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Vascular changes in the human endometrium following the administration of the progesterone antagonist RU 486
CONTRACEPTION
VASCULARCHANGESINTHEHUYANBWDOYBTRIUM FOLLOIINGTHEADMINISTRATIONOFTHE PROGESTERONEANTAGONISTRU486
Rlisabeth Johannisson. M.D.l , Martin Oberholzer, M.D.2, Maria-Liisa Swahn. M.D.3and Marc Elygdeman, M.D.3. 1ILaboratory of Analytical and Quantitative Cytology, Geneva, Switzerland 2)Department of Pathology, Kantonsspital, Basel, Switzerland 3)Department of Obstetrics and Gynecology,Karolinska Hospital, S- 104 0 I Stockholm, Sweden
ABSTRACT Eleven healthy women were assigned to one of two groups. They received 50 mg RU486 orally per day either on cycle days 7 to 10 ( preovulatory group n-5) or on cycle days 20 to 23 (postovulatory groupn-6). An endometrial biopsy was taken on the fourth day of the RU-treatment in the preovulatory group and on the second (n-2) or fourth (n-4) treatment day in the postovulatory group. Biopsies from 34 untreated women representing matched samples from early and mid preovulatory phase (n-10) and mid and late postovulatory phase (n=24) were used as control. The ultrastructure of the endometrial capillaries was investigated by morphometric methods. The administration of RU 486 during the preovulatory phase did not modify the vascular structure. However, when given in the postovulatory phase, necrosis occurred in the capillary endothelial cells with and without regressive changes of the adjacent stroma. The area and diameter of the capillary lumen and the area of the adventitia was smaller than in the control material (pc 0.0 1). The result of the study suggests that RU486, when administered in the postovulatory phase, directly affects the capillary vessels of the endometrium. Submitted for publication October 5, 1988 Accepted for publication October 17, 1988
JANUARY
1989 VOL. 39 NO. 1
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INTRODUCTION Studies in monkeys (1,2,3,4) and in women (5,6) have shown that RU 486 has a strong antiprogestational effect on the, endometrium, inducing menstrual bleeding when given in the luteal phase or terminating early pregnancy. When given in the luteal phase, RU486 has been reported to cause regressive changes and an almost complete shedding of the upper functional layer of the endometrium (4.7). On the other hand, it has been reported that RU486 has a weak progestational effect when given to monkeys in the proliferative phase (3) or when administered to estrogentreated postmenopausal women (8). Although there seem to be some similarities between the regressive changes taking place in the endometrium during the onset of a normal menstruation and those occurring in the secretory endometrium after RU486 administration, the effect on the endometrial vessels may not be the same. Koering et al. (3) reported that the blood vessels appeared intact in castrated monkeys that received RU 486 during combined treatment with estradiol and progesterone. Nevertheless extravascular red blood cells were present in the stroma and a menstruation-like bleeding took place. During physiological menstruation the onset of menstrual bleeding is believed to be preceded by endometrial shrinking and a constriction of the endometrial arteries (9). The induced anemia is likely to cause necrotic changes of the vessels and extravasation of red blood cells. The mechanism of RU486-induced menstrual-like bleeding is still not fully known.The aim of the present study was to investigate the effect of RU486 on the ultrastructure of the endometrial capillaries when given in the mid proliferative phase and in the mid and late luteal phase and to compare these findings with the vascular structure observed in biopsies of matched control groups of untreated women.
MATERIALANDMETHODS Subjects
Eleven healthy women, aged 25 to 45 years, with regular menstrual cycles participated in the study. All women were of proven fertility and none of them had used steroidal contraceptives or an IUDduring at least 3 months prior to the study. On admission a gynecologic examination was performed and the women were instructed to use barrier methods for contraception during the entire study period.
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The study included one control and one treatment cycle in the experimental group. During the treatment cycle the women received 50 mg RU486 orally per day between 8 a.m. and 10 a.m. either on cycle days 7 to 10 ( preovulatory group; n- 5) or on cycle days 20 to 23 ( postovulatory group; n- 6). During the control and treatment cycle daily morning urine (the first void upon rising) was collected and kept frozen until assayed. An endometrial biopsy was taken on cycle day 10 in the preovulatory group. In the postovulatory group the endometrial biopsy was initially planned to be taken on the 23rd day of the cycle. However, since the first four subjects in this group had already started to bleed on that day, the biopsies in the remaining two women were obtained on day 2 1. prior to the onset of bleeding. The endometrial biopsies were taken with a Randall curette from the anterior and lateral walls of the uterine cavity without prior cervical dilatation or local anaesthesia. To obtain a comparison between untreated and treated biopsy samples, endometrial material was selected from 90 women with normal menstrual cycles to match the groups of women treated with RU486. Out of this control group 10 women represented early and mid preovulatory phase and 24 women mid and late postovulatory phase, The control biopsies were obtained and processed in the same way as the material of the experimental group.
Half of the biopsy material was immediately fixed in Elouin’ssolution and used for light microscopic examination after embedding in paraffin, sectioning and staining in haematoxylin-eosin. The other half of the biopsy was fixed in 3 X glutaraldehyde in 0.1 mol/l sodium cacodylate buffer (pH 7.3 - 7.4) containing 2%calcium chloride. The material for electron microscopy was further processed by postfixation in 0904 ( 1XIfor two hours at 4QC,dehydration in graded ethanol solutions and embedding in propylenoxyde/Epon. The material prepared for light microscopy was assessed by morphometrical methods as described by Johannisson et al. ( IO). In the electron micrographs a detailed morphometric analysis was carried out on the capillary structure of the upper functional layer of the endometrium using the method described by Oberholzer (11). The following measurements JANUARY 1989 VOL. 39 NO. 1
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were made: the area of the capillary lumen, the area of the entire capillary, the largest diameter of the capillary lumen, the area of the endothelial cells, the area of the adventitia. the thickness of the endothelial cells and the thickness of the adventitia. A morphometric instrument type Leitz A.S.M.system ( Leitz Wet&r GmBH, WetzlarGermany) was used. The statistical analyses of the results from the electron microscopic study were carried out using the Mann-Whitney non-parametric test. Harmone assays LH and cortisol were estimated by radioimmunoassay techniques described by Sufi et al. ( 121.Radioimmunoassays of estradiol and progesterone in urine were performed according to Beksac et al. ( 13, 14).
The study was approved by the local Ethics Committee of the Karolinska Hospital, Stockholm, Sweden. RESULTS fieovutatory
phase
In women treated during the preovulatory phase the urinary concentration of estradiol during the first 14 days of the treatment cycle was significantly lower than during the control cycle (pr0.05). The LH surge was also postponed when compared with the control cycle. The biopsy was taken on cycle day 10. but due to the delay of the LH peak as a mean on day LH-IO. The details of the changes of the hormonal pattern following the administration of RU 486 in the preovulatory phase are described in a previous publication (7). Endometrial biopsies from 5 women were analyzed morphometrically. The light microscopic appearance of the endometrium was compatible with a preovulatory phase and did not significantly differ from the morphologic pattern of the 10 control specimens.The size of the glands remained small and did not vary from that observed in the controls. No basal vacuolization or other manifestations of a progesterone effect were observed. In the electron microscopic assessment of the capillaries the administration of RU 486 in the preovulatory phase did not significantly alter the vascular structure as indicated in Table I. 106
JANUARY 1989 VOL. 39 NO. I
30.9k4.8 2.1kO.4 0.6tO.6
Area of adventitia (p2)
Thickness of endothelial cells (p)
Thickness of adventitia (~1
1) p c 0.0 1; 2) p < 0.05 when compared with the postovulatory
89.0222.0
Area of endothelial cells (~1~1
0.6tO.l
1.920.4
36.82 14.0
97.2t34.7
13.823.6
198.Ok66.0
60.3t32.4
phase of the normal cycle.
0.620.03
1.9tl.l
27.024.8
10.321.8 83.5’15.1
Largest diameter of capillary lumen (CL) 10.922.7
141.1t35.8
153.92357
Area of the entire capillary (p2)
27.721 1.1
35.42 14.8
Postovmse Normal cycle (n-24)
h-6)
0.5?0.082)
25.8+5.3l) 2.020.4
86.22 12.9
10.8+1.52)
163.5~29.0
32.3+10.41)
RU 486
capillary structure when given in the pre- and
Preovulatorv ohase Normal cycle h-10) RU 486 (n-5)
phases (Mean+SE)
on the endometrial
Area of capillary lumen Qt21
Indices
postovulatory
Table I. Effect of the RLJ486 treatment
CONTRACEPTION
htovufatwy phase When RU 486 was administered in the postovulatory phase all women started to bleed on the third or fourth day of treatment, despite the presence of luteal progesterone levels. It is noteworthy that in patients with only one bleeding episode during the treatment cycle,the plasma progesterone values were significantly lower (pc 0.05) than in the group of women with two bleeding episodes. In the latter group the concentration remained elevated until the second bleeding episode as described in a previous publication (7). In the endometrial material obtained from two women on the second day of treatment the lightmicroscopic picture corresponded to late secretory changes and a limited number of extravascular red blood cells was found in the stroma. All four endometrial specimens obtained on the fourth day of RU 486 treatment revealed strong regressive changes with extravasation of red blood cells and a heavy infiltration of neutrophils in the stroma. These light microscopic changes were similar to those observed premenstrually or in early menstruating endometrium of the normal cycle. It is noteworthy that these regressive changes, which usually do not occur before the 12th day after the LH surge (LH+12) of the normal menstrual cycle, were observed already on days LH +7 and +8.
The capillaries revealed an important heterogeneity in their ultrastructure varying from capillaries with well preserved adventitia to vessels with clear breaks into the surrounding stroma. In most specimens, degenerative changes were found in the endothelial cells of the capillaries. Fig. 1 shows a capillary of an endometrium on the fourth day of RU 486 administration. Strong degenerative changes are present in the endothelial cells whereas less regressive changes appear in the pericytes and in the surrounding stroma. The endoplasmic reticulum of the endothelial cells revealed an extensive dilatation, occasionally forming irregular cisternae or vacuoles in the cytoplasm (Fig.1). In some of the endothelial cells the cytoplasmic matrix had lost its normal structure. The mitochondria also were swollen and showed degenerative changes, In some biopsies the regressive changes were evident not only in the endothelial cells but also in the surrounding stroma (Fig. 2). The control material from 24 women representing endometrium from LH +7 to LH + 11 did not not reveal any degenerative changes of the endothelial cells as illustrated in Fig. 3. Nor did the endometrium obtained from women treated with RU486 in the preovulatory phase reveal 108
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Pig. 1 Endometrial capillary K) from a biopsy obtained on the fourth day of the RU486 treatment in the postovulatory phase. The endotheiial cells (El show signs of necrosis, whereas the adjacent stroma cells 6) are less degenerated. Note the extravasal red blood cells (RI. x 56 IO. JANUARY 1989 VOL. 39 NO. 1
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Pig. 2 Endometrial capillary from a biopsy obtained on the fourth day of the RU 486 treatment in the postovulatory phase. The entire capillary 1C)as well as the surrounding stroma (S) show strong regression. I 56 IO.
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Fig. 3 Endometrial capillary of a control biopsy obtained on day LH +7.
The capillary (0 is intact and the endothelial cells (El show no signs of regression. The capillary adventitia (A) is surrounding the endothelium. P - pericytes. x 56 IO.
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similar degeneration of the capillary endothelial cells (Fig.4). To evaluate statistically the difference between the postovulatory RU 486-
treated group and the matched control group in respect of the vascular structure of the endometrial capillarieqa detailed morphometric study was carried out on a number of indices reflecting the vascular dynamics of the capillaries. As indicated in Table I these measurements revealed a significant decrease in the capillary lumen area (~(0.01) and in the largest capillary diameter (p(O.05)when compared with the matched controls. The area of the adventitia ( p
The results of the present study indicate that RU 486 induced uterine bleeding when administered for four days in the late luteal phase whereas the same treatment did not have any effect on the endometrium when given on days 7 to 10 of the preovulatory phase. These findings agree with the general concept that RU 486 acts as a potent antiprogesterone and binds to the progesterone receptor (1, 15, 16). In competing with endogeneous progesterone RU486 is capable of inhibiting the activity of this steroid on the endometrium. thereby inducing bleeding despite the presence of elevated levels of plasma progesterone (5, 17). However, it has also been reported that RU486 has some agonistic properties both at the endometrial and the pituitary level (8, 18). In the present study we did not observe a progestational effect on the endometrium when RU 486 was given on days 7- 10 of the cycle. However, estrogen secretion was suppressed and the LH surge delayed in these women (7). Therefore. it can not be excluded that the endometrium had not reached its maximum of estrogen stimulation at the time of RU486 administration. Previous studies of our group have shown that the administration of progesterone on cd 2 to 6 of the preovulatory phase had little or no effect on the morphology of the endometrium, whereas administration of the same amount of progesterone on cd 7 to 11 clearly induced a progestational effect in the mucosa (19). The duration and magnitude of exposure to estrogens may therefore be important for the maturation of the endometrium and its reaction to progesterone. This may be reIevant also to the studies where RU486 had a weak progestational effect on the proliferative endometrium. In one study exogenous estradiol 112
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Pig. 4 Endometrial capillary of a biopsy obtained on the fourth day of the RU 486 treatment in the preovulatory phase. The endothelial cells (El show no signs of necrotic changes. R = red blood cell. x 56 10.
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was given to castrated monkeys for 13 days before RU 486 administration (3) whereas in the other postmenopausal women were treated daily with 0.625 mg estradiol benzoate for 9 days before additional RU 486 administration (8). Treatment with RU486 during the postovulatory phase resulted in significant changes in the endometrial morphology, not only in the glands and stroma but also in the vascularization of the upper functional layer. Signs of cell injury and cell death were found in the capillary endothelial cells already on the second day of treatment. These became even more evident in the biopsies obtained on the fourth day of treatment when also the surrounding stroma showed important regressive changes. As for the changes observed in the endometrial vessels, studies in monkeys have reported extravascular red blood cells 32 hours after RU486 treatment, even though at the light microscopic examination the blood vessels appeared intact (3). Since the presence of extravascular red blood cells is suggestive of vascular damages, it is probable that minor cell injuries, visible only in the electron microscope, existed in the blood vessels of the monkey endometrium described above. The present electron microscopic examination of the endometrial material obtained after RU 486 treatment revealed signs of cell injury and cell death of the capillary endothelial cells with and without regressive changes of the surrounding endometrial stroma. No such changes were found in the matched control samples or in the samples obtained after preovulatory RU 486 treatment. Furthermore, the capillary adventitia underwent a rapid degradation reflected by a significant decrease in its area and thickness when compared to the control samples, It is likely that the intracellular changes observed in the endothelial cells after RU486 treatment reflect an altered homeostasis. If the zero level of homeostatic ability is set at the point of the cell death, than the degradative cellular reactions observed in the present study would be compatible with necrosis. This necrosis may then explain the extravasation of red blood cells and the endometrial bleeding. The early degenerative changes which seem to affect mainly the endothelial cells within the first 48 hours after the administration of RU 486 therefore support the theory that the antiprogesterone blocks the progesterone action at the vascular level. It is noteworthy that the administration of RU 486 in the postovulatory phase also decreased significantly the area of the capillary lumen suggesting a decreased blood flow. This decrease in blood flow could be 114
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due to a constriction of the arteries suggested by Markee (9) as a possible mechanism of action for the onset of a menstrual bleeding. It is noteworthy in this respect that progesterone receptors have been demonstrated in the cells composing the coiled arteries (20). RU 486- induced bleeding is generally ascribed to a direct effect of the compound on the endometrial cells which are known to have high concentration of progesterone receptors (1,2,3,4). The observations of the present study suggest that the vasculature may be an important, perhaps primary, target of the compound, although the possibility cannot be excluded that these changes are secondary to regressive changes in the function al layer. To settle the question, further studies are needed on the presence and distribution of progesterone receptors in the vessels and on the effect of RU 486on vascular morphology. Such studies may well throw light upon the mechanisms involved also in the normal menstruation. ACKNOWLEDGEMENTS
The present study was supported by the WHOSpecial Programme of Research, Development and Research Training in Human Reproduction, Geneva, Switzerland, and by the Swiss National Research Foundation, grant 3.955-0.84. RU 486 was kindly supplied by Roussel-Uclaf,Romainville, France. The expert technical assistance of Miss Rita Schwendimann of Basle. Mrs Erika Jacobsson-Strom and Mrs Fabienne Simon of Geneva, and Mrs A&rid Haggblad of Stockholm is gratefully acknowledged. We are also indebted to Dr Paul van Look, WHO.Geneva, who read the manuscript and generously gave us most valuable criticism
1, Healy, D.L.,Baulieu. E.E.and Hodgen GD.Induction of menstruation by an antiprogesterone steroid (RU486) in primates. Sited action, doseresponse relationships, and hormonal effects. Fertil. Steril. 40: 253 (1983) 2. Shortle, B.,Dyrenfurth, I. and Ferin. M. Effects of an antiprogesterone agent, RU486, on the menstrual cycle of the rhesus monkey J. Clin. Endocrinol. Metab. 60: 731 (1985)
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3. Koering, M.J.,Healy, D.L.and Hodgen, G.D..Morphologic response of endometrium to a progesterone receptor antagonist, RU 486, in monkeys. Fertil. Steril. 45280 (1986) 4. Chillik,C.F.,Hsiu, J.G.,Acosta, A.A.,van Uem, J.F.H.M.and Hodgen G.D.RU 486-induced menses in cynomolgus monkeys: uniformity of endometrial sloughing. Fertil. Steril. 45: 708 (1986) 5. Schindler, A.M.,Zanon, P., Obradovic, D.,Wyss, R.,Graff, P. and Herrmann W.. Early ultrastructural changes in RU-486-exposed decidua. Gynecolobstetlnvest. 20: 62 (1985) 6. Kovacs,L., Sas, M.,Resch, B.A.,Ugocsai,G.,Swahn, M.L.and Bygdeman M. Termination of very early pregnancy by RU 486-an antiprogestational compound. Contraception 29: 399 (1984) 7. Swahn, M.L.,Johannisson, E.,Daniore, V., de la Terre, 8. and Bygdeman, M: The effect of RU486 administered during the proliferative and secretory phase of the cycle on the bleeding pattern, hormonal parameters and the endometrium. Human Reproduction. (Submitted for publication) 8. Gravanis. A., Schaison, G.,George, M., de Brux, J.. Satyaswaroop, P.G.and Baulieu, E.E.and Robel, P. Endometrial and pituitary responses to the steroidal antiprogestin RU486 in postmenopausal women. J. Clin. Endocrinol. Metab. 60:156 (1985) 9. Markee. J.E.Menstruation in intraocular endometrial transplants in the rhesus monkey. Contrib. Embryol. 28: 2 19 (1940) 10. Johannisson, E..Landgren. B.M..Rohr, H.P.and DiczfalusyE.:Endometrial morphology and peripheral hormone levels in women with regular menstrual cycles. Fertil. Steril. 48: 40 1 (1987) 11. Oberholzer, M. Morphometrie in der klinischen Pathologie. Allgemeine Grundlagen. Springer Verlag, Berlin, 1983. 12. Sufi, S.B..Donaldson, A., and JeffcoateSL. Method Manual, WHO Programme for the Provision of Matched AssayReagents, 10th Ed., London,1986.
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13. Beksac, MS., Wang, Y. and Cekan, S. Validity of the radioimmunoassay of unconjugated estradiol in urine. Acta Reprod. Turcica 555 (1984) 14. Beksac, MS. and Cekan. S. Reliability of progesterone measurements in urine. J. Clin.Chem. Clin.B&hem. 23:183 (1985) 15. Philibert, D.,Deraedt, T., Teutsch. G..Tournemine, C. and Sakiz, E. RU 38486 a new lead for steroidal antihormones. Presented at the 64th Annual Meeting of the Endocrine Society, San Francisco, June 16 to 18,1982,Abstract No. 668. 16. Baulieu, E.E.RU486. An antiprogestin steroid with contragestive activity in women. InThe Antiprogestin Steroid RU486 and Human Fertility Control (EEBaulieu and SJ Segal, Editors). Plenum Press, New York, 1985, p.l. 17. Schaison, G.,George, M.,Lestrat, N.,Reinberg, A. and Baulieu. E.E.Effects of the antiprogesterone steroid RU486 during midluteal phase in normal women. J. Clin.Endocrinol. Metab. 6 1:484 (1985) 18. Collins,R.L.and Hodgen, G.D.Blockade of the spontaneous midcycle gonadotropin surge in monkeys by RU486: a progesterone antagonist or agonist? J. Clin.Endocrinol. Metab. 63:1270 (1986) 19. King,S.,Johannisson, E.,Landgren, B.M.and Diczfalusy,E. Pituitary, ovarian and endometrial effects of progesterone released prematurely during the proliferative phase. Contraception 27: 177 i 1983) 20. Bergqvist, A., Carlstrom. K.and Ljungberg, 0.. Histochemical localization of estrogen and progesterone receptors. J. Histochem. Cytochem. 32: 493 (19841
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VASCULARCHANGESINTHEHUYANBWDOYBTRIUM FOLLOIINGTHEADMINISTRATIONOFTHE PROGESTERONEANTAGONISTRU486
Rlisabeth Johannisson. M.D.l , Martin Oberholzer, M.D.2, Maria-Liisa Swahn. M.D.3and Marc Elygdeman, M.D.3. 1ILaboratory of Analytical and Quantitative Cytology, Geneva, Switzerland 2)Department of Pathology, Kantonsspital, Basel, Switzerland 3)Department of Obstetrics and Gynecology,Karolinska Hospital, S- 104 0 I Stockholm, Sweden
ABSTRACT Eleven healthy women were assigned to one of two groups. They received 50 mg RU486 orally per day either on cycle days 7 to 10 ( preovulatory group n-5) or on cycle days 20 to 23 (postovulatory groupn-6). An endometrial biopsy was taken on the fourth day of the RU-treatment in the preovulatory group and on the second (n-2) or fourth (n-4) treatment day in the postovulatory group. Biopsies from 34 untreated women representing matched samples from early and mid preovulatory phase (n-10) and mid and late postovulatory phase (n=24) were used as control. The ultrastructure of the endometrial capillaries was investigated by morphometric methods. The administration of RU 486 during the preovulatory phase did not modify the vascular structure. However, when given in the postovulatory phase, necrosis occurred in the capillary endothelial cells with and without regressive changes of the adjacent stroma. The area and diameter of the capillary lumen and the area of the adventitia was smaller than in the control material (pc 0.0 1). The result of the study suggests that RU486, when administered in the postovulatory phase, directly affects the capillary vessels of the endometrium. Submitted for publication October 5, 1988 Accepted for publication October 17, 1988
JANUARY
1989 VOL. 39 NO. 1
103
CONTRACEPTION
INTRODUCTION Studies in monkeys (1,2,3,4) and in women (5,6) have shown that RU 486 has a strong antiprogestational effect on the, endometrium, inducing menstrual bleeding when given in the luteal phase or terminating early pregnancy. When given in the luteal phase, RU486 has been reported to cause regressive changes and an almost complete shedding of the upper functional layer of the endometrium (4.7). On the other hand, it has been reported that RU486 has a weak progestational effect when given to monkeys in the proliferative phase (3) or when administered to estrogentreated postmenopausal women (8). Although there seem to be some similarities between the regressive changes taking place in the endometrium during the onset of a normal menstruation and those occurring in the secretory endometrium after RU486 administration, the effect on the endometrial vessels may not be the same. Koering et al. (3) reported that the blood vessels appeared intact in castrated monkeys that received RU 486 during combined treatment with estradiol and progesterone. Nevertheless extravascular red blood cells were present in the stroma and a menstruation-like bleeding took place. During physiological menstruation the onset of menstrual bleeding is believed to be preceded by endometrial shrinking and a constriction of the endometrial arteries (9). The induced anemia is likely to cause necrotic changes of the vessels and extravasation of red blood cells. The mechanism of RU486-induced menstrual-like bleeding is still not fully known.The aim of the present study was to investigate the effect of RU486 on the ultrastructure of the endometrial capillaries when given in the mid proliferative phase and in the mid and late luteal phase and to compare these findings with the vascular structure observed in biopsies of matched control groups of untreated women.
MATERIALANDMETHODS Subjects
Eleven healthy women, aged 25 to 45 years, with regular menstrual cycles participated in the study. All women were of proven fertility and none of them had used steroidal contraceptives or an IUDduring at least 3 months prior to the study. On admission a gynecologic examination was performed and the women were instructed to use barrier methods for contraception during the entire study period.
104
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CONTRACEPTION
The study included one control and one treatment cycle in the experimental group. During the treatment cycle the women received 50 mg RU486 orally per day between 8 a.m. and 10 a.m. either on cycle days 7 to 10 ( preovulatory group; n- 5) or on cycle days 20 to 23 ( postovulatory group; n- 6). During the control and treatment cycle daily morning urine (the first void upon rising) was collected and kept frozen until assayed. An endometrial biopsy was taken on cycle day 10 in the preovulatory group. In the postovulatory group the endometrial biopsy was initially planned to be taken on the 23rd day of the cycle. However, since the first four subjects in this group had already started to bleed on that day, the biopsies in the remaining two women were obtained on day 2 1. prior to the onset of bleeding. The endometrial biopsies were taken with a Randall curette from the anterior and lateral walls of the uterine cavity without prior cervical dilatation or local anaesthesia. To obtain a comparison between untreated and treated biopsy samples, endometrial material was selected from 90 women with normal menstrual cycles to match the groups of women treated with RU486. Out of this control group 10 women represented early and mid preovulatory phase and 24 women mid and late postovulatory phase, The control biopsies were obtained and processed in the same way as the material of the experimental group.
Half of the biopsy material was immediately fixed in Elouin’ssolution and used for light microscopic examination after embedding in paraffin, sectioning and staining in haematoxylin-eosin. The other half of the biopsy was fixed in 3 X glutaraldehyde in 0.1 mol/l sodium cacodylate buffer (pH 7.3 - 7.4) containing 2%calcium chloride. The material for electron microscopy was further processed by postfixation in 0904 ( 1XIfor two hours at 4QC,dehydration in graded ethanol solutions and embedding in propylenoxyde/Epon. The material prepared for light microscopy was assessed by morphometrical methods as described by Johannisson et al. ( IO). In the electron micrographs a detailed morphometric analysis was carried out on the capillary structure of the upper functional layer of the endometrium using the method described by Oberholzer (11). The following measurements JANUARY 1989 VOL. 39 NO. 1
105
CONTRACEPTION
were made: the area of the capillary lumen, the area of the entire capillary, the largest diameter of the capillary lumen, the area of the endothelial cells, the area of the adventitia. the thickness of the endothelial cells and the thickness of the adventitia. A morphometric instrument type Leitz A.S.M.system ( Leitz Wet&r GmBH, WetzlarGermany) was used. The statistical analyses of the results from the electron microscopic study were carried out using the Mann-Whitney non-parametric test. Harmone assays LH and cortisol were estimated by radioimmunoassay techniques described by Sufi et al. ( 121.Radioimmunoassays of estradiol and progesterone in urine were performed according to Beksac et al. ( 13, 14).
The study was approved by the local Ethics Committee of the Karolinska Hospital, Stockholm, Sweden. RESULTS fieovutatory
phase
In women treated during the preovulatory phase the urinary concentration of estradiol during the first 14 days of the treatment cycle was significantly lower than during the control cycle (pr0.05). The LH surge was also postponed when compared with the control cycle. The biopsy was taken on cycle day 10. but due to the delay of the LH peak as a mean on day LH-IO. The details of the changes of the hormonal pattern following the administration of RU 486 in the preovulatory phase are described in a previous publication (7). Endometrial biopsies from 5 women were analyzed morphometrically. The light microscopic appearance of the endometrium was compatible with a preovulatory phase and did not significantly differ from the morphologic pattern of the 10 control specimens.The size of the glands remained small and did not vary from that observed in the controls. No basal vacuolization or other manifestations of a progesterone effect were observed. In the electron microscopic assessment of the capillaries the administration of RU 486 in the preovulatory phase did not significantly alter the vascular structure as indicated in Table I. 106
JANUARY 1989 VOL. 39 NO. I
30.9k4.8 2.1kO.4 0.6tO.6
Area of adventitia (p2)
Thickness of endothelial cells (p)
Thickness of adventitia (~1
1) p c 0.0 1; 2) p < 0.05 when compared with the postovulatory
89.0222.0
Area of endothelial cells (~1~1
0.6tO.l
1.920.4
36.82 14.0
97.2t34.7
13.823.6
198.Ok66.0
60.3t32.4
phase of the normal cycle.
0.620.03
1.9tl.l
27.024.8
10.321.8 83.5’15.1
Largest diameter of capillary lumen (CL) 10.922.7
141.1t35.8
153.92357
Area of the entire capillary (p2)
27.721 1.1
35.42 14.8
Postovmse Normal cycle (n-24)
h-6)
0.5?0.082)
25.8+5.3l) 2.020.4
86.22 12.9
10.8+1.52)
163.5~29.0
32.3+10.41)
RU 486
capillary structure when given in the pre- and
Preovulatorv ohase Normal cycle h-10) RU 486 (n-5)
phases (Mean+SE)
on the endometrial
Area of capillary lumen Qt21
Indices
postovulatory
Table I. Effect of the RLJ486 treatment
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htovufatwy phase When RU 486 was administered in the postovulatory phase all women started to bleed on the third or fourth day of treatment, despite the presence of luteal progesterone levels. It is noteworthy that in patients with only one bleeding episode during the treatment cycle,the plasma progesterone values were significantly lower (pc 0.05) than in the group of women with two bleeding episodes. In the latter group the concentration remained elevated until the second bleeding episode as described in a previous publication (7). In the endometrial material obtained from two women on the second day of treatment the lightmicroscopic picture corresponded to late secretory changes and a limited number of extravascular red blood cells was found in the stroma. All four endometrial specimens obtained on the fourth day of RU 486 treatment revealed strong regressive changes with extravasation of red blood cells and a heavy infiltration of neutrophils in the stroma. These light microscopic changes were similar to those observed premenstrually or in early menstruating endometrium of the normal cycle. It is noteworthy that these regressive changes, which usually do not occur before the 12th day after the LH surge (LH+12) of the normal menstrual cycle, were observed already on days LH +7 and +8.
The capillaries revealed an important heterogeneity in their ultrastructure varying from capillaries with well preserved adventitia to vessels with clear breaks into the surrounding stroma. In most specimens, degenerative changes were found in the endothelial cells of the capillaries. Fig. 1 shows a capillary of an endometrium on the fourth day of RU 486 administration. Strong degenerative changes are present in the endothelial cells whereas less regressive changes appear in the pericytes and in the surrounding stroma. The endoplasmic reticulum of the endothelial cells revealed an extensive dilatation, occasionally forming irregular cisternae or vacuoles in the cytoplasm (Fig.1). In some of the endothelial cells the cytoplasmic matrix had lost its normal structure. The mitochondria also were swollen and showed degenerative changes, In some biopsies the regressive changes were evident not only in the endothelial cells but also in the surrounding stroma (Fig. 2). The control material from 24 women representing endometrium from LH +7 to LH + 11 did not not reveal any degenerative changes of the endothelial cells as illustrated in Fig. 3. Nor did the endometrium obtained from women treated with RU486 in the preovulatory phase reveal 108
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Pig. 1 Endometrial capillary K) from a biopsy obtained on the fourth day of the RU486 treatment in the postovulatory phase. The endotheiial cells (El show signs of necrosis, whereas the adjacent stroma cells 6) are less degenerated. Note the extravasal red blood cells (RI. x 56 IO. JANUARY 1989 VOL. 39 NO. 1
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Pig. 2 Endometrial capillary from a biopsy obtained on the fourth day of the RU 486 treatment in the postovulatory phase. The entire capillary 1C)as well as the surrounding stroma (S) show strong regression. I 56 IO.
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Fig. 3 Endometrial capillary of a control biopsy obtained on day LH +7.
The capillary (0 is intact and the endothelial cells (El show no signs of regression. The capillary adventitia (A) is surrounding the endothelium. P - pericytes. x 56 IO.
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similar degeneration of the capillary endothelial cells (Fig.4). To evaluate statistically the difference between the postovulatory RU 486-
treated group and the matched control group in respect of the vascular structure of the endometrial capillarieqa detailed morphometric study was carried out on a number of indices reflecting the vascular dynamics of the capillaries. As indicated in Table I these measurements revealed a significant decrease in the capillary lumen area (~(0.01) and in the largest capillary diameter (p(O.05)when compared with the matched controls. The area of the adventitia ( p
The results of the present study indicate that RU 486 induced uterine bleeding when administered for four days in the late luteal phase whereas the same treatment did not have any effect on the endometrium when given on days 7 to 10 of the preovulatory phase. These findings agree with the general concept that RU 486 acts as a potent antiprogesterone and binds to the progesterone receptor (1, 15, 16). In competing with endogeneous progesterone RU486 is capable of inhibiting the activity of this steroid on the endometrium. thereby inducing bleeding despite the presence of elevated levels of plasma progesterone (5, 17). However, it has also been reported that RU486 has some agonistic properties both at the endometrial and the pituitary level (8, 18). In the present study we did not observe a progestational effect on the endometrium when RU 486 was given on days 7- 10 of the cycle. However, estrogen secretion was suppressed and the LH surge delayed in these women (7). Therefore. it can not be excluded that the endometrium had not reached its maximum of estrogen stimulation at the time of RU486 administration. Previous studies of our group have shown that the administration of progesterone on cd 2 to 6 of the preovulatory phase had little or no effect on the morphology of the endometrium, whereas administration of the same amount of progesterone on cd 7 to 11 clearly induced a progestational effect in the mucosa (19). The duration and magnitude of exposure to estrogens may therefore be important for the maturation of the endometrium and its reaction to progesterone. This may be reIevant also to the studies where RU486 had a weak progestational effect on the proliferative endometrium. In one study exogenous estradiol 112
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Pig. 4 Endometrial capillary of a biopsy obtained on the fourth day of the RU 486 treatment in the preovulatory phase. The endothelial cells (El show no signs of necrotic changes. R = red blood cell. x 56 10.
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was given to castrated monkeys for 13 days before RU 486 administration (3) whereas in the other postmenopausal women were treated daily with 0.625 mg estradiol benzoate for 9 days before additional RU 486 administration (8). Treatment with RU486 during the postovulatory phase resulted in significant changes in the endometrial morphology, not only in the glands and stroma but also in the vascularization of the upper functional layer. Signs of cell injury and cell death were found in the capillary endothelial cells already on the second day of treatment. These became even more evident in the biopsies obtained on the fourth day of treatment when also the surrounding stroma showed important regressive changes. As for the changes observed in the endometrial vessels, studies in monkeys have reported extravascular red blood cells 32 hours after RU486 treatment, even though at the light microscopic examination the blood vessels appeared intact (3). Since the presence of extravascular red blood cells is suggestive of vascular damages, it is probable that minor cell injuries, visible only in the electron microscope, existed in the blood vessels of the monkey endometrium described above. The present electron microscopic examination of the endometrial material obtained after RU 486 treatment revealed signs of cell injury and cell death of the capillary endothelial cells with and without regressive changes of the surrounding endometrial stroma. No such changes were found in the matched control samples or in the samples obtained after preovulatory RU 486 treatment. Furthermore, the capillary adventitia underwent a rapid degradation reflected by a significant decrease in its area and thickness when compared to the control samples, It is likely that the intracellular changes observed in the endothelial cells after RU486 treatment reflect an altered homeostasis. If the zero level of homeostatic ability is set at the point of the cell death, than the degradative cellular reactions observed in the present study would be compatible with necrosis. This necrosis may then explain the extravasation of red blood cells and the endometrial bleeding. The early degenerative changes which seem to affect mainly the endothelial cells within the first 48 hours after the administration of RU 486 therefore support the theory that the antiprogesterone blocks the progesterone action at the vascular level. It is noteworthy that the administration of RU 486 in the postovulatory phase also decreased significantly the area of the capillary lumen suggesting a decreased blood flow. This decrease in blood flow could be 114
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due to a constriction of the arteries suggested by Markee (9) as a possible mechanism of action for the onset of a menstrual bleeding. It is noteworthy in this respect that progesterone receptors have been demonstrated in the cells composing the coiled arteries (20). RU 486- induced bleeding is generally ascribed to a direct effect of the compound on the endometrial cells which are known to have high concentration of progesterone receptors (1,2,3,4). The observations of the present study suggest that the vasculature may be an important, perhaps primary, target of the compound, although the possibility cannot be excluded that these changes are secondary to regressive changes in the function al layer. To settle the question, further studies are needed on the presence and distribution of progesterone receptors in the vessels and on the effect of RU 486on vascular morphology. Such studies may well throw light upon the mechanisms involved also in the normal menstruation. ACKNOWLEDGEMENTS
The present study was supported by the WHOSpecial Programme of Research, Development and Research Training in Human Reproduction, Geneva, Switzerland, and by the Swiss National Research Foundation, grant 3.955-0.84. RU 486 was kindly supplied by Roussel-Uclaf,Romainville, France. The expert technical assistance of Miss Rita Schwendimann of Basle. Mrs Erika Jacobsson-Strom and Mrs Fabienne Simon of Geneva, and Mrs A&rid Haggblad of Stockholm is gratefully acknowledged. We are also indebted to Dr Paul van Look, WHO.Geneva, who read the manuscript and generously gave us most valuable criticism
1, Healy, D.L.,Baulieu. E.E.and Hodgen GD.Induction of menstruation by an antiprogesterone steroid (RU486) in primates. Sited action, doseresponse relationships, and hormonal effects. Fertil. Steril. 40: 253 (1983) 2. Shortle, B.,Dyrenfurth, I. and Ferin. M. Effects of an antiprogesterone agent, RU486, on the menstrual cycle of the rhesus monkey J. Clin. Endocrinol. Metab. 60: 731 (1985)
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3. Koering, M.J.,Healy, D.L.and Hodgen, G.D..Morphologic response of endometrium to a progesterone receptor antagonist, RU 486, in monkeys. Fertil. Steril. 45280 (1986) 4. Chillik,C.F.,Hsiu, J.G.,Acosta, A.A.,van Uem, J.F.H.M.and Hodgen G.D.RU 486-induced menses in cynomolgus monkeys: uniformity of endometrial sloughing. Fertil. Steril. 45: 708 (1986) 5. Schindler, A.M.,Zanon, P., Obradovic, D.,Wyss, R.,Graff, P. and Herrmann W.. Early ultrastructural changes in RU-486-exposed decidua. Gynecolobstetlnvest. 20: 62 (1985) 6. Kovacs,L., Sas, M.,Resch, B.A.,Ugocsai,G.,Swahn, M.L.and Bygdeman M. Termination of very early pregnancy by RU 486-an antiprogestational compound. Contraception 29: 399 (1984) 7. Swahn, M.L.,Johannisson, E.,Daniore, V., de la Terre, 8. and Bygdeman, M: The effect of RU486 administered during the proliferative and secretory phase of the cycle on the bleeding pattern, hormonal parameters and the endometrium. Human Reproduction. (Submitted for publication) 8. Gravanis. A., Schaison, G.,George, M., de Brux, J.. Satyaswaroop, P.G.and Baulieu, E.E.and Robel, P. Endometrial and pituitary responses to the steroidal antiprogestin RU486 in postmenopausal women. J. Clin. Endocrinol. Metab. 60:156 (1985) 9. Markee. J.E.Menstruation in intraocular endometrial transplants in the rhesus monkey. Contrib. Embryol. 28: 2 19 (1940) 10. Johannisson, E..Landgren. B.M..Rohr, H.P.and DiczfalusyE.:Endometrial morphology and peripheral hormone levels in women with regular menstrual cycles. Fertil. Steril. 48: 40 1 (1987) 11. Oberholzer, M. Morphometrie in der klinischen Pathologie. Allgemeine Grundlagen. Springer Verlag, Berlin, 1983. 12. Sufi, S.B..Donaldson, A., and JeffcoateSL. Method Manual, WHO Programme for the Provision of Matched AssayReagents, 10th Ed., London,1986.
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13. Beksac, MS., Wang, Y. and Cekan, S. Validity of the radioimmunoassay of unconjugated estradiol in urine. Acta Reprod. Turcica 555 (1984) 14. Beksac, MS. and Cekan. S. Reliability of progesterone measurements in urine. J. Clin.Chem. Clin.B&hem. 23:183 (1985) 15. Philibert, D.,Deraedt, T., Teutsch. G..Tournemine, C. and Sakiz, E. RU 38486 a new lead for steroidal antihormones. Presented at the 64th Annual Meeting of the Endocrine Society, San Francisco, June 16 to 18,1982,Abstract No. 668. 16. Baulieu, E.E.RU486. An antiprogestin steroid with contragestive activity in women. InThe Antiprogestin Steroid RU486 and Human Fertility Control (EEBaulieu and SJ Segal, Editors). Plenum Press, New York, 1985, p.l. 17. Schaison, G.,George, M.,Lestrat, N.,Reinberg, A. and Baulieu. E.E.Effects of the antiprogesterone steroid RU486 during midluteal phase in normal women. J. Clin.Endocrinol. Metab. 6 1:484 (1985) 18. Collins,R.L.and Hodgen, G.D.Blockade of the spontaneous midcycle gonadotropin surge in monkeys by RU486: a progesterone antagonist or agonist? J. Clin.Endocrinol. Metab. 63:1270 (1986) 19. King,S.,Johannisson, E.,Landgren, B.M.and Diczfalusy,E. Pituitary, ovarian and endometrial effects of progesterone released prematurely during the proliferative phase. Contraception 27: 177 i 1983) 20. Bergqvist, A., Carlstrom. K.and Ljungberg, 0.. Histochemical localization of estrogen and progesterone receptors. J. Histochem. Cytochem. 32: 493 (19841
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