A new method of histologic dating of human endometrium in the luteal phase

Transcription

1 FERTILITY AND STERILITY Copyright o 1988 The American Fertility Society Vol. 5, No. 1, July 1988 Printed in U.S.A. A new method of histologic dating of human endometrium in the luteal phase Tin-Chiu Li, M.R.C.P., M.R.C.O.G.*tt Andrew W. Rogers, Ph.D., D.Sc. Peter Dockery, Ph.D. II Elizabeth A. Lenton, Ph.D.* II Ian D. Cooke, F.R.C.O.G.*II University of Sheffield, Jessop Hospital for Women, Sheffield, England Morphometric analysis was performed on 7 endometrial biopsy specimens collected from a population of fertile women. Of the 17 morphometric measurements that were performed on each endometrial biopsy, only 5 were required to achieve a highly significant correlation with chronologie dating based on luteinizing hormone surge (R =.99). The result of histologic dating, based on morphometric analysis of endometrial biopsies collected from a separate, unbiased population, correlated strongly and significantly with chronologie dating (r =.98, P <.1). The correlation was better than that achieved when histologic dating was performed according to the conventional dating criteria (r =.88, P <.1). Fertil Steril 5:52, 1988 In 195, Noyes et ap published their classic description of the histologic changes of the endometrium throughout the menstrual cycle. Since then, their description has been widely accepted as a set of standard criteria for the histologic dating of human endometrium. Recently, their paper was found to be the most frequently cited article ever published in. 2 Using such dating criteria to study endometrial development in the luteal phase of women with infertility has, however, generated much controversy. For example, the reported incidence of retarded endometrial development (on which the Received December 1, 1987; revised and accepted, March 17, Department of Obstetrics & Gynaecology, University of Sheffield. t Research fellow supported by a grant from Roussel Laboratories Ltd, U.K. Grant No t Reprint requests: Tin-Chiu Li, Jessop Hospital for Women, Sheffield, S3 7RE, England. Department of Anatomy and Cell Biology, University of Sheffield. II Harris Birthright Center for Reproductive Medicine, Jessop Hospital for Women, Sheffield. 52 Li et al. Histologic dating of endometrium diagnosis of luteal phase defect 3 has been commonly based) among women with infertility has varied from 4% to 65%. 4-8 While some investigators reported the presence of significant correlation between endometrial development and peripheral progesterone (P) concentration, 4 9 many others found a lack of correlation between the two The concentration of P receptors in those with retarded endometrial development has been found to be higher than, the same as/ 5 or lower than 16 in those with normal endometrial development. Not surprisingly, there is also a lack of consensus on whether treatment of retarded endometrial development would improve fertility or on the best form such treatment should take. We hypothesize that these controversial literature reports are in part due to a lack of precision in the measurement of endometrial development by the subjective dating criteria of Noyes et ap To test this hypothesis, we have applied morphometric analysis, a quantitative and objective technique, to the dating of endometrial biopsy specimens (EBs) obtained from a population of fertile women and compared the results to those obtained by the criteria of Noyes et ap on the same population.

2 Subjects MATERIALS AND METHODS In this study, 73 normal fertile women were recruited from those attending the gynaecologic outpatient clinic of the University Department of Obstetrics and Gynaecology at the Jessop Hospital for Women, Sheffield. Most of them attended requesting sterilization or reversal of sterilization. Normal fertile women are defined as those who (1) had regular cycles of 25 to 35 days; (2) had not received any steroid hormones (including oral contraceptives) or used an intrauterine contraceptive device for at least 3 months; (3) were aged between 18 and 4 years; (4) had no known history of menstrual disorder; and (5) had one or more prior successful pregnancies. Endometrial Biopsy Endometrial biopsy (EB) was performed as an outpatient procedure. The woman undergoing the procedure was put in the dorsal position, and her external genitalia were cleaned with Savlodil (.15% w/v chlorhexidine gluconate and.15% w/v cetrimide; ICI, Macclesfield, UK). A pelvic examination was performed to exclude any pelvic pathology and to determine the position and size of the uterus. A speculum was then inserted into the vagina to expose the cervix, which was then cleaned with Savlodil. With a Sharman's currette (Downs Surgical Ltd, Sheffield, UK), a single endometrial specimen was obtained from the fundus and upper part of the body of the uterus. Each specimen was fixed immediately in 2% glutaraldehyde in cacodylate buffer (ph 7.4) for 4 to 6 hours. It was then washed in buffer, dehydrated, and embedded in JB-4 (Polysciences, Inc., Warrington, PA), a plastic polymer. After this, it was sectioned at 2 #lm, and the sections were stained with acid fuchsin and toluidine blue for examination by light microscopy. Chronologie Dating of EB All the EBs were timed by reference to the luteinizing hormone (LH) surge. The day of the LH surge (designated LH + ) was determined by daily LH assay on a specimen either of morning urine or of plasma, starting on day 9 of the menstrual cycle, as described elsewherep Vol. 5, No. 1, July 1988 Histologic Study of EB All EBs were studied histologically by one observer (T.C.L.), who was unaware of the chronologie dating at the time of the study. Two different methods were used: (1) dating criteria of Noyes et au; and (2) morphometric analysis/s- 2 in which 17 morphologic features were measured for each EB. In glands, the features measured were (1) number of mitoses per 1 gland cells (G1); (2) gland cell height (G2); (3) amount of secretion in gland lumen (G3); (4) maximal gland diameter (G4); (5) volume fraction of gland occupied by gland cell (G5); (6) volume fraction of endometrium occupied by gland (G6); (7) volume fraction of gland cell occupied by nucleus (G7); (8) number of supranuclear secretory vacuoles per 1 gland cells (G8); (9) number of subnuclear vacuoles per 1 gland cells (G9); (1) amount of pseudostratification of gland cells (G1); and (11) number of apoptotic bodies 21 per 1 gland cells (Gll). In stroma, the features measured were (1) number of mitoses per 1 stromal cells (S1); ~2) amount of stromal edema (S2); (3) amount of predecidual reaction (S3); (4) diameter ofvenules (S4); (5) amount of leukocytic infiltration (S5); and (6) amount of extravasation (S6). Four different methods were used for the measurement of the above 17 morphologic features. They include (1) point-counting, with the use of square lattices with 121 intersection points (G5, G6, and G7); (2) cell counting 1 S- 21 (G1, G8, G9, Gll, and S1); (3) linear measurement (G2, G4, and S4); (4) semiquantitative measurement, 2 with a 4- point scoring system, from to 3: = absent; 1 =mild; 2 =moderate; 3 =marked (G3, G1, S2, S3, S5, and S6). The problems of mitotic counts were discussed by Scully, 22 Kempson, 23 and Norris. 24 In order to minimize errors in mitotic counts, the following points were observed throughout the study: (1) thin sections of 2 #LID were used; (2) the section had to be well stained; (3) at least 1 fields were examined; (4) at least 1 cells were examined; (5) only definite mitotic figures were included in the count, the criterion used for identifying mitosis being the absence of nuclear membrane; 18 and (6) the count was always performed at high magnification under oil immersion. The measurement of cross sectional diameter in tubular structures such as endometrial glands (G4) and stromal vessels (S4) may be subject to error due to tangential or longitudinal cuts through the Li et al. Histologic dating of endometrium 53

3 structure. In these situations, the true diameters are likely to be overestimated. To overcome this problem, a systematic biased sampling method 19 was used. For glandular diameter, the cross-sectional diameter of gland was measured only when epithelial cells on each side of the lumen were in the plane of section from basement membrane to ciliated luminal surface. For venule diameter, it was not easy to be sure if a venule had been cut transversely or not. The assumption made was that in a round tubular structure such as the venule, the cross-sectional diameter would be represented by the least diameter in any two-dimensional cut section. Similarly, the measurement of gland cell height may be subject to error, unless the gland cell is cut parallel to its longitudinal axis. Hence a systematic biased sampling method was similarly employed, selecting cells for the measurement of gland cell height only if the same cell extended in the section from cilia to basement membrane. For each EB, a minimum of 2 fields were examined from 4 sections, 2 each from a separate block. Statistical Analysis In order to evaluate the correlation between these 17 morphometric measurements on the one hand and chronologie dating based on the LH surge on the other, multiple regression analysis was performed with the statistical package for Social Science (SPSS-X). The chronologie dating was entered as the dependent variable, and the 17 morphometric measurements were entered as independent variables. The method of stepwise selection of variables for inclusion or exclusion was used in constructing the regression model. The population of 7 EBs was divided into two subpopulations by random selection (random number table), so that the first one consisted of 6 EBs (subpopulation A) and the second one consisted of 1 EBs (subpopulation B). Only subpopulation A (n = 6) was subjected to multiple regression analysis as described above. Subpopulation B provided Table 1 The Age, Body Weight, and Usual Cycle Length of68 Normal Fertile Women Whose EBs Were Analyzed Morphometrically Standard Mean deviation Range Age (years) Body weight (kg) Usual cycle length (days) Li et al. Histologic dating of endometrium Table 2 The Distribution of Chronologie Date Based on LH Surge of 7 EBs Collected From Normal Fertile Women Chronologie date (days from LH surge) LH+O LH+ 1 LH+2 LH+3 LH+4 LH+5 LH+6 LH+7 LH+8 LH+9 LH + 1 LH + 11 LH+ 12 LH + 13 LH+ 14 Frequency an unbiased population to test the validity of the regression model established by subpopulation A. RESULTS A total of 73 women was initially recruited. However, three of them did not produce a clear surge of LH based on daily urine LH dipstick tests and were therefore excluded from the study. In another two women, the specimen obtained from EB was insufficient for histologic assessment. Of the remaining 68 women, two of them had two EBs, each from a different cycle. Thus, there were 7 EBs collected from 68 normal fertile women in which histologic analysis was performed. The age, body weight, and usual cycle length of these 68 women are shown in Table 1, and the distribution of chronologie dating based on the LH surge of these 7 EBs are shown in Table 2. Individual Morphometric Measurements The result of each morphometric measurement throughout the luteal phase is shown in Figure 1. Multiple Regression Analysis Initially, all 17 measurements were entered without any prior transformation of data. Subsequent analysis of residual plots suggested that G 1, G3, G9, G1, Gll, S3, and S5 had uneven distribution of residuals and that log transformation should be performed. Thus, a subsequent entry of the 17 measurements was made with log transformation of the seven measurements mentioned

4 : G... a 7S ;:i:~ 3 :: : 15.,'.::. 2 :: I~~ ':' ~' I 1 'I I:. 2 : ~:,... I' 'I... 1 I:. I... ~. r i... o~~~ 5 II.... :... on... i:;.,. " 5,'i.,. 1, 'I;,,, I JS ;, ".;.,... ' ,...,... ;i : +:-... ; I : ; ~; :~:: ~::: I... T 11. #Sf.? 2 4 e a 2 4 e a 112, a a 111 M Days from LH peak Figure 1 The results of individual morphometric measurements throughout the luteal phase, of 7 EBs collected from a fertile population. Gland: G 1 = number of mitoses per 1 gland cells; G2 = gland cell height (~tm); Ga = amount of secretion in gland lumen (score to a); G4 = maximal gland diameter (~tm); G5 = volume fraction of gland occupied by gland cell; G6 = volume fraction of endometrium occupied by gland; G7 = volume fraction of gland cell occupied by nucleus; G8 = number of supranuclear secretory vacuoles per 1 gland cells; G9 = number of subnuclear vacuoles per 1 gland cells; G1 = amount of pseudostratification of gland cell (score to a); Gll =number of apoptotic bodies per 1 gland cells. Stroma: S1 = number of mitoses per 1 stromal cells; S2 = amount of stromal edema (score to a); sa= amount ofpredecidual reaction (score to a); S4 = diameter of venule (~tm); S5 = amount ofleukocytic infiltration (score to a); S6 = amount of extravasation (score to a). above. The measurement selected for inclusion by the computer in each step and the multiple regression coefficient (R) achieved for each step are shown in Table 3. At the first step, the R was calculated for each of the 17 potential predictor variables. The one with the largest R, i.e., best predictive property (G5) was then selected. At the second step, the R was computed for the variable first selected (G5), paired with each of the remaining 16 predictor variables, the pair with the highest R being the best predictors (G5, log 83). Subsequent steps were performed in a similar manner, each time taking the previous step with each of the remaining variables in turn, the best set being those with the highest R. The procedure stopped when the R could no longer be improved upon significantly. Vol. 5, No. 1, July 1988 The final regression equation and the coefficient of each measurement is shown below: Chronologie dating= 7.81 (constant) X log G X log G3-.33 X G5 Histologic Dating X log G X log 83 To test the validity of the regression model so established for histologic dating, the equation was applied to an unbiased subpopulation, subpopulation B (n = 1), as defined earlier. The results of the five selected morphometric measurements were fitted into the regression equation to predict the dating of each of these 1 EBs. The results of the predicted date were then correlated to the actual chronologie data as shown in Figure 2, in which a strong and significant correlation (r =.98, P <.1) was found. Also shown in Figure 2, for comparison, is the correlation between histologic dating according ~o the criteria of Noyes et au of the same population of EBs and the chronologie date (r =.88, P <.1). DISCUSSION Johannison et al. 2 first employed the techniques of morphometric analysis, an objective and quantitative technique, to the study of endometrium and related the results to peripheral hormone levels. In this study, we applied morphometric analysis to the histologic dating of endometrium in the luteal phase. The results indicated that only 5 out of the 17 morphometric measurements were required to Table 3 The Measurements Selected by Stepwise Method of Multiple Regression Analysis, SPSS-X, Among the 17 Morphometric Measurements Entered Multiple Measurement regression Significance selected or coefficient (P) off Step added (R) achieved value 1 G5.91 <.1 2 log sa.9a <.1 a log Ga.97 <.1 4 log G1.98 <.1 5 log G1.99 <.1 No further measurement selected Li et al. Histologic dating of endometrium 55

5 14 A 12.II: 1 Ill Q) Q. 8 ::1: 6..J E 2 II) >o Ill Q) -Ill " B iii u 8 c;, II) :f 2 / / Chronological date (Days from LH peak) Figure 2 The correlations between chronologie dating and histological dating by two different methods. (A) Histologic dating by morphometric analysis (r =.98, P <.1). (B) Histologic dating by the criteria of Noyes et al. 1 (r =.88, P <.1). achieve a strong and significant correlation (R =.99, P <.1) to the chronologie date as defined by the LH surge. When the regression model is applied to another, unbiased population to predict the chronologie date, the correlation between predicted date and actual date was again strong and significant (r =.98, P <.1). Thus, it can be concluded that the results of morphometric analysis can be used reliably to date the endometrium with considerable accuracy. All the EBs were embedded in JB-4 (Polysciences, Inc, Warrington, PA), a plastic medium, because this enables semi-thin sections of 2 ~m to be cut. This is important because morphometric measurements are more accurate if thin sections are used. 19 The conventional method of preparation of histologic specimens employs paraffin wax for embedding, and the sections obtained would be at least 5-6 ~m thick. As a result, they would not be as suitable for morphometric analysis. Comparison of Morphometric Analysis with the Criteria of Noyes et al. When the two methods of histologic dating (morphometric analysis, dating criteria of Noyes et al. 1 ) are compared directly to each other in the same, unbiased population, it is found that the results of histologic dating by morphometric analysis correlate better with chronologie dating than do the results of histologic dating based on the criteria of Noyes et at.l (Fig. 2). Although the criteria of Noyes et at.l have been accepted as the standard method for dating of the endometrium, three criticisms may be directed at them. (1) Population Used to Establish the Dating Criteria. These criteria were based on the results of the analysis of endometrial biopsy specimens collected mainly from infertile women, in whom the development of the endometrium may be abnormal. It is more valid to base one's criteria on the study of endometrial biopsy specimens collected from normal, fertile women. So far, there have been only two published reports on detailed morpholbgic study of endometrium from normal, fertile women, both from Johannisson et al., 2 25 but they did not apply their results to histologic dating. In our study, a normal fertile population of 68 women was used to establish the dating criteria. (2) Precision of Chronologie Dating. The analysis of Noyes et al. 1 relied mainly on the onset of the next menstru.al period (NMP) to calculate the chronologie date, on the assumption that the luteal phase lasts 14 days. However, this retrospective method of chronologie dating has three important drawbacks. These have recently been reviewed by Li et al., 17 who have also demonstrated that the use of the LH peak to determine chronologie dating had significantly better correlation with histologic dating than did the use of the NMP to calculate chronologie dating. Noyes et at.l also used basal body temperature (BBT) to estimate the time of ovulation in some of the cases. However, the lack of precision of this particular method in timing ovulation has since been reported in several studies (for review, see Li et al. 17 ). Thus, the dating criteria of Noyes et al. 1 suffered from a lack of precision of chronologie dating. In our study, in contrast, all the EBs were timed by the use of the LH surge. (3) Subjectivity of Histologic Dating. The dating criteria of Noyes et al. 1 were based on subjective morphologic assessment of the endometrium. Subjective histologic assessment involving certain dis- 56 Li et al. Histologic dating of endometrium

6 tinctive features may be highly reproducible, but subjective assessment involving a grading process such as grading of neoplasia and dating of endometrium often suffers from considerable inter-observer and intra-observer variability and diagnostic errors (or shifts), which may be either random or systematic. Noyes et al. in their original work 1 did encounter such a problem and they commented that "... some of the high percentage of error was due to individual interpretation...." They then improved the accuracy of the proposed criteria for dating from 38% to 6% by reducing the number of observers down to one. In this study, an objective method of analyzing endometrial morphology, morphometric analysis, has been used. It has the theoretical advantage of being more reproducible and less prone to diagnostic error, and quality control is possible. Reappraisal of Dating Criteria of Noyes et al. Although the histologic dating criteria of Noyes et al., 1 which have been in use for more than 3 years, have their limitations, no formal reappraisal of these criteria has ever been made. The quantitative data presented in this study on endometrial specimens that were accurately timed by LH surge and collected from a normal fertile population (n = 7) provide an opportunity to update the dating criteria of Noyes et al.,l which could be modified and restated as follows: DayLH+2 Both glandular and stromal mitoses are moderately common, and there is little evidence of secretion either in the gland cells or gland lumen. The gland lumen appears closed, and pseudostratification of gland cells is marked. The proportion of endometrium occupied by glands to stroma is low. DayLH+3 There is still a moderate amount of mitotic activity in both the glandular and stromal cells. Subnuclear vacuoles are present in 5% or more of gland cells, pushing the gland cell nuclei to a more central position. As a result, the gland cells appear taller, and pseudostratification is less marked. A few supranuclear vacuoles may be seen in gland cells, but secretory material in the gland lumen is still absent or scanty. Thus, the gland lumen still appears closed. DayLH+4 The mitotic activity in both glandular and stromal cells has further diminished, so that only occasional mitoses are seen. The number of subnuclear vacuoles and supranuclear vacuoles is maximal. The gland cells also achieve their maximal height. Pseudostratification has virtually disappeared. There is now a small but definite amount of secretion in the gland lumen, the diameter of which has started to increase. DayLH + 5 Glandular mitotic activity is now negligible, although occasional stromal mitoses are still seen and remain throughout the rest of the cycle. As the secretory vacuoles start to discharge their contents into the gland lumen, the number of subnuclear and supranuclear vacuoles gradually decreases, as does the height of the gland cell. On the other hand, more secretory material is present in the lumen, the diameter of which continues to increase. DayLH+6 Glandular mitotic activity is virtually absent. The number of secretory vacuoles continues to decrease as more and more of them are discharged into the lumen, which in turn accumulates more and more secretion. About a quarter of the whole endometrium is now occupied by glands. DayLH+ 7 A moderate amount of secretion has now accumulated in the gland lumen, the diameter of which continues to increase. The number of secretory vacuoles is negligible. The gland cell has started to assume a low cuboidal appearance, becoming both shorter and broader than those earlier in the luteal phase. In the stroma, edema has started to appear. DayLH+B Both the amount of secretion in the gland lumen and stromal edema have reached a maximum. As a result of the latter, the proportion of endometrium occupied by glands has dropped to less than a quarter. DayLH+9 The amount of secretion in the gland lumen remains plentiful, and the glandular diameter con- Vol. 5, No. 1, July 1988 Li et al. Histologic dating of endometrium 57

7 tinues to increase. In the stroma, the edema has become less marked, and a predecidual reaction has started to occur around blood vessels, which appear to have increased in number. DayLH + 1 The appearance may be rather similar to that of day LH + 9 except that stromal edema has further subsided and the predecidual reaction becomes more definite but remains confined to areas around the blood vessels. As a result of the decrease in edema, the proportion of endometrium occupied by glands has increased to about one third. On the other hand, the proportion of gland cells occupied by the nucleus has started to decrease, probably a result of an increase in the amount of gland cell cytoplasm. DayLH+ 11 The appearance of gland and gland cells is on the whole similar to that of day LH + 1, although the gland has continued its gradual increase in diameter. In the stroma, the predecidual change may still be confined to the perivascular region, or it may be seen occasionally adjacent to glands as well. Starting day LH + 11, an increasing amount of small lymphocytic cells may be seen in the stroma. DayLH+ 12 The amount of cytoplasm in the gland cells continues to increase, so that some gland cells may appear "blown up." Whereas previously the number of apoptotic bodies amongst gland cells is small, it has now started to increase. In the stroma, the predecidual reaction may be seen around glands and beneath the luminal epithelium, and the number of lymphocytic cells continues to increase. DayLH+ 13 There is further increase in the number of apoptotic bodies among gland cells. Within the gland lumen, a few granulocytic cells may be seen. The predecidual change has now extended outside the perivascular and periglandular regions. The number of lymphocytic cells in the stroma also continues to increase. DayLH + 14 The number of apoptotic bodies among gland cells and the number of granulocytic cells within the gland lumen have further increased. Predecidual change is now marked, and sheets of predecidual cells may be seen across the stroma. The number of lymphocytic cells within the stroma has reached a maximum, and disintegration of the stroma, along with extravasation, may be evident. Analysis of the Regression Model Although 17 morphometric measurements were made in this study, the result of multiple regression analysis indicated that only 5 among these 17 were necessary to achieve an accurate histologic dating. These were G1, G3, G5, G1, and S3. Certain morphometric measurements were excluded from the final regression model (Fig. 1), despite the fact that they had strong and significant correlations with chronologie dating, e.g., G4, r =.8, P <.1. The explanation for this is that among the 17 morphometric measurements, some of them are strongly correlated to each other. In the case of G4, its correlation with G5, a measurement included in the final regression model,. was It is therefore not surprising that the addition of G4 would not have conferred any significant improvement to the regression model. Similarly, the exclusions of G 11 and S5 could be explained for by their significant correlation to S3. On the other hand, some measurements that appeared to be of unique discriminating value in the dating of endometrium were excluded for a different reason. For example, in the case of G9, the number of subnuclear vacuoles per 1 gland cells rises rapidly from day LH + 2 to a maximum of more than 5 on days LH + 3 to LH + 4, then falls rapidly to less than 5 after day LH + 7. However, such a distinct biologic event, which is one of the most useful dating criteria of Noyes et al.,l could not be readily expressed by a linear regression relationship to chronologie date and was therefore excluded from the regression model. The same explanation applies to G2, G1, G8, and S2. The remaining measurements, G6, S1, S4, and S6, were not included because their changes throughout the luteal phase did not bear strong or significant linear correlations with chronologie dating. It is of interest to note that the first measurement selected for inclusion in the regression model was G5, the volume fraction of gland occupied by gland cells. This is a quantitative measurement, and the regression coefficient achieved by this mea- 58 Li et al. Histologic dating of endometrium

8 surement alone was.91. The addition of each of the four subsequent measurements refined and improved the regression model, but after a total of five measurements had helen selected, no further improvement of the regression model was possible from further addition of other measurements. Of the five measurements considered to be important in the regression model, four relate to glands and only one relates to stroma. This is not surprising as most of the morphologic changes in the endometrium, especially in the first half of the luteal phase, are related to glands rather than stroma. Furthermore, the four measurements relating to glands measure different aspects of glandular morphology: one measures mitotic activity (G1), two measure secretory activity (G3, G5), and one measures structural arrangement of gland cells (G1). Areas for Improvement This study is the first one to attempt to apply the technique of morphometric analysis to histologic dating of the endometrium. Although it has produced encouraging results, it is possible that improvement could be made in a number of areas. First, not all the measurements were quantitative; some of them were semiquantitative ones based on a scoring system. They may be criticized as being subjective and not necessarily superior to the corresponding criteria of Noyes et ap In this study, we demonstrated that the quantification of some (11 out of 17) measurements had already produced results of histologic dating better than those of the criteria of Noyes et ap It is hoped that refinement in techniques of morphometric analysis may be able to quantify all measurements and lead to further improvement of its use in histologic dating of endometrium. Second, the statistical method used to analyze these measurements has not been able to include certain highly discriminating morphologic features such as the number of subnuclear vacuoles and the change in gland cell height. It is hoped that future analysis of results may benefit from a statistical approach that could maximize the use of these discriminating morphologic features. Third, one of the potential disadvantages of morphometric analysis is that it is a time-consuming technique. From our experience, to perform morphometric analysis on all the 17 features required approximately 2 hours. However, if we consider only the measurement of the 5 features that were required to achieve accurate histologic dating, the time required is considerably shortened to about half an hour per endometrial biopsy analyzed. It may be possible that the considerable time required to perform morphometric analysis may be further reduced by applying the technique of image analysis, so that certain measurements could be performed by a programmed automated counter. Acknowledgments. We are grateful to Dr. Shirley Hill, F.R.C.Path., Mr. Peter Thomas, B.Sc., Mrs. Christine Pigott, Miss Carol Richardson, and Mrs. Linda Highfield for their valuable help with various aspects of this project. REFERENCES 1. Noyes RW, Hertig AT, Rock J: Dating the endometrial biopsy. Fertil Steril1:3, Key JD, Kempers RD: Citation classics: most cited articles from. Fertil Steril 47:91, Jones GS: The luteal phase defect. Fertil Steril 27:351, Cook CL, Rao, Ch V, Yussman MA: Plasma gonadotrophin and sex steroid hormone levels during early, midfollicular, and midluteal phases of women with luteal phase defects. Fertil Steril 4:45, Cumming DC, Honore LH, Scott JZ, Williams KP: The late luteal phase in infertile women: Comparison of simultaneous endometrial biopsy and progesterone levels. Fertil Steril 43:715, Huang KE: The primary treatment of luteal phase inadequacy: progesterone versus clomiphene citrate. Am J Obstet Gynecol 155:824, Jones GS, Pourmand K: An evaluation of etiological factors and therapy in 555 private patients with primary infertility. Fertil Steril 13:398, Wentz AC: Endometrial biopsy in the evaluation of infertility. Fertil Steril 33:121, Gautray JP, Debrux J, Tajchner G, Robel P, Mouren M: Clinical investigation of the menstrual cycle. III. Clinical, endometrial and endocrine aspects of luteal defect. Fertil Steril 35:296, Annos T, Thompson IE, Taymor ML: Luteal phase deficiency and infertility: difficulties encountered in diagnosis and treatment. Obstet Gynecol 55:75, Cooke ID, Morgan CA, Parry TE: Correlation of endometrial biopsy and plasma progesterone levels in infertile women. J Obstet Gynaecol Br Commonw 76:647, Shangold M, Berkeley A, Gray J: Both midluteal serum progesterone levels and late luteal endometrial histology should be assessed in all infertile women. Fertil Steril 4:627' Gravanis A, Zorn J-R, Tanguy G, Nessmann C, Cedard L, Robel P: The 'dysharmonic luteal phase' syndrome: endometrial progesterone receptor and estradiol dehydrogenase. Fertil Steril 42:73, Saracoglu OF, Aksel S, Yeoman RR, Wiebe RH: Endometrial estradiol and progesterone receptors in patients with Vol. 5, No. 1, July 1988 Li et al. Histologic dating of endometrium 59

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