Administration of an antiprogesterone up-regulates estrogen receptors in the endometrium of women using Norplant : a pilot study

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1 FERTILITY AND STERILITY VOL. 77, NO. 2, FEBRUARY 2002 Copyright 2002 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Administration of an antiprogesterone up-regulates estrogen receptors in the endometrium of women using Norplant : a pilot study Anna F. Glasier, M.D., a,b Hong Wang, Ph.D., a Jane E. Davie, M.B.Ch.B., b R. W. Kelly, Ph.D., c and Hilary O. D. Critchley, M.D. a University of Edinburgh; Lothian Primary Care NHS Trust; and MRC Human Reproductive Sciences Unit, Edinburgh, Scotland, United Kingdom Received June 22, 2001; revised and accepted September 26, Levonorgestrel-releasing implant system is manufactured by Hoechst Marion Roussel. The study was undertaken by the Contraceptive Development Network, which is funded by the Medical Research Council and Department for International Development (G ). Hong Wang was supported by a grant from the Rockefeller Foundation. Reprint requests: Anna Glasier, M.D., University of Edinburgh, Department of Obstetrics and Gynaecology, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh, EH9 3ET, Scotland (FAX: ; a.glasier@ ed.ac.uk). a Department of Obstetrics and Gynaecology, University of Edinburgh. b Lothian Primary Care NHS Trust. c MRC Human Reproductive Sciences Unit /02/$22.00 PII S (01) Objective: To determine the effect of a single dose of mifepristone (200 mg) on endometrial estrogen and progesterone receptors in Norplant users. Design: A prospective single-blind placebo-controlled pilot study. Setting: Women were recruited from a large family planning clinic and were studied either at the clinic or in a clinical research unit attached to a teaching hospital gynecology department. Patient(s): Eight women using Norplant and experiencing vaginal bleeding more often than once every 24 days. All completed the study. Intervention(s): Endometrial biopsies were taken after treatment with both placebo and 200 mg of mifepristone, both given at the start of a bleeding episode. Main Outcome Measure(s): Expression of endometrial progesterone (PR) and estrogen (ER) receptors, ovulation, and vaginal bleeding. Result(s): Mifepristone administration was associated with down-regulation of PR receptor subtype B and up-regulation of ER. Women treated with mifepristone showed a tendency to increased ovulation rates and reduced vaginal bleeding. Conclusion(s): The effect of mifepristone on endometrial steroid receptors was consistent with functional inhibition of progesterone. The findings warrant further investigation of this regimen as a strategy to reduce frequent bleeding. (Fertil Steril 2002;77: by American Society for Reproductive Medicine.) Key Words: Norplant, mifepristone, bleeding, endometrium, contraception Irregular or breakthrough bleeding is perhaps the most frequent side effect of oral contraceptives (1); in some parts of the world, it is the commonest reason for discontinuation (2). Irregular bleeding is particularly associated with low-dose progestogen-only contraception (pills, implants, and progestogen-releasing intrauterine systems), which in most women do not completely inhibit endogenous ovarian activity. In a recent review of first-year continuation rates of Norplant contraceptive implants in the United Kingdom (3), over 50% of women who had the implants removed cited menstrual change as the reason. By 18 months after insertion, 20% of women attending a large family planning clinic in Scotland (4) had stopped using Norplant, with 43% of them attributing discontinuation to irregular bleeding. The mechanisms responsible for irregular bleeding are not well understood. The occurrence of erratic bleeding episodes does not clearly correlate with fluctuations in circulating concentrations of endogenous or exogenous steroids, nor with any particular pattern of endometrial histology. It is thought that breakthrough bleeding is probably related to molecular changes in the endometrium that disrupt vascular growth, function, and repair and disturb hemostatic mechanisms (5). Estrogen receptors have now been clearly demonstrated in the endothelium of the endometrial vasculature (6), and thus these changes may be mediated by 366

2 a direct or indirect effect of steroids on the vasculature acting via cytokines or chemokines (7). Whatever the cause of breakthrough bleeding, it is well recognized that the addition of ethinyl estradiol to a progestogen-only contraceptive regimen reliably improves bleeding patterns (8, 9). More recently it has been demonstrated that the addition of an antiprogesterone to progestogen-only contraception reduces the incidence of unscheduled bleeding in monkeys (10), and we have shown that once a month administration of mifepristone improves bleeding patterns in Chinese women using the levonorgestrel-releasing sino-implant (11). The mechanism for this effect of both estrogen and mifepristone is unknown. In the normal cycle, after menstruation, a period of unopposed estrogen stimulation (during the follicular phase) appears to be necessary for the regeneration of endometrial progesterone receptors. Although the endometrium of women using Norplant has been shown to have an increased concentration of progesterone receptors (PR) (12, 13), exposure to progestogens can lead to refractoriness to the steroid (see 14 for review). It is possible that both add-back estrogen, acting directly on the endometrium, and antiprogesterone, through a direct effect on steroid receptors allowing the regeneration of functional PR, mimic this physiological process. In an attempt to determine whether the addition of an antiprogesterone to a progestogen-only regimen of contraception might alter the expression of endometrial steroid receptors, we undertook a small pilot study of women experiencing troublesome bleeding while using the contraceptive implant Norplant. None of the authors has any vested interest of a commercial nature relevant to this study. PATIENTS AND METHODS Eight women who had been using Norplant for at least 4 months and who were experiencing vaginal bleeding more than once every 24 days were recruited to the study from a large family planning clinic in Edinburgh. All eight women took both placebo and mifepristone treatment, and all completed the study. None complained of any side effects associated with either treatment. Women kept a daily diary of menstrual bleeding and spotting throughout the study. Ovarian activity was monitored for 8 weeks by measuring estrone and pregnanediol/creatinine ratios in early morning specimens of urine collected three times every week. Samples from individual women were run in a single batch. Urinary estrone-3-glucuronide was measured using a specific radioimmunoassay and pregnanediol-3-glucuronide using an enzyme-linked immunosorbent assay (ELISA) (for full details see 15). A pregnanediol/creatinine ratio of 0.5 or more was taken as an indication of ovulation. On the third day after the onset of the first bleeding episode to occur after 8 weeks of baseline monitoring, a single placebo tablet (vitamin C, 50 mg) was given and urine collection was increased to daily (Fig. 1). In a similar manner, 200 mg of mifepristone was taken on day 3 of the first bleeding episode to occur at least 3 weeks after placebo treatment. Daily urine collection continued until 4 weeks after the administration of mifepristone. Participants were blinded to the treatments. Between 3 and 5 days after administration of both placebo and mifepristone an endometrial biopsy was taken using a Pipelle endometrial sampler. On the day of the biopsy a single venous blood sample (10 ml) was taken for the measurement of estradiol and progesterone by RIA using methods that have previously been published from our center (16). A sample of cervical mucus was also collected using a tuberculin syringe (15). Cervical mucus score was assessed using the criteria established by the World Health Organization (17) wherein scores of 0 to 3 were given for mucus volume, consistency, ferning, spinnbarkeit, and cellularity (total score possible 15). All endometrial tissue samples were fixed overnight in 10% neutral buffered formalin at 4 C, rinsed, and stored in 70% ethanol and thereafter routinely wax embedded. Sections 5 m thick were cut for routine histopathology (hematoxylin and eosin staining) and immunolocalization of progesterone receptor (subtypes A and B), progesterone receptor subtype B, and estrogen receptor. Local ethics committee approval was obtained for the study and all participants gave written, informed consent. Differences in bleeding patterns were analyzed using the Wilcoxon signed rank test and by paired t test. Immunohistochemical Procedures Localization of progesterone receptors was undertaken with the use of two antibodies, a rabbit polyclonal antibody against PR B subtype, and a monoclonal antibody that detects both A and B subtypes. It is not possible to raise a specific subtype antibody against the A form of the receptor. Hence, we refer to PR A B as the receptor detected by the antibody that recognizes both subtypes of the PR, and PR B as the receptor detected by antibody specific to the B form of the receptor. Furthermore, we assume PR A is the subtype responsible for positive immunostaining when the B subtype cannot be detected. (For full details of the immunohistochemical protocols used in our study, see 18, 19). Briefly, paraffin-embedded tissue sections were treated with primary antibodies raised against the A and B subtype of the progesterone receptor (1:40 Novocastra Laboratories, Newcastle, United Kingdom), the B form of the progesterone receptor (1:200 in house rabbit anti-human antibody [18]), and the estrogen receptor (1 in 25 ER1D5, DAKO Laboratories, High Wycombe, United Kingdom). An antigen-retrieval step (tissue sections microwaved in citrate buffer, ph 6.0 [18]) was necessary for localization of epitope representing the A and B form of the progesterone receptor and the estrogen receptor. Standard immunohistochemical FERTILITY & STERILITY 367

3 FIGURE 1 Study design. detection methods were employed (avidin-biotin-peroxidase: Vectastain, PK-4002, DAB kit, SK-4100, Vector Laboratories, Peterborough, United Kingdom). Negative controls were included by replacing the primary antibody with nonimmune serum of equivalent concentration. Scoring of Immunohistochemical Staining and Statistical Analysis Immunostaining intensity and distribution of epitopes in all tissue sections were assessed semiquantitatively, on a four-point scale where 0 no staining, 1 mild staining, 2 moderate staining, and 3 intense staining. Scoring was performed blind by a single observer. As we reported elsewhere, in endometrial tissue sections, a high correlation (0.963) of immunostaining was measured objectively by computerized image analysis and by subjective semiquantitative scoring of immunoreactivity (18). These published data support the subsequent statistical analyses performed on semiquantitative scores of sex steroid immunostaining. As the data were noncontinuous, analysis employed a Mann- Whitney test. RESULTS The characteristics of the women and their responses to treatment with mifepristone and placebo are shown in Table 1. The amount of tissue from one woman following placebo TABLE 1 Menstrual bleeding pattern reported with mifepristone and placebo. Treatment Bleeding/spotting days Dry days Mean Range Mean Range Women ovulated After placebo After mifepristone Glasier et al. RU486 and Norplant treated endometrium Vol. 77, No. 2, February 2002

4 FIGURE 2 Semiquantitative immunostaining scores (median value) of progesterone receptor (PgR) (A and B isoforms), progesterone receptor B (PgR B ) isoform, and estrogen receptor (ER) in endometrium after placebo and after mifepristone. 0 no immunostaining; 3 intense immunostaining. treatment, and from the same woman and one other following mifepristone administration was inadequate for analysis; thus, seven biopsies were available after placebo treatment and six after mifepristone. Steroid Receptor Immunostaining The response of receptor expression to the administration of mifepristone in endometrium exposed to Norplant is shown in Figure 2. Levonorgestrel-exposed endometrium displayed intense immunostaining for PR subtype A B in both stromal and glandular cells. Administration of mifepristone was not associated with any change in this pattern. Endometrium exposed to levonorgestrel displayed a lower level of immunostaining for PR subtype B than for PR A B in both glands and stroma; PR B expression was significantly further reduced following mifepristone (P.05) (Fig. 3). Estrogen receptor immunoreactivity was intense in both glandular and stromal components of the Norplant (levonorgestrel-treated) endometrium. Mifepristone administration was followed by a significant increase in ER immunoreactivity in glandular cells (P.05). Bleeding Patterns, Ovarian Activity, and Cervical Mucus Characteristics Both placebo and mifepristone were administered on the third day of an episode of vaginal bleeding. The mean number of days during which women continued to experience bleeding or spotting after treatment was slightly less (4 days, range: 0 to 6) following mifepristone than after placebo treatment (6 days, range: 0 to 21). The mean number of days when no bleeding or spotting occurred (dry days) following the end of the treatment episode until the onset of the next episode of bleeding or spotting was slightly longer following administration of mifepristone (17 days, range: 8 to 25) than after placebo (14 days, range: 8 to 39). Neither of these observations reached the level of statistical significance. Because the study ended 4 weeks after mifepristone treatment, it was not possible to determine the total number of dry days in every woman; two had experienced no further bleeding by the time the study ended. Following mifepristone administration (see Table 1), three women ovulated and two others had an increase in pregnanediol/creatinine ratio, which did not reach a value consistent with normal ovulation ( 0.5). In comparison, only two women showed signs of ovulation during the FERTILITY & STERILITY 369

5 FIGURE 3 Strong positive nuclear progesterone receptor (PgR) immunostaining (brown) was observed in glands and stroma of endometrium treated with (a) Norplant and (b) mifepristone-norplant. (c), Lower positive progesterone receptor B (PgR B ) immunostaining was apparent in glands and stroma of Norplant treated endometrium. (d), Positive progesterone receptor B (PgR B ) immunostaining was present in few cells of glands and stroma of mifepristone-norplant treated endometrium. (e), Strong estrogen receptor (ER) immunostaining was observed in glands and stroma of Norplant treated endometrium. (f), Intense estrogen receptor (ER) immunostaining was observed in glands and stroma of endometrium treated with mifepristone- Norplant. 8-week pretreatment period and only one woman ovulated in the 4 weeks following placebo. There were no clear differences identifiable in endometrial steroid receptor immunostaining between the women who did or did not ovulate. In no woman did ovulation occur before the time of the biopsy. No cervical mucus was detectable in any of the women on the day of placebo treatment. On the day of mifepristone administration, cervical mucus was detectable in only one woman, in whom the total score was 6. There were no pregnancies during the course of the study. 370 Glasier et al. RU486 and Norplant treated endometrium Vol. 77, No. 2, February 2002

6 DISCUSSION The findings of this pilot study lend support to our hypothesis that the administration of an antiprogesterone might functionally inhibit the actions of levonorgestrel, despite its continued delivery from Norplant implants. A possible mechanism for this is as follows. The predominant steroid receptor pattern in levonorgestrel-exposed endometrium is characterized by maximal expression of subtype A with a reduction in expression of both PR subtype B and estrogen receptors. This is a similar pattern to that observed during the luteal phase of the normal cycle and one where it is associated with endometrial breakdown and vaginal bleeding. Thus, the chronic exposure to low-dose levonorgestrel is associated with an endometrium constantly liable to degenerate. Mifepristone administration causes further reduction of the expression of PR B receptors, amplifying the lack of progesterone action and inducing menstruation. The simultaneous up-regulation of estrogen receptors induces the factors responsible for endometrial proliferation, causing cessation of bleeding and a period of bleed-free days until endometrial shedding recurs as the background pattern of endometrial receptor status associated with Norplant (high PR A, low PR B, and low ER) is restored. The actual mechanism by which the steroids act on the endometrial vasculature is unknown. However, increased immunostaining for matrix metalloproteinases (MMPs), which are involved with tissue breakdown, has been described in the endometrium of Norplant users (20) and these enzymes have been implicated in endometrial breakdown at the onset of menstruation in the normal cycle (7). Like other investigators (12, 13), we have shown in this study that PR concentrations are high in both the glandular and stromal cells of the endometrium of women exposed to levonorgestrel delivered by Norplant. We have also demonstrated, for the first time, the presence of PR subtype B receptors in endometrium exposed to Norplant. It is clear from Figure 3 that PR B immunoreactivity in glandular and stromal cells is much lower than the immunoreactivity for PR A B, which we infer to represent PR subtype A (18). Mifepristone administration resulted in further depression of the B subtype receptor. The simultaneous up-regulation of estrogen receptors as a result of mifepristone administration may be an indication of the functional withdrawal of progesterone and its effects on the endometrium. There are no data on endometrial vessel changes in women using Norplant, who have been exposed to an antigestogen. We and others have reported that the PR A isoform is likely to be the dominant isoform in the secretory phase of the cycle (18, 21). Moreover, PR A is the dominant form in differentiating stromal cells in vitro. The abundance of PR A declines in vitro during the decidualization process (22). A decline in PR levels, either PR A or PR B, will be of functional importance as there is differential gene activation. Moreover, PR A may act as a repressor of PR B function, and the expression ratios of the two subtypes will determine the response to progesterone/progestogen (21, 23). Mifepristone may also have an effect on the hypothalamus, stimulating positive feedback by blocking the inhibitory action of progesterone and inducing ovulation (16), an action which, independent of any direct effect on the endometrium, would be sufficient to improve bleeding patterns. Certainly there was a suggestion in our study that ovulation was more likely to occur during the 4 weeks after mifepristone administration than during the weeks before and after placebo. Cheng et al. (11) demonstrated an improvement in bleeding patterns when mifepristone was administered to women experiencing bleeding dysfunction while using the levonorgestrel contraceptive implant. In that study ovarian activity was not monitored, so we do not know whether the improvement was due to induction of regular ovulation or to some other direct effect on the endometrium as described above. Bleeding patterns tend to improve with the duration of use of Norplant (11, 24), probably because as circulating concentrations of levonorgestrel fall ovulation becomes more frequent about one-third of women ovulate regularly. The altered endometrial vessel morphology described by Hickey et al. (25) coincides with the time when bleeding problems are most common. In our study, the duration of Norplant use varied between 4 and 24 months, but only two women showed evidence of ovulation during the pretreatment period (8 weeks) and all were experiencing frequent bleeding episodes. It is possible that mifepristone may have a different effect depending on the duration of Norplant use, but the sample size in this small pilot study was insufficient to assess this. It is possible then that the regular administration of an antiprogesterone for example, once each month might be a useful strategy for reducing the side effects of low-dose progestogen-only contraceptives, thereby increasing both acceptability and continuation rates. Norplant is a highly effective method of contraception that has three mechanisms of action. It inhibits ovulation, prevents normal sperm transport through the female genital tract (particularly through the cervix), and is associated with endometrial atrophy (24). If mifepristone administration increases the number of ovulatory cycles, it might reduce the contraceptive efficacy of Norplant at the expense of inducing more tolerable bleeding patterns. Ovulation is infrequent during the first 2 years of Norplant, but it increases with time as discussed earlier. However, the cumulative pregnancy rate at the end of 5 years is only 1.1%, and thus it would seem unlikely that an increase in the frequency of ovulation following mifepristone would significantly increase the failure rate of Norplant. In the clinical trial of once a month mifepristone admin- FERTILITY & STERILITY 371

7 istration (11), there were no pregnancies during 300 womanmonths of use. Contraceptive efficacy could also be reduced if antiprogesterone improved the quality of cervical mucus. No woman showed any evidence of having fertile mucus at the time when placebo and mifepristone were given. It is however possible that the functional inhibition of progesterone by mifepristone might induce fertile mucus, which we failed to detect as we did not collect samples within days of mifepristone administration. Although ours was only a small pilot study, our observations suggest that further investigation of the effects of an antiprogesterone on the endometrium, and on vaginal bleeding patterns, in women using long-acting low-dose progestogen-only contraceptives would be worthy of future study. Acknowledgments: The authors thank Mrs. Ann Mayo for assistance with sample collection, Mrs. Teresa Henderson for technical support, Mrs. Audrey Duncan for secretarial help, and Tom McFetters and Ted Pinner for help with the illustrations. References 1. Anderson FD. Bleeding and contraceptive steroids. In: Alexander NJ, d Arcangues C, eds. Steroid hormones and uterine bleeding. Washington DC: AAAS Press, 1992: Findlay JK. Future direction for research on endometrial bleeding. Hum Reprod 1996;11(suppl 2): Peers T, Stevens JE, Graham J, Davey A. Norplant Implants in the UK: first year continuation and removals. Contraception 1996;53: Davie J, Hiremath K, Glasier A. The introduction of a new contraceptive: two years experience with Norplant. Health Bulletin 1996;54: Fraser IS. Bleeding arising from the use of exogenous steroids. Baillieres Best Pract Res Clin Obstet Gynaecol 1999;13: Critchley HOD, Brenner RM, Henderson TA, Williams K, Nayak NR, Slayden OVD, et al. 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