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Midluteal phase endometrial biopsy does not accurately predict luteal function*
FERTILITY AND STERILITY
Vol. 59, No.2, February 1993
Printed on acid-free paper in U.S.A.
Copyright © 1993 The American Fertility Society
Midluteal phase endometrial biopsy does not accurately predict luteal function*
Marcelo C_ Batista, M_D.t:l: Tannia P.Cartledge, R.N-§ Maria J _ Merino II Constantine Axiotis, M_D.II
Maria P. Platia, M.D.t George R. Merriam, M.D.t D. Lynn Loriaux, M.D., Ph.D.t Lynnette K. Nieman, M.D. tll
National Institute of Child Health and Human Development, Warren Grant Magnuson Clinical Center, and National Cancer Institute, National Institutes of Health, Bethesda, Maryland
Objective: To investigate whether a midluteal phase endometrial biopsy accurately predicts luteal function. Design: One nonpregnant menstrual cycle was evaluated in a prospective fashion. Setting: Outpatient Clinic of the Clinical Center of the National Institutes of Health. Participants: Fifty healthy, normally cycling women. Interventions: Serum progesterone (P) was measured daily throughout the luteal phase. An endometrial biopsy was performed 7 to 9 days after the luteinizing hormone (LH) surge, as detected by rapid plasma assays, and dated histologically according to Noyes' criteria. Main Outcome Measure: To correlate endometrial maturation with luteal P secretion. Results: Mean integrated P measurements were reduced only when the lag between histologic and chronological dating was ;?:3 days or ;?:4 days, depending on whether chronological dates were assigned prospectively from the LH surge or retrospectively from the onset of next menses, respectively. However, these lags did not consistently predict deficient luteal function because subnormal integrated P secretion was seen in only 14% of women with these delays in endometrial maturation. Conclusions: Midluteal phase endometrial biopsy provides a crude test of luteal function that does not precisely distinguish luteal insufficiency. Fertil Steril 1993;59:294-300 Key Words: Progesterone, endometrium, endometrial biopsy, luteal phase, luteal insufficiency, luteal phase defect
Corpus luteum (CL) insufficiency is characterized by deficient progesterone (P) secretion resulting in
Received January 6, 1992; revised and accepted October 21, 1992. * Supported in part by grant 204688/88.4, Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Sao Paulo, Brazil. t Developmental Endocrinology Branch, National Institute of Child Health and Human Development. :j: ·Present address: Department of Endocrinology, Hospital das Clinicas, University of Sao Paulo, Sao Paulo, Brazil. § Nursing Department, Warren Grant Magnuson Clinical Center. II Laboratory of Pathology, Division of Cancer Biology and Diagnostics, National Cancer Institute. ~ Reprint requests: Lynnette K. Nieman, M.D_, National Institutes of Health, Building 10, Room 10N262, Bethesda, Maryland 20892. 294
Batista et al.
Endometrial histology versus P
inadequate development ofthe endometrium to sustain implantation of an embryo (1, 2). Although usually sporadic, it may occur as a recurrent event in women with infertility and habitual abortion (2). Luteal insufficiency is most commonly diagnosed by performing an endometrial biopsy in the latter part of the luteal phase (1-4). The development of the endometrium should, in theory, reflect the end-organ response to P and, as such, provide a bioassay that is independent of fluctuations in serum hormone levels (3,4). Endometrial maturation is traditionally assessed by correlating the histologic dating of the biopsy, as defined by the criteria of Noyes et al. (5), with the chronological dating, representing the day of the cycle when the biopsy is obtained. Although the latter date is usually assigned retrospectively by counting backward from the onset of the next Fertility and Sterility
menses (1), several investigators believe that this should be done prospectively by counting forward from the day of the luteinizing hormone (LH) surge or day of ovulation (6, 7). Several inconsistencies exist in the definition of CL insufficiency, mainly because the endometrial biopsy has not been completely validated as a test ofluteal function (3). Different delays in endometrial maturation, such as a lag of 2, 3, 4, or more days between histologic and chronological dating, have been considered indicative of luteal insufficiency by various authors (3,8). However, the diagnostic accuracy of these criteria, as defined by comparison with the gold standard test of luteal function, the integrated P secretion, has never been carefully evaluated (3). To address this issue, we correlated endometrial maturation, as assessed by a midluteal phase biopsy, with the integrated P secretion, as determined by daily luteal hormone measurements, in a single cycle in 50 healthy women. Both the prospective and retrospective methods of chronological dating were used to evaluate the development of the endometrium and to define the accuracy of the different criteria used to diagnose luteal insufficiency. MATERIALS AND METHODS Subjects
Fifty healthy nonpregnant women (mean age, 34.4 ± 1.0 years; range, 22 to 47) participated in this study. All had regular menstrual cycles and were within 30% of ideal body weight (mean, 65.7 ± 1.9 kg; range, 44.6 to 95). Sixteen were between 40 and 47 years of age. Thirty-three had spontaneous pregnancies in the past, whereas the remainder had never attempted conception. No woman had taken hormonal contraceptives or used intrauterine devices during the previous 3 months. All subjects used barrier methods or abstained from sexual intercourse during the study. Pregnancy was excluded by a serum beta human chorionic gonadotropin concentration.:os; 2.0 ng/mL (the threshold for detection of pregnancy in this assay) at the beginning and end of the study cycles. The study was approved by the Clinical Research Subpanel of the National Institute of Child Health and Human Development. Informed written consent was obtained from each woman.
terminations of plasma LH starting on cycle days 6 to 10. Serum P levels were measured daily throughout the entire luteal phase. An endometrial biopsy was scheduled prospectively and obtained 7 to 9 days after the LH peak. Forty-three women participated in an additional study cycle that included daily LH measurements, as in the first cycle, but no endometrial biopsy. One to five recovery cycles elapsed between the two study cycles, except in one subject who underwent a biopsy cycle immediately after a nonbiopsy cycle. Endometrial Biopsy
An endometrial biopsy was obtained from the uterine fundus using the Pipelle (Prodimed, Neuillyen-TheIle, France). This suction curette provides specimens of similar quality with less discomfort than the more traditional instruments, as demonstrated in a previous study of late luteal phase biopsies (9). At the end of the study, all biopsies were reviewed independently by two pathologists (M.M. and C.A.) who had no previous knowledge of the specific day of the cycle when the specimen was obtained. Histologic dating was done according to the criteria of Noyes et al. (5), using a 2-day reading, e.g., secretory day 22 to 23 (10). Chronological dating was assigned both prospectively, by counting forward from the day of the LH surge (taken as cycle day 14) until the day of the biopsy, and retrospectively, by counting backward from the onset of next menses (taken as cycle day 28) (6,7). Endometrial maturation was assessed by correlating the most advanced histologic date with the chronological date of the biopsy, in keeping with Noyes' recommendation that dating should rely primarily on the morphology of the most advanced portion or feature of the endometrium (5). Hormone Assays
Plasma LH was determined by a rapid commercial kit (Diagnostic Products Corporation, Los Angeles, CA). Serum P was measured by radioimmunoassay after extraction of the samples (11). Recovery was 50% to 110%, and sensitivity was 0.4 to 0.8 nmol/L. The intraassay and interassay coefficients of variation were below 12% and 17%, respectively. All P samples for a given cycle were included in the same assay. Analysis
Protocol
All 50 women underwent one study cycle. The midcycle LH surge was identified by using rapid deVol. 59, No.2, February 1993
The follicular phase was defined as the days elapsed between the onset of menses and the day of the LH peak. The luteal phase was defined as the Batista et al.
Endometrial histology versus P
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days after the LH peak until the day before next menses. The integrated P secretion was defined as the sum of daily serum measurements obtained during the luteal phase. Results are expressed as means ± SE. Simple linear regression was used to correlate variables. Because the endometrial biopsy may shorten but not lengthen the luteal phase (5), biopsy and nonbiopsy cycles were compared by one-tailed paired t-tests. Because P is necessary for the differentiation of the endometrium (4), delayed endometrial maturation should be associated with reduced but not with increased P secretion. Thus, one-tailed t-tests for unpaired variables were used to compare data between women with normal and delayed biopsies. Differences were considered significant at P < 0.05.
RESULTS Cycle Length
The duration of the biopsy cycle was 26.8 ± 0.4 days (range, 19 to 35), the follicular phase was 14.9 ± 0.4 days (range, 9 to 24), and the luteal phase was 11.9 ± 0.2 days (range, 8 to 15). In 43 women who underwent two study cycles, a shorter luteal phase was seen in the biopsy cycle (12.0 ± 0.2 days) compared with the nonbiopsy cycle (13.1 ± 0.3 days, P = 0.004). 296
Batista et al. Endometrial histology versus P
Endometrial Dating
Histologic dates assigned by both pathologists were identical in 76%, within 1 day in 92%, and within 2 days of each other in 96% of biopsies. Because of the relatively high percentage of agreement between the two pathologists, all data analysis presented below were carried out using only one set of histologic dates, as assigned by one pathologist. The results did not change significantly when the other pathologist's reading was used instead. Retrospective compared with prospective dating resulted in earlier chronological dates that deviated more from the corresponding histologic dates (Fig. 1). Progesterone Versus Endometrial Maturation
The correlation between integrated P measurements and endometrial maturation was poor (Fig. 2), regardless of whether chronological dating was assigned prospectively (r = 0.167) or retrospectively (r = 0.292). Likewise, a poor correlation was found when integrated values were calculated up to the day of the biopsy instead of throughout the entire luteal phase (data not shown). To define the magnitude of the delay in endometrial maturation that might be associated with deficient luteal function, women were divided in groups of normal and delayed endometrial development according to different criteria, and their mean integrated P secretion was compared. These criteria involved the use of proFertility and Sterility
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gressively larger lags between histologic and chronological dating to classify a biopsy as delayed. A significant reduction in mean integrated P values was first seen when endometrial histology lagged 3 or more days behind the chronological dating of the biopsy, as assigned prospectively from the LH surge (Fig. 3A). When retrospective dating was used, however, a significant difference was only observed when biopsies had a lag of 4 or more days (Fig. 3B). This latter group comprised 86% of biopsies included in the former group. Based on these findings, a "delayed" biopsy, suggestive of CL insufficiency, was defined as the one in which endometrial histology lagged at least 3 or 4 days behind the prospective or retrospective chronological dating, respectively. Using these criteria, the incidence of delayed biopsies was the same (28%) regardless of the method of chronological dating. All delayed biopsies were obtained on days 7 or 8 after the LH surge. Three or five women 40 to 47 years of age had delayed endometrial maturation, depending on whether prospective or retrospective chronological dating was used, respectively. Although mean integrated P measurements were reduced in cycles with delayed biopsies, only two individual values fell below the range seen in women with normal endometrial maturation (Fig. 3A and B right). Mean daily P levels were similar in these two groups on the first 4 to 5 days after the LH surge (Fig. 4). Concentrations started to decrease in the Vol. 59, No.2, February 1993
midluteal phase and tended to be lower throughout the remainder of the luteal phase in women with delayed biopsies. Although significant differences were seen between the two groups on several days of the luteal phase, neither these values, alone or in combination, nor peak luteal P levels accurately predicted a delayed biopsy in any given woman (data not shown). DISCUSSION
The endometrial biopsy has never been carefully validated as a diagnostic test of luteal function (3). Previous studies failed to show a good correlation between serum P measured on the day of the biopsy and the degree of endometrial maturation (8, 1214). Furthermore, mean daily P concentrations overlapped in women with normal and delayed endometrial development, although they were in generallower in the latter subjects (15-17). These findings may be because of the pulsatile secretion pattern of P leading to wide variation in serum concentrations of this hormone (18). To reduce this problem in the present study, we correlated endometrial maturation with the integrated P secretion, as determined by daily serum hormone measurements throughout the luteal phase. This parameter reflects the total P output by the CL and is believed by many to be the gold standard for the evaluation of luteal function (3). Other measures such as urinary free P Batista et al.
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or pregnanediol excretion may also reflect endogenous P production but have so far not been completely validated (3). Traditionally, endometrial maturation is considered delayed and suggestive of deficient luteal function when the histologic reading lags at least 2 or 3 days behind the chronological dating of the biopsy (3). These criteria were arbitrarily defined based solely on Noyes' demonstration that histologic and chronological dating agree within 2 days of each other in approximately 80% of cycles (19). However, they were never shown to precisely discriminate between normal and deficient luteal function. Our data indicated that only biopsies exhibiting more severe delays in endometrial maturation were associated with low P secretion, i.e., those with a lag ~ 3 days or ~4 days between histologic and chronological dating, depending on whether chronological dates were assigned prospectively from the day of the LH surge or retrospectively from the onset of next menses' respectively. In these cycles, mean daily serum P concentrations were reduced in the midluteal and late luteal phase, suggesting that the CL started to fail at the time of its maximal activity and continued to do so throughout the remainder of the luteal phase. A high frequency (28%) of delayed biopsies was found in our normal population, a figure similar to the one (19%) reported by Israel in fertile women 298
Batista et al.
Endometrial histology versus P
(see Andrews [2]). Although several women 40 to 47 years of age were enrolled in our protocol, they did not exhibit a higher incidence of delayed endometrial maturation than the younger group and thus cannot account for the increased number of abnormal biopsies seen in our study. It is possible that the timing ofthe biopsy, which was performed in the midluteal phase, influenced our results because specimens obtained >5 days before the onset of next menses may be more frequently associated with delayed endometrial development (4). In this study, the definition of a delayed biopsy was based on a group of women with low P secretion. It is important to note that, although mean integrated values were decreased in this group, individual measurements overlapped significantly in women with normal and delayed endometrial maturation and were definitely reduced in only 2 of 14 cycles with delayed biopsies, regardless of the method of chronological dating. A similar overlap was seen between these two groups when peak luteal P levels were compared. These results suggest that the endometrial biopsy can detect a group of women with low P secretion but does not precisely distinguish luteal insufficiency in a given cycle. Use of the biopsy appears to result in a higher rate of diagnosis of CL insufficiency, as evidenced by several women with delayed endometrial maturation and apparently normal P secretion. This may explain why a signifFertility and Sterility
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Day after LH Surge Figure 4 Mean (±SE) P levels plotted relative to the day of the LH surge (day 0) in 50 regularly cycling women with normal (0) or delayed (e) endometrial maturation. An endometrial biopsy was considered delayed when the histologic dating lagged :2:3 days (A) or :2:4 days (B) behind the chronological dating, as assigned prospectively from the day of the LH surge or retrospectively from the onset of next menses, respectively. Asterisks indicate significant differences (P < 0.05) between groups.
icant percentage of infertile women with recurrent luteal phase defects, as diagnosed by abnormal biopsies, do not respond to P replacement therapy (20). It is also possible that in these patients, as well as in some of our volunteers, the endometrium was refractory to P stimulation or perhaps an additional maturational factor besides P was missing. Alternatively, the low predictive value of a delayed biopsy may be related to inaccuracies in the standard histologic criteria of Noyes et al. (5). Morphometric analysis of the endometrium may improve the accuracy of this method and provide a more quantitative test of luteal function (17, 21). Following Noyes' recommendation (22), all biopsies were performed in the midluteal phase, i.e., 7 to 9 days after the LH surge, rather than in the late luteal phase, as advocated by other investigators (2, 4). The specific timing of biopsies performed in our study may be responsible for the higher proportion of agreement (76% to 96%) observed between our two pathologists compared with others (6, 19). Recent studies have shown that histologic dating of the endometrium using morphometric analysis (23) or Noyes' criteria (24) is more precise in the first half of the luteal phase, i.e., up to post-LH surge day 7 to 8. During this part of the cycle, endometrial changes occur with a high degree of regularity and precision, exhibiting less variance than later in the luteal phase (23). Furthermore, histologic dates assigned to midluteal phase biopsies agree more closely with chronological dates than those obtained in the Vol. 59, No.2, February 1993
late luteal phase (19). It is possible, however, that a late luteal phase biopsy would reflect more precisely the entire steroidogenic activity of the CL and thus provide a clearer separation between normal and deficient luteal function than the one seen in our study. Chronological dating of an endometrial biopsy is conventionally assigned retrospectively from the onset of next menses (1). However, this method has recently been challenged by several investigators who believe that prospective dating from the day of the LH surge or day of ovulation is more precise (6, 7). This latter view was confirmed in our study because histologic and chronological dates agreed more closely when prospective dating was used. Differences observed between the two methods of chronological dating may be explained by our observation that the luteal phase was shortened by approximately 1 day in biopsy cycles. This was reported by Noyes et al. (5) in his original paper but not confirmed in a later study (19). It may be related to luteolytic factors, i.e., prostaglandins, released during the biopsy, or to partial disruption of the endometrium caused by the procedure (5). This effect of the biopsy, together with the intrinsic variation in the length of the luteal phase observed in the current and previous studies (25); may introduce inaccuracies when endometrial maturation is assessed by retrospective dating, because this method assumes a fixed 14-day luteal span (6). In summary, our results show that histologic dating of the endometrium according to Noyes' criteria Batista et al.
Endometrial histology versus P
299
can detect a group of women with low P secretion but does not precisely distinguish luteal insufficiency in a given cycle. Chronological dating should be assigned prospectively rather than retrospectively to avoid inaccuracies caused by the variable length of the luteal phase and to the accelerated onset of menses induced by the procedure. A deficient CL should be suspected only when endometrial histology lags at least 3 days behind the prospective chronological dating of the biopsy. However, the true incidence of luteal insufficiency, as defined by integrated P measurements, is probably much less, accounting for only 13% of delayed biopsies in our series.
10.
11.
12.
13. 14.
Acknowledgments. We are indebted to the nursing staff of the Ninth Floor Clinic of the Clinical Center of the National Institutes of Health for supporting this study. We also thank Ms. Barbara Filmore, Mr. Geoffrey Jackson, and Mrs. Helena Fundament for technical assistance.
15.
REFERENCES
17.
1. Jones GS. The luteal phase defect. Fertil Steril 1976;27: 351-6. 2. Andrews WC. Luteal phase defects. Fertil Steril 1979;32: 501-9. 3. McNeely MJ, Soules MR. The diagnosis of luteal phase deficiency: a critical review. Fertil SteriI1988;50:1-15. 4. Brodie BL, Wentz AC. An update on the clinical relevance of luteal phase inadequacy. Semin Reprod Endocrinol 1989;7: 138-54. 5. Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Fertil Steril 1950;1:1-25. 6. Li T-C, Rogers AW, Lenton EA, Dockery P, Cooke I. Acomparison between two methods of chronological dating of human endometrial biopsies during the luteal phase, and their correlation with histologic dating. Fertil Steril 1987;48:92832. 7. Shoupe D, Mishell DR Jr, Lacarra M, Lobo RA, Horenstein J, d' Ablaing G, et al. Correlation of endometrial maturation with four methods of estimating day of ovulation. Obstet Gynecol 1989;73:88-92. 8. Cooke ID, Morgan CA, Parry TE. Correlation of endometrial biopsy and plasma progesterone levels in infertile women. J Obstet Gynaecol Br Comm 1972;79:647-50. 9. Hill GA, Herbert III CM, Parker RA, Wentz AC. Comparison of late luteal phase endometrial biopsies using the Novak
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curette or Pipelle endometrial suction curette. Obstet Gynecol 1989;73:443-5. Scott RT, Snyder RR, Strickland DM, Tyburski CC, Bagnall JA, Reed KR, et al. The effect of interobserver variation in dating endometrial histology on the diagnosis of luteal phase defects. Fertil Steril 1988;50:888-92. Abraham GE, Swerdloff R, Tulchinsky D, Odell WD. Radioimmunoassay of plasma progesterone. J Clin Endocrinol Metab 1971;32:619-24. Shepard MK, Senturia YD. Comparison of serum progesterone and endometrial biopsy for confirmation of ovulation and evaluation of luteal function. Fertil Steril 1977;28: 54l-8. Rosenfeld DL, Chudow S, Bronson RA. Diagnosis of luteal phase inadequacy. Obstet Gynecol 1980;56:193-6. 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 1985;43:715-9. Jones GS, Aksel S, Wentz AC. Serum progesterone values in the luteal phase defects. Effect of chorionic gonadotropin. Obstet GynecoI1974;44:26-34. Soules MR, McLachlan RI, Ek M, Dahl KD, Cohen NL, Bremner WJ. Luteal phase deficiency: characterization of reproductive hormones over the menstrual cycle. J Clin Endocrinol Metab 1989;69:804-12. Li T-C, Lenton EA, Dockery P, Rogers AW, Cooke ID. The relation between daily salivary progesterone profile and endometrial development in the luteal phase of fertile and infertile women. Br J Obstet Gynaecol 1989;96:445-53. Filicori M, Butler JP, Crowley WF Jr. Neuroendocrine regulation of the corpus luteum in the human. Evidence for pulsatile progesterone secretion. J Clin Invest 1984;73:1638-47. Noyes RW, Haman JO. Accuracy of endometrial dating: correlation of endometrial dating with basal body temperature and menses. Fertil Steril 1953;4:504-17. Balash J, Vanrell JA. Corpus luteum insufficiency and fertility: a matter of controversy. Hum Reprod 1987;2:557-67. Li T-C, Rogers AW, Dockery P, Lenton EA, Cooke ID. A new method of histologic dating of human endometrium in the luteal phase. Fertil Steril 1988;50:52-60. Noyes RW. The underdeveloped secretory endometrium. Am J Obstet Gynecol 1959;77:929-45. Johannisson E, Landgren B-M, Rohr HP, Diczfalusy E. Endometrial morphology and peripheral hormone levels in women with regular menstrual cycles. Fertil Steril 1987;48: 401-8. Li T-C, Dockery P, Rogers AW, Cooke ID. How precise is histologic dating of endometrium using the standard dating criteria? Fertil Steril 1989;51:759-63. Lenton EA, Landgren BM. Normal variation in the length of the luteal phase of the menstrual cycle: identification of the short luteal phase. Br J Obstet GynaecoI1984;91:685-9.
Fertility and Sterility
Vol. 59, No.2, February 1993
Printed on acid-free paper in U.S.A.
Copyright © 1993 The American Fertility Society
Midluteal phase endometrial biopsy does not accurately predict luteal function*
Marcelo C_ Batista, M_D.t:l: Tannia P.Cartledge, R.N-§ Maria J _ Merino II Constantine Axiotis, M_D.II
Maria P. Platia, M.D.t George R. Merriam, M.D.t D. Lynn Loriaux, M.D., Ph.D.t Lynnette K. Nieman, M.D. tll
National Institute of Child Health and Human Development, Warren Grant Magnuson Clinical Center, and National Cancer Institute, National Institutes of Health, Bethesda, Maryland
Objective: To investigate whether a midluteal phase endometrial biopsy accurately predicts luteal function. Design: One nonpregnant menstrual cycle was evaluated in a prospective fashion. Setting: Outpatient Clinic of the Clinical Center of the National Institutes of Health. Participants: Fifty healthy, normally cycling women. Interventions: Serum progesterone (P) was measured daily throughout the luteal phase. An endometrial biopsy was performed 7 to 9 days after the luteinizing hormone (LH) surge, as detected by rapid plasma assays, and dated histologically according to Noyes' criteria. Main Outcome Measure: To correlate endometrial maturation with luteal P secretion. Results: Mean integrated P measurements were reduced only when the lag between histologic and chronological dating was ;?:3 days or ;?:4 days, depending on whether chronological dates were assigned prospectively from the LH surge or retrospectively from the onset of next menses, respectively. However, these lags did not consistently predict deficient luteal function because subnormal integrated P secretion was seen in only 14% of women with these delays in endometrial maturation. Conclusions: Midluteal phase endometrial biopsy provides a crude test of luteal function that does not precisely distinguish luteal insufficiency. Fertil Steril 1993;59:294-300 Key Words: Progesterone, endometrium, endometrial biopsy, luteal phase, luteal insufficiency, luteal phase defect
Corpus luteum (CL) insufficiency is characterized by deficient progesterone (P) secretion resulting in
Received January 6, 1992; revised and accepted October 21, 1992. * Supported in part by grant 204688/88.4, Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Sao Paulo, Brazil. t Developmental Endocrinology Branch, National Institute of Child Health and Human Development. :j: ·Present address: Department of Endocrinology, Hospital das Clinicas, University of Sao Paulo, Sao Paulo, Brazil. § Nursing Department, Warren Grant Magnuson Clinical Center. II Laboratory of Pathology, Division of Cancer Biology and Diagnostics, National Cancer Institute. ~ Reprint requests: Lynnette K. Nieman, M.D_, National Institutes of Health, Building 10, Room 10N262, Bethesda, Maryland 20892. 294
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inadequate development ofthe endometrium to sustain implantation of an embryo (1, 2). Although usually sporadic, it may occur as a recurrent event in women with infertility and habitual abortion (2). Luteal insufficiency is most commonly diagnosed by performing an endometrial biopsy in the latter part of the luteal phase (1-4). The development of the endometrium should, in theory, reflect the end-organ response to P and, as such, provide a bioassay that is independent of fluctuations in serum hormone levels (3,4). Endometrial maturation is traditionally assessed by correlating the histologic dating of the biopsy, as defined by the criteria of Noyes et al. (5), with the chronological dating, representing the day of the cycle when the biopsy is obtained. Although the latter date is usually assigned retrospectively by counting backward from the onset of the next Fertility and Sterility
menses (1), several investigators believe that this should be done prospectively by counting forward from the day of the luteinizing hormone (LH) surge or day of ovulation (6, 7). Several inconsistencies exist in the definition of CL insufficiency, mainly because the endometrial biopsy has not been completely validated as a test ofluteal function (3). Different delays in endometrial maturation, such as a lag of 2, 3, 4, or more days between histologic and chronological dating, have been considered indicative of luteal insufficiency by various authors (3,8). However, the diagnostic accuracy of these criteria, as defined by comparison with the gold standard test of luteal function, the integrated P secretion, has never been carefully evaluated (3). To address this issue, we correlated endometrial maturation, as assessed by a midluteal phase biopsy, with the integrated P secretion, as determined by daily luteal hormone measurements, in a single cycle in 50 healthy women. Both the prospective and retrospective methods of chronological dating were used to evaluate the development of the endometrium and to define the accuracy of the different criteria used to diagnose luteal insufficiency. MATERIALS AND METHODS Subjects
Fifty healthy nonpregnant women (mean age, 34.4 ± 1.0 years; range, 22 to 47) participated in this study. All had regular menstrual cycles and were within 30% of ideal body weight (mean, 65.7 ± 1.9 kg; range, 44.6 to 95). Sixteen were between 40 and 47 years of age. Thirty-three had spontaneous pregnancies in the past, whereas the remainder had never attempted conception. No woman had taken hormonal contraceptives or used intrauterine devices during the previous 3 months. All subjects used barrier methods or abstained from sexual intercourse during the study. Pregnancy was excluded by a serum beta human chorionic gonadotropin concentration.:os; 2.0 ng/mL (the threshold for detection of pregnancy in this assay) at the beginning and end of the study cycles. The study was approved by the Clinical Research Subpanel of the National Institute of Child Health and Human Development. Informed written consent was obtained from each woman.
terminations of plasma LH starting on cycle days 6 to 10. Serum P levels were measured daily throughout the entire luteal phase. An endometrial biopsy was scheduled prospectively and obtained 7 to 9 days after the LH peak. Forty-three women participated in an additional study cycle that included daily LH measurements, as in the first cycle, but no endometrial biopsy. One to five recovery cycles elapsed between the two study cycles, except in one subject who underwent a biopsy cycle immediately after a nonbiopsy cycle. Endometrial Biopsy
An endometrial biopsy was obtained from the uterine fundus using the Pipelle (Prodimed, Neuillyen-TheIle, France). This suction curette provides specimens of similar quality with less discomfort than the more traditional instruments, as demonstrated in a previous study of late luteal phase biopsies (9). At the end of the study, all biopsies were reviewed independently by two pathologists (M.M. and C.A.) who had no previous knowledge of the specific day of the cycle when the specimen was obtained. Histologic dating was done according to the criteria of Noyes et al. (5), using a 2-day reading, e.g., secretory day 22 to 23 (10). Chronological dating was assigned both prospectively, by counting forward from the day of the LH surge (taken as cycle day 14) until the day of the biopsy, and retrospectively, by counting backward from the onset of next menses (taken as cycle day 28) (6,7). Endometrial maturation was assessed by correlating the most advanced histologic date with the chronological date of the biopsy, in keeping with Noyes' recommendation that dating should rely primarily on the morphology of the most advanced portion or feature of the endometrium (5). Hormone Assays
Plasma LH was determined by a rapid commercial kit (Diagnostic Products Corporation, Los Angeles, CA). Serum P was measured by radioimmunoassay after extraction of the samples (11). Recovery was 50% to 110%, and sensitivity was 0.4 to 0.8 nmol/L. The intraassay and interassay coefficients of variation were below 12% and 17%, respectively. All P samples for a given cycle were included in the same assay. Analysis
Protocol
All 50 women underwent one study cycle. The midcycle LH surge was identified by using rapid deVol. 59, No.2, February 1993
The follicular phase was defined as the days elapsed between the onset of menses and the day of the LH peak. The luteal phase was defined as the Batista et al.
Endometrial histology versus P
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days after the LH peak until the day before next menses. The integrated P secretion was defined as the sum of daily serum measurements obtained during the luteal phase. Results are expressed as means ± SE. Simple linear regression was used to correlate variables. Because the endometrial biopsy may shorten but not lengthen the luteal phase (5), biopsy and nonbiopsy cycles were compared by one-tailed paired t-tests. Because P is necessary for the differentiation of the endometrium (4), delayed endometrial maturation should be associated with reduced but not with increased P secretion. Thus, one-tailed t-tests for unpaired variables were used to compare data between women with normal and delayed biopsies. Differences were considered significant at P < 0.05.
RESULTS Cycle Length
The duration of the biopsy cycle was 26.8 ± 0.4 days (range, 19 to 35), the follicular phase was 14.9 ± 0.4 days (range, 9 to 24), and the luteal phase was 11.9 ± 0.2 days (range, 8 to 15). In 43 women who underwent two study cycles, a shorter luteal phase was seen in the biopsy cycle (12.0 ± 0.2 days) compared with the nonbiopsy cycle (13.1 ± 0.3 days, P = 0.004). 296
Batista et al. Endometrial histology versus P
Endometrial Dating
Histologic dates assigned by both pathologists were identical in 76%, within 1 day in 92%, and within 2 days of each other in 96% of biopsies. Because of the relatively high percentage of agreement between the two pathologists, all data analysis presented below were carried out using only one set of histologic dates, as assigned by one pathologist. The results did not change significantly when the other pathologist's reading was used instead. Retrospective compared with prospective dating resulted in earlier chronological dates that deviated more from the corresponding histologic dates (Fig. 1). Progesterone Versus Endometrial Maturation
The correlation between integrated P measurements and endometrial maturation was poor (Fig. 2), regardless of whether chronological dating was assigned prospectively (r = 0.167) or retrospectively (r = 0.292). Likewise, a poor correlation was found when integrated values were calculated up to the day of the biopsy instead of throughout the entire luteal phase (data not shown). To define the magnitude of the delay in endometrial maturation that might be associated with deficient luteal function, women were divided in groups of normal and delayed endometrial development according to different criteria, and their mean integrated P secretion was compared. These criteria involved the use of proFertility and Sterility
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gressively larger lags between histologic and chronological dating to classify a biopsy as delayed. A significant reduction in mean integrated P values was first seen when endometrial histology lagged 3 or more days behind the chronological dating of the biopsy, as assigned prospectively from the LH surge (Fig. 3A). When retrospective dating was used, however, a significant difference was only observed when biopsies had a lag of 4 or more days (Fig. 3B). This latter group comprised 86% of biopsies included in the former group. Based on these findings, a "delayed" biopsy, suggestive of CL insufficiency, was defined as the one in which endometrial histology lagged at least 3 or 4 days behind the prospective or retrospective chronological dating, respectively. Using these criteria, the incidence of delayed biopsies was the same (28%) regardless of the method of chronological dating. All delayed biopsies were obtained on days 7 or 8 after the LH surge. Three or five women 40 to 47 years of age had delayed endometrial maturation, depending on whether prospective or retrospective chronological dating was used, respectively. Although mean integrated P measurements were reduced in cycles with delayed biopsies, only two individual values fell below the range seen in women with normal endometrial maturation (Fig. 3A and B right). Mean daily P levels were similar in these two groups on the first 4 to 5 days after the LH surge (Fig. 4). Concentrations started to decrease in the Vol. 59, No.2, February 1993
midluteal phase and tended to be lower throughout the remainder of the luteal phase in women with delayed biopsies. Although significant differences were seen between the two groups on several days of the luteal phase, neither these values, alone or in combination, nor peak luteal P levels accurately predicted a delayed biopsy in any given woman (data not shown). DISCUSSION
The endometrial biopsy has never been carefully validated as a diagnostic test of luteal function (3). Previous studies failed to show a good correlation between serum P measured on the day of the biopsy and the degree of endometrial maturation (8, 1214). Furthermore, mean daily P concentrations overlapped in women with normal and delayed endometrial development, although they were in generallower in the latter subjects (15-17). These findings may be because of the pulsatile secretion pattern of P leading to wide variation in serum concentrations of this hormone (18). To reduce this problem in the present study, we correlated endometrial maturation with the integrated P secretion, as determined by daily serum hormone measurements throughout the luteal phase. This parameter reflects the total P output by the CL and is believed by many to be the gold standard for the evaluation of luteal function (3). Other measures such as urinary free P Batista et al.
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or pregnanediol excretion may also reflect endogenous P production but have so far not been completely validated (3). Traditionally, endometrial maturation is considered delayed and suggestive of deficient luteal function when the histologic reading lags at least 2 or 3 days behind the chronological dating of the biopsy (3). These criteria were arbitrarily defined based solely on Noyes' demonstration that histologic and chronological dating agree within 2 days of each other in approximately 80% of cycles (19). However, they were never shown to precisely discriminate between normal and deficient luteal function. Our data indicated that only biopsies exhibiting more severe delays in endometrial maturation were associated with low P secretion, i.e., those with a lag ~ 3 days or ~4 days between histologic and chronological dating, depending on whether chronological dates were assigned prospectively from the day of the LH surge or retrospectively from the onset of next menses' respectively. In these cycles, mean daily serum P concentrations were reduced in the midluteal and late luteal phase, suggesting that the CL started to fail at the time of its maximal activity and continued to do so throughout the remainder of the luteal phase. A high frequency (28%) of delayed biopsies was found in our normal population, a figure similar to the one (19%) reported by Israel in fertile women 298
Batista et al.
Endometrial histology versus P
(see Andrews [2]). Although several women 40 to 47 years of age were enrolled in our protocol, they did not exhibit a higher incidence of delayed endometrial maturation than the younger group and thus cannot account for the increased number of abnormal biopsies seen in our study. It is possible that the timing ofthe biopsy, which was performed in the midluteal phase, influenced our results because specimens obtained >5 days before the onset of next menses may be more frequently associated with delayed endometrial development (4). In this study, the definition of a delayed biopsy was based on a group of women with low P secretion. It is important to note that, although mean integrated values were decreased in this group, individual measurements overlapped significantly in women with normal and delayed endometrial maturation and were definitely reduced in only 2 of 14 cycles with delayed biopsies, regardless of the method of chronological dating. A similar overlap was seen between these two groups when peak luteal P levels were compared. These results suggest that the endometrial biopsy can detect a group of women with low P secretion but does not precisely distinguish luteal insufficiency in a given cycle. Use of the biopsy appears to result in a higher rate of diagnosis of CL insufficiency, as evidenced by several women with delayed endometrial maturation and apparently normal P secretion. This may explain why a signifFertility and Sterility
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icant percentage of infertile women with recurrent luteal phase defects, as diagnosed by abnormal biopsies, do not respond to P replacement therapy (20). It is also possible that in these patients, as well as in some of our volunteers, the endometrium was refractory to P stimulation or perhaps an additional maturational factor besides P was missing. Alternatively, the low predictive value of a delayed biopsy may be related to inaccuracies in the standard histologic criteria of Noyes et al. (5). Morphometric analysis of the endometrium may improve the accuracy of this method and provide a more quantitative test of luteal function (17, 21). Following Noyes' recommendation (22), all biopsies were performed in the midluteal phase, i.e., 7 to 9 days after the LH surge, rather than in the late luteal phase, as advocated by other investigators (2, 4). The specific timing of biopsies performed in our study may be responsible for the higher proportion of agreement (76% to 96%) observed between our two pathologists compared with others (6, 19). Recent studies have shown that histologic dating of the endometrium using morphometric analysis (23) or Noyes' criteria (24) is more precise in the first half of the luteal phase, i.e., up to post-LH surge day 7 to 8. During this part of the cycle, endometrial changes occur with a high degree of regularity and precision, exhibiting less variance than later in the luteal phase (23). Furthermore, histologic dates assigned to midluteal phase biopsies agree more closely with chronological dates than those obtained in the Vol. 59, No.2, February 1993
late luteal phase (19). It is possible, however, that a late luteal phase biopsy would reflect more precisely the entire steroidogenic activity of the CL and thus provide a clearer separation between normal and deficient luteal function than the one seen in our study. Chronological dating of an endometrial biopsy is conventionally assigned retrospectively from the onset of next menses (1). However, this method has recently been challenged by several investigators who believe that prospective dating from the day of the LH surge or day of ovulation is more precise (6, 7). This latter view was confirmed in our study because histologic and chronological dates agreed more closely when prospective dating was used. Differences observed between the two methods of chronological dating may be explained by our observation that the luteal phase was shortened by approximately 1 day in biopsy cycles. This was reported by Noyes et al. (5) in his original paper but not confirmed in a later study (19). It may be related to luteolytic factors, i.e., prostaglandins, released during the biopsy, or to partial disruption of the endometrium caused by the procedure (5). This effect of the biopsy, together with the intrinsic variation in the length of the luteal phase observed in the current and previous studies (25); may introduce inaccuracies when endometrial maturation is assessed by retrospective dating, because this method assumes a fixed 14-day luteal span (6). In summary, our results show that histologic dating of the endometrium according to Noyes' criteria Batista et al.
Endometrial histology versus P
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can detect a group of women with low P secretion but does not precisely distinguish luteal insufficiency in a given cycle. Chronological dating should be assigned prospectively rather than retrospectively to avoid inaccuracies caused by the variable length of the luteal phase and to the accelerated onset of menses induced by the procedure. A deficient CL should be suspected only when endometrial histology lags at least 3 days behind the prospective chronological dating of the biopsy. However, the true incidence of luteal insufficiency, as defined by integrated P measurements, is probably much less, accounting for only 13% of delayed biopsies in our series.
10.
11.
12.
13. 14.
Acknowledgments. We are indebted to the nursing staff of the Ninth Floor Clinic of the Clinical Center of the National Institutes of Health for supporting this study. We also thank Ms. Barbara Filmore, Mr. Geoffrey Jackson, and Mrs. Helena Fundament for technical assistance.
15.
REFERENCES
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Fertility and Sterility