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Timing of the endometrial biopsy may be critical for the accurate diagnosis of luteal phase deficiency*†
FERTILITY AND STERILITY
Vol. 61, No.3, March 1994
Printed on acid-free paper in U. S. A.
Copyright" 1994 The American Fertility Society
Timing of the endometrial biopsy may be critical for the accurate diagnosis of luteal phase deficiency*t
Arthur J. Castelbaum, M.D.:!: James Wheeler, M.D.§ Christos B. Coutifaris, M.D., Ph.D.:!:
Luigi Mastroianni, Jr., M.D.:!: Bruce A. Lessey, M.D., Ph.D.:!: II
University of Pennsylvania Medical Center, Philadelphia, Pennsylvania
Objective: To determine the optimal time to perform the endometrial biopsy for the detection of "out-of-phase" endometrium. Design: Two endometrial biopsies were performed during a single menstrual cycle in each subject. The patient's chronological day was determined by counting forward from the midcycle LH surge, as assessed by urinary LH detection. The "early" biopsy was done on day LH + 7.4 ± 0.8, and the "late" biopsy on day LH + 11.6 ± 0.7. Each biopsy was independently read by two pathologists and was considered out of phase if the histologic date was ~3 days delayed compared with the chronological date . Setting: Infertility practice of an academic teaching hospital. Patients: Thirty-three ovulatory women seeking evaluation for infertility. Main Outcome Measure: Number of patients with out-of-phase endometrium detected by the early versus the late biopsy. Results: There was a significantly greater detection rate for out-of-phase endometrium using the early biopsy (12.1 % to 18.2% incidence depending on the observer) compared with the later biopsy (6.1 % to 9.1 % incidence). A majority of the early out-of-phase biopsies corrected by the time of the later biopsy. Conclusion: Our findings indicate that an endometrial biopsy performed in the midluteal phase may detect a greater number of women with delayed endometrial maturation during the temporal window of embryo implantation. The observation that most ofthe women with out-of-phase midluteal biopsies had normal late luteal endometrium may represent a cryptic form of luteal phase deficiency. Fertil Steril1994;61:443-7 Key Words: Endometrial biopsy, timing, diagnosis, luteal phase deficiency, endometrium
In the 45 years since Dr. Georgeanna Jones first proposed the existence of a luteal phase defect (LPD) (1), controversy persists about its existence, diagnosis, pathophysiology, and treatment. Evalua-
Received July 12, 1993; revised and accepted
* Supported by the National Institutes of Health grant HD30476-l(BAL), Philadelphia, Pennsylvania. t Presented at the 49th Annual Meeting of The American Fertility Society, Montreal, Canada, October 9 to 14, 1993. :j: Department of Obstetrics and Gynecology. § Department of Pathology. II Reprint requests and present address: Bruce A. Lessey, M.D., Ph.D, University of North Carolina, Department of Obstetrics and Gynecology, MacNider Building, CB 7570, Chapel Hill, North Carolina, 27599-7570. Vol. 61, No.3, March 1994
tion of the luteal phase by endometrial histology remains the gold standard for the diagnosis of this disorder. Endometrial histology is thought to best reflect the hormonal milieu of the luteal phase, permissive for embryo attachment and implantation. Although P is the principal steroidogenic modulator of endometrial maturation, measurement of plasma P has not proven to be a good predictor of LPD because of the overlap of LPD patients with normal women (2). Integrated daily plasma P levels (3) correlate poorly with midluteal endometrial histology (4). The ideal time to perform the endometrial biopsy during the luteal phase is unsettled. It has been suggested that the biopsy be done a few days before Castelbaum et al. Timing of endometrial biopsy
443
the start of the next menstrual period as a maximal and cumulative bioassay of estrogen and Paction (5). Although several studies have noted that early and midluteal phase biopsies are poorer predictors of the next menstrual period when compared with a late luteal biopsy due to a wider variation in the histologic dating (5-9), dating referenced to the urinary LH surge have demonstrated no increased interobserver (10, 11) or intraobserver (12) variation between an early and late luteal biopsy for evaluation ofthe luteal phase. In fact, Li et al. (12) noted greater precision using endometrial biopsies obtained before day 22 of the menstrual cycle compared with those obtained after day 22. In addition, morphometric analysis has demonstrated higher error rates in reading biopsies as they moved away from the LH surge (13). It is now clear that the window of uterine receptivity to embryo implantation spans postovulatory days 6 through 10 (14, 15) and that it is day 7 human embryos that implant (15). These observations suggest that even though the window of uterine receptivity is wide, human embryos are capable of implanting within a much narrower time frame in their development. Therefore, it can be hypothesized that an out-of-phase endometrium between postovulatory days 6 and 10 may contribute to early embryonic loss, even if the late luteal endometrial histology is in phase. To address whether the timing of the endometrial biopsy is critical in detecting out-of-phase endometrium, we prospectively biopsied 33 ovulatory women twice in the same luteal phase.
MATERIALS AND METHODS Patients
Thirty-three women (age, 33.9 ± 4.5 years; range, 24 to 42 years; mean ± SD) seeking infertility evaluation at an academic teaching hospital enrolled in the study. The protocol was approved by the Institutional Review Board of the University of Pennsylvania, and each patient gave informed written consent. All subjects were ovulatory. Protocol
Each patient was evaluated during a single cycle. The day of ovulation (LH + 0) was determined by a sensitive home urine LH kit (Ovuquick; Quidel, San Diego, CA), and the cycle day was determined by counting forward from the day of ovulation. The early biopsy was performed on day LH + 7.4 ± 0.8 444
Castelbaum et al.
Timing of endometrial biopsy
(range, LH + 5 to + 9) whereas the late biopsy was performed on day LH + 11.7 ± 0.8 (range, LH + 10 to + 13). The second biopsy was performed 4.2 ± 0.8 days after the first biopsy (range, 3 to 6 days). All biopsies were done by two reproductive endocrinologists, using a suction curette (Z-sampler; Zinnanti Surgical Instruments, Chatsworth, CA). Biopsy material was obtained from the uterine fundus, and then was fixed and processed. Biopsies were read according to the criteria of Noyes et al. (16). Biopsy specimens were examined twice: first by a board-certified pathologist with knowledge of the patient's last menstrual period (LMP) routinely reading out specimens that day (pathologist 1) and, later, by a single senior pathologist with a special interest in gynecological pathology without knowledge of the LMP (pathologist 2). An out-ofphase biopsy was defined as 2':3 day lag between the chronological and histologic day. Statistical Analysis
Wilcoxon Signed Rank Test was used to compare the interobserver variation of the biopsy readings between pathologists. Chi -square analysis was performed to compare the detection rate of out-ofphase endometrium in early versus late biopsies. Significance was defined as P < 0.05.
RESULTS
Overall, out-of-phase endometrium was detected in 24.2% of cases (8 of 33) by pathologist 1 compared with 18.2% (6 of 33) for pathologist 2. There was a 12.1 % to 18.2% incidence (depending on the observer) of an out-of-phase early biopsy compared with a 6.1% to 9.1% incidence using the later biopsy. Interestingly, 80% (pathologist 1) to 100% (pathologist 2) of the early out-of-phase biopsies corrected by the time of the later biopsy. One late biopsy, deemed insufficient for diagnosis by pathologist 1, was read as "in phase" by pathologist 2. Two biopsies that were in phase in the early biopsy became out of phase on the later biopsy. Figure 1 displays a paired scattergram of the chronological and associated histologic day for each biopsy by each pathologist. Surprisingly, the majority of early out-of-phase biopsies (represented by asterisks in Fig. 1) demonstrated normal endometrial maturation at the time of the later biopsy. One case of acute endometritis was noted on the late biopsy. Figure 2 displays the interobserver variation between the two pathologists' histologic readings for early and late biopsies. Agreement within 2 days Fertility and Sterility
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Figure 1 The chronological day and associated histologic reading for each biopsy read by pathologist 1 (A) and pathologist 2 (B). The dotted line represents idealized synchrony between chronological and histologic days. A solid line connects each patient's two biopsies. Out-of-phase biopsies are identified by asterisks.
was noted in 78% ofthe early compared with 97% of the late biopsies. There was no significant interobserver variation between the early and late luteal biopsies (P = 0.42, Wilcoxon Signed Rank Test). The percentage of patients designated as out of phase based on the early biopsy versus the later biopsy is shown in Figure 3. For each pathologist, the detection rate for out-of-phase endometrium was significantly· higher in the earlier biopsy (pathologist 1, P = 0.0001, X2 = 14.85; pathologist 2, P < 0.0001, X2 = 15.43). Vol. 61, No.3, March 1994
As a better understanding of uterine receptivity emerges, the issue of when to perform the endometrial biopsy becomes ever more important. There is a distinct temporal window spanning postovulatory days 6 through 10 (cycle days 20 to 24) during which the endometrium is thought to be receptive to embryo attachment and implantation (14, 15). Many have suggested performing the endometrial biopsy closer to the next menses as a cumulative bioassay of Paction (5) whereas others have argued that the midluteal phase biopsy, corresponding to the window of implantation, is a more physiologically important time to examine uterine receptivity (17). We now present evidence supporting this latter concept. In the current study, the greater number of outof-phase endometrial samples identified by the earlier biopsy suggests that the timing of sampling may be important for the detection of LPD. By relying on a late luteal phase biopsy alone, only two or three out-of-phase cycles from 33 women seeking evaluation of infertility would have been diagnosed (6.1 % to 9.1 %). Alternatively, a biopsy performed during the putative time of implantation identified four or six out-of-phase cycles (12.1% to 18.2%). These findings are consistent with those of Cummings and colleagues (8) who noted an out-of-phase biopsy rate of 38.6% in infertile patients during cycle days 25 to 28 and a significantly higher rate of 53.6% among infertile women biopsied before cycle day 25. The number and distribution of out-of-phase biopsies noted by both pathologists were quite simi1ar. Their concordance in histologic dating (78% and 97% agreement within 2 days for the early and late biopsies, respectively) is comparable to those reported by Noyes and Haman (18). The ideal method for accurately defining chronological dating remains unsettled, although counting forward from the day of ovulation previously has been proposed (19). We used the urinary LH surge to detect ovulation and to time the biopsies. Several studies have demonstrated that this method results in a higher correlation with histologic dating in parous (20) and infertile (21) women compared with use of the next menstrual period. Clinical practice has been influenced by the belief that dating of the early and midluteal biopsy is less accurate compared with late luteal material whereas the data in the literature are contradictory. The present study demonstrates no statistically significant difference in interobserver variability Castelbaum et at.
Timing of endometrial biopsy
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between the two biopsies. A wider interobserver variation previously has been reported on biopsies done during the early or midluteal versus late luteal phase (5). Other reports (10, 11) demonstrate negligible interobserver variation throughout the luteal phase. Gibson and colleagues (10) examined 25 duplicate endometrial biopsies from women seeking infertility evaluation. Each slide was read by five different observers. The interobserver variation between endometrium examined before cycle day 22 and after day 22 was not different. Similar results were obtained by Scott et al. (11). Although Li et al. (12) demonstrated no statistical difference in intraobserver variation in endometrial dating before cycle day 22 compared with specimens from day 22 onward, the accuracy (incidence of exact agreement) was better for the earlier biopsies. The evaluation
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of morphometric analyses has also demonstrated that early and midluteal endometrial biopsies are more consistent in their parameters compared with later biopsies (22). The difference in the percentages of out-of-phase endometrium between the early and late biopsies in the current study, therefore, cannot be attributed to errors in the histologic interpretation of the biopsy specimens. Because the two biopsies in the current study were performed in the same cycle, the effect the first endometrial biopsy may have had on the second is unknown. The majority of patients demonstrated normal endometrial maturation on both biopsies (76% to 82%) and only 1 of 33 late biopsy specimens revealed endometritis. On one occasion, the second biopsy in one cycle revealed focal histologic changes consistent with a previous biopsy site. Although there were reactive changes in the stroma of the involved tissue fragment, with a mild neutrophilic infiltrate, superficial fibrin, and red blood cells, the glands showed only minor changes. Other endometrial fragments in the specimen showed no reactive changes and can be dated readily by standard criteria (16). There is no reason, therefore, to suspect that the explanation for the "catch up" demonstrated by the women with an out-of-phase early, but normal late biopsy rests with consequences of the first biopsy per se. Although the physiologic consequences of an out-of-phase early biopsy are uncertain, asynchrony between endometrial maturation and embryonic development is associated with defects of implantation (23). In humans, there appears to be a specific time in the luteal phase when the endometrium is permissive to implantation by the trophectoderm of Fertility and Sterility
the embryo. The existence of a window of implantation in humans is based on studies of Hertig et al. (14) and more recent studies using hypersensitive hCG assay of maternal serum after ET, demonstrating initial hCG rise occurs during cycle days LH + 6 to LH + 10 (15). Our findings indicate that a subgroup of women with in-phase endometrium late in the luteal phase are out of phase during the window of implantation. This may represent a cryptic form of LPD. Specifically, these women would have been missed had only the later biopsy been performed. Recently, 15 women who conceived during the cycle of their endometrial biopsy were evaluated (24). All 10 women biopsied on postovulatory day 6 to 11 had in-phase endometrium. Ironically, four of the five pregnant women biopsied on postovulatory day 12 had varying degrees of gland-stromal dyssynchrony, again supporting the utility of an earlier endometrial biopsy. Our study of dual biopsies in the same luteal phase suggests that endometrial biopsies performed for the evaluation of infertility may detect a larger number of out-of-phase cycles when performed during the early and mid compared with late luteal phase ofthe menstrual cycle. Based on the preponderance of the evidence in the literature, as well as our own observations, we propose that a biopsy be performed during the window of implantation as a more appropriate time for scrutiny of endometrial histology. Additional larger studies involving both infertile and fertile women are needed to confirm our findings and to determine the true incidence of LPD. Acknowledgment. We thank David Guzick, M.D., Ph.D., Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, for his assistance in data analysis.
REFERENCES 1. Jones GES. Some newer aspects of the management of infertility. JAMA 1949; 141:1123-8. 2. Gibson M. Clinical evaluation of luteal function. Semin Reprod EndocrinoI1990;8:130-41. 3. Wu CH, Minassian SS. The integrated luteal progesterone: an assessment ofluteal function. Fertil SteriI1987;48:93740. 4. Batista MC, Cartledge TP, Merino MJ, Axiotis C, Platia MP, Merriam GR, et al. Midluteal phase endometrial biopsy does not accurately predict luteal function. Fertil SteriI1993;59:294.-300.
Vol. 61, No.3, March 1994
5. Wentz AC, Kossoy LR, Parker RA. The impact of luteal phase inadequacy in an infertile population. Am J Obstet Gynecol 1990; 162:937-45. 6. Rosenfeld DL, Chudow S, Bronson RA. Diagnosis of luteal phase inadequacy. Obstet GynecoI1980;56:193-6. 7. Huang KE. The primary treatment of luteal phase inadequacy: progesterone versus clomiphene citrate. Am J Obstet Gynecol 1986; 155:824-8. 8. Cummings 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. 9. Wentz AC. Endometrial biopsy in the evaluation ofinfertility. Fertil Steril1980;33:121-4. 10. Gibson M, Badger GJ, Byrn F, Lee KR, Korson R, Trainer TD. Error in histologic dating of secretory endometrium: variance component analysis. Fertil Steril 1991; 56:242-7. 11. 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. 12. Li T-C, Dockery P, Rogers AW, Cooke ID. How precise is histologic dating of endometrium using the standard dating criteria? Fertil SteriI1989;51:759-63. 13. Johannisson E, Parker RA, Landgren B-M, Diczfalusy E. Morphometric analysis of the human endometrium in relation to peripheral hormone levels. Fertil Steril1982;38:56471. 14. Hertig AT, Rock J, Adams EC. A description of 34 human ova within the first 17 days of development. Am J Anat 1956; 98:435-93. 15. Bergh PA, Navot D. The impact of embryonic development and endometrial maturity on the timing of implantation. Fertil Steril1992;58:537-42. 16. Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Fertil Steril 1950; 1:3-25. 17. Li T -C, Cooke ID. Evaluation of the luteal phase. Hum Reprod 1991;6:484-99. 18. Noyes RW, Haman JO. Accuracy of endometrial dating: correlation of endometrial dating with basal body temperature and menses. Fertil SteriI1953;4:504-17. 19. Rosenfeld DL, Garcia CoR. A comparison of endometrial histology with simultaneous plasma progesterone determinations in infertile women. Fertil Steril1976;27:1256-66. 20. Shoupe D, Mishell DR, 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. 21. Li T-C, Rogers AW, Lenton EA, Dockery P, Cooke I. A comparison between two methods of chronological dating of human endometrial biopsies during the luteal phase, and their correlation with histologic dating. Fertil Steril 1987; 48:928-32. 22. 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. 23. Pope WF. Uterine asynchrony: a cause of embryonic loss. Bioi Reprod 1988;39:999-1003. 24. Andoh K, Mizunuma H, Nakazato Y, Yamada K, Michishita M, Ibuki Y. Endometrial dating in the conception cycle. Fertil SteriI1992;58:1127-30.
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Vol. 61, No.3, March 1994
Printed on acid-free paper in U. S. A.
Copyright" 1994 The American Fertility Society
Timing of the endometrial biopsy may be critical for the accurate diagnosis of luteal phase deficiency*t
Arthur J. Castelbaum, M.D.:!: James Wheeler, M.D.§ Christos B. Coutifaris, M.D., Ph.D.:!:
Luigi Mastroianni, Jr., M.D.:!: Bruce A. Lessey, M.D., Ph.D.:!: II
University of Pennsylvania Medical Center, Philadelphia, Pennsylvania
Objective: To determine the optimal time to perform the endometrial biopsy for the detection of "out-of-phase" endometrium. Design: Two endometrial biopsies were performed during a single menstrual cycle in each subject. The patient's chronological day was determined by counting forward from the midcycle LH surge, as assessed by urinary LH detection. The "early" biopsy was done on day LH + 7.4 ± 0.8, and the "late" biopsy on day LH + 11.6 ± 0.7. Each biopsy was independently read by two pathologists and was considered out of phase if the histologic date was ~3 days delayed compared with the chronological date . Setting: Infertility practice of an academic teaching hospital. Patients: Thirty-three ovulatory women seeking evaluation for infertility. Main Outcome Measure: Number of patients with out-of-phase endometrium detected by the early versus the late biopsy. Results: There was a significantly greater detection rate for out-of-phase endometrium using the early biopsy (12.1 % to 18.2% incidence depending on the observer) compared with the later biopsy (6.1 % to 9.1 % incidence). A majority of the early out-of-phase biopsies corrected by the time of the later biopsy. Conclusion: Our findings indicate that an endometrial biopsy performed in the midluteal phase may detect a greater number of women with delayed endometrial maturation during the temporal window of embryo implantation. The observation that most ofthe women with out-of-phase midluteal biopsies had normal late luteal endometrium may represent a cryptic form of luteal phase deficiency. Fertil Steril1994;61:443-7 Key Words: Endometrial biopsy, timing, diagnosis, luteal phase deficiency, endometrium
In the 45 years since Dr. Georgeanna Jones first proposed the existence of a luteal phase defect (LPD) (1), controversy persists about its existence, diagnosis, pathophysiology, and treatment. Evalua-
Received July 12, 1993; revised and accepted
* Supported by the National Institutes of Health grant HD30476-l(BAL), Philadelphia, Pennsylvania. t Presented at the 49th Annual Meeting of The American Fertility Society, Montreal, Canada, October 9 to 14, 1993. :j: Department of Obstetrics and Gynecology. § Department of Pathology. II Reprint requests and present address: Bruce A. Lessey, M.D., Ph.D, University of North Carolina, Department of Obstetrics and Gynecology, MacNider Building, CB 7570, Chapel Hill, North Carolina, 27599-7570. Vol. 61, No.3, March 1994
tion of the luteal phase by endometrial histology remains the gold standard for the diagnosis of this disorder. Endometrial histology is thought to best reflect the hormonal milieu of the luteal phase, permissive for embryo attachment and implantation. Although P is the principal steroidogenic modulator of endometrial maturation, measurement of plasma P has not proven to be a good predictor of LPD because of the overlap of LPD patients with normal women (2). Integrated daily plasma P levels (3) correlate poorly with midluteal endometrial histology (4). The ideal time to perform the endometrial biopsy during the luteal phase is unsettled. It has been suggested that the biopsy be done a few days before Castelbaum et al. Timing of endometrial biopsy
443
the start of the next menstrual period as a maximal and cumulative bioassay of estrogen and Paction (5). Although several studies have noted that early and midluteal phase biopsies are poorer predictors of the next menstrual period when compared with a late luteal biopsy due to a wider variation in the histologic dating (5-9), dating referenced to the urinary LH surge have demonstrated no increased interobserver (10, 11) or intraobserver (12) variation between an early and late luteal biopsy for evaluation ofthe luteal phase. In fact, Li et al. (12) noted greater precision using endometrial biopsies obtained before day 22 of the menstrual cycle compared with those obtained after day 22. In addition, morphometric analysis has demonstrated higher error rates in reading biopsies as they moved away from the LH surge (13). It is now clear that the window of uterine receptivity to embryo implantation spans postovulatory days 6 through 10 (14, 15) and that it is day 7 human embryos that implant (15). These observations suggest that even though the window of uterine receptivity is wide, human embryos are capable of implanting within a much narrower time frame in their development. Therefore, it can be hypothesized that an out-of-phase endometrium between postovulatory days 6 and 10 may contribute to early embryonic loss, even if the late luteal endometrial histology is in phase. To address whether the timing of the endometrial biopsy is critical in detecting out-of-phase endometrium, we prospectively biopsied 33 ovulatory women twice in the same luteal phase.
MATERIALS AND METHODS Patients
Thirty-three women (age, 33.9 ± 4.5 years; range, 24 to 42 years; mean ± SD) seeking infertility evaluation at an academic teaching hospital enrolled in the study. The protocol was approved by the Institutional Review Board of the University of Pennsylvania, and each patient gave informed written consent. All subjects were ovulatory. Protocol
Each patient was evaluated during a single cycle. The day of ovulation (LH + 0) was determined by a sensitive home urine LH kit (Ovuquick; Quidel, San Diego, CA), and the cycle day was determined by counting forward from the day of ovulation. The early biopsy was performed on day LH + 7.4 ± 0.8 444
Castelbaum et al.
Timing of endometrial biopsy
(range, LH + 5 to + 9) whereas the late biopsy was performed on day LH + 11.7 ± 0.8 (range, LH + 10 to + 13). The second biopsy was performed 4.2 ± 0.8 days after the first biopsy (range, 3 to 6 days). All biopsies were done by two reproductive endocrinologists, using a suction curette (Z-sampler; Zinnanti Surgical Instruments, Chatsworth, CA). Biopsy material was obtained from the uterine fundus, and then was fixed and processed. Biopsies were read according to the criteria of Noyes et al. (16). Biopsy specimens were examined twice: first by a board-certified pathologist with knowledge of the patient's last menstrual period (LMP) routinely reading out specimens that day (pathologist 1) and, later, by a single senior pathologist with a special interest in gynecological pathology without knowledge of the LMP (pathologist 2). An out-ofphase biopsy was defined as 2':3 day lag between the chronological and histologic day. Statistical Analysis
Wilcoxon Signed Rank Test was used to compare the interobserver variation of the biopsy readings between pathologists. Chi -square analysis was performed to compare the detection rate of out-ofphase endometrium in early versus late biopsies. Significance was defined as P < 0.05.
RESULTS
Overall, out-of-phase endometrium was detected in 24.2% of cases (8 of 33) by pathologist 1 compared with 18.2% (6 of 33) for pathologist 2. There was a 12.1 % to 18.2% incidence (depending on the observer) of an out-of-phase early biopsy compared with a 6.1% to 9.1% incidence using the later biopsy. Interestingly, 80% (pathologist 1) to 100% (pathologist 2) of the early out-of-phase biopsies corrected by the time of the later biopsy. One late biopsy, deemed insufficient for diagnosis by pathologist 1, was read as "in phase" by pathologist 2. Two biopsies that were in phase in the early biopsy became out of phase on the later biopsy. Figure 1 displays a paired scattergram of the chronological and associated histologic day for each biopsy by each pathologist. Surprisingly, the majority of early out-of-phase biopsies (represented by asterisks in Fig. 1) demonstrated normal endometrial maturation at the time of the later biopsy. One case of acute endometritis was noted on the late biopsy. Figure 2 displays the interobserver variation between the two pathologists' histologic readings for early and late biopsies. Agreement within 2 days Fertility and Sterility
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Figure 1 The chronological day and associated histologic reading for each biopsy read by pathologist 1 (A) and pathologist 2 (B). The dotted line represents idealized synchrony between chronological and histologic days. A solid line connects each patient's two biopsies. Out-of-phase biopsies are identified by asterisks.
was noted in 78% ofthe early compared with 97% of the late biopsies. There was no significant interobserver variation between the early and late luteal biopsies (P = 0.42, Wilcoxon Signed Rank Test). The percentage of patients designated as out of phase based on the early biopsy versus the later biopsy is shown in Figure 3. For each pathologist, the detection rate for out-of-phase endometrium was significantly· higher in the earlier biopsy (pathologist 1, P = 0.0001, X2 = 14.85; pathologist 2, P < 0.0001, X2 = 15.43). Vol. 61, No.3, March 1994
As a better understanding of uterine receptivity emerges, the issue of when to perform the endometrial biopsy becomes ever more important. There is a distinct temporal window spanning postovulatory days 6 through 10 (cycle days 20 to 24) during which the endometrium is thought to be receptive to embryo attachment and implantation (14, 15). Many have suggested performing the endometrial biopsy closer to the next menses as a cumulative bioassay of Paction (5) whereas others have argued that the midluteal phase biopsy, corresponding to the window of implantation, is a more physiologically important time to examine uterine receptivity (17). We now present evidence supporting this latter concept. In the current study, the greater number of outof-phase endometrial samples identified by the earlier biopsy suggests that the timing of sampling may be important for the detection of LPD. By relying on a late luteal phase biopsy alone, only two or three out-of-phase cycles from 33 women seeking evaluation of infertility would have been diagnosed (6.1 % to 9.1 %). Alternatively, a biopsy performed during the putative time of implantation identified four or six out-of-phase cycles (12.1% to 18.2%). These findings are consistent with those of Cummings and colleagues (8) who noted an out-of-phase biopsy rate of 38.6% in infertile patients during cycle days 25 to 28 and a significantly higher rate of 53.6% among infertile women biopsied before cycle day 25. The number and distribution of out-of-phase biopsies noted by both pathologists were quite simi1ar. Their concordance in histologic dating (78% and 97% agreement within 2 days for the early and late biopsies, respectively) is comparable to those reported by Noyes and Haman (18). The ideal method for accurately defining chronological dating remains unsettled, although counting forward from the day of ovulation previously has been proposed (19). We used the urinary LH surge to detect ovulation and to time the biopsies. Several studies have demonstrated that this method results in a higher correlation with histologic dating in parous (20) and infertile (21) women compared with use of the next menstrual period. Clinical practice has been influenced by the belief that dating of the early and midluteal biopsy is less accurate compared with late luteal material whereas the data in the literature are contradictory. The present study demonstrates no statistically significant difference in interobserver variability Castelbaum et at.
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Figure 3 Comparison of detection rate for out-of-phase endometrium in patients biopsied early versus late in the same menstrual cycle as judged by two pathologists. Castelbaum et al.
. 0
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between the two biopsies. A wider interobserver variation previously has been reported on biopsies done during the early or midluteal versus late luteal phase (5). Other reports (10, 11) demonstrate negligible interobserver variation throughout the luteal phase. Gibson and colleagues (10) examined 25 duplicate endometrial biopsies from women seeking infertility evaluation. Each slide was read by five different observers. The interobserver variation between endometrium examined before cycle day 22 and after day 22 was not different. Similar results were obtained by Scott et al. (11). Although Li et al. (12) demonstrated no statistical difference in intraobserver variation in endometrial dating before cycle day 22 compared with specimens from day 22 onward, the accuracy (incidence of exact agreement) was better for the earlier biopsies. The evaluation
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Figure 2 Histograms demonstrating the interobserver difference, in days, between the readings of the two pathologists for the early versus late endometrial biopsies.
of morphometric analyses has also demonstrated that early and midluteal endometrial biopsies are more consistent in their parameters compared with later biopsies (22). The difference in the percentages of out-of-phase endometrium between the early and late biopsies in the current study, therefore, cannot be attributed to errors in the histologic interpretation of the biopsy specimens. Because the two biopsies in the current study were performed in the same cycle, the effect the first endometrial biopsy may have had on the second is unknown. The majority of patients demonstrated normal endometrial maturation on both biopsies (76% to 82%) and only 1 of 33 late biopsy specimens revealed endometritis. On one occasion, the second biopsy in one cycle revealed focal histologic changes consistent with a previous biopsy site. Although there were reactive changes in the stroma of the involved tissue fragment, with a mild neutrophilic infiltrate, superficial fibrin, and red blood cells, the glands showed only minor changes. Other endometrial fragments in the specimen showed no reactive changes and can be dated readily by standard criteria (16). There is no reason, therefore, to suspect that the explanation for the "catch up" demonstrated by the women with an out-of-phase early, but normal late biopsy rests with consequences of the first biopsy per se. Although the physiologic consequences of an out-of-phase early biopsy are uncertain, asynchrony between endometrial maturation and embryonic development is associated with defects of implantation (23). In humans, there appears to be a specific time in the luteal phase when the endometrium is permissive to implantation by the trophectoderm of Fertility and Sterility
the embryo. The existence of a window of implantation in humans is based on studies of Hertig et al. (14) and more recent studies using hypersensitive hCG assay of maternal serum after ET, demonstrating initial hCG rise occurs during cycle days LH + 6 to LH + 10 (15). Our findings indicate that a subgroup of women with in-phase endometrium late in the luteal phase are out of phase during the window of implantation. This may represent a cryptic form of LPD. Specifically, these women would have been missed had only the later biopsy been performed. Recently, 15 women who conceived during the cycle of their endometrial biopsy were evaluated (24). All 10 women biopsied on postovulatory day 6 to 11 had in-phase endometrium. Ironically, four of the five pregnant women biopsied on postovulatory day 12 had varying degrees of gland-stromal dyssynchrony, again supporting the utility of an earlier endometrial biopsy. Our study of dual biopsies in the same luteal phase suggests that endometrial biopsies performed for the evaluation of infertility may detect a larger number of out-of-phase cycles when performed during the early and mid compared with late luteal phase ofthe menstrual cycle. Based on the preponderance of the evidence in the literature, as well as our own observations, we propose that a biopsy be performed during the window of implantation as a more appropriate time for scrutiny of endometrial histology. Additional larger studies involving both infertile and fertile women are needed to confirm our findings and to determine the true incidence of LPD. Acknowledgment. We thank David Guzick, M.D., Ph.D., Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, for his assistance in data analysis.
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5. Wentz AC, Kossoy LR, Parker RA. The impact of luteal phase inadequacy in an infertile population. Am J Obstet Gynecol 1990; 162:937-45. 6. Rosenfeld DL, Chudow S, Bronson RA. Diagnosis of luteal phase inadequacy. Obstet GynecoI1980;56:193-6. 7. Huang KE. The primary treatment of luteal phase inadequacy: progesterone versus clomiphene citrate. Am J Obstet Gynecol 1986; 155:824-8. 8. Cummings 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. 9. Wentz AC. Endometrial biopsy in the evaluation ofinfertility. Fertil Steril1980;33:121-4. 10. Gibson M, Badger GJ, Byrn F, Lee KR, Korson R, Trainer TD. Error in histologic dating of secretory endometrium: variance component analysis. Fertil Steril 1991; 56:242-7. 11. 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. 12. Li T-C, Dockery P, Rogers AW, Cooke ID. How precise is histologic dating of endometrium using the standard dating criteria? Fertil SteriI1989;51:759-63. 13. Johannisson E, Parker RA, Landgren B-M, Diczfalusy E. Morphometric analysis of the human endometrium in relation to peripheral hormone levels. Fertil Steril1982;38:56471. 14. Hertig AT, Rock J, Adams EC. A description of 34 human ova within the first 17 days of development. Am J Anat 1956; 98:435-93. 15. Bergh PA, Navot D. The impact of embryonic development and endometrial maturity on the timing of implantation. Fertil Steril1992;58:537-42. 16. Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Fertil Steril 1950; 1:3-25. 17. Li T -C, Cooke ID. Evaluation of the luteal phase. Hum Reprod 1991;6:484-99. 18. Noyes RW, Haman JO. Accuracy of endometrial dating: correlation of endometrial dating with basal body temperature and menses. Fertil SteriI1953;4:504-17. 19. Rosenfeld DL, Garcia CoR. A comparison of endometrial histology with simultaneous plasma progesterone determinations in infertile women. Fertil Steril1976;27:1256-66. 20. Shoupe D, Mishell DR, 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. 21. Li T-C, Rogers AW, Lenton EA, Dockery P, Cooke I. A comparison between two methods of chronological dating of human endometrial biopsies during the luteal phase, and their correlation with histologic dating. Fertil Steril 1987; 48:928-32. 22. 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. 23. Pope WF. Uterine asynchrony: a cause of embryonic loss. Bioi Reprod 1988;39:999-1003. 24. Andoh K, Mizunuma H, Nakazato Y, Yamada K, Michishita M, Ibuki Y. Endometrial dating in the conception cycle. Fertil SteriI1992;58:1127-30.
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