Prevention of endometrial hyperplasia by progesterone during long-term estradiol replacement: influence of bleeding pattern and secretory changes*

FERTILITY AND STERILITY Vol. 59, No.5, May 1993 Copyright e 1993 The American Fertility Society Printed on acid-free paper in U.S.A. Prevention of endometrial hyperplasia by progesterone during long-term estradiol replacement: influence of bleeding pattern and secretory changes* Dean L. Moyer, M.D.t:j: Bruno de Lignieres, M.D. Pierre Driguez, M.D. Jean Pierre Pez, University of Southern California Medical Center, Los Angeles, California, and Hopital Necker, Paris, France Objective: To determine the relative influences of induction of withdrawal bleeding, secretory transformation, and reduction of mitosis in glands on prevention of endometrial hyperplasia during long-term hormonal replacement therapy. Design: Observational expanded clinical case report. Setting: Reproductive Endocrine Department of Hopital Necker, Paris, France, and Pathology Department of Women's Hospital, Los Angeles County and University of Southern California Medical Center, Los Angeles, California. Patients: Postmenopausal women seeking treatment for symptomatic menopause. Interventions: Endometrial biopsy and/or ambulatory hysteroscopy. Main Outcome Measure: Endometrial histology including progestational maturation patterns and glandular epithelial mitosis rates. Macroscopic endometrial appearance. Results: The use of larger doses of E 2 and P induced more marked secretory changes and more frequent withdrawal bleeding than the lower doses. There was no evidence of endometrial hyperplasia after 5 years of E 2/P replacement therapy independently of bleeding pattern or progestational maturation. Consistent reduction of mitosis rates in glandular epithelium was found after 9 or more days of P administration in each cycle. Conclusions: Control of endometrial growth is mainly related to control of mitosis in glands by a relatively low doses of P. Induction of withdrawal bleeding and endometrial secretory transformation, which require larger doses of Progesterone, do not provide additional benefit for prevention of hyperplasia. Induction of amenorrhea with a relatively low dose of P may be offered to women seeking hormone replacement therapy with similar levels of safety. Fertil Steril 1993;59:992-7 Key Words: Hormone replacement therapy, progesterone, endometrial hyperplasia Exogenous estrogens (Es) have been successfully prescribed to postmenopausal women to alleviate Received October 20, 1992; revised and accepted December 23, 1992. * Supported in part by Besins-Iscovesco Laboratories, Paris, France. t Department of Pathology, University of Southern California Medical Center. t Reprint requests: Dean L. Moyer, M.D., 21018 Osborne 3, Canoga Park, California 91304. Reproductive Endocrine Department, Hopital Necker. the symptoms of menopause and to prevent osteoporotic fractures. However, when E is the only hormone used in replacement therapy, the incidence of endometrial pathology is increased and is typified by the occurrence of endometrial hyperplasia with short-term use and adenocarcinoma after long-term use (1, 2). The addition of natural P or a synthetic progestogen to E replacement therapy reduces the risk for developing both endometrial hyperplasia and carcinoma when the dose, duration, and cycle timing of the progestogen are optimal (3, 4). Presently, the optimal type and dosage of progestogen remains 992 Moyer et al. Prevention of endometrial hyperplasia Fertility and Sterility

unclear because of the metabolic side effects of some progestogens (5, 6). The minimal mean E dose needed to prevent osteoporosis has been identified, and the amount of estrogenic stimulation similar to the early to midfollicular phase is thought to be sufficient (7). To identify the optimal amount of progestogen or P formulations, it is necessary to determine the minimal antiproliferative effect that prevents endometrial hyperplasia. It is relevant that the antiproliferative phenomenon occurring in endometrial glands should be completely separated from secretory glandular maturation. The withdrawal bleeding dreaded by many older women may be eliminated by using lower doses of a progestogen that are most always associated with amenorrhea. In this series the characteristics of endometrial morphology that coincide with long-term safety were documented in postmenopausal women receiving different combinations of E 2 and P over a period of 5 or more years. Patients MATERIALS AND METHODS A combined E 2/P treatment was offered to 236 symptomatic postmenopausal women seeking hormone replacement in the Reproductive Endocrinology Department of the Hopital Necker in Paris between 1980 and 1982. All these nonhysterectomized women had amenorrhea for 12 months or more, a plasma E 2 level< 147 pmol/dl and plasma FSH and LH > 20 IU /L. Seventy-six women started hormone replacement therapy in 1980, 68 in 1981, and 92 in 1982. Hormone Treatment These women were treated with daily percutaneous 17/)-E 2 (Oestrogel; Besins-Iscovesco Laboratories, Paris, France). The initially proposed daily dose was 1.5 mg E 2 (low E 2), with a treatment schedule administered for 21 days out of 28. Within the first 6 treated months, this initial dose was increased to 3 mg/d (high E 2) if optimal improvement in clinical menopause symptoms was not obtained. The initial monthly duration of treatment was also increased to 25 days out of 28 if there was a recurrence of clinical symptoms during the week off treatment. Estradiol treatment remained unchanged during the rest of the survey. The initial dose of oral micronized P (Utrogestan; Besins-Iscovesco Laboratories) was 200 mg (low P) given at bedtime the last 14 days of E 2 treatment. Within the first 6 treated months, this dose was increased to 300 mg/d (high P) in women who preferred cyclic withdrawal bleeding and who did not get it with the 200-mg/d dose. The monthly duration of treatment was shortened to 10 or 12 days when early uterine bleeding occurred before the end of the treatment cycle. Progesterone treatment remained unchanged during the rest of the survey. A precise record of bleeding schedule was obtained from each patient during the last 12 months of the study before biopsy, done in 1987 or 1988 after a minimum of 5 years of unchanged treatment. E 2 Plasma Levels Estradiol plasma levels were performed by RIA once a year. Plasma sampling was scheduled during the last 10 days of the treatment cycle, and in 1987 or 1988, not more than 3 days before or after the endometrial biopsies. The E 2 assay was performed by RIA using specific tritium-labeled antibody after steroid extraction and selective chromatography using a Sephadex LH20 column. The sensitivity was <5 pg/ml, the intraassay coefficient of variation (CV) was <5%, and the interassay CV was <10%. The cross-reactivity with estrone was <1 %. Endometrial Examinations An endometrial sampling was scheduled during days 2 to 14 of progestogen treatment and in any woman with irregular uterine bleeding. Biopsy was performed in every women after at least 5 years of hormone replacement therapy without reference to her bleeding pattern. All endometrial tissue recovered was fixed in Bouin's solution followed by standard histologic methods using hematoxylin and eosin staining. All endometrial tissue samples were evaluated morphologically for abnormalities and for the level of secretory maturation. All biopsy samples had mitoses counts performed by light microscopic examination of multiple endometrial glands. The mitotic figures were counted while observing 1,000 or more glandular cells in four different areas of the biopsy (total, 4,000 cells or more). The mitotic index was expressed as a mean number of mitoses observed per 1,000 glandular epithelial cells. Hysteroscopic Evaluations In each woman who had insufficient endometrial tissue removed at the time of biopsy, including those Vol. 59, No.5, May 1993 Moyer et al. Prevention of endometrial hyperplasia 993

who showed only lower uterine segment, an ambulatory gas hysteroscopy was performed. A second biopsy was attempted at this time. Statistical Analysis Nonparametric test of Mann-Whitney was used for the comparisons of E2 values, age, and age at menopause. Single regression analysis was used to calculate the correlation coefficient between mitoses and the duration of P exposure. Dropouts RESULTS Of the initial group of 236 women starting percutaneous E2 and oral micronized P treatment in 1980, 1981, or 1982, 79 women dropped out during the first 5 years of the treatment. The primary reasons for not continuing the treatment were the lack of recurrence of initial clinical symptoms after several years of replacement therapy and fear of cancer commonly raised by the lay press. No one stopped the treatment because of occurrence of irregular bleeding or because of symptomatic endometrial patho logy. Treatment Regimens Each woman in the different treatment regimens was followed for at least 5 years. The mean duration of treatment was 5. 7 years at the time of endometrial biopsy or hysteroscopy. One hundred twenty-six women of 157 remained on the initial cyclic treatment of 1.5 mg percutaneous E2 (low E2) for 21 days out of 28 combined with 200 mg oral P (low P) for 14 days out of 28. This group was classified as low E2/low P. Twenty-three women were changed to 3 mg percutaneous E2 daily (high E2) and to 300 mg oral P (high P) to induce cyclic bleeding. In these women the duration of P treatment was shortened to 10 days, and they were classified as high E2/high P. A small group of women received intermediate doses of E2 and P in response to clinical symptoms. Three women remained on 1.5 mg/d E2, but P was increased to 300 mg to induce cyclic bleeding (low E2/high P), and in five women the daily E2 was increased to 3 mg because of an insufficient response to the clinical symptoms and the daily dose of P was 200 mg for 14 out of 28 days (high E2/low P). E 2 Plasma Levels In the 129 patients using low E2 treatment, mean plasma E2 level during the last 5 years of treatment exposure was 78 ± 1.8 pg/ml (mean± SEM). At each of the last 5 yearly examinations the mean plasma levels were 89 ± 2.6 pgjml (year 1), 71 ± 1.2 pg/ml (year 2), 68 ± 1.3 pgjml (year 3), 86 ± 2.2 pg/ml (year 4), and 80 ± 1.7 pg/ml (year 5). All these values were significantly different (P < 0.001) from E2 baseline before treatment, which was 18 ± 2 pg/ml. The low E2 group had significantly different E2levels (P < 0.01) compared with the values of the high E2 treatment group (123 ± pg/ml). Bleeding Patterns Cyclic withdrawal bleeding was defined as any blood-stained discharge passed per vagina between the last 2 days of a treatment cycle and the first 2 days of the next treatment cycle. Irregular bleeding was defined as any blood discharge passed per vagina beginning after the first 2 days of the treatment cycle but before the last 2 days ofthis cycle. No bleeding means that no detectable bloody vaginal discharge was noted at any time during the cycle. During the last 12 months of this series, 4 of 157 women (2%) presented with at least two episodes of irregular bleeding, 34 (21%) presented with regular cyclic bleeding, and 119 (77%) experienced no bleeding (Fig. 1). Demographic characteristics were not related to the bleeding pattern. The ages at time of biopsy of women with or without cyclic bleeding 100 80 % D Amenorrhea 60 Cyclic withdrawal bleeding 40 20 0 High E 2/P Low E.{P Irregular bleeding Figure 1 Bleeding patterns during the last 12 months of the 5.7-year study compared with high or low E,JP treatments. 994 Moyer et al. Prevention of endometrial hyperplasia Fertility and Sterility

did not differ (58.7 ± 3 and 59.3 ± 4) nor did the years since menopause (9.3 ± 3 versus 8.4 ± 3). The bleeding patterns appeared to be related to the doses of the ovarian hormone used for treatment. Of the 34 women with regular cyclic bleeding, 21 (61.7%) used a high E 2 dose, 13 (38.2%) used a low E 2 dose, and 26 of 34 patients (76.4%) used a high P dose. The highest incidence of cyclic bleeding (83%) was observed in the high E 2/high P group (Fig. 1); conversely, the highest incidence of amenorrhea (88%) was observed in the low E 2/low P group. Endometrial Biopsy In four women who developed irregular bleeding while under treatment, a dilation and curettage was performed under general anesthesia. The tissue morphology showed benign endometrial polyp in three of them, and the fourth woman was diagnosed as having a submucosal leiomyoma. None of these four women showed either endometrial hyperplasia or carcinoma or even an adequate amount of endometrial tissue outside the polyps. In the 153 other women who remained in the study for >5 years, an endometrial biopsy was attempted but only in 66 was any tissue obtained. Of the 66 endometrial biopsies, 53 showed an adequate amount of endometrial tissue for histologic examination. The other 13 samples showed either low uterine segment endometrium or endocervical tissue and were not included in the histologic analysis. The recovery of an adequate amount of endometrial tissue at the time of the biopsy attempt appears to be related to both the doses of E 2/P and the bleeding pattern but not to demographic characteristics. There was 100% recovery of endometrial tissue in the high E 2/high P group but only 18% recovery in the low E 2/low P group (P < 0.001). No endometrial tissue was available in 83% of amenorrheic women, but in 93% of women experiencing regular withdrawal bleeding, biopsy produced an adequate amount of tissue. The 53 women with adequate endometrium in the biopsy were 58.1 ± 4 years old, 8.8 ± 3 years after menopause, and under hormone replacement therapy for the past 5.7 ± 1 years. None of the characteristics was significantly different from the group with unproductive biopsies. Except for the 4 women with irregular bleeding, all of the endometrial biopsies showed various aspects of normal glands and stroma without evidence of hyperplasia or carcinoma. Secretory maturation was variable ranging from minimal to moderate in amount, but in none of the biopsies did the quantity of secretory maturation equal a fully developed gland and stroma pattern equivalent to the late luteal phase. Moderate secretory maturation was observed in 78% of the high E 2 /high P group but only in 8% of the low E 2/ low P group. The 53 adequate endometrial samples showed the glandular epithelium to have a mean of 1. 7 mitoses (ranging from 0 to 21) per 1,000 epithelia cells. Women receiving 9 days or more of P therapy at the time of biopsy had significantly lower (P < 0.001) mitotic activity, 0.3 mitoses per 1,000 gland cells (range, 0 to 2) compared with women receiving 8 or less days of P treatment at the time of biopsy, who had a mean of 4.3 mitoses per 1,000 gland cell (range, 0 to 21). Single regression analysis showed a significant negative correlation (r = -0.52) between duration of P exposure and mitotic activity (Fig. 2). This decrease in mitotic activity was similar in endometrial samples with or without moderate secretory changes. In those women in whom the first endometrial biopsy was not successful in producing adequate amounts of endometrial tissue, an ambulatory hysteroscopy was performed and consistently showed macroscopic atrophy of the endometrial mucosa with no sufficient tissue for a second biopsy attempt. There was no evidence of widespread or localized endometrial hyperplasia nor adenocarcinoma. DISCUSSION The use of E-only replacement therapy results in endometrial hyperstimulation with the subsequent development of endometrial hyperplasia in many 24 20 16 Mitotic 12 Index 8 4 0 0 [I -..., ~ [I [I ll ~ ~.!! 101 '" 3 6 9 12 15 Duration (days) of progesterone administration Figure 2 Regression of mitotic index compared with days of P administration. D Vol. 59, No.5, May 1993 Moyer et al. Prevention of endometrial hyperplasia 995

women (1, 8). Endometrial carcinoma has been reported to be greater in women receiving unopposed E when compared with those who have never received E therapy (9). With the use of unopposed E for> 1 year, the increased risk for endometrial carcinoma may last> 10 years after the therapy is withdrawn (10). These risks are not acceptable to menopausal women when alternative treatment forms are available. Current strategy to reduce the risk of developing endometrial carcinoma is the addition of synthetic progestogen or natural P to thee therapy, in doses large enough to induce both full secretory transformation and withdrawal bleeding (11). However, this strategy is poorly accepted on a long-term basis by most users who do not like recurrence of "periods" (12) and the induction of dose-related side effects (13). The present series evaluates the long-term relationship between the expected antiproliferative effect of a formulation of oral micronized P with the level of secretory maturation of the endometrium and induced withdrawal bleeding. Evidence of dissociation between these different effects of progestogens on endometrium already exist. Sequential oral contraceptive (OC) pills with relatively short progestin exposure ( <7 days each cycle) have been clearly associated with abnormally high incidence of endometrial hyperplasia and adenocarcinoma, despite their ability to induce secretory changes and regular withdrawal bleeding (14). On the other hand, combined OC pills have clearly shown a protective effect against the risks of endometrial hyperplasia and adenocarcinoma (15) even though these compounds do not induce significant physiological secretory changes. Previously, we showed that semiquantitative changes in both glands and stroma summarize the level of progestational maturation responding to changes in the dosages of either an E or a progestogen (16). In the present study, the lack of complete glandular and stromal progestational changes was not associated with any detectable impairment in antiproliferative effect. When the biopsy was taken at 8 days or less of P administration, the reduction in mitotic activity was inconsistent. However, after 8 days of oral micronized P exposure, mitotic activity in the endometrial tissue was decreased in all samples even in those showing the lowest level of secretory maturation and in amenorrheic patients. The maximal reduction of mitoses was noted on biopsies taken after 11 days of P treatment. In both the high and the low E 2/P, the antiproliferative effect seems to be sufficient because no hyperplasia was observed after 5 to 7 years exposure during treatment either by biopsy when endometrial tissue was available or by hysteroscopy when no endometrial tissue was available for biopsy. In the present series, most of the menopausal women (82%) were totally relieved of their clinical symptoms with low E 2 treatment, which increased E 2 plasma levels to a mean of 78 ± 1.8 pg/ml. Using the same percutaneous treatment, this plasma level has been shown to be sufficient in preventing bone loss and could therefore be recommended for replacement in postmenopausal women. We have shown evidence that P demonstrates an antiproliferative effect. Inhibition of endometrial proliferation (decrease in epithelial mitotic activity) may be dissociated from secretory maturational changes (stromal pseudostratification and glandular secretion) and related withdrawal bleeding by relatively low dose combinations of E 2/P. For the majority of women, the least amount ofe and P that is necessary to prevent osteoporosis on one hand and to prevent endometrium to proliferate on the other hand is the preferred treatment. The higher doses inducing withdrawal bleeding and completely developed secretory changes do not substantially improve safety but are likely to increase noncompliance in majority of users. REFERENCES 1. Paterson MEL, Wade-Evans T, Sturdee DW. Endometrial disease after treatment with oestrogen and progestogens in the climacteric. Br Med J 1980;280:822-35. 2. Mack TM, Pike MC, Henderson BE. Estrogen and endometrial cancer in a retirement community. N Eng! J Med 1976;294:1262-7. 3. 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10. Paganini-Hill A, Ross RK, Henderson BE. Endometrial cancer and patterns of oestrogen replacement therapy: a cohort study. Br J Cancer 1989;59:445-7. 11. Padwick ML, Pryse-Davies J, Whitehead MI. A simple method for determining the optimal dosage of progestin in postmenopausal women receiving estrogens. N Eng! J Med 1986;315:930-4. 12. Hahn RG. Compliance considerations with estrogen replacement: withdrawal bleeding and other factors. Am J Obstet Gynecol1989;161:1854-8. 13. Fraser D, Whitehead MI, Endacott J, Morton J, Ryder TA, Pryse-Davies J. Are fixed-dose estrogen progestogen com- binations ideal for all HRT users? Br J Obstet Gynaecol 1989;96:776-82. 14. Silverberg SG, Markowski EL, Roche WD. Endometrial carcinoma in women under 40 years of age. Cancer 1977;39:592-8. 15. Org HW, Layde PM, Webster LA, Wingo PA. Centers of disease control cancer and steroid hormone study: oral contraceptive use and the risk of endometrial cancer. JAMA 1983;249:1600-4. 16. Gibbons WE, Moyer DL, Lobo RA, Roy S, Mishell DR. Biochemical and histologic effects of sequential estrogen/progestin therapy on the endometrium of postmenopausal women. Am J Obstet Gynecol 1986;154:456-61. Vol. 59, No.5, May 1993 Moyer et al. Prevention of endometrial hyperplasia 997