* Reprint requests: Dr. T. C. Li, Jessop Hospital for Women,

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1 FERTILITY AND STERILITY Copyright 1987 The American Fertility Society Vol 48, No6, Decemher 1987 Printed in USA A comparison between two methods of chronological dating of human endometrial biopsies during the luteal phase, and their correlation with histologic dating Tin-Chiu Li, MRCOG*t:j: Andrew W Rogers, PhD, DSc Elizabeth A Lenton, PhD*11 Peter Dockery, PhDII Ian Cooke, FRCOG*II University of Sheffield, Jessop Hospital for Women, Sheffield, England This prospective study was performed on 61 infertile women to examine the correlation between histologic dating using the same criteria by two independent observers and chronological dating by two different methods: (1) determination of luteinizing hormone (LH) peak by daily LH assay, (2) calculation based on the onset of the next menstrual period (NMP) The correlation between histologic dating and chronological dating was found to be significantly better if the LH peak was used to determine the chronological date than if the NMP was used (r = 7 and 37, respectively) Fertil Steril48:928, 1987 The human endometrium is examined histologically, not only to diagnose pathologic conditions but, in infertile couples, to determine whether ovulation has occurred and to evaluate the function of the corpus luteum In the luteal phase defect/ the diagnosis is primarily based on the discrepancy between the observed endometrial pattern (histologic dating) and the expected endometrial pattern according to the time at which the endometrial biopsy (EB) was taken (chronological dating) The histologic dating is performed conventionally by the criteria of Noyes et a1 2 For many years, the chronological Received May 4, 1987; revised and accepted August 5, 1987 Department of Obstetrics and Gynaecology, University of Sheffield t Research fellow supported by a grant from Roussel Laboratories Ltd, UK, grant no 327 * Reprint requests: Dr T C 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 date has been assigned retrospectively from the onset of the next menstrual period (NMP)3-5 It is now well established, however, that the luteal phase may vary considerably in length,6,7 so that the use of the onset of the NMP as a guide to dating may introduce an undesirable variability Some investigators have addressed this problem by using the basal body temperature (BBT) to identify the time of ovulation 8 or by combining the BBT with the onset ofthe NMP 9 Others determine the day of the luteinizing hormone (LH) surgelo,n The present study examines the accuracy and reliability of histologic dating of endometrial biopsies, when correlated, respectively, with two methods of chronological dating: prospectively from the LH peak, and retrospectively from the onset of the NMP MATERIALS AND METHODS EBs were obtained from 65 women attending the infertility clinic at the University Department of Obstetrics and Gynaecology in the Jessop Hospital for Women, Sheffield The day of the LH surge 928 Li et al Endometrial dating

2 (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 All EBs were taken between luteal days LH + 4 and LH + 11 All of these patients produced a definite LH peak Each patient recorded the day of onset of the NMP EBs were performed as an outpatient procedure, with or without Entonox (BOC Ltd, South Yorkshire, England) analgesia Using a Sharman's curette (Downs Surgical Ltd, Sheffield, England), a single 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 74) for 4 to 6 hours The specimen then was washed in buffer and postfixed in osmium tetroxide After dehydration, the specimen was embedded in araldite, sectioned at 1 ~m, and the sections stained with toluidine blue for examination by light microscopy Sections were examined independently by two observers, both of whom were unaware of the timing of the biopsy specimen Each observer assigned a date to the biopsy, expressed as days after the LH peak, using the histologic criteria of Noyes et al 2 Each biopsy specimen then was assigned chronological dates by the following two methods The first indicated the number of days between the LH peak and the taking of the biopsy The second assumed a luteal phase of 14 days, and used the number of days between the taking of the biopsy and the onset of the NMP to derive a date For example, the day that immediately precedes the onset of menstruation (M - 1) would be taken as LH + 14 Care was taken to exclude premenstrual spotting in determining the onset of menstruation, since spotting may follow EB, and failure to recognize its presence may spuriously advance the date taken as the start of menstruation The 65 women were a consecutive series of patients, un selected except that women with a known history of anovulatory or irregular cycles were excluded from the study Of the 65 patients, 3 were subsequently excluded because inadequate tissue specimens were obtained at EB, and another patient was excluded because the specimen demonstrated proliferative changes Hormone Assays Daily LH levels were determined by two methods, radioimmunoassay (RIA) on plasma samples or enzyme immunoassay on early morning Vol 48, No6, December 1987 urine specimens In 53 patients, both methods were used The two methods agreed exactly on the date of the LH peak in 43 cases (81%), and differed by 1 day in 7 cases (13%) In an additional 3 cases (6%), no clear LH peak was seen in the urine assay, despite a clear surge being present in plasma Statistics Comparisons were made between the histologic dates assigned by the two observers, and between these dates and the chronological dates found by the two methods described previously, using the regression model by Deming 12 and by Cornbleet and Gochman 13 RESULTS Of the 61 biopsies, 1 was taken in the cycle of conception In the woman in whom the EB was taken in the cycle of conception and three other women in whom the onset of the NMP was not recorded, the determination of a chronological date from the onset of the NMP was not possible The mean luteal phase length was 128 days The correlation of histologic dating between the two observers (Fig 1) was highly significant (r = 7, P < 1) Correlations also were made between histologic dating (by the two observers) and chronological dating (by the two methods), as shown in Figure 2 The correlation coefficients and their significance are shown in Table 1 The difference between histologic dating and chronological dating was analyzed as in Figure 3 1 'i ~ B ::J III c( J: J 6 Ii E > Ii ~ III Q 4 - III >- IV 2 m \ o!, : B Observer B (Days from LH surge) Figure 1 The correlation of histologic dating between the two observers, A and B Li et al Endometrial dating 929 1

3 Figure 2 $Ẏ,! : o,,, r-r,,2 / ' : I I,, ~ :! : ~ 1'1 _, o ~,,---~---is---ra -'''''""""2 _,: -'2 -,' -a -is _~ _~ ;, LH Oat M ot Chronological Dating (day) A comparison of the correlations between histologic dating (by two observers) and chronological dating (using two reference points) The observers (A and B) individually assessed the same slides and their observations were correlated separately to either the LH surge (LH date) or the onset of the NMP (M date) For each biopsy, the histologic dating by the two independent observers was used to derive a mean value from which the chronological date was subtracted (A positive result would mean that the histologic date is relatively more advanced than the chronological date, whereas a negative result would indicate a retarded endometrial development) When the chronological date was based on the determination of the LH surge, the difference between histologic and chronological dating appeared to be slightly skewed toward retardation (mean value = -7 ± 13, skewness = -63, standard deviation [SD] = 13) When a normal curve is fitted to the histogram,14 there appears to be a subpopulation with pathologically retarded endometrial development In comparison, when the chronological date was derived from the onset of the NMP, the skewness was lost (-4), but the variance was increased Table 1 The Correlation Coefficients and Their Significance, for the Correlation Between Histologic Dating (Two Different Observers) and Chronological Dating (Two Different Methods) Histologic dating by observer A Histologic dating by observer B Chronological dating by LH surge r = 65 P < 1 r = 75 P < 1 93 Li et al Endometrial dating Chronological dating by the onset of the next menstrual period r = 28 P<5 r = 41 P < : 1 U i a :::I i a :: 4 2 o 1 : a u i a :::I ~ 4 &I 2 o -a -a H Date - LH Date (days) Figure 3 The frequency distribution of the difference between histologic dating and chronological dating by each method H date, mean value of the histologic dating by the two observers LH date, the chronological dating derived from the LH surge M date, the chronological dating derived from the onset of the next menstrual period A normal curve has been fitted to the frequency distribution according to the bar chart (SD = 214) The mean of the difference was -193 ± 28 When a normal curve is fitted to the histogram, 14 1 't IS no I onger apparent that a subpopulation with retarded endometrial development exists DISCUSSION Several sources of error can complicate any attempt to correlate the histologic appearance of an EB with the time during the luteal phase at which it was taken These include observer error in assigning a histologic date, error in assigning a correct time for the biopsy in the luteal phase, and the possibility that the endometrium is failing to respond normally to circulating hormones In addition, the variance of these measurements is affected by the size of the population of women studied and the criteria used in their selection Histologic Dating In this study, two independent observers assigned histologic dates to the same sets of slides, using agreed criteria It is interesting that the correlation between them was no better than r = 7 Any such judgment based on the subjective evaluation of histologic patterns is likely to introduce some degree of error into experimental data Mor- 4

4 phometry, the objective measurement of defined parameters in histologic material, should reduce this source of variability Work is in progress in our departments to develop morphometric methods of assigning histologic dates to endometrial biopsies Chronological Dating Errors in determining the appropriate chronological date for an EB are likely to be small using the prospective method of dating from the LH peak, particularly if RIA of serum specimens is used in preference to assays based on early morning samples of urine In our hands, the latter method occasionally failed to identify a clear LH peak, which could be seen by RIA of plasma It is reasonable to expect that endometrial changes early in the luteal phase are linked to ovulation in a programmed sequence The retrospective method of dating from the start of the NMP, however, has three important features that increase its contribution to the overall variance of any experiment First, it is based on the assumption that the luteal phase lasts approximately 14 days Lenton et al 7 have shown that only 266% of 327 regularly cycling women had luteal phases of exactly 14 days and 31 % had luteal phases of 12 days or less, or 16 days or more Johannisson et al 6 have presented similar findings Second, an EB in the late luteal phase is often followed by premenstrual spotting, making it difficult to identify with certainty the day of onset of the NMP Third, it is possible that EB causes the local release of prostaglandins in the endometrium, which in turn might shorten the luteal phase It is clear from our results that the correlation between histologic and chronological dating is much better using the LH peak than when using the retrospective method of dating from the onset of the NMP The mean observed luteal phase length in our study was 128 days, which was 12 days short ofthe assumed luteal phase length of 14 days This would explain the relatively large mean difference between the histologic date and chronological date based on the NMP (-193) compared with that when the chronological date was based on the LH peak (-7) In clinical practice, it is important to remember that, when the NMP is used for chronological dating, apparent retardation of endometrial development may result if the actual luteal phase length is shorter than the assumed luteal phase length In a study correlating 3 pairs of EB taken from 3 women in two different cycles, Balasch et al 15 used the onset of the NMP to determine the chronological date and found that, if the initial EB was out-of-phase (OOP), 49% of the second EB taken in a subsequent cycle were again OOP However, if the initial EB was normal, 18% of the second EB were OOP This rather poor correlation between the first and second EB might be due to the sporadic nature of the abnormality of the luteal phase endometrium (heterogeneity of cycles), but may well be, as we discussed earlier, due to a lack of precision in dating the endometrium when the onset of the NMP is used to calculate the chronological date The use of a more accurate method, such as the determination of LH peak for chronological dating, should improve the precision of the observation, and may avoid the necessity of obtaining at least two retarded biopsies in order to make the diagnosis of luteal phase defect Correlation Varies in Different Populations The correlation between histologic structure and the days in the luteal phase on which EBs were taken will clearly be poorer if some of the endometrial samples are failing to respond normally to circulating levels of ovarian hormones Lundy et au 6 achieved a correlation of r = 97 in a series of 8 normal, healthy women In our series of 61 women, the best correlation we achieved was r = 75, but our group consisted of patients attending an infertility clinic who, on the evidence of the skewed distribution, probably included a subgroup with luteal phase defect These figures compare well with Tredway et al1o whose correlation using the LH peak was r = 79 in a group of 11 women of unspecified fertility Noyes and Haman 9 achieved remarkably good correlation (r from 61 to 81) using a combination of BBT and the onset of the NMP They undoubtedly had greater experience at histologic dating than we do, but they also excluded from their original sample some 5% of patients, largely on the grounds of doubt about the accuracy or completeness of the data leading to chronological dating For all of these reasons, it is difficult to compare directly the results of the several published studies (with different populations) on methods of dating endometrial biopsies These difficulties may account for the continued acceptance of retrospective chronological dating using the onset of the NMP In our study, prospective and retrospective methods are compared directly in the same group of patients for the first time It is clear that the correlation between histologic and chronological Vol 48, No6, December 1987 Li et al Endometrial dating 931

5 dating is much more precise with the prospective method, based on the day of the LH peak Clinical Aspects If the onset of the NMP period is used for the chronological dating, 42% of all the EBs will have a discrepancy between histologic date and chronological date of more than 2 days (OOP) However, if the LH peak is used for chronological dating, only 1% of the EBs would be regarded as OOP This means that the use of the onset of the NMP to calculate the chronological date will lead to significant (x 2 = 339, P < 1) overestimation of the incidence of OOP endometrium and of luteal phase defect In addition, we have evidence 17 to support the fact that women with shorter or longer luteal phases will continue to show that particular pattern Thus, if the NMP is used for chronological dating, they will continue to give the same result without necessarily having any pathology at all Acknowledgments We would like to thank Shirley Hill, FRCPath, Christine Pigott, Linda Highfield, Helen King, MBBS, and all the staff of the Research Clinic at Jessop Hospital for Women, Sheffield, for their valuable help with different aspects of this project REFERENCES 1 Jones GS: The luteal phase defect Fertil Steril 27:351, Noyes RW, Hertig AT, Rock J: Dating the endometrial biopsy Fertil Steril1:3, Cooke ID, Morgan CA, Parry TE: Correlation of endometrial biopsy and plasma progesterone levels in infertile women J Obstet Gynaecol Br Commnw 76:647, Jones GS: The clinical evaluation of ovulation and the luteal phase defect J Reprod Med 18:139, Wentz AC: Endometrial biopsy in the evaluation ofinfertility Fertil Steril 33:121, Johannisson E, Parker RA, Landgren B-M, Diczfalusy E: Morphometric analysis of the human endometrium in relation to peripheral hormone levels Fertil Steril 38:564, Lenton EA, Landgren B-M, Sexton L: Normal variation in the length ofthe luteal phase of the menstrual cycle: identification of the short luteal phase Br J Obstet Gynaecol 91:685, Gautray JP, De Brux 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, Noyes RW, Haman JO: Accuracy of endometrial dating: correlation of endometrial dating with basal body temperature and menses Fertil Steril 4:54, Tredway DR, Mishell DR, Moyer DL: Correlation of endometrial dating with luteinizing hormone peak Am J Obstet GynecoI117:13, Koninckx PR, Goddeeris PG, Lauweryns JM, de Hertogh RC, Brosens IA: Accuracy of endometrial biopsy dating in relation to the midcycle luteinizing hormone peak Fertil Steril 28:443, Deming WE: Systematic computation for fitting curves by least squares In Statistical Adjustment of Data New York, John Wiley & Sons, 1943, p Cornbleet PJ, Gochman N: Incorrect least-squares regression coefficients in method-comparison analysis Clin Chem 25:432, Diem K, Lentner C: Scientific Tables Basel, Switzerland, Ciba-Geigy, 197, p Balasch J, Vanrell JA, Creus M, Marquez M, Gonzalez Merlo J: The endometrial biopsy for diagnosis of luteal phase deficiency Fertil Steril 44:699, Lundy LE, Lee SG, Levy W, Woodruff JD, Wu CH, Abdalla M: The ovulatory cycle: a histologic, thermal, steroid and gonadotrophin correlation Obstet GynecoI44:14, Lenton EA, Lawrence GF, Coleman RA, Cooke ID: Individual variation in gonadotrophin and steroid concentrations and in lengths of follicular and luteal phases in women with regular menstrual cycles Clin Reprod Fertil 2:143, Li et al Endometrial dating