretory endometrium. Predecidualized activity recurs in the stromal cells. Stromal granular stromal cells in the late secretory phase appear oval lymphocytes are scattered throughout the stroma. to polygonal with a moderate amount of pale cyto- These cells have dark, often lobulated nuclei. Histologic Dating of the Normal, Cycling Endometrium 19

Figure 2.12. Late secretory endometrium, days and show a variable amount of intraluminal secre- 26–27. The stroma consists of sheets of predecidual- tion. In the stratum compactum beneath the surface ized cells with a heavy infiltrate of granular lympho- epithelium the glands are small and tubular. cytes. The glands in the functionalis remain tortuous mononuclear and bilobate cells with faintly their base. On day 28 fibrin thrombi begin to granular cytoplasm (Fig. 2.11). Others have form in small vessels, and hemorrhage follows small round and dense nuclei. Variably termed with extravasation of erythrocytes into the “stromal granulocytes,” “granulated lympho- stroma. cytes,” “K cells,” “leukocytes,” or “neutrophils,” these cells are not polymorphonuclear leuko- Menstrual Endometrium cytes (neutrophils).2;3;14;15 The latter occur normally only in menstrual endometrium. Recent Menstrual endometrium shows glandular and immunohistochemical studies reveal that most stromal breakdown that rapidly affects all the of these cells are T lymphocytes.16–18 We there- functionalis by the end of day 28. This stage fore prefer the designation “granular lympho- shows fibrin thrombi in small vessels, con- cytes.” They are normally present in small densed and collapsed stroma, and necrotic numbers earlier in the cycle but become promi- debris (Figs. 2.13 and 2.14). With this necrosis, nent by the late secretory phase. a true neutrophilic infiltrate becomes a part of At day 27 the endometrium is premenstrual. the physiologic process.19 When the bleeding is Predecidua is present in sheets with many inter- extensive, it may not be possible to assess the spersed granular lymphocytes. The glands are development of the glands or stroma or the highly convoluted and saw-toothed. The glands “normality” of the tissue. Once breakdown begin to show apoptosis with nuclear dust at starts, the stromal cells coalesce into aggregates Figure 2.13. Menstrual endometrium. Hemorrhage into the stroma forms lakes of erythrocytes. The hemorrhage disrupts the glands and stroma, although the tortuosity of the glands persists.

Figure 2.14. Menstrual endometrium.With stromal hemorrhage, the predecidual cells collapse and they lose their abundant cytoplasm. Pitfalls in Dating 21

Figure 2.15. Menstrual endometrium. Glands and shapes but show nuclear dust accumulating in the stroma near the basalis undergo collapse as the subnuclear cytoplasm (arrowheads). Predecidual superﬁcial tissue sloughs. The glands retain tortuous change in the stroma has become indistinct.

and clusters that often show little cytoplasm. menstrual phase before breakdown becomes With extensive stromal collapse during men- extensive.4 struation, the predecidual change in the stromal cells becomes indistinct (Figs. 2.14 and 2.15). Pitfalls in Dating The extensive breakdown also can result in striking morphologic alterations with artifac- The preceding description summarizes the tual glandular crowding. As a result, menstrual basic histologic changes of endometrial devel- endometrium can be confused with hyperplasia opment. In addition to understanding the or even carcinoma if the background bleeding normal morphology in ideal situations, one pattern is not recognized. Conversely, hyper- needs to consider a number of practical points plasia and carcinoma are proliferative pro- when interpreting the endometrial biopsy. cesses that rarely show extensive breakdown There are several caveats and potential pitfalls, of the type displayed by menstrual endo- knowledge of which assists in accurate diagno- metrium. Because of the artifacts induced sis of normal endometrium and helps avoid by the breakdown and bleeding of the men- errors in dating. The following, in our opinion, strual phase, this tissue is not suitable for eval- are especially important aspects to consider in uation of glandular and stromal development. evaluating this biopsy material: Some advocate biopsy at the onset of bleeding to be certain that the procedure does not inter- 1. Endometrium with surface epithelium is rupt an early pregnancy, but this tissue is best for interpretation. Absence of surface not optimal unless obtained very early in the epithelium compromises the interpretation. 22 2. Normal Endometrium and Infertility Evaluation

2. Tissue from the lower uterine segment This artifact can result in back-to-back glands or basalis is not satisfactory for dating. that do not represent hyperplasia. Endometrium from these regions does not 7. Focal cystic glands or nonreactive glands respond fully to hormones. can occur in normal endometrium and have no 3. Straight, tubular glands beneath the sur- significance by themselves. face are normal and not a sign of irregularity in 8. Patchy stromal edema is normal by days maturation in the late secretory phase. 17 to 18 of the secretory phase and does not 4. Scattered subnuclear vacuoles in glands signify irregular maturation. are not sufficient evidence of ovulation. To be 9. Identifying very early pregnancy based on certain that ovulation has occurred, more than endometrial changes alone is very difficult. 50% of the glands must show subnuclear Apparent “hypersecretory” late secretory vacuoles. phase glands with vacuolated cytoplasm usually 5. The presence of secretions in the glandu- are a variation of normal development and do lar lumen does not indicate secretory endome- not, by themselves, indicate early pregnancy trium. Proliferative, hyperplastic, and neoplastic (see Chapter 3). glands can contain luminal secretions. It is the 10. Compact predecidua with spindle- glandular cytoplasm and nuclear changes that shaped stromal cells may not be appreciated as are most important for determining the pres- a true predecidual reaction. Directing attention ence or absence of secretory changes. to stromal changes around spiral arteries assists 6. Focal glandular crowding caused by tan- in the identification of predecidua. gential sectioning can occur in proliferative or 11. Lymphocytes and granular lymphocytes secretory endometrium (Figs. 2.16 and 2.17). normally become prominent in the stroma of

Figure 2.16. Artifactual crowding of late secretory pattern of focal glandular crowding. This artifact has endometrium. Tangential sectioning of normal late no signiﬁcance and should not be misinterpreted as secretory endometrium near the basalis yields a hyperplasia. Artifacts and Contaminants 23

Figure 2.17. Artifactual crowding of late secretory hyperplastic nor “hypersecretory,” however. Identi- endometrium. This is another example of late se- fying surface epithelium elsewhere in the sections cretory phase glands that appear crowded due to often helps to avoid misinterpretation of these tangential sectioning. This pattern is neither normal glands. the late secretory phase.These do not represent detached from the surrounding stroma, and inflammation. fragmented glands become randomly oriented, 12. If the tissue is difficult to date because of often appearing closely spaced (Fig. 2.18). This apparent discordance in features, the possibil- artifact should not be mistaken for real crowd- ity of chronic endometritis or a polyp should be ing that occurs in hyperplasia or carcinoma. considered. Fragmentation and close apposition of dis- 13. The endometrium cannot be dated parate tissues such as cervical epithelium and accurately when polyps, inflammation, or other functionalis also lead to confusing patterns. abnormalities are present. Artifactually crowded glands lack a continuous investment of tissue and are not connected by intervening stroma. These latter features help Artifacts and Contaminants in recognition of the artifact. Fragmentation also is a common feature of atrophy (see Besides variations in the normal anatomy, such Chapter 5). as the basalis and lower uterine segment, Another frequent change is so-called tele- several artifacts of the biopsy often complicate scoping of glands.20 Telescoping may occur the histologic patterns. One frequent artifact is in either proliferative or secretory phase tissue fragmentation caused by mechanical dis- endometrium (Fig. 2.19), but it also complicates ruption of the tissue. As a result, glands are many nonphysiologic conditions. Telescoping Figure 2.18. Artifactual fragmentation. Normal pro- ard arrangement that should not be confused with a liferative phase endometrium is fragmented as a significant abnormality. Focal “telescoping” artifact result of the procedure. The glands have a haphaz- also is present (arrow).

Figure 2.19. Telescoping artifact. Normal secretory is an apparent result of the biopsy procedure and has endometrium shows telescoping artifact with a no significance. gland-in-gland appearance. This common alteration Artifacts and Contaminants 25 results in a pattern of an apparent gland within extracellular mucin are common (Fig. 2.21).The the lumen of another gland and can mimic extracellular mucin may contain neutrophils, hyperplasia or neoplasia (Fig. 2.20). This arti- cell debris, macrophages, or giant cells that are fact seems to be a result of mechanical disrup- normal components with no pathologic signifi- tion and “snap back” of the gland during cance in the absence of inflammation in the curettage, resulting in intussusception which endometrial stroma. Occasionally, benign cervi- rarely occurs in hysterectomy specimens. Tan- cal contaminants become more complex and gential sectioning of tortuous glands also con- troublesome in biopsies. Endocervical glands tributes to this phenomenon. Fortunately, with squamous metaplasia or microglandular telescoping rarely presents difficulty in inter- hyperplasia yield complex patterns, but these pretation once the observer understands the elements are cytologically bland and usually phenomenon. In questionable cases, the cytol- blend into more typical cervical epithelium (Fig. ogy of the glandular cells and comparison with 2.22). In questionable cases, continuity with surrounding tissue establishes this change as an endometrial surface epithelium may help to artifact. establish origin in the corpus. It is also helpful to Endometrial biopsies also often contain con- look at the surrounding stroma and see if it is of taminants from the cervix. Most of these conta- endometrial or endocervical type. Rarely, an minants are obvious. Strips of bland squamous endometrial biopsy also may reveal an admix- or mucinous epithelium and irregular pools of ture of fragments of tissue from cervical dyspla-

Figure 2.20. Telescoping with focal artifactual gland fragmentation. This is a common artifact seen in crowding. Focus of disrupted secretory glands shows normal proliferative and secretory endometrium apparent gland crowding caused by telescoping and that should not be mistaken for hyperplasia. 26 2. Normal Endometrium and Infertility Evaluation

Figure 2.21. Cervical contaminants. Left: Fragmen- glands. Right: Amorphous endocervical mucus with tation has resulted in a strip of mucinous glandular a few macrophages may be admixed with endome- epithelium adjacent to detached proliferative phase trial tissues. This finding has no clinical significance. sia, squamous carcinoma, or adenocarcinoma. and macrophages that are filled with lipid from Chapter 10 addresses the differential diagnosis erythrocytes in areas of chronic nonphysiologic of endocervical versus endometrial carcinoma. bleeding.22 Separate fragments of adipose tissue Occasionally, curettage yields sheets of histi- with clearly identifiable fat cells in an endome- ocytes with no associated mucin or other tissue trial biopsy almost always represent omentum (Fig. 2.23). These histiocytes apparently reside or extrauterine pelvic soft tissue and indicate in the endometrial cavity, and show the typical perforation of the uterus. We have also occa- histiocyte cytology with a lobulated nucleus and sionally seen colonic mucosa in endometrial amphophilic cytoplasm. We have seen them in biopsies. In these circumstances the clinician association with hydrometra and with benign should be notified immediately. bleeding patterns. They apparently represent a response to intracavitary debris and have been referred to as “nodular histiocytic hyperplasia” Luteal Phase Defect and because they can represent a nodular accumulation of histiocytes in sections.21 They are Abnormal Secretory benign but can mimic endometrial stromal cells Phase Patterns or stromal cell lesions. Immunohistochemical stains for histiocyte markers, such as lysozyme Luteal phase defect (LPD), or inadequate or KP 1, can facilitate their recognition. Stromal luteal phase, is a recognized cause of infertility, foam cells, in contrast, represent stromal cells so-called ovulatory infertility.4;23–25 This disorder Luteal Phase Defect and Abnormal Secretory Phase Patterns 27 is sporadic and relatively common, but it is a der,27;30;34;35 although some authors suggest that significant factor for infertility in fewer than 5% endometrial biopsy with histologic dating has of patients.4;25 LPD also has been implicated as limited utility in luteal phase evaluation.9;36;37 a factor in early habitual spontaneous abortion In LPD, ovulation occurs, but the subsequent and in abnormal uterine bleeding.26 The etiol- luteal phase does not develop appropriately. ogy of LPD is obscure. Usually the abnormal- There is insufficient progesterone production ity appears to arise as a result of hypothalamic to support development of the endometrium to or pituitary dysfunction that causes decreased histologic day 28 of the cycle. Usually, this levels of follicle-stimulating hormone (FSH) abnormality is recognized clinically when the in the follicular phase, abnormal luteinizing histologic date lags more than 2 days from the hormone (LH) secretion, decreased levels of actual postovulatory date.23;34 In this circum- LH and FSH at the time of ovulation, or ele- stance LPD is clinically significant only if the vated prolactin levels.4;23;27 Hypothyroidism also abnormal lag in maturation occurs in at least may be a factor in LPD.4 Women with apparent two consecutive biopsies.34;35;38 Using the crite- LPD often have relatively low progesterone rion for a lag in secretory phase development levels during the luteal phase,7;28–32 and moni- by dates, there usually is no morphologic abnor- toring mid-luteal phase progesterone levels can mality. Clinical evaluation, including the basal be effective for diagnosing this abnormality.33 body temperature, the time of the LH surge, or Endometrial biopsy plays an important role in the onset of menses after biopsy establishes the the diagnosis and management of this disor- diagnosis in this situation.

Figure 2.22. Cervical contaminant. Fragmented showing breakdown. Other ﬁelds in the section endocervical epithelium with microglandular hyper- showed proliferative endometrium. plasia lies adjacent to a fragment of endometrium 28 2. Normal Endometrium and Infertility Evaluation

Figure 2.23. Histiocytes. A detached aggregate of tiocytes with oval, folded nuclei and faintly vacuo- histiocytes superﬁcially resembles endometrial lated cytoplasm. A lack of intrinsic vasculature helps stroma. Inset: The cells have cytologic features of his- to indicate that this is a contaminant.

Because of inadequate progesterone produc- ized. LPD may cause discordance in the devel- tion, LPD may also cause abnormalities in the opment of the glands and the stroma.35;39;41–43 development of secretory endometrium.35;39 In The resulting pattern is that of irregular matu- fact, experimental evidence shows that varia- ration, with different areas showing a marked tions in the relative amounts of the sex steroid (greater than 4 days) variation in development. hormones, estradiol and progesterone, affect Although LPD can be reflected in endometrial endometrial development.40 In these experi- morphologic abnormalities such as irregular mental conditions, relatively low doses of estro- maturation, there are no large-scale studies that gen and progesterone result in glandular and have clearly identified specific pathologic fea- stromal hypoplasia. Higher doses of estrogen tures of this condition. but low doses of progesterone result in stromal On occasion, endometrial biopsies show inadequacy, while high levels of progesterone abnormal secretory phase patterns.35;39;41;43–45 In and low doses of estrogen lead to glandular such cases the endometrium typically shows inadequacy. These data suggest that variations secretory changes that cannot be assigned to in follicle and corpus luteum development with any day of the normal cycle (Figs. 2.24 and decreases in hormone production alter the 2.25). The pattern may show true irregular development of secretory endometrium follow- maturation with a large variation in the pattern ing ovulation. of endometrial development from field to field. Morphologic features of LPD other than a For example, some areas may show early secre- lag in the histologic date are poorly character- tory changes characteristic of about days 18 to Clinical Queries and Reporting 29

Figure 2.24. Abnormal secretory phase pattern. show secretory changes but have tubular outlines Endometrial biopsy in a premenopausal woman and lack appropriate tortuosity. Further, some glands being evaluated for infertility shows secretory lack secretory vacuoles. The stroma shows edema in changes that cannot be assigned to a speciﬁc histo- this ﬁeld but other areas lack this stromal response logic date of the normal menstrual cycle. The glands (see Fig. 2.25).

19, while other areas show foci of predecidua developed secretory pattern. For this latter consistent with at least day 24. Pathologic group of cases the abnormality may be dys- processes other than LPD can cause abnormal functional, related to abnormal development of secretory phase development, however (Table the corpus luteum, or the aberration may be 2.4). One study of “deficient secretory phase” secondary to underlying pathology that is not endometrium found the changes of elongated, adequately sampled. Such cases can be classi- hyperchromatic gland nuclei, diminished secre- fied only as abnormal secretory phase pattern tory activity, and poorly developed stroma.45 (see later). The etiology of these “deficient” secretory pattens was not known although immunohistochemical expression of estrogen and proges- Clinical Queries and Reporting terone receptor content of gland cells appeared decreased. Using endometrial biopsy or curettage in the When abnormal secretory phase patterns infertility workup, the gynecologist seeks the are the result of specific abnormalities such as following information: (1) histologic evidence inflammation or polyps, the primary abnormal- of ovulation, (2) histologic date of secretory ity may be evident. In other cases, there may be phase specimens, and (3) presence or absence no identifiable etiology for the abnormally of endometrial abnormalities that may be 30 2. Normal Endometrium and Infertility Evaluation

Figure 2.25. Abnormal secretory phase pattern. stroma that lacks edema. The cause of this type of Another area from the specimen shown in Fig. 2.24 abnormality cannot be determined by morphology shows poorly developed secretory glands and dense alone.

Table 2.4. Causes of undatable endometrium. responsible for infertility. Secretory phase Hormonal effects changes indicate that ovulation has occurred. Anovulation Dating the secretory phase gives a general Luteal phase defect assessment of progesterone production by the Persistent corpus luteum corpus luteum and the ability of the endo- Exogenous hormones metrium to respond to progesterone. Pregnancy In practice, a span of up to 3 days in dates Organic lesions from field to field is acceptable as normal, espe- Polyps cially in mid- to late secretory endometrium Leiomyomas Chronic inflammation where areas of edematous stroma alternate Hyperplasia with areas of predecidualized stroma. Also, the Carcinoma glandular changes in the late secretory phase Atrophy can be highly variable. The pathologist should Sampling problems date the tissue on the basis of the most Fragmentation advanced changes using a 2-day span (e.g., days Lack of surface epithelium 24 to 25). Dating the secretory phase is some- what subjective and neither completely exact nor reproducible, and therefore it is also important that everyone involved in the interpreta- References 31 tion and clinical application of histologic dating changes (e.g., “secretory with irregular matura- understand the limitations of this morphologic tion” or “abnormal secretory phase pattern”) assessment.46;47 Studies of interobserver varia- with a description of the abnormality is suffi- tion show that 60% to 80% or more of endome- cient to indicate that the secretory develop- trial biopsies for dating are within 2 days of ment is not normal. The gynecologist can use each other when evaluated by experienced this information in combination with other clin- pathologists.47 Furthermore, 80% of the time ical observations to determine its significance the dates are within 2 days of the expected day and possible cause. Descriptive diagnoses compared with basal body temperature and should be used carefully, however. The term menstrual dates.48 Some investigators find that “dyssynchronous endometrium” has been used the criteria for histologic dating do not have to describe apparent alterations in secretory sufficient precision to determine the degree of phase development. “Dyssynchronous” is not a corpus luteum function in the late secretory word with a specific connotation and therefore phase.49 its use can be confusing unless there is clear The field-to-field variations in normal late communication between the pathologist and secretory endometrium probably lead to some the gynecologist regarding its meaning. of the interobserver and intraobserver varia- Recently, morphometric analysis has been tions found in dating.With experience, a pathol- attempted to increase the accuracy of endome- ogist should be able to provide a reasonable trial histologic dating.50 It was found that five assessment of the endometrial development in morphometric measurements, including mitotic the secretory phase. rate in gland cells, amount of luminal secretion, Obviously for the infertility patient every volume fraction of gland occupied by gland cell, attempt should be made to provide an accurate amount of pseudostratification of gland cells, date, but there are cases in which precise histo- and amount of predecidual reaction, added logic dating is not possible. When accurate precision to histologic dating.50 Furthermore, dating cannot be done, it is important, if pos- immunohistochemical analysis for specific sible, to indicate why (Table 2.4). Hormonal secretory products of the endometrium and effects, various organic lesions, and sampling integrins may help to identify LPDs.51–54 Other problems all can make dating difficult or impos- cell products such as cyclin E and p27 also may sible. Other organic factors such as inflamma- be useful in assessing secretory phase develop- tion, adhesions, or polyps may interfere with ment.55 These data indicate that further refine- pregnancy (see Chapter 7).These abnormalities ments in evaluation of normal endometrium affect fertility by altering the development of may evolve that have clinical utility.At present, the glands and stroma, thereby preventing however, routine histologic evaluation remains normal implantation or mechanically disrupt- a cost-effective method of determining the ing the early implanting placenta. Therefore, relative degree of endometrial development when present these abnormalities should be through the menstrual cycle. reported. If factors preclude reporting a specific date, an attempt should be made to decide whether the endometrium is proliferative or References secretory, because secretory phase development generally indicates that ovulation has (1) Noyes RW, Hertig AT, Rock J. Dating the occurred. endometrial biopsy. Fertil Steril 1950; 1:3–25. (2) Dallenbach-Hellweg G. Histopathology of On occasion the biopsy shows an abnormal the endometrium. 4th ed. New York: Springer- secretory pattern that cannot be histologically Verlag; 1987. dated. In such cases, the abnormal pattern may (3) Hendrickson MR, Kempson RL. Surgical be due to an LPD or to some other pathologic pathology of the uterine corpus (Major prob- factor that is not identifiable in the specimen. lems in pathology series). Volume 12. Philadel- In practice, a descriptive diagnosis of the phia: W.B. Saunders; 1980. 32 2. Normal Endometrium and Infertility Evaluation

(4) Speroff L, Glass RH, Kase NG. Clinical gyne- (17) King A, Wellings V, Gardner L, Loke YW. cologic endocrinology and infertility. 6th ed. Immunocytochemical characterization of the Baltimore: Lippincott Williams & Wilkins; unusual large granular lymphocytes in human 1999. endometrium throughout the menstrual cycle. (5) Merrill JA. The interpretation of endometrial Hum Immunol 1989; 24:195–205. biopsies. Clin Obstet Gynecol 1991; 34:211–221. (18) Bulmer JN, Hollings D, Ritson A. Immunocy- (6) Hill GA, Herbert CMI, Parker RA, Wentz AC. tochemical evidence that endometrial stromal Comparison of late luteal phase endometrial granulocytes are granulated lymphocytes. biopsies using the Novak curette or Pipelle J Pathol 1987; 153:281–288. endometrial suction curette. Obstet Gynecol (19) Poropatich C, Rojas M, Silverberg SG. Poly- 1989; 73:443–446. morphonuclear leukocytes in the endometrium (7) Hecht BR, Bardawil WA, Khan-Dawood FS, during the normal menstrual cycle. Int J Dawood MY. Luteal insufficiency: Correlation Gynecol Pathol 1987; 6:230–234. between endometrial dating and integrated (20) Numers CV. On the traumatic effect of curet- progesterone output in clomiphene citrate- tage on the endometrial biopsy,with special ref- induced cycles. Am J Obstet Gynecol 1990; erence to so-called invagination pictures and 163:1986–1991. the “crumbling endometrium.” Acta Obstet (8) Shoupe D, Mishell DRJ, Lacarra M, Lobo RA, Gynecol Scand 1942; 28:305–313. Horenstein J, d’Ablaing G et al. Correlation of (21) Kim KR, Lee YH, Ro JY. Nodular histiocytic endometrial maturation with four methods of hyperplasia of the endometrium. Int J Gynecol estimating day of ovulation. Obstet Gynecol Pathol 2002; 21:141–146. 1989; 73:88–92. (22) Silver SA, Sherman ME. Morphologic and (9) Peters AJ, Lloyd RP,Coulam CB. Prevalence of immunophenotypic characterization of foam out-of-phase endometrial biopsy specimens. cells in endometrial lesions. Int J Gynecol Am J Obstet Gynecol 1992; 166:1738–1746. Pathol 1998; 17:140–145. (10) Masterson R, Armstrong EM, More IAR. The (23) Jones GS. Luteal phase insufficiency. Clin cyclical variation in the percentage of ciliated Obstet Gynecol 1972; 16:255–273. cells in the normal human endometrium. (24) Wentz AC. Diagnosing luteal phase inadequacy. J Reprod Fertil 1975; 42:537–540. Fertil Steril 1982; 37:334. (11) Hendrickson MR, Kempson RL. Ciliated carci- (25) Wentz AC, Kossoy LR, Parker RA. The impact noma-a variant of endometrial adenocarci- of luteal phase indaequacy in an infertile noma. A report of 10 cases. Int J Gynecol population. Am J Obstet Gynecol 1990; Pathol 1983; 2:1–12. 162:937–945. (12) Ronnett BM, Kurman RJ. Precursor lesions of (26) Sherman ME, Mazur MT, Kurman RJ. Benign endometrial carcinoma. In: Kurman R, ed. diseases of the endometrium. In: Kurman RJ, Blaustein’s pathology of the female genital ed. Blaustein’s pathology of the female genital tract. 5th ed. New York: Springer-Verlag; 2002: tract. 5th ed. New York: Springer-Verlag; 2002: 467–500. 421–466. (13) Silverberg SG, Kurman RJ. Tumors of the (27) Daly DC, Walters CA, Soto-Albors CE, uterine corpus and gestational trophoblastic Riddick DH. Endometrial biopsy during treat- disease. Atlas of tumor pathology, 3rd series, ment of luteal phase defects is predictive of Fascicle 3. Washington, DC: Armed Forces therapeutic outcome. Fertil Steril 1983; 40: Institute of Pathology; 1992. 305–310. (14) Buckley CH, Fox H. Biopsy pathology of the (28) Rosenfeld DL, Garcia C. A comparison of endometrium. 2nd ed. London: Arnold, 2002. endometrial histology with simultaneous (15) Mutter GL, Ferenczy A. Anatomy and histol- plasma progesterone determinations in infer- ogy of the uterine corpus. In: Kurman RJ, ed. tile women. Fertil Steril 1976; 27:1256–1266. Blaustein’s pathology of the female genital (29) Annos T, Thompson IE, Taymor ML. Luteal tract. 5th ed. New York: Springer-Verlag; 2002. phase deficiency and infertility: Difficulties (16) Bulmer JN, Lunny DP, Hagin SV. Immunohis- encountered in diagnosis and treatment. Obstet tochemical characterization of stromal leuco- Gynecol 1980; 55:705–710. cytes in nonpregnant human endometrium. (30) Rosenfeld DL, Chudow S, Bronson RA. Diag- Am J Reprod Immmunol Microbiol 1988; nosis of luteal phase inadequacy. Obstet 17:83–90. Gynecol 1980; 56:193–196. References 33

(31) Cooke ID, Morgan CA, Parry TE. Correlation (44) Noyes RW. The underdeveloped secretory of endometrial biopsy and plasma proges- endometrium. Am J Obstet Gynecol 1959; 77: terone levels in infertile women. J Obstet 929–945. Gynaecol Br Commonw 1972; 79:647–650. (45) Thornburgh I, Anderson MC. The endometrial (32) Cumming DC, Honore LH, Scott JZ, Williams deﬁcient secretory phase. Histopathology 1997; KP. The late luteal phase in infertile women: 30:11–15. Comparison of simultaneous endometrial (46) Zaino R, Novotney D, Ireland D, Meyer W, biopsy and progesterone levels. Fertil Steril Fritz M, Lessey B, et al. Dating the endometrial 1985; 43:715–719. biopsy: A re-evaluation. Mod Pathol 2003; 16: (33) Jordan J, Craig K, Clifton DK, Soules MR. 216A. Luteal phase defect: The sensitivity and speci- (47) Duggan MA, Brashert P, Ostor A, Scurry J, ﬁcity of diagnostic methods in common clinical Billson V, Kneafsey P, et al. The accuracy and use. Fertil Steril 1994; 62:54–62. interobserver reproducibility of endometrial (34) Wentz AC. Endometrial biopsy in the evalua- dating. Pathology 2001; 33:292–297. tion of infertility. Fertil Steril 1980; 33:121– (48) Noyes RW, Haman JO. The accuracy of 124. endometrial dating. Fertil Steril 1953; (35) Witten BI, Martin SA. The endometrial biopsy 4:504–517. as a guide to the management of luteal phase (49) Li T-C, Dockery P,Rogers AW, Cooke ID. How defect. Fertil Steril 1985; 44:460–465. precise is histologic dating of endometrium (36) Balasch J, Fabregues F, Creus M, Vanrell JA. using the standard dating criteria? Fertil Steril The usefulness of endometrial biopsy for luteal 1989; 51:759–763. phase evaluation in infertility. Hum Reprod (50) Li TC, Rogers AW, Dockery P, Lenton EA, 1992; 7:973–977. Cooke ID. A new method of histologic dating (37) Scott RT, Snyder RR, Strickland DM, Tyburski of human endometrium in the luteal phase. CC, Bagnall JA, Reed KR, et al. The effect of Fertil Steril 1988; 50:52–60. interobserver variation in dating endometrial (51) Graham RA, Seif MW,Aplin JD, Li TC, Cooke histology on the diagnosis of luteal phase ID, Rogers AW, et al. An endometrial factor defects. Fertil Steril 1988; 50:888–892. in unexplained infertility. Br Med J 1990; (38) Downs KA, Gibson M. Basal body temperature 300:1428–1431. graph and the luteal phase defect. Fertil Steril (52) Seif MW, Aplin JD, Buckley CH. Luteal phase 1983; 40:466–468. defect:The possibility of an immunohistochem- (39) Dallenbach-Hellweg G. The endometrium of ical diagnosis. Fertil Steril 1989; 51:273–279. infertility. A review. Pathol Res Pract 1984; (53) Lessey BA, Castelbaum AJ, Wolf L, Greene W, 178:527–537. Paulson M, Meyer WR, et al. Use of integrins (40) Good RG, Moyer DL. Estrogen-progesterone to date the endometrium. Fertil Steril 2000; relationships in the development of secretory 73:779–787. endometrium. Fertil Steril 1968; 19:37–45. (54) Ordi J, Creus M, Ferrer B, Fabregues F, (41) Gillam JS. Study of the inadequate secretion Carmona F, Casamitjana R, et al. Midluteal phase endometrium. Fertil Steril 1955; 6: endometrial biopsy and alphavbeta3 integrin 18–36. expression in the evaluation of the endo- (42) Deligdisch L. Hormonal pathology of the metrium in infertility: implications for fecun- endometrium. Mod Pathol 2000; 13:285–294. dity. Int J Gynecol Pathol 2002; 21:231–238. (43) Dallenbach-Hellweg G. Changes in the (55) Dubowy RL, Feinberg RF, Keefe DL, Doncel endometrium caused by endogenous hormonal GF, Williams SC, McSweet JC, et al. Improved dysfunction. Verh Dtsch Ges Pathol 1997; 81: endometrial assessment using cyclin E and p27. 213–218. Fertil Steril 2003; 80:146–156.