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The effects of progesterone receptor blockade in the luteal phase of normal fertile women
FERTILITY AND STERILITY
Vol. 50, No.5, November 1988
Printed in U.S.A.
Copyright" 1988 The American Fertility Society
The effects of progesterone receptor blockade in the luteal phase of normal fertile women Tin-Chiu Li, M.R.C.P., M.R.C.O.G.*t:j: Peter Dockery, Ph.D.§~ Peter Thomas, B.Sc.ll
Andrew W. Rogers, Ph.D, D.Sc.§ Elizabeth A. Lenton, Ph.D.t~ Ian D. Cooke, F.R.C.O.G.t~
University of Sheffield, Jessop Hospital for Women, Sheffield, and Roussel Laboratories Ltd., Uxbridge, England
The effects of a single, variable dose (5 to 200 mg) ofRU38486 (RU486) (Roussel-Uclaf, Paris, France) in the first half of the luteal phase of the menstrual cycle were studied in 30 normal fertile volunteers. Quantitative histologic techniques were employed, and the results were compared with normal ranges derived from a separate, normal, fertile population. It was found that RU486 inhibited glandular secretory activity, accelerated degenerative changes, induced various vascular changes, increased stromal but not glandular mitotic activity, and did not affect the predecidual reaction. The superiority of morphometric analysis over traditional dating criteria was demonstrated in this study of endometrial responses to an exogenously administered agent. In addition, it was found that temperature drop occurred in 56%, menstrual induction in 43%, luteolysis in 24%, mood change in 23%, and thirst sensation in 3% of women studied. Both menstrual induction and changes in hypothalamic function after the administration of RU486 occurred independently of luteolysis and so were likely to be the direct result of progesterone receptor blockade in these organs. Menstrual induction was significantly related to the dose given and the day on which RU486 was administered. Mood change was significantly related to the day on which RU486 was given. Most of the effects of RU486 occurred around 48 hours after its administration. Fertil Steril 50:732 1988
Antagonists have been developed to the three classes of reproductive steroid hormones: estrogens, androgens and progestogens. Antiestrogens include clomiphene citrate and tamoxifen, which are important and commonly used in the induction of ovulation and treatment of breast cancer. Antiandrogens such as cyproterone acetate are curReceived April5, 1988; revised and accepted July 14, 1988. * Research Fellow supported by a grant from Roussel Laboratories Ltd. t Department of Obstetrics and Gynaecology, University of Sheffield. :j: Reprint requests: Tin-Chiu Li, M.R.C.P., M.R.C.O.G., Jessop Hospital for Women, Sheffield, S3 7RE, England. § Department of Anatomy and Cell Biology, University of Sheffield. 'If Harris Birthright Centre for Reproductive Medicine, Jessop Hospital for Women. \1 Statistician, Roussel Laboratories Ltd.
732
Li et al.
Effects of P receptor blockade
rently available for the treatment of hirsutism in the female and hypersexuality in the male. The earlier progesterone (P) antagonists such anordrin and ORF 9371 suffered the important disadvantages of having either androgenic side effects or low efficacy and were therefore not considered clinically useful. A breakthrough occurred in 1982 when RU38486 or RU486 (Roussel-Uclaf, Paris, France) was synthesized from norethisterone. A leading article from the British Medical Journal 1 addressed the potential clinical applications of P antagonists and highlighted the particular value of RU486. The major clinical application ofRU486 is likely to be in contraception and contragestion. RU486 may be given in the late follicular phase to inhibit the luteinizing hormone (LH) surge and suppress ovulation, 2 •3 in the early luteal phase as postcoital Fertility and Sterility
contraception,3 in the mid and late luteal phase to prevent successful implantation and induce menstruation,4-6 or in the first trimester of pregnancy to achieve abortion, either alone 7•8 or in combination with a prostaglandin. 9 Other potential clinical applications of RU486 include treatment of breast cancer, 10 induction of labor11 including cases with intrauterine death/ 2 treatment of Cushing's syndrome, 13 and treatment of open -angle glaucoma. 14 In this volunteer study, the aim was to investigate the effects of P-receptor blockade, by single variable doses of RU486 on endometrial development, menstrual induction, and hypothalamic and corpus luteum function simultaneously in the luteal phase of a group of normal fertile women.
MATERIALS AND METHODS Subjects
All women participating in this study were healthy volunteers who had previously had, or were seeking, tubal sterilization. In order to obtain a homogeneous population of women with normal endometrium prior to the administration of the drug, women were only considered for entry into the study if they fulfilled the following criteria: (1) previously fertile; (2) had normal regular menstruation between 25 and 35 days; (3) aged between 18 and 40 years; (4) did not have uterine pathology, e.g., fibroid, or abnormal menstrual bleeding; (5) had not used an intrauterine contraceptive device, oral contraception, or any hormone that might disturb steroid hormone concentrations or endometrial morphology, for at least two months prior to the study; and (6) in good health. In addition, it was considered that women recruited for the study should have a baseline endometrial biopsy (EB-1) performed just before the administration of RU486 to ensure that they had normal endometrium before the study. Should this biopsy specimen be found to be abnormal, the woman would be excluded from the study. Administration of RU 486
A single dose of RU 486 was given in the first half of the luteal phase, between days 2 and 6 after the LH surge. In order that the administration of RU486 could be timed precisely, all women participating in the study had daily LH monitoring, startVol. 50, No.5, November 1988
ing from the mid follicular phase, to identify the LH surge, as described elsewhere. 15 As there was no information in the literature about the morphologic response of the endometrium to different doses of RU486, an arbitrary dose range, from 5 mg to 400 mg was proposed. The possible doses included 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, and 400 mg. An arbitrary, starting dose of 200 mg was used. In order to determine the range of effective doses of RU486 more efficiently, an up-and-down design was used. One of the single response methods reviewed by Bolognese16 was modified, so that patients were dosed in groups of three. The number of responders in each group would govern the next dose to be given, as follows: if number of responders = 3, then dose increment = -2; if number of responders = 2; then dose increment = -1; if number of responders = 1, then dose increment = +1; if number of responders = 0, then dose increment
= +2. The endometrium was considered to have responded to RU486 if it was significantly advanced or retarded from its expected structure by more than 2 days, according to the criteria of Noyes et al. 17 Analysis of Endometrial Response
An experimental endometrial biopsy (EB-2) was performed 3 days after the administration of RU486. This procedure has been described elsewhere.15 Histologic analysis of these EB-2s was initially carried out by using the dating criteria of Noyes et al. 17 However, during the course of the study, this particular method of analysis was found to be unsatisfactory (see discussion). As a result, the technique of morphometric analysis 18 was later added to the histologic assessment of all the endometrial specimens. Control Experiment on Endometrial Response
The study described above involved taking an EB-1 just before the administration of RU486, and then taking another biopsy specimen (EB-2) 3 days later to evaluate the impact of RU486 on endometrial development. A question arose as to whether the first EB might have any effect on endometrial development 3 days later? For this reason, a separate control experiment was performed in which four women who were regularly cycling had two EBs taken 3 days apart in the luteal phase, without the administration of RU486. Li et al.
Effects of P receptor blockade
733
Daily Salivary Progesterone Assay
Women participating in the study were asked to start collecting daily samples of saliva from day 9 of the cycle. They were asked to expectorate 3 ml of saliva at approximately the same time each day. Progesterone concentration in the saliva was determined by a direct radioimmunoassay method19 similar to that described by Chearskul et al., 20 using 125I progesterone (ER-205, Steranti Research Laboratory Ltd., St. Albans, England). Basal Body Temperature
Women participating in the study were asked to record their daily morning temperature starting from day 9 of the cycle for up to 28 days. Menstrual Diary
Each woman recruited to the study was asked to keep a daily record of any vaginal bleeding for 3 months, using the following categories: no bleeding, spotting, light bleeding, moderate bleeding, and heavy bleeding.
For each morphometric measurement, as 30 individual results were compared with the nomogram, it was possible that 5% of these 30 results (1.5) might fall outside the normal range by chance alone. With the Poisson distribution for rare events, 21 it was calculated that the chance of up to 4 of the 30 results falling outside the normal range in normal subjects was <6%, but for 5 or more of the 30 results to fall outside the normal range in normal subjects was <0.019%. Therefore, one may consider that if 5 or more of the 30 results were outside the normal range, this would be unlikely to have arisen by chance, but would more likely be due to the effect of RU486. In addition, results of an individual parameter falling outside the normal range by chance would have equal probability of lying above or below the normal range. However, reference to Figure 1 demonstrates that most measurements deviated from · the normal range in a consistent manner, i.e., either below or above the normal range. Such patterns of deviation may be taken as evidence of a response to RU486.
Symptom Check List
A symptom check list including mood changes such as irritability, depression, insomnia, and others including dizziness, fatigue, nausea, vomiting, and diarrhea was used both before and after the administration ofRU486. When present, the severity of each was recorded and coded: mild, moderate, and severe. Any additional symptom not included in the check list was also asked for, and the time of onset of any of these symptoms was noted. Ethical Considerations
This study was approved by the Southern Hospitals Ethics Committee of Sheffield, United Kingdom. All volunteers participating in the study had explained to them the purpose of the study, what would be involved, the possible side effects of RU486, and their freedom to withdraw from the study at any time. Written consent was obtained from each of the participating volunteers. Statistics
The normal range of morphologic features throughout the luteal phase was derived from 2.5 to 97 .5th percentile of 70 EBs collected from a normal, fertile population as reported earlier by Li et al. 18 734
Li et al.
Effects of P receptor blockade
RESULTS Control Experiment
The results of the control experiment demonstrated that in all four cases, histologic dating of the endometrium for EB-2 was in good agreement with chronologie dating from the LH surge. The only unusual histologic finding of note was the presence of increased leucocytic infiltration, in one subject. As leukocytic infiltration is one of the original dating criteria proposed by Noyes et al., 17 this observation must be considered in the subsequent analysis of data in the main study. Main Study
In this study, a total of 76 normal, fertile women who requested sterilization or reversal of sterilization were initially interviewed with a view to recruitment; however, only 37 fulfilled the inclusion criteria and agreed to participate in the study. Of these, three did not produce a clear LH surge in the urine samples and were therefore excluded. The remaining 34 women who participated in the study all received a dose ofRU486, but 4 were eventually excluded because the histologic dating of their EB1s showed significant disparity with the chronologie dating. Thus, 30 women were included in the Fertility and Sterility
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Days from LH peak Figure 1 The effects of RU486 administration on endometrial morphology. Results of individual morphometric measurements 3 days after the administration of a variable dose of RU486 (e, 5 to 50 mg, /::;, 75 to 200 mg) are shown on a background of the normal range established from 70 fertile women as described by Li et al. 18 G1, number of mitoses per 1000 gland cells; G2, gland cell height (~m); G3, amount of secretion in gland lumen (score 0 to 3); G4, maximal gland diameter (~m); G5, volume fraction of gland occupied by gland cell; G6, volume fraction of endometrium occupied by gland; G 7, volume fraction of gland cell occupied by nucleus; G8, number of supranuclear secretory vacuoles per 100 gland cells; G9, number of subnuclear vacuoles per 100 gland cells; G 10, amount of pseudostratification of gland cell (score 0 to 3); G 11, number of apoptotic bodies per 1000 gland cells; S1, number of mitoses per 1,000 stromal cells; S2, amount of stromal oedema (score 0 to 3); S3, amount of predecidual reaction (score 0 to 3); S4, diameter of venule (~m); S5, amount of leukocyte infiltration (score 0 to 3); S6, amount of extravasation (score 0 to 3). Vol. 50, No.5, November 1988
Li et al.
Effects of P receptor blockade
735
final analysis of results. Among those included in the study, three received 200 mg, three received 150 mg, two received 100 mg, three received 75 mg, six received 50 mg, seven received 25 mg, three received 10 mg, and three received 5 mg of RU486. Among these 30 women, 5 received RU486 on day LH +2, 5 on day LH +3, 5 on day LH +4, 4 on day LH +5, and 11 on day LH +6. The mean age of this group of woman was 33.2 years (standard deviation [SD] 3.6, range 24 to 39). Traditional Dating Criteria
Of the 30 cases, 12 were considered to have normal histologic dating according to the dating criteria of Noyes et aLP 8 were considered to be retarded, and the remaining 10 were considered to be advanced. All 10 classified as advanced showed a "menstrual" structure by these criteria. Morphometric Analysis: Histologic Dating
The use of morphometric analysis in conjunction with multiple regression analysis to carry out histologic dating of endometrium has been described by Li et al. 18 Among the 30 cases, dating by morphometric analysis suggested that endometrial development was retarded in 17 cases (57%), normal in 13 cases (43%), whereas none of them was considered to have advanced endometrial development. Morphometric Analysis: Individual Morphologic Features
The effects of RU486 on the various morphologic features 3 days after its administration are shown in Figure 1. According to the Poisson distribution for rare events, significant changes (P < 0.05) were observed in 15 out of 17 morphologic features (Table 1). Menstrual Induction
After the administration of RU486, 13 out of 30 (43%) women had menstruation induced 2 days later. Of these 13 women, 6 had a further menstruationa few days later, around the time expected for that cycle, whereas 5 did not and the remaining 2 defaulted from follow-up. The time between the administration of RU486 and the onset of menstruation was found to have a narrow range of 40 to 57 hours with a median of 43 hours. 736
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Effects of P receptor blockade
Table 1 The Frequency of Positive Response (see text) of Individual Morphometric Measurements After the Administration of a Variable Dose of RU486, in Descending Order of Frequency Morphometric measurement
Frequency of positive response
86 G10 G3 Gll 82 G8 85 G9 84 G4 G5 G7 G6 81 G2 83 G1
25/30 (83%) 4 24/30 (80%) 4 18/30 (60%) 4 18/30 (60%) 4 17/30(57%) 4 16/30 (53%) 4 16/30 (53%) 4 15/30 (50%) 4 8/18 (44%) 4 13/30 (43%) 4 11/30 (37%) 4 9/30 (30%) 4 8/30 (27%) 4 8/30 (27%) 4 6/30 (20%) 4 3/30 (10%) 0/30 (0%)
a Likely that the response was due to RU486, according to Poisson distribution for rare events (P < 0.05).
Salivary Progesterone Profile
Of the 30 women who were included in the study, 17 completed daily saliva collections for progesterone assay. Among these 17 women, 4 (24%) had a fall of salivary progesterone concentration within 3 days after the administration of RU486 to below the normal range established in our laboratory (Fig. 2) and may be considered to have had luteolysis induced by RU486. Luteolysis induced by RU486 was correlated to menstruation induced by this agent in Table 2. Temperature Regulation
Sixteen women completed daily basal temperature records in the cycle of study. Among these women, nine (56%) had a significant fall in the temperature within 3 days after the administration of RU486, examples of which are shown in Figure 3. Among those who had a significant fall in temperature, six also had simultaneous daily salivary progesterone profile for comparison. Only one out of these six women had both a fall in temperature and progesterone profile (luteolysis), whereas the remaining five had a fall in temperature but did not have luteolysis. All the observed falls in temperature occurred within 3 days after the administration ofRU486. Mood Change and Thirst Sensation
After the administration of RU486, 7 of the 30 women (23%) had mood change, in the formofirriFertility and Sterility
A
Table 3 The Relationship Between the Presence or Absence of a Response of the Various Endometrial Features (Measurements) to the Dose of RU486 Given (Dose), or the Day of the Luteal Phase on Which RU486 Was Given (Date)"
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Figure 2 The effects of a single, variable dose (5 to 200 mg) of RU486 on daily saliva progesterone profile. Arrow indicates the day of administration of RU486. The continuous line represents the profile of women who had received a dose of RU486. The dotted line represents the 5th centile of a normal population of women and so the lower limit of the normal range. (A) An example (subject 28) demonstrating that a significant fall of saliva progesterone concentration occurred after the administration of RU486 (luteolysis) in some cases. (B) An example (subject number 22) demonstrating that no apparent fall of saliva progesterone concentration was observed after the administration of RU486 in other cases.
tability or depression, whereas the remaining 23 women (73%) did not have any noticeable mood change. Analysis of the relationship between mood change and luteolysis by 2 X 2 contingency table demonstrated no significant association between the two. Further analysis suggests that mood change could occur in the absence of luteolysis induced by RU486.
One of the 30 women experienced marked thirst sensation after the administration of RU486. This was not associated with luteolysis. Again, all these changes were reported to occur on days 1 and 2 (mainly day 2) after the administration ofRU486, and none of them persisted after day3. 37.0 36.6 36.2
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Table 2 2 X 2 Contingency Table Showing the Relationship Between Luteolysis and Menstruation Induced by RU486"
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Vol. 50, No.5, November 1988
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Days from LH peak Figure 3 The effects of a single, variable dose (5 to 200 mg) of RU486 on basal body temperature. Arrow indicates the day of administration of RU486. A, Subject 17; B, Subject 22; C, Subject 28. The responses in A and B were not associated with luteolysis but C was.
Li et al.
Effects of P receptor blockade
737
Factors Affecting the Response
The relationship between the presence or absence of a response, the dose of RU486 administered, and the day of the luteal phase on which RU486 was given were analyzed by probit-logistic regression analysis. 22 The results of endometrial response are shown in Table 3. A significant relationship was defined when the absolute value of the ratio of the regression coefficient to standard error (z-value) was 1.96 or more. In addition, it was found that significant endometrial response of one or more of the 17 morphologic features were observed throughout the dose range studied (5 to 200mg). Menstrual induction by RU486 was found to be significantly (P < 0.05) related both to the dose and the date, being more likely if the dose was larger and if RU486 was given later in the luteal phase. The drop in salivary progesterone profile or basal body temperature after the administration of RU486 was found to have no significant relationship to the dose or the date. On the other hand, mood change was significantly related to the date (negatively) but not to the dose, i.e., the earlier in the luteal phase RU486 was given, the more likely mood change would be present. DISCUSSION
The effects of RU486 administration in the luteal phase from days LH +2 to LH +6 for a dose range of 5 mg to 200 mg were examined in this study. Glandular Secretory Activities
Preceptor blockade by RU486 in the first half of the luteal phase reduces the amount of glandular secretory activity, as evidenced by reductions in the amount of secretory material in the gland lumen, the maximum gland diameter, and the volume fraction of gland occupied by gland lumen. It is interesting to note the two different effects of RU486 on the number of secretory vacuoles in the gland cells. When RU486 is given on LH +2, before the formation of secretory vacuoles, it suppresses the formation of these vacuoles 3 days later. However, when RU486 is given on days LH +3 to LH +5, when the secretory vacuoles have already been formed, it has the effect of keeping these vacuoles in the gland cells and prevents them from discharging into the lumen. Thus, it is likely that P is involved both in the formation and release 738
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Effects of P receptor blockade
of secretory vacuoles in gland cells. In both cases, progesterone receptor blockade results in reduction of the amount of secretory material being delivered to the gland lumen. It is probable that the observed increase in gland cell height and pseudostratification are both related to reduction in the amount of secretion in the gland lumen. With the lack of secretory material distending the lumen, the glands tend to be partially collapsed, as a result of which the gland cells are more closely packed, thus giving rise to the observed appearance of being taller and narrower, as well as pseudostratified. Degenerative Changes
This study suggests that Preceptor blockade increases the number of apoptotic bodies per 1,000 gland cells. 18 An apoptotic body signifies pro- . grammed cell death and as such represents degenerative changes. Thus, P receptor blockade causes degenerative changes in gland cells. It is likely that the increased number of apoptotic bodies seen in glands, either physiologically toward the end of the luteal phase or pharmacologically via the blockade ofP receptors by RU486, shares a common mechanism, which is P withdrawal. The finding that progesterone receptor blockade causes degenerative changes and reduces glandular secretory activities suggests that RU486 may be an effective postcoital contraceptive agent. Stromal Vascular Changes
P receptor blockade by RU486 induces several changes in the vessels of the stroma. Venule Diameter
Among the 30 EB-2s, the diameter of venules could not be measured in 12 cases, in view of the significant amount of extravasation present in association with induction of menstruation. In the remaining 18 cases, 8 of them (44%) were found to have a significant increase in venule diameter. Such an observation may help to explain some cases of heavy vaginal bleeding when RU486 is used to achieve medical abortion. 3 •8 Stromal Edema
Edema should be regarded as a vascular event. The possible mechanisms underlying the formation of stromal edema include increase of vascular permeability, or increase in oncotic pressure secFertility and Sterility
ondary to constriction in venules. After the administration of RU486, there is a reduction in the amount of stromal edema. It is likely that the reduction of stromal edema by RU486 is, at least partly, explained by its vasodilator effect on venules as described earlier. With dilatation of venules, the oncotic pressure in the arterial end of the capillaries is reduced, thereby reducing the formation of edema. Whether RU486 has an additional effect on the reduction of vascular permeability, which could also lead to reduction in stromal edema, is not known at present. With reduction in stromal edema, the total volume occupied by the stromal component of the endometrium is reduced. As a result, this would give rise to an increase in the volume fraction of the endometrium occupied by glands. In this study, seven out of eight observed cases with an increase in such a volume fraction were associated with, and therefore likely to be explained by, reduction in stromal edema. Stromal Extravasation
Excessive extravasation was seen in 25 out of 30 cases after the administration of RU486. Of these, 13 were associated with successful induction of menstruation, and the presence of extravasation was therefore not surprising. A further four cases were associated with spotting and could similarly be accounted for. The remaining eight cases had evidence of stromal extravasation in the absence of any vaginal bleeding. It is possible that smaller degrees of stromal extravasation may be caused by RU486 without any clinical evidence of vaginal bleeding. It is only when vascular change or extravasation is significant that bleeding from the endometrium results. Leukocytic Infiltration
Although 16 out of 30 cases had excessive leukocytic infiltration in the stroma, such a finding is difficult to interpret for two reasons. Firstly, as shown in the control experiment, leukocytic infiltration may be increased by a prior EB (EB-1) performed 3 days earlier. Thus, the increase in leukocytic infiltration observed may or may not be due to RU486. Secondly, in the presence of extravasation, as in most of these cases, the assessment of leukocytic infiltration became difficult and the results might not be accurate. Mechanism of Vascular Changes
The vascular changes described above may be related to increase in local prostaglandin production Vol. 50, No.5, November 1988
or increased sensitivity of the vessels to prostaglandins. 23- 24 Mitotic Activity
Glandular Mitosis
It is perhaps rather unexpected to find that RU486 has no effect on the number of mitoses seen per 1,000 glandular cells. This is in contrast to the conventional view that P suppresses glandular mitotic activity, which is the basis of its use in the treatment of endometrial hyperplasia and carcinoma. However, the finding in this study that progesterone receptor blockade by RU486 did not increase mitotic activity in gland cells in all the 30 cases rather suggests that the mechanism controlling glandular mitotic activity, at least in the early luteal phase, involves more than P. It is possible that the rapid decline in mitotic activity in the early luteal phase of normal menstrual cycles is related mainly to the parallel decrease in estrogen and intracellular estrogen receptors. The effect of P during this time may be relatively unimportant. Stromal Mitosis
RU486 increased stromal mitotic activity in 8 out of 30 cases. Such an effect of RU486 is more likely if it is administered later in the luteal phase, rather than earlier. This is in contrast to glandular mitosis, which does not seem to be affected by the administration of RU486. Together, such findings suggest that mitotic activity in glands and stroma may be under the influence of different controlling factors. A similar conclusion was reached by Johannisson et al., 25 who observed a discrepancy in the peak frequency of mitoses of the glandular epithelium and the stroma. This is further supported by the observation that, in normal fertile women, glandular mitosis disappears after day LH +4, whereas stromal mitosis persists, although with reduced activity, throughout the rest of the luteal phase. Predecidual Reaction
Preceptor blockade by RU486 had no demonstrable effect on the predecidual reaction in this study. However, as RU486 was given on days when the predecidual reaction is normally absent, a more appropriate conclusion is that RU486 has not been shown to induce the predecidual reaction. The reLi et al.
Effects of P receptor blockade
739
sult of this study would not permit one to conclude whether RU486 blocks predecidual reaction, which requires a separate study to administer RU486 in the latter half of the luteal phase. Volume Fraction of Gland Cell Occupied by Nucleus
An effect ofRU486 is to increase the relative volume of nucleus to cytoplasm. This may be due to two possibilities: (1) the volume of the nucleus is increased, or (2) the volume of the cytoplasm is decreased. However, it is not possible from this study to deduce which is the more likely possibility. Histologic Dating
The results of histologic dating of EBs taken 3 days after the administration ofRU486 (EB-2s) by the two methods (criteria of Noyes et al. 19 and morphometric analysis 20 ) differed significantly. For example, whereas 10 of the 30 EBs (33%) were considered to be significantly advanced according to the criteria of Noyes et al./ 7 none was considered to be significantly advanced according to morphometric analysis. 18 Although it has been shown by Li et al. 18 that histologic dating by morphometric analysis is better than by the criteria of Noyes et al./ 7 we feel that histologic dating, which aims at measuring the overall maturity of the endometrium, suffers from several important disadvantages in studies of this kind. Firstly, the only limited conclusion that could be derived is whether endometrial development is normal, advanced or retarded. As such, it would not have provided a detailed description of how each morphological feature is altered. Secondly, these dating criteria are both unweighted; in situations such as the one after P receptor blockade by RU486, when certain morphologic features appear retarded (e.g., glandular secretory activity), others remain unchanged (e.g., glandular mitotic count) and still others appear advanced (e.g., stromal extravasation), the dating of endometrium then becomes difficult, arbitrary, and highly subjective. In addition, when the dating criteria of Noyes et alP are used, confidence limits of an observation cannot be established and results cannot be quantified. Importance of Baseline EB (EB-1)
In this study, out of 34 women initially recruited, 4 women (12%) were excluded from analysis be740
Li et al.
Effects of P receptor blockade
cause EB-1 demonstrated that endometrial morphology was different from that expected from the LH surge. Two subjects had retarded endometrial development and might well have had an intrinsic receptor defect as an underlying cause of their retarded endometria; thus, their exclusion from the study was considered justified. One subject had a much more advanced endometrium: analysis of her salivary progesterone profile revealed that she probably had a cycle with a very short follicular phase and the urinary "LH surge" was a false-positive result in her luteal phase. Another subject, in retrospect, actually did not produce a clear urinary LH surge, but the result was misjudged. If an EB-1 had not been taken before the administration of RU486, these four cases would have been included in the analysis and would have distorted the results reported. Thus, a strong case is made for performing a biopsy before the administration of RU486 as a means of improving the homogeneity of the population being studied. In contrast, the result of the control experiment demonstrated that a prior biopsy did not influence subsequent morphologic development of the endometrium, apart from an increase in the amount of leukocytic infiltration observed. Menstrual Induction and Luteolysis
The result of this study suggested that there was a significant association between menstrual induction and luteolysis after the administration of RU486. The presence of luteolysis was always associated with menstrual induction, but menstrual induction might occur in the absence of luteolysis. This would suggest that menstrual induction by RU486 is not dependent on luteolysis and provides evidence to support the hypothesis that menstrual induction is due to the direct effect of RU486 on the endometrium. It is still not clear whether luteolysis induced by RU486 in the human is due to the direct effect of the drug on the ovary or indirectly via its effect on the pituitary. A further prospective study designed to examine the simultaneous ovarian and pituitary response to RU486 would be required to answer this question. Hypothalamic Functions
The findings in this study that the administration of RU486 affected the various functions of the hypothalamus (the regulation of temperature, thirst, and mood) provide evidence to support the Fertility and Sterility
fact that hypothalamic functions in the luteal phase of normal fertile women are under the direct influence of P. The observation that temperature fall was associated with luteolysis only in one out of six cases, but not in the remaining five cases, suggests that temperature fall after the administration of RU486 is not dependent on luteolysis (fall in P concentration) but is likely to be a direct result of P receptor blockade in the temperature regulation center of the hypothalamus. Similar conclusions could be drawn of the mechanisms of mood change and thirst sensation after the administration of RU486. Factors Affecting the Various Responses
The endometrial response to P receptor blockade, including menstrual induction and changes in endometrial morphology were both significantly related to the dose of RU486 administered and the day of the luteal phase on which RU486 was given. Mood change was significantly related to the date but not the dose of RU486 administered. In contrast, the response of the corpus luteum and the hypothalamic temperature regulation centre were unrelated to the date or the dose. Such a difference indicates that the response of various target organs (hypothalamus, corpus luteum, and endometrium) to P receptor blockade is modified by different factors.
between endometrial development and P withdrawal (caused by Preceptor blockade). From the results reported in this study, one may speculate that RU486 could be an effective postcoital contraceptive agent. However, proof of this would require a separate clinical study. This study is the first ever reported which employs quantitative morphometric techniques to investigate the effects of an exogenous compound (in this case, an antiprogesterone, RU486) on endometrial development, with reference to normal ranges derived from a controlled normal population. The detailed, quantitative information obtained from such a method of analysis is undoubtedly superior to the traditional method of histologic analysis, i.e., criteria of Noyes et at.I 7 Future studies on the response of endometrium to exogenous agents should benefit from such an improved, rigorous approach to analysis. In this study, the simultaneous observation of the various effects of RU486 in the same group of women provided a unique opportunity to analyse the relationship among the responses of different target tissues to P receptor blockade by RU486 . Acknowledgments. The authors are grateful to Mrs. Christine Piggott, Ian Roberts, M.B.B.S., Jane Stevens, B.Sc., Mrs. Gillian Burkinshaw, Mrs. Linda Highfield, and the nursing staff of the University Department of Obstetrics and Gynaecology at Jessop Hospital for Women, Sheffield, for their valuable help in this study.
Temporal Relationship
It is of interest to note that after the administration of a single dose of RU486, most effects occurred around 48 hours. Menstruation induced by RU486 occurred over a narrow range of 40 to 57 hours, with a median of 43 hours. All 4 cases of observed luteolysis started 2 to 3 days after the administration of RU486. The same applied to the fall in basal body temperature. Mood change experienced by women also occurred almost exclusively on day 2 after the administration of RU 486, and none of them persisted for more than 72 hours after the administration of RU486. It is likely that such a finding will have important clinical applications in further studies of the various effects resulting from P receptor blockade.
REFERENCES 1. Healy DL, Fraser HM: The antiprogesterones are coming:
2.
3.
4.
Clinical Aspects
The findings in this study have provided a detailed and quantitative account of the relationship Vol. 50, No.5, November 1988
5.
menses induction, abortion and labour? Br Med J 290:580, 1985 Collins RL, Healy DL, Hodgen GD: Effects of RU486 on pulsatile gonadotropin secretion in monkeys throughout the ovarian menstrual cycle. (Abstr. 72). Presented at The Forty-First Annual Meeting of The American Fertility Society, Chicago, Illinois, September 28 to October 2, 1985. Published by the American Fertility Society, Birmingham Alabama, p 40 Herrmann WL, Schindler AM, Wyss R, Bischof P: Effects of the antiprogesterone RU486 in early pregnancy and during the menstrual cycle. In The Antiprogestin Steroid RU486 and Human Fertility Control, Edited by EE Baulieu, SJ Segal. New York, Plenum Press, 1985, p 179 Shoupe D, Mishell DR, Lacarra M, Gutierrez E, Lahteenmaki P, Spitz IM: Endocrinologic effects of the antiprogesterone RU486 in the luteal phase of normal women. In The Antiprogestin Steroid RU486 and Human Fertility Control, Edited by EE Baulieu, SJ Segal. Plenum Press, New York, 1985, p 285 Nieman LK, Healy DL, Spitz IM, Merriam GR, Bardin CW, Loriaux DL, Brousos GP: Use of single doses of the
Li et al.
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741
antiprogesterone steroid RU486 for induction of menstruation in normal women. In The Antiprogestin Steroid RU486 and Human Fertility Control, Edited by EE Baulieu, SJ Segal. New York, Plenum Press, 1985, p 279 6. Schaison G, George M, Lestrat N, Baulieu EE: RU486 in women with normal or anovulatory cycles. In The Antiprogestin Steroid RU486 and Human Fertility Control, Edited by EE Baulieu, SJ Segal. New York, Plenum Press, 1985, p 271, 1985 7. Kovacs L, Sas M, Resch BA, Ugocsai G, Swahn ML, Bygdeman M, Rowe RJ: Termination of very early pregnancy by RU486-an antiprogesterone compound. Contraception 29:399, 1984 8. Vervest HAM, Haspels AA: Preliminary results with the antiprogestational compound RU-486 (mifepristone) for interruption of early pregnancy. Fertil Steril44:627, 1985 9. Bygdeman M, Swah M-L: Progesterone receptor blockade. Effect on uterine contractility and early pregnancy. Contraception 32:45, 1985 10. Bardon S, Vignon F, Chalbos D, Rochefort H: RU486, a progestin and glucocorticoid antagonist, inhibits the groWth of breast cancer cells via the progesterone receptor. J Clin Endocrinol Metab 50:692, 1985 11. Baulieu EE, Ulmann A: Antiprogesterone. activity of RU486 and its contragestive and other applications. Hum Reprod 1:107, 1986 12. Cabrol D, Bouvier d'Yvoire M, Mermet E, Cedard L, Sureau C, Baulieu EE: Induction of labour with mifepristone after intrauterine fetal death. Lancet 2:1019, 1985 13. Nieman LK, Crousos GP, Kellner C, Spitz IM, Nisula BC, Cutler GB, Merriam GR, Bardin CW, Loriaux DL: Successful treatment of Cushing's syndrome with the glucocorticoid antagonist RU486. J Clin Endocrinol Metab 61:536, 1985 14. Philips CI, Green K, Gore SM, Cullen PM, Campbell M: Eye drops of RU486-6, a peripheral steroid blocker, lower intra-ocular pressure in rabbits. Lancet 1:767, 1984 15. Li T-C, Rogers AW, Lenton EA, Dockery P, Cooke ID: A comparison between two methods of chronological dating
742
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of human endometrial biopsies during the luteal phase, and their correlation with histologic dating. Fertil Steril48:928, 1987 16. Bolognese JA: A Monte Carlo comparison of three up-anddown designs for dose ranging. Controlled Clinical Trials 4: 187,1983 17. Noyes RW, Hertig AT, Rock J: Dating the endometrial biopsy. Fertil Steril1:3, 1950 18. Li T -C, Rogers AW, Dockery P, Lenton EA, Cooke ID: A new method of hist
Fertility and Sterility
Vol. 50, No.5, November 1988
Printed in U.S.A.
Copyright" 1988 The American Fertility Society
The effects of progesterone receptor blockade in the luteal phase of normal fertile women Tin-Chiu Li, M.R.C.P., M.R.C.O.G.*t:j: Peter Dockery, Ph.D.§~ Peter Thomas, B.Sc.ll
Andrew W. Rogers, Ph.D, D.Sc.§ Elizabeth A. Lenton, Ph.D.t~ Ian D. Cooke, F.R.C.O.G.t~
University of Sheffield, Jessop Hospital for Women, Sheffield, and Roussel Laboratories Ltd., Uxbridge, England
The effects of a single, variable dose (5 to 200 mg) ofRU38486 (RU486) (Roussel-Uclaf, Paris, France) in the first half of the luteal phase of the menstrual cycle were studied in 30 normal fertile volunteers. Quantitative histologic techniques were employed, and the results were compared with normal ranges derived from a separate, normal, fertile population. It was found that RU486 inhibited glandular secretory activity, accelerated degenerative changes, induced various vascular changes, increased stromal but not glandular mitotic activity, and did not affect the predecidual reaction. The superiority of morphometric analysis over traditional dating criteria was demonstrated in this study of endometrial responses to an exogenously administered agent. In addition, it was found that temperature drop occurred in 56%, menstrual induction in 43%, luteolysis in 24%, mood change in 23%, and thirst sensation in 3% of women studied. Both menstrual induction and changes in hypothalamic function after the administration of RU486 occurred independently of luteolysis and so were likely to be the direct result of progesterone receptor blockade in these organs. Menstrual induction was significantly related to the dose given and the day on which RU486 was administered. Mood change was significantly related to the day on which RU486 was given. Most of the effects of RU486 occurred around 48 hours after its administration. Fertil Steril 50:732 1988
Antagonists have been developed to the three classes of reproductive steroid hormones: estrogens, androgens and progestogens. Antiestrogens include clomiphene citrate and tamoxifen, which are important and commonly used in the induction of ovulation and treatment of breast cancer. Antiandrogens such as cyproterone acetate are curReceived April5, 1988; revised and accepted July 14, 1988. * Research Fellow supported by a grant from Roussel Laboratories Ltd. t Department of Obstetrics and Gynaecology, University of Sheffield. :j: Reprint requests: Tin-Chiu Li, M.R.C.P., M.R.C.O.G., Jessop Hospital for Women, Sheffield, S3 7RE, England. § Department of Anatomy and Cell Biology, University of Sheffield. 'If Harris Birthright Centre for Reproductive Medicine, Jessop Hospital for Women. \1 Statistician, Roussel Laboratories Ltd.
732
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Effects of P receptor blockade
rently available for the treatment of hirsutism in the female and hypersexuality in the male. The earlier progesterone (P) antagonists such anordrin and ORF 9371 suffered the important disadvantages of having either androgenic side effects or low efficacy and were therefore not considered clinically useful. A breakthrough occurred in 1982 when RU38486 or RU486 (Roussel-Uclaf, Paris, France) was synthesized from norethisterone. A leading article from the British Medical Journal 1 addressed the potential clinical applications of P antagonists and highlighted the particular value of RU486. The major clinical application ofRU486 is likely to be in contraception and contragestion. RU486 may be given in the late follicular phase to inhibit the luteinizing hormone (LH) surge and suppress ovulation, 2 •3 in the early luteal phase as postcoital Fertility and Sterility
contraception,3 in the mid and late luteal phase to prevent successful implantation and induce menstruation,4-6 or in the first trimester of pregnancy to achieve abortion, either alone 7•8 or in combination with a prostaglandin. 9 Other potential clinical applications of RU486 include treatment of breast cancer, 10 induction of labor11 including cases with intrauterine death/ 2 treatment of Cushing's syndrome, 13 and treatment of open -angle glaucoma. 14 In this volunteer study, the aim was to investigate the effects of P-receptor blockade, by single variable doses of RU486 on endometrial development, menstrual induction, and hypothalamic and corpus luteum function simultaneously in the luteal phase of a group of normal fertile women.
MATERIALS AND METHODS Subjects
All women participating in this study were healthy volunteers who had previously had, or were seeking, tubal sterilization. In order to obtain a homogeneous population of women with normal endometrium prior to the administration of the drug, women were only considered for entry into the study if they fulfilled the following criteria: (1) previously fertile; (2) had normal regular menstruation between 25 and 35 days; (3) aged between 18 and 40 years; (4) did not have uterine pathology, e.g., fibroid, or abnormal menstrual bleeding; (5) had not used an intrauterine contraceptive device, oral contraception, or any hormone that might disturb steroid hormone concentrations or endometrial morphology, for at least two months prior to the study; and (6) in good health. In addition, it was considered that women recruited for the study should have a baseline endometrial biopsy (EB-1) performed just before the administration of RU486 to ensure that they had normal endometrium before the study. Should this biopsy specimen be found to be abnormal, the woman would be excluded from the study. Administration of RU 486
A single dose of RU 486 was given in the first half of the luteal phase, between days 2 and 6 after the LH surge. In order that the administration of RU486 could be timed precisely, all women participating in the study had daily LH monitoring, startVol. 50, No.5, November 1988
ing from the mid follicular phase, to identify the LH surge, as described elsewhere. 15 As there was no information in the literature about the morphologic response of the endometrium to different doses of RU486, an arbitrary dose range, from 5 mg to 400 mg was proposed. The possible doses included 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, and 400 mg. An arbitrary, starting dose of 200 mg was used. In order to determine the range of effective doses of RU486 more efficiently, an up-and-down design was used. One of the single response methods reviewed by Bolognese16 was modified, so that patients were dosed in groups of three. The number of responders in each group would govern the next dose to be given, as follows: if number of responders = 3, then dose increment = -2; if number of responders = 2; then dose increment = -1; if number of responders = 1, then dose increment = +1; if number of responders = 0, then dose increment
= +2. The endometrium was considered to have responded to RU486 if it was significantly advanced or retarded from its expected structure by more than 2 days, according to the criteria of Noyes et al. 17 Analysis of Endometrial Response
An experimental endometrial biopsy (EB-2) was performed 3 days after the administration of RU486. This procedure has been described elsewhere.15 Histologic analysis of these EB-2s was initially carried out by using the dating criteria of Noyes et al. 17 However, during the course of the study, this particular method of analysis was found to be unsatisfactory (see discussion). As a result, the technique of morphometric analysis 18 was later added to the histologic assessment of all the endometrial specimens. Control Experiment on Endometrial Response
The study described above involved taking an EB-1 just before the administration of RU486, and then taking another biopsy specimen (EB-2) 3 days later to evaluate the impact of RU486 on endometrial development. A question arose as to whether the first EB might have any effect on endometrial development 3 days later? For this reason, a separate control experiment was performed in which four women who were regularly cycling had two EBs taken 3 days apart in the luteal phase, without the administration of RU486. Li et al.
Effects of P receptor blockade
733
Daily Salivary Progesterone Assay
Women participating in the study were asked to start collecting daily samples of saliva from day 9 of the cycle. They were asked to expectorate 3 ml of saliva at approximately the same time each day. Progesterone concentration in the saliva was determined by a direct radioimmunoassay method19 similar to that described by Chearskul et al., 20 using 125I progesterone (ER-205, Steranti Research Laboratory Ltd., St. Albans, England). Basal Body Temperature
Women participating in the study were asked to record their daily morning temperature starting from day 9 of the cycle for up to 28 days. Menstrual Diary
Each woman recruited to the study was asked to keep a daily record of any vaginal bleeding for 3 months, using the following categories: no bleeding, spotting, light bleeding, moderate bleeding, and heavy bleeding.
For each morphometric measurement, as 30 individual results were compared with the nomogram, it was possible that 5% of these 30 results (1.5) might fall outside the normal range by chance alone. With the Poisson distribution for rare events, 21 it was calculated that the chance of up to 4 of the 30 results falling outside the normal range in normal subjects was <6%, but for 5 or more of the 30 results to fall outside the normal range in normal subjects was <0.019%. Therefore, one may consider that if 5 or more of the 30 results were outside the normal range, this would be unlikely to have arisen by chance, but would more likely be due to the effect of RU486. In addition, results of an individual parameter falling outside the normal range by chance would have equal probability of lying above or below the normal range. However, reference to Figure 1 demonstrates that most measurements deviated from · the normal range in a consistent manner, i.e., either below or above the normal range. Such patterns of deviation may be taken as evidence of a response to RU486.
Symptom Check List
A symptom check list including mood changes such as irritability, depression, insomnia, and others including dizziness, fatigue, nausea, vomiting, and diarrhea was used both before and after the administration ofRU486. When present, the severity of each was recorded and coded: mild, moderate, and severe. Any additional symptom not included in the check list was also asked for, and the time of onset of any of these symptoms was noted. Ethical Considerations
This study was approved by the Southern Hospitals Ethics Committee of Sheffield, United Kingdom. All volunteers participating in the study had explained to them the purpose of the study, what would be involved, the possible side effects of RU486, and their freedom to withdraw from the study at any time. Written consent was obtained from each of the participating volunteers. Statistics
The normal range of morphologic features throughout the luteal phase was derived from 2.5 to 97 .5th percentile of 70 EBs collected from a normal, fertile population as reported earlier by Li et al. 18 734
Li et al.
Effects of P receptor blockade
RESULTS Control Experiment
The results of the control experiment demonstrated that in all four cases, histologic dating of the endometrium for EB-2 was in good agreement with chronologie dating from the LH surge. The only unusual histologic finding of note was the presence of increased leucocytic infiltration, in one subject. As leukocytic infiltration is one of the original dating criteria proposed by Noyes et al., 17 this observation must be considered in the subsequent analysis of data in the main study. Main Study
In this study, a total of 76 normal, fertile women who requested sterilization or reversal of sterilization were initially interviewed with a view to recruitment; however, only 37 fulfilled the inclusion criteria and agreed to participate in the study. Of these, three did not produce a clear LH surge in the urine samples and were therefore excluded. The remaining 34 women who participated in the study all received a dose ofRU486, but 4 were eventually excluded because the histologic dating of their EB1s showed significant disparity with the chronologie dating. Thus, 30 women were included in the Fertility and Sterility
o,
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Days from LH peak Figure 1 The effects of RU486 administration on endometrial morphology. Results of individual morphometric measurements 3 days after the administration of a variable dose of RU486 (e, 5 to 50 mg, /::;, 75 to 200 mg) are shown on a background of the normal range established from 70 fertile women as described by Li et al. 18 G1, number of mitoses per 1000 gland cells; G2, gland cell height (~m); G3, amount of secretion in gland lumen (score 0 to 3); G4, maximal gland diameter (~m); G5, volume fraction of gland occupied by gland cell; G6, volume fraction of endometrium occupied by gland; G 7, volume fraction of gland cell occupied by nucleus; G8, number of supranuclear secretory vacuoles per 100 gland cells; G9, number of subnuclear vacuoles per 100 gland cells; G 10, amount of pseudostratification of gland cell (score 0 to 3); G 11, number of apoptotic bodies per 1000 gland cells; S1, number of mitoses per 1,000 stromal cells; S2, amount of stromal oedema (score 0 to 3); S3, amount of predecidual reaction (score 0 to 3); S4, diameter of venule (~m); S5, amount of leukocyte infiltration (score 0 to 3); S6, amount of extravasation (score 0 to 3). Vol. 50, No.5, November 1988
Li et al.
Effects of P receptor blockade
735
final analysis of results. Among those included in the study, three received 200 mg, three received 150 mg, two received 100 mg, three received 75 mg, six received 50 mg, seven received 25 mg, three received 10 mg, and three received 5 mg of RU486. Among these 30 women, 5 received RU486 on day LH +2, 5 on day LH +3, 5 on day LH +4, 4 on day LH +5, and 11 on day LH +6. The mean age of this group of woman was 33.2 years (standard deviation [SD] 3.6, range 24 to 39). Traditional Dating Criteria
Of the 30 cases, 12 were considered to have normal histologic dating according to the dating criteria of Noyes et aLP 8 were considered to be retarded, and the remaining 10 were considered to be advanced. All 10 classified as advanced showed a "menstrual" structure by these criteria. Morphometric Analysis: Histologic Dating
The use of morphometric analysis in conjunction with multiple regression analysis to carry out histologic dating of endometrium has been described by Li et al. 18 Among the 30 cases, dating by morphometric analysis suggested that endometrial development was retarded in 17 cases (57%), normal in 13 cases (43%), whereas none of them was considered to have advanced endometrial development. Morphometric Analysis: Individual Morphologic Features
The effects of RU486 on the various morphologic features 3 days after its administration are shown in Figure 1. According to the Poisson distribution for rare events, significant changes (P < 0.05) were observed in 15 out of 17 morphologic features (Table 1). Menstrual Induction
After the administration of RU486, 13 out of 30 (43%) women had menstruation induced 2 days later. Of these 13 women, 6 had a further menstruationa few days later, around the time expected for that cycle, whereas 5 did not and the remaining 2 defaulted from follow-up. The time between the administration of RU486 and the onset of menstruation was found to have a narrow range of 40 to 57 hours with a median of 43 hours. 736
Li et al.
Effects of P receptor blockade
Table 1 The Frequency of Positive Response (see text) of Individual Morphometric Measurements After the Administration of a Variable Dose of RU486, in Descending Order of Frequency Morphometric measurement
Frequency of positive response
86 G10 G3 Gll 82 G8 85 G9 84 G4 G5 G7 G6 81 G2 83 G1
25/30 (83%) 4 24/30 (80%) 4 18/30 (60%) 4 18/30 (60%) 4 17/30(57%) 4 16/30 (53%) 4 16/30 (53%) 4 15/30 (50%) 4 8/18 (44%) 4 13/30 (43%) 4 11/30 (37%) 4 9/30 (30%) 4 8/30 (27%) 4 8/30 (27%) 4 6/30 (20%) 4 3/30 (10%) 0/30 (0%)
a Likely that the response was due to RU486, according to Poisson distribution for rare events (P < 0.05).
Salivary Progesterone Profile
Of the 30 women who were included in the study, 17 completed daily saliva collections for progesterone assay. Among these 17 women, 4 (24%) had a fall of salivary progesterone concentration within 3 days after the administration of RU486 to below the normal range established in our laboratory (Fig. 2) and may be considered to have had luteolysis induced by RU486. Luteolysis induced by RU486 was correlated to menstruation induced by this agent in Table 2. Temperature Regulation
Sixteen women completed daily basal temperature records in the cycle of study. Among these women, nine (56%) had a significant fall in the temperature within 3 days after the administration of RU486, examples of which are shown in Figure 3. Among those who had a significant fall in temperature, six also had simultaneous daily salivary progesterone profile for comparison. Only one out of these six women had both a fall in temperature and progesterone profile (luteolysis), whereas the remaining five had a fall in temperature but did not have luteolysis. All the observed falls in temperature occurred within 3 days after the administration ofRU486. Mood Change and Thirst Sensation
After the administration of RU486, 7 of the 30 women (23%) had mood change, in the formofirriFertility and Sterility
A
Table 3 The Relationship Between the Presence or Absence of a Response of the Various Endometrial Features (Measurements) to the Dose of RU486 Given (Dose), or the Day of the Luteal Phase on Which RU486 Was Given (Date)"
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2.53 -2.21 -2.51 2.93 3.12 2.20 2.06 2.03
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• Only those with a significant relationship (definition see text) are shown.
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Figure 2 The effects of a single, variable dose (5 to 200 mg) of RU486 on daily saliva progesterone profile. Arrow indicates the day of administration of RU486. The continuous line represents the profile of women who had received a dose of RU486. The dotted line represents the 5th centile of a normal population of women and so the lower limit of the normal range. (A) An example (subject 28) demonstrating that a significant fall of saliva progesterone concentration occurred after the administration of RU486 (luteolysis) in some cases. (B) An example (subject number 22) demonstrating that no apparent fall of saliva progesterone concentration was observed after the administration of RU486 in other cases.
tability or depression, whereas the remaining 23 women (73%) did not have any noticeable mood change. Analysis of the relationship between mood change and luteolysis by 2 X 2 contingency table demonstrated no significant association between the two. Further analysis suggests that mood change could occur in the absence of luteolysis induced by RU486.
One of the 30 women experienced marked thirst sensation after the administration of RU486. This was not associated with luteolysis. Again, all these changes were reported to occur on days 1 and 2 (mainly day 2) after the administration ofRU486, and none of them persisted after day3. 37.0 36.6 36.2
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Induced Menstruation • Fisher's exact test, P
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Vol. 50, No.5, November 1988
0
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Days from LH peak Figure 3 The effects of a single, variable dose (5 to 200 mg) of RU486 on basal body temperature. Arrow indicates the day of administration of RU486. A, Subject 17; B, Subject 22; C, Subject 28. The responses in A and B were not associated with luteolysis but C was.
Li et al.
Effects of P receptor blockade
737
Factors Affecting the Response
The relationship between the presence or absence of a response, the dose of RU486 administered, and the day of the luteal phase on which RU486 was given were analyzed by probit-logistic regression analysis. 22 The results of endometrial response are shown in Table 3. A significant relationship was defined when the absolute value of the ratio of the regression coefficient to standard error (z-value) was 1.96 or more. In addition, it was found that significant endometrial response of one or more of the 17 morphologic features were observed throughout the dose range studied (5 to 200mg). Menstrual induction by RU486 was found to be significantly (P < 0.05) related both to the dose and the date, being more likely if the dose was larger and if RU486 was given later in the luteal phase. The drop in salivary progesterone profile or basal body temperature after the administration of RU486 was found to have no significant relationship to the dose or the date. On the other hand, mood change was significantly related to the date (negatively) but not to the dose, i.e., the earlier in the luteal phase RU486 was given, the more likely mood change would be present. DISCUSSION
The effects of RU486 administration in the luteal phase from days LH +2 to LH +6 for a dose range of 5 mg to 200 mg were examined in this study. Glandular Secretory Activities
Preceptor blockade by RU486 in the first half of the luteal phase reduces the amount of glandular secretory activity, as evidenced by reductions in the amount of secretory material in the gland lumen, the maximum gland diameter, and the volume fraction of gland occupied by gland lumen. It is interesting to note the two different effects of RU486 on the number of secretory vacuoles in the gland cells. When RU486 is given on LH +2, before the formation of secretory vacuoles, it suppresses the formation of these vacuoles 3 days later. However, when RU486 is given on days LH +3 to LH +5, when the secretory vacuoles have already been formed, it has the effect of keeping these vacuoles in the gland cells and prevents them from discharging into the lumen. Thus, it is likely that P is involved both in the formation and release 738
Li et al.
Effects of P receptor blockade
of secretory vacuoles in gland cells. In both cases, progesterone receptor blockade results in reduction of the amount of secretory material being delivered to the gland lumen. It is probable that the observed increase in gland cell height and pseudostratification are both related to reduction in the amount of secretion in the gland lumen. With the lack of secretory material distending the lumen, the glands tend to be partially collapsed, as a result of which the gland cells are more closely packed, thus giving rise to the observed appearance of being taller and narrower, as well as pseudostratified. Degenerative Changes
This study suggests that Preceptor blockade increases the number of apoptotic bodies per 1,000 gland cells. 18 An apoptotic body signifies pro- . grammed cell death and as such represents degenerative changes. Thus, P receptor blockade causes degenerative changes in gland cells. It is likely that the increased number of apoptotic bodies seen in glands, either physiologically toward the end of the luteal phase or pharmacologically via the blockade ofP receptors by RU486, shares a common mechanism, which is P withdrawal. The finding that progesterone receptor blockade causes degenerative changes and reduces glandular secretory activities suggests that RU486 may be an effective postcoital contraceptive agent. Stromal Vascular Changes
P receptor blockade by RU486 induces several changes in the vessels of the stroma. Venule Diameter
Among the 30 EB-2s, the diameter of venules could not be measured in 12 cases, in view of the significant amount of extravasation present in association with induction of menstruation. In the remaining 18 cases, 8 of them (44%) were found to have a significant increase in venule diameter. Such an observation may help to explain some cases of heavy vaginal bleeding when RU486 is used to achieve medical abortion. 3 •8 Stromal Edema
Edema should be regarded as a vascular event. The possible mechanisms underlying the formation of stromal edema include increase of vascular permeability, or increase in oncotic pressure secFertility and Sterility
ondary to constriction in venules. After the administration of RU486, there is a reduction in the amount of stromal edema. It is likely that the reduction of stromal edema by RU486 is, at least partly, explained by its vasodilator effect on venules as described earlier. With dilatation of venules, the oncotic pressure in the arterial end of the capillaries is reduced, thereby reducing the formation of edema. Whether RU486 has an additional effect on the reduction of vascular permeability, which could also lead to reduction in stromal edema, is not known at present. With reduction in stromal edema, the total volume occupied by the stromal component of the endometrium is reduced. As a result, this would give rise to an increase in the volume fraction of the endometrium occupied by glands. In this study, seven out of eight observed cases with an increase in such a volume fraction were associated with, and therefore likely to be explained by, reduction in stromal edema. Stromal Extravasation
Excessive extravasation was seen in 25 out of 30 cases after the administration of RU486. Of these, 13 were associated with successful induction of menstruation, and the presence of extravasation was therefore not surprising. A further four cases were associated with spotting and could similarly be accounted for. The remaining eight cases had evidence of stromal extravasation in the absence of any vaginal bleeding. It is possible that smaller degrees of stromal extravasation may be caused by RU486 without any clinical evidence of vaginal bleeding. It is only when vascular change or extravasation is significant that bleeding from the endometrium results. Leukocytic Infiltration
Although 16 out of 30 cases had excessive leukocytic infiltration in the stroma, such a finding is difficult to interpret for two reasons. Firstly, as shown in the control experiment, leukocytic infiltration may be increased by a prior EB (EB-1) performed 3 days earlier. Thus, the increase in leukocytic infiltration observed may or may not be due to RU486. Secondly, in the presence of extravasation, as in most of these cases, the assessment of leukocytic infiltration became difficult and the results might not be accurate. Mechanism of Vascular Changes
The vascular changes described above may be related to increase in local prostaglandin production Vol. 50, No.5, November 1988
or increased sensitivity of the vessels to prostaglandins. 23- 24 Mitotic Activity
Glandular Mitosis
It is perhaps rather unexpected to find that RU486 has no effect on the number of mitoses seen per 1,000 glandular cells. This is in contrast to the conventional view that P suppresses glandular mitotic activity, which is the basis of its use in the treatment of endometrial hyperplasia and carcinoma. However, the finding in this study that progesterone receptor blockade by RU486 did not increase mitotic activity in gland cells in all the 30 cases rather suggests that the mechanism controlling glandular mitotic activity, at least in the early luteal phase, involves more than P. It is possible that the rapid decline in mitotic activity in the early luteal phase of normal menstrual cycles is related mainly to the parallel decrease in estrogen and intracellular estrogen receptors. The effect of P during this time may be relatively unimportant. Stromal Mitosis
RU486 increased stromal mitotic activity in 8 out of 30 cases. Such an effect of RU486 is more likely if it is administered later in the luteal phase, rather than earlier. This is in contrast to glandular mitosis, which does not seem to be affected by the administration of RU486. Together, such findings suggest that mitotic activity in glands and stroma may be under the influence of different controlling factors. A similar conclusion was reached by Johannisson et al., 25 who observed a discrepancy in the peak frequency of mitoses of the glandular epithelium and the stroma. This is further supported by the observation that, in normal fertile women, glandular mitosis disappears after day LH +4, whereas stromal mitosis persists, although with reduced activity, throughout the rest of the luteal phase. Predecidual Reaction
Preceptor blockade by RU486 had no demonstrable effect on the predecidual reaction in this study. However, as RU486 was given on days when the predecidual reaction is normally absent, a more appropriate conclusion is that RU486 has not been shown to induce the predecidual reaction. The reLi et al.
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sult of this study would not permit one to conclude whether RU486 blocks predecidual reaction, which requires a separate study to administer RU486 in the latter half of the luteal phase. Volume Fraction of Gland Cell Occupied by Nucleus
An effect ofRU486 is to increase the relative volume of nucleus to cytoplasm. This may be due to two possibilities: (1) the volume of the nucleus is increased, or (2) the volume of the cytoplasm is decreased. However, it is not possible from this study to deduce which is the more likely possibility. Histologic Dating
The results of histologic dating of EBs taken 3 days after the administration ofRU486 (EB-2s) by the two methods (criteria of Noyes et al. 19 and morphometric analysis 20 ) differed significantly. For example, whereas 10 of the 30 EBs (33%) were considered to be significantly advanced according to the criteria of Noyes et al./ 7 none was considered to be significantly advanced according to morphometric analysis. 18 Although it has been shown by Li et al. 18 that histologic dating by morphometric analysis is better than by the criteria of Noyes et al./ 7 we feel that histologic dating, which aims at measuring the overall maturity of the endometrium, suffers from several important disadvantages in studies of this kind. Firstly, the only limited conclusion that could be derived is whether endometrial development is normal, advanced or retarded. As such, it would not have provided a detailed description of how each morphological feature is altered. Secondly, these dating criteria are both unweighted; in situations such as the one after P receptor blockade by RU486, when certain morphologic features appear retarded (e.g., glandular secretory activity), others remain unchanged (e.g., glandular mitotic count) and still others appear advanced (e.g., stromal extravasation), the dating of endometrium then becomes difficult, arbitrary, and highly subjective. In addition, when the dating criteria of Noyes et alP are used, confidence limits of an observation cannot be established and results cannot be quantified. Importance of Baseline EB (EB-1)
In this study, out of 34 women initially recruited, 4 women (12%) were excluded from analysis be740
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cause EB-1 demonstrated that endometrial morphology was different from that expected from the LH surge. Two subjects had retarded endometrial development and might well have had an intrinsic receptor defect as an underlying cause of their retarded endometria; thus, their exclusion from the study was considered justified. One subject had a much more advanced endometrium: analysis of her salivary progesterone profile revealed that she probably had a cycle with a very short follicular phase and the urinary "LH surge" was a false-positive result in her luteal phase. Another subject, in retrospect, actually did not produce a clear urinary LH surge, but the result was misjudged. If an EB-1 had not been taken before the administration of RU486, these four cases would have been included in the analysis and would have distorted the results reported. Thus, a strong case is made for performing a biopsy before the administration of RU486 as a means of improving the homogeneity of the population being studied. In contrast, the result of the control experiment demonstrated that a prior biopsy did not influence subsequent morphologic development of the endometrium, apart from an increase in the amount of leukocytic infiltration observed. Menstrual Induction and Luteolysis
The result of this study suggested that there was a significant association between menstrual induction and luteolysis after the administration of RU486. The presence of luteolysis was always associated with menstrual induction, but menstrual induction might occur in the absence of luteolysis. This would suggest that menstrual induction by RU486 is not dependent on luteolysis and provides evidence to support the hypothesis that menstrual induction is due to the direct effect of RU486 on the endometrium. It is still not clear whether luteolysis induced by RU486 in the human is due to the direct effect of the drug on the ovary or indirectly via its effect on the pituitary. A further prospective study designed to examine the simultaneous ovarian and pituitary response to RU486 would be required to answer this question. Hypothalamic Functions
The findings in this study that the administration of RU486 affected the various functions of the hypothalamus (the regulation of temperature, thirst, and mood) provide evidence to support the Fertility and Sterility
fact that hypothalamic functions in the luteal phase of normal fertile women are under the direct influence of P. The observation that temperature fall was associated with luteolysis only in one out of six cases, but not in the remaining five cases, suggests that temperature fall after the administration of RU486 is not dependent on luteolysis (fall in P concentration) but is likely to be a direct result of P receptor blockade in the temperature regulation center of the hypothalamus. Similar conclusions could be drawn of the mechanisms of mood change and thirst sensation after the administration of RU486. Factors Affecting the Various Responses
The endometrial response to P receptor blockade, including menstrual induction and changes in endometrial morphology were both significantly related to the dose of RU486 administered and the day of the luteal phase on which RU486 was given. Mood change was significantly related to the date but not the dose of RU486 administered. In contrast, the response of the corpus luteum and the hypothalamic temperature regulation centre were unrelated to the date or the dose. Such a difference indicates that the response of various target organs (hypothalamus, corpus luteum, and endometrium) to P receptor blockade is modified by different factors.
between endometrial development and P withdrawal (caused by Preceptor blockade). From the results reported in this study, one may speculate that RU486 could be an effective postcoital contraceptive agent. However, proof of this would require a separate clinical study. This study is the first ever reported which employs quantitative morphometric techniques to investigate the effects of an exogenous compound (in this case, an antiprogesterone, RU486) on endometrial development, with reference to normal ranges derived from a controlled normal population. The detailed, quantitative information obtained from such a method of analysis is undoubtedly superior to the traditional method of histologic analysis, i.e., criteria of Noyes et at.I 7 Future studies on the response of endometrium to exogenous agents should benefit from such an improved, rigorous approach to analysis. In this study, the simultaneous observation of the various effects of RU486 in the same group of women provided a unique opportunity to analyse the relationship among the responses of different target tissues to P receptor blockade by RU486 . Acknowledgments. The authors are grateful to Mrs. Christine Piggott, Ian Roberts, M.B.B.S., Jane Stevens, B.Sc., Mrs. Gillian Burkinshaw, Mrs. Linda Highfield, and the nursing staff of the University Department of Obstetrics and Gynaecology at Jessop Hospital for Women, Sheffield, for their valuable help in this study.
Temporal Relationship
It is of interest to note that after the administration of a single dose of RU486, most effects occurred around 48 hours. Menstruation induced by RU486 occurred over a narrow range of 40 to 57 hours, with a median of 43 hours. All 4 cases of observed luteolysis started 2 to 3 days after the administration of RU486. The same applied to the fall in basal body temperature. Mood change experienced by women also occurred almost exclusively on day 2 after the administration of RU 486, and none of them persisted for more than 72 hours after the administration of RU486. It is likely that such a finding will have important clinical applications in further studies of the various effects resulting from P receptor blockade.
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