Barbieri R. L.

Carlstrom K.

Dmowski W. P.

Ferenczy A.

Flamigni C.

Halme J.

Ikegami H.

Kokko E.

Kreige H.

Lamb M. P.

Luciano A. A.

Osteen K. G.

Zullo F.

MONTEMAGNO, U.

DE PLACIDO, G.

COLACURCI, N.

ZULLO, F.

Archivio della ricerca - Università degli studi di Napoli Federico II

PDFlib PLOP: PDF Linearization, Optimization, ProtectionPage inserted by evaluation versionwww.pdflib.com – sales@pdflib.comEndometrial Effects of Danazol in Perimenopausal Abnormal Bleeding U. MONTEMAGNO, G. DE PLACIDO, N. COLACURCI, AND F. ZULLO Department of Obstetrics and Gynecology I1 School of Medicine of Naples Naples, Italy INTRODUCTION Danazol (D), a 2-3 isoxazol derivative of 17-ethinyltestosterone, is routinely used for the treatment of endometriosis' and is known to suppress menstrual bleeding.* Endometrial effects of D are not only related to the well-known inhibition of the hypo- thalamic pituitary function and ovarian steroid synthesis, but they seem to be primarily determined by a direct action on the end~metrium.~ In fact, D binds to progesterone receptors (PR) and inhibits 17-P-hydroxy-steroid-dehydrogena~e~ and steroid sulfatase activity5 in human endometrium. The effectiveness of D in treatment of perimeno- pausal abnormal bleeding has been confirmed in many trials.C7 The demonstration of antiproliferative activity on human endometrial cancer cells in vivos and on ade- nomatous hyperplasia in vivo,9 together with the histologic observation that D induces rapid atrophic changes whereas a marked decidual reaction is found through progestin treatment, suggest a difference in the effect of D and typical progestins on endometrial tissue. In this regard we have considered useful to evaluate clinically, hysteroscopically and histopathologically the efficacy of D 200 mg daily for 3 months in the treatment of perimenopausal abnormal bleeding sustained by endometrial hyperplasia without cytologic atypiatO in patients at their first therapeutic approach (group A) and in women already treated by progestin with persistance of the disease or recurrence within two months (group B). In addition a further endometrial antiproliferative mechanism of action of D, probably in relation with its immunosuppressive activity is indirectly shown by means of a primary endometrial cell culture conditioned by medium from peritoneal macrophages cultured in vitro. MATERIALS AND METHODS Subjects and Clinical Protocols We have treated by 200 mg daily of D 42 patients with perimenopausal abnormal bleeding underlying an endometrial hyperplasia without cytologic atypia and not pre- viously treated (group A) and 23 women with uterine bleeding already treated by Norethisterone and/or Medroxyprogesterone but with a persistance or recurrence of symptoms within two months (group B). Each patient underwent a hysteroscopic ex- 283 284 ANNALS NEW YORK ACADEMY OF SCIENCES amination with guided endometrial biopsy before starting treatment. The treatment was monitored by: 1) a subjective semiquantitative evaluation of bleeding during treatment and in a follow up to 12 months; 2) hysteroscopically before, at the end of treatment, and in a follow up to 12 months; 3) histopathologically before and after treatment. Side effects and adverse reactions were carefully evaluated. Experimental Section On the basis of a previous experience" in which we showed that D, at a concen- tration of 10-6M, significantly reduced the phagocytic ability of peritoneal macro- phages in vitro, we wanted to see if the steroid is able to decrease the secretion of growth factors12J3 by these immune cells and how such an immunosuppressive effect can be related to endometrial proliferation in vitro. Macrophages derived from peri- toneal fluid collected laparoscopically with an average yield of 5 x 10s cells/ml. Cells were put in RPMI medium with 5% FBS steroid free, washed, centrifuged, and re- suspended to a final concentration of 1 x 106 per dish using 24-well tissue culture plates in 1.5 ml of medium. Cultures were washed 2 hours after plating to separate nonadherent cells, and then incubated for 48 hours. Then supernatants were recovered and frozen at -80°C or immediately used. In some of these cultures, after separating nonadherent cells, D was added at different concentrations washing and changing medium 24 hours later. Then macrophages were cultured for 48 hours when super- natants were recovered. Endometrial cells derived from biopsy specimens sampled in the luteal phase and immediately put in ice-cold 1 : 1 mixture of DMEM and Ham's- F-12. Samples were dissected and a separation between stromal and epithelial cells was performed following the Osteen technique14 with some slight modifications. 15 Es- sentially the method was based on three consecutive incubations in medium containing 0.5% collagenase and 0.05% DNAase followed by differential sedimentation of epi- thelial cells at unit gravity and by selective attachment of stromal cells to plastic cul- tureware. At the end of separation both epithelial and stromal cells were plated at a final density of 5 x lo5 cells/ml in 24-well plates in DMEM-F-12 with 10% char- coal extracted FBS for 48 hours (TO). Cultures were washed and incubated in medium with 2% steroid free FBS for 48 hours (TI). Cell counts were performed on day 2 and day 4 of culture. At TO different amounts (1040% vol/vol) of supernatant from macrophage culture previously treated or not with different concentrations of D were added to the endometrial cell cultures. Viability of cell cultures were assessed by Trypan blue exclusion. RESULTS AND DISCUSSION In group A (TABLE 1) there was a subjective significant decrease of bleeding in 38 out of 41 patients (88.2%), and this result remained unchanged (84.3%) 2 months after discontinuation of treatment, while 4 months after (24.9% of recurrence) and up to 12 months the abnormal bleeding reappeared in 56.2% of women. In TABLE 2 is shown how in group B (patients not responding to progestins) the persistence of heavy blood loss at the end of treatment was 22.7%; this can be considered a very good result considering patients as a control for themselves. This result was practically unchanged 2 months later (27%), with a significant increase in recurrence at 4 (49.9%) MONTEMAGNO et al.: ENDOMETRIAL EFFECTS OF DANAZOL 285 TABLE 1. Blood Loss and Days of Bleeding in Group A Months Posttreatment End of Pretreatment Treatment 2 4 up to 12 n ('3%) n (%) n (X) n (%) n (%) Blood Loss 1 (3) 2 (7) 4 (25) Flooding 19 (45) - Heavy 23 (55) 3 (7) 3 (9) 6 (18) 6 (31) 33 (81) 25 (79) 18 (69) 6 (38) Normal - 1 (8) Amenorrhea - Number of Patients 42 41 32 28 16 Days of Bleeding - 2 ( 5 )  1 (3) 1 (3) 3 (7) 2 (8) 1 (3) Spotting - (mean * SD) 8.9 f 3.7 5.2 f 3.4 5.4 f 3.8 6.3 f 4.8 6.9 f 3.7 and up to 12 months (55.3%). Hysteroscopically in only two patients in group A (4.8%) and two patients in group B (9%) did the hyperplastic picture not revert at the end of treatment, and one of these four patients was a false positive on the basis of the biopsy result (TABLE 3). The hysteroscopic follow-up showed a rapid appearance of hypoatrophic pictures with a progressive increase in the normal findings both prolif- erative and secretive, but also with a meaningful reappearance of hyperplasia up to 60% at 4 and 12 months (TABLE 4). The comparison of hysteroscopic findings with histopathologic pictures shows clearly a good correlation with just 1 hysteroscopic false positive, 2 false negative, and 2 not diagnostic exams (TABLE 5) .  The most interesting aspect of this comparison is the invaluable importance of the hysteroscopy in the diagnosis of hypoatrophia, which is very often undiagnosed bioptically for inadequate sampling. The most frequent side effects during this treatment were weight gain, muscle cramps, headache and acne, but in only two cases was it necessary to stop treatment (TABLE 6). Our data clearly show the efficacy of the regimen of D 200mg daily in the treatment of perimenopausal abnormal bleeding, as indicated in previous reports but TABLE 2. Blood Loss and Davs of Bleedine in GrouD B Months Posttreatment End of Pretreatment Treatment 2 4 up to 12 n (56) n (%) n (%) n (%) n ( % )  Blood Loss Flooding 8 (38) 1 ( 5 )  2 (9) 3 (17) 3 (26) Heavy 14 (81) 4 (18) 4 (18) 6 (33) 8 (53) 3 (28) 14 (63) 14 (83) 8 (44) Normal - 1 (5 )  1 (5 )  2 (9) 1 ( 5 )  1 (6) 1 (7) Amenorrhea - Number of Patients 23 22 22 18 15 Days of Bleeding - - Spotting - (mean f SD) 8 . 5 f 4 . 1  5 . 9 k 2 . 2  6 . 4 f 3 . 6  6 . 8 f 4 . 3  7 . 6 f 3 . 8  286 ANNALS NEW YORK ACADEMY OF SCIENCES TABLE 3. Hysteroscopic Findings before (TO) and at the End of Treatment (TI) with Danazol 200 mg Daily for 3 Months To TI Hysteroscopic Group A Group B Group A Group B Finding n (%) n (96) n (%) n (%) Hyperplasia 39 (92.8) 22 (95.6) 2 (4.8) 2 (9.0) Hypo-atrophy - - 32 (78.0) 16 (72.7) Normotrophy 2 (4.7) - 6 (14.6) 4 (18.1) Not diagnostic 1 (2.3) 1 (4.3) I (2.4) - TOTAL 42 23 41 22 TABLE 4. Hysteroscopic Follow-up at 2 Months (Tz), 4 Months (T3) and up to 12 Months (T4) after Treatment with Danazol 200 mg Daily for 3 Months T2 T3 T4 Hysteroscopic Group A Group B Group A Group B Group A GroupB Finding n (%) n (%) n (%) n (%) n (I) n (%) Hyperplasia 2 (11.1) 3 (20.0) 4 (28.5) 3 (27.2) 9 (60.0) 5 (62.5) Hypo-atrophy 10 (55.5) 8 (53.3) 4 (28.5) 2 (18.1) 2 (13.3) 1 (12.5) Normotrophy Proliferative 4 (22.2) 3 (50.0) 4 (28.5) 4 (36.3) 3 (20.0) 2 (25.0) Secretory 2 (11.1) 1 (6.6) 2 (14.2) 2 (18.1) 5 (33.3) - Not diagnostic - - - - - - TOTAL 18 15 14 11 15 8 TABLE 5. Comparison between Hysteroscopic Findings and Histopathologic Pic- tures before (TO) and after Treatment (TI) Hysteroscopic Finding Histopathologic Appearance Adenomatous hyperplasia Glandular hyperplasia Gland. cystic hyperplasia Proliferative (a) with fibrotio stroma (b) Irregular maturation Atrophic Atrophic with stroma Inadequate sampling pseudodecidualization Hyper- plasia Hermo- trophy Hypo- atrophy Not diagnostic TOTAL To TI 9 2  21 1 31 - - la - -  - -  To TI 2 -  - -  - 7b - 2  - 1  - -  - I1 - 19 To TI - -  2 -  To TI 9 2  25 1 31 - - 8  - 3  - 18 - 12 - 19 TOTAL 61 4 2 10 - 48 2 1 65 63 MONTEMAGNO et al.: ENDOMETRIAL EFFECTS OF DANAZOL 287 loo- t 90- Q 80.  70 K '1E 60- f 20. X 50 1 40- 50-  m lo= TABLE 6. Side Effects during Treatment Month 1 Month 3 n ( 7 % )  n ( 7 % )  Weight gain Headache Nausea or vomiting Acne Hirsutism Muscle cramps Hot flushes Skin rash Voice change Edema Decreased breast size TOTAL 7 (10.7) 3 (4.6) 3 (4.6) 3 (4.6) 1 (1.5) 4 (6.1) 3 (4.6) 1 (1.5) 1 (1.5) 2 (3.0) 3 (4.6) 65 5 (7.9) 3 (4.7) 4 (6.3) l (1 .5 )  2 (3.1) 3 (4.7) - - l (1 .5)  4 (6.3) - 63 they also point out the effectiveness of this steroid in those forms not responding to progestins, confirming indirectly the existence of additional mechanisms of antipro- liferative action on the endometrium if compared to progestogens. In order to verify the hypothesis of an antiproliferative effect mediated by the immunosuppressive ac- tivity of this steroid, we have used an experimental model of primary endometrial cell cultures conditioned by supernatants from peritoneal macrophages cultured in vitro with or without D. The rationale underlying this hypothesis was to test the ability of D to inhibit the secretion of growth factors stimulating the endometrial proliferation by uterine immune cells. m COX rol frol  Y S  + 40% 20% n lox a c t ? (  FIGURE 1. Epithelial cells growth with the addiction of different concentration of supernatant from a peritoneal macrophage culture (MS). 288 130 120 110 100. 8 K 90. x 70 t 1 60. li c 50-  3 40. 30 9 20 10 ANNALS NEW YORK ACADEMY OF SCIENCES 80% voltvol U S  4- 40% I 20% A ftrl FIGURE 2. Stromal cells growth with the addiction of different concentration of supernatant from a peritoneal macrophage culture (MS). 100- 90 Q 80.  70 2 60 K 50 ? 40. 30. z 5 20 * K - - t 3    2 5 0 10-4 Y OanaxoI + 10-5 - lo-= x 10-7 lo-' A C t r l  Day o f  culturo FIGURE 3. Epithelial cells with the addiction of 40% vollvol of supernatant from a peritoneal macro- phage culture previously treated with different concentration of Danazol. MONTEMAGNO et a/.: ENDOMETRIAL EFFECTS OF DANAZOL 289 Our results with this model have shown a marked increase in proliferation when adding to endometrial epithelial cells an aliquot of 40% in volume of supernant from the peritoneal macrophage culture (FIG. 1 ) .  We have used peritoneal samples as a source of macrophages because they can be easily collected and because they seem to be strictly related to the resident immune cells in human endometrium.'5 Using endometrial stromal cells the increase induced by macrophage supernatant was not so evident, probably in relation to the more active early proliferation to confluence of these cells in virro (FIG. 2). When we have used supernatants of macrophages pre- viously exposed to different concentrations of D, we have demonstrated a significant decrease in the proliferation with the peak at 10-6M concentration of D (FIG. 3). In conclusion, our clinical data strongly suggest the presence of other mechanisms of endometrial antiproliferative effect of D in addition to the progestin-like ones. Among these further mechanisms, our preliminary results using a conditioned endometrial primary cell culture seem to show the importance of the inhibition in the production of growth factors by local immune cells exerted by D. REFERENCES 1 .  DMOWSKI, W.P. 1971. Endocrine properties and clinical application of D. Fertil. Steril. 31(3): 237-251. 2. LUCIANO, A. A. 1982. A guide to managing endometriosis. Contemp. Obstet. Gynecol. 19(5): 211-234. 3. 4. 5 .  6. 7. 8. 9. 10. 1 1 .  12. 13. 14. 15. BARBIERI, .  L. & K. J. RYAN. 1981. D endocrine pharmacology and therapeutic applications. Am. J. Obstet. Gynecol. 149(4): 453-463. KOKKO, E., 0. JANNE, A. KAUPPILA, L. RONNBERG & R. VIHKO. 1982. D has progestin like actions on the human endometrium. Acta Endocrinol. 99: 588-593. CARLSTROM, K., A. DOBERL, A. POUSETTE, G. RANNEVIK & N. WILKING. 1984. Inhibition of steroid sulfatase activity by D. Acta Obstet. Gynecol. Scand. U3: 107. LAMB, M. P. 1987. D in menorrhagia: A double-blind placebo-controlled study. J. Obstet. Gynecol. I 212-216. FLAMICNI, C.  V. IASONNI, C. BULLETTI & G. FUSCHINI. 1986. Efficacia e tollerabilita' del D nel trattamento della sindrome menorragica: studio multicentrico. Minerva Ginecol. 38: 1-8. IKEGAMI, H., N. TARAKAWA, I. SHINIZU, T. AONO, 0. TANIZAWA & K. MATSUMOTO. 1986. D binds to progesterone receptors and inhibits the growth of human endometrial cancer cells in vitro. Am. J. Obstet. Gynecol. 155(4): 857-861. TERAKAWA, N.,  M. INOUE, I. SHIMIZU, H. IKEGAMI, T. MIZUTANI. M. SAKATA, D. TANIZAWA & K. MATSUMOTO. 1988. Preliminary report on the use of D in the treatment of endometrial adenornatous hyperplasia. Cancer 62: 2618-2621. FERENCZY, A. & M. GELFAND. 1989. The biologic significance of cytologic atypia in progestogen- treated endometrial hyperplasia. Am. J. Obstet. Gynecol. 1: 126. ZULLO. F.. D. L. FULGHAM, S. I. ROH, G. DE PLACIDO & N. J. ALEXANDER. 1989. Decreased sperm phagocytic ability of peritoneal macrophages from women with endometriosis, using danazol and 6-methyl-prednisolone. Gamete Physiology, Serono Symposia, Newport Beach, CA, USA, November 6-10, 1988. Abstract Book, Abst. 56. HALME, J., M. G. HAMMOND & C. S. SYROP. 1985. Macrophages modulate human granulosa luteal progesterone production. J. Clin. Endocrinol. Metab. 61: 912. HALME, J., S. BECKER & S. HASKILL. 1987. Altered maturation and function of peritoneal macro- phages: Possible role in pathogenesis of endometriosis. Am. J. Obstet. Gynecol. 156: 783. OSTEEN, K. G., G. A.  HILL, J. T. HARGREVE & F. GASTEIN. 1989. Development of a method to isolate and culture highly purified populations of stromal and epithelial cells from human endometrial biopsy specimens. Fertil. Steril. 52(6): 965. ZULLO, F., K. G. OSTEEN, G. D. HODCEN & T. L. ANDERSON. 1989. Development of a model 290 ANNALS NEW YORK ACADEMY OF SCIENCES for isolation and culture of endometrial epithelial and stromal cells from the non-human pri- mate. Society for Gynecological Investigation. San Diego, CA, USA, March 15-18, 1989. Abstract book, Abst. 77. KREICE, H., H. J. RADZUN, K.  HEIDORN, M. R. PARWORESCH & L. METTLER. 1989. Expression of the M-CSF-gene and C-FMS-proto-oncogene in human tissue macrophages. J. Reprod. Immunol. July 1989 (Suppl.): 184. 16. 

Endometrial Effects of Danazol in Perimenopausal Abnormal Bleeding

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Endometrial Effects of Danazol in Perimenopausal Abnormal Bleeding

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