Direct effects of medroxyprogesterone acetate, danazol, and leuprolide acetate on endometrial stromal cell proliferation in vitro*†

FERTILITY AND STERILITY

Vol. 58, No.2, August 1992

Printed on acid-free paper in U.S.A.

Copyright cD 1992 The American Fertility Society

Direct effects of medroxyprogesterone acetate, danazol, and leuprolide acetate on endometrial stromal cell proliferation in vitro*t

Eric S. Surrey, M.D.:} Jouko Halme, M.D., Ph.D.§ Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Cedars-Sinai Medical Center, and University of California-Los Angeles School of Medicine, Los Angeles, California

Objective: To assess the direct effects of three endometriosis chemotherapeutic agents (medroxyprogesterone acetate [MPA], danazol, and leuprolide acetate [LA]) on endometrial cell proliferation. Design: Analysis of cell proliferation in vitro. Setting: Proliferative phase endometrial stromal cells isolated from biopsy specimens and grown in short-term culture served as a model for stromal components of endometriotic implants. Patients: Biopsies obtained from volunteers with regular cycles and without endometriosis or endometrial pathology. Interventions: Medroxyprogesterone acetate, danazol, and LA were added to nutrient media with the following supplements: 2.5% calf serum (CS) only; 2.5% CS + estradiol (E 2) 110 pmol/L; 2.5% CS + E2 550 pmol/L; 10% preovulatory serum (net E2 624 pmol/L). Main Outcome Measures: Cumulative [3HJ-thymidine incorporation as a reflection of cell proliferation. Results: Medroxyprogesterone acetate and danazol exerted significant antiproliferative effects on stromal cell proliferation (P < 0.05), effects that were enhanced by an absence of exogenous E 2. Leuprolide acetate exerted no consistent effects. Conclusions: These data imply that MP A and danazol but not LA exert direct effects, suppressing Fertil Steril 1992;58:273-8 growth of endometriotic implants. Key Words: Endometriosis, stromal cells, danazol, progestins, gonadotropin-releasing hormone agonist

Various chemotherapeutic agents have been employed in the medical management of endometriosis. Those that have been used most commonly in clinReceived November 15, 1991; revised and accepted April 3, 1992.

* Supported in part by grant HD21546 from the National Institutes of Health, Bethesda, Maryland. t Presented in part as the First Prize Winning Poster at the 45th Annual Meeting of The American Fertility Society, San Francisco, California, November 11 to 16, 1989. t Reprint requests: Eric S. Surrey, M.D., Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Suite 1720, Los Angeles, California 90048. § Present address: Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of North Carolina, Chapel Hill, North Carolina. Vol. 58, No.2, August 1992

ical practice include danazol, gonadotropin-releasing hormone agonists (GnRH-a), and progestins such as medroxyprogesterone acetate (MPA) (1-5). The effectiveness of these medications has been reported in terms of such indirect parameters as improvement in symptoms, increase in pregnancy rates, and changes in laparoscopic extent or histologic appearance of disease. Clear evidence of a direct action of these agents on growth patterns of endometriotic implants has not been consistently identified. In the present investigation, we have attempted to demonstrate the direct effects of these three drugs on proliferation of endometriotic implants. The role of ovarian steroids and sustained growth of ectopic endometrium in the pathophysiology and mainte-

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nance of endometriosis has been well established (6). Accordingly, isolated endometrial stromal cells grown in short-term culture with varying steroid hormone supplements were used as a model.

MATERIALS AND METHODS

Endometrial biopsies were obtained from regularly cycling reproductive age women between 20 and 35 years immediately before laparoscopic tubal sterilization. All patients gave informed consent as approved by the University Human Subjects Committee. Patients with endometrial pathology, endometriosis, irregular bleeding, or who had received hormonal preparations <30 days before biopsy were excluded. All biopsies were obtained during the late proliferative phase. The tissue was immediately placed into culture medium and processed within 60 minutes of collection. The basic medium employed throughout these experiments was Hepes-buffered Ham's F-10 medium (pH 7.4; Irvine Scientific, Irvine, CA) supplemented with 0.1 % L-glutamine, 4 mg/mL D-glucose, and 0.1% gentamycin (Sigma Laboratories, St. Louis, MO). The technique used for separation and isolation of endometrial stromal cells has been previously described in detail (7, 8). The mean total stromal cell yield for the experiments described in this investigation was 5.97 X 106 cells/biopsy (range 2.4 X 106 to 9 X 106 ). The cells were suspended in medium supplemented with 2.5% calf serum (CS; Colorado Calf Serum Co., Denver, CO) and plated into microwell plates (30 mm 2 wells; ACS Nunclon, Roskild, Denmark) in a concentration of 20,000 cells/well. We have described previously the effects of cell number and CS concentrations on proliferative characteristics for this model (7). The plates were incubated at 37°C in 5% CO 2and 95% air under sterile tissue culture conditions. The medium was aspirated after 24 hours. Subsequently, chemotherapeutic agents were added in quadruple replicates to supplemented culture medium: MPA (Provera; Upjohn, Kalamazoo, MI) in concentrations of 3, 30, and 300 mg/mL; the GnRH-a leuprolide acetate (LA, Lupron; TAP Pharmaceutical Corp., North Chicago, IL) in concentrations of 1, 10, and 100 ng/mL; danazol (Danocrine, Winthrop, New York, NY) in concentrations of 10, 100, and 1000 ng/mL. The range of concentrations were chosen according to previously established therapeutic plasma levels for each of these agents (9-11 and TAP Pharmaceutical Corp., unpublished observations, 1988). 274

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In an effort to demonstrate the effect of physiological steroid hormone levels, each of these agents was added to culture medium with one of four supplements. Group A: Ten percent human preovulatory serum pooled from five women without endometriosis undergoing controlled ovarian hyperstimulation with human menopausal gonadotropins for assisted reproductive technologies. Serum was obtained on the day of but before administration of human chorionic gonadotropins. The pooled sample had a 17{3-estradiol (E 2) concentration of 1,700 pg/mL (6,240 pmoljL) and progesterone (P) concentration of 0.7 ng/mL (2.22 nmol/L) as measured by previously described radioimmunoassay (RIA) techniques (12). Thus, the net E2 concentration in this medium was 170 pg/mL (624 pmoljL), compatible with expected midfollicular phase values in unstimulated cycles. Group B: 2.5% CS alone. Group C: 2.5% CS in combination with E2 30 pg/mL (110 pmoljL). Group D: 2.5% CS in combination with E2 150 pg/mL (550 pmoljL). Ethanol 0.25% was employed as a solvent for E2 as well as the three chemotherapeutic agents. No detectable quantities of E 2, estrone, or P were identified in either the basic medium or CS by standard RIA techniques (12). Cells grown in each of the aforementioned nutrient media with 0.25% ethanol only were employed as controls. Identical plates for all assays were prepared for harvesting 24, 48, and 72 hours after addition of chemotherapeutic agents. Stromal cell proliferation as reflected by deoxyribonucleic acid synthesis was measured by calculating [3H]-thymidine incorporation by stromal cells using previously described techniques (7). One microcurie of [3H]-thymidine (New England Nuclear, Boston, MA) was added to each microwell 24 hours before harvesting. Immediately before harvesting, one drop of 0.1 M ethylenediaminetetraacetic acid (Sigma) was added to each well to detach the cells that were then trapped onto glass fiber filter paper by using a semiautomatic cell harvester (Skatron, Sterling, VA). Each filter disc paper corresponded to an individual well on the culture plates. These were air dried and placed in liquid scintillation counting tubes to which 2.0 mL of scintillation cocktail had been added (Beckman, Fullerton, CA). Incorporation of radioactivity was measured with a liquid scintillation counter (Beckman). Statistical analysis of the results was performed by Student's grouped t-tests with Buonferroni corrections for multiple comparisons. A calculated P value < 0.05 was considered statistically significant unless otherwise indicated. Each assay was performed in quadruplicate with cells obtained from a

Endometrial stromal cells and chemotherapy

Fertility and Sterility

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single biopsy. Each experiment was repeated three times with cells obtained from different individuals. Results are expressed as cumulative counts per minute. Because of the contributory effect of preceding data points, statistical analyses were performed only on the cumulative 72-hour data representing the summation of 3H-thymidine incorporation for plates harvested at 24, 48, and 72 hours. Cells from different patients were never mixed during individual experiments. Because of the qualitative but not quantitative similarity of results among assays, representative data are presented.

culture media are displayed in Figure 2. No consistently significant effects on cumulative stromal cell proliferation were observed with any of the concentrations of LA regardless of culture medium employed. The results from similar experiments performed with the addition of danazol to stromal cells incubated in each of the four different media are presented in Figure 3. After 72 hours, cumulative endometrial stromal cell proliferation was significantly lower than controls (P < 0.05) when danazol 100 ngjmL and 1000 ngjmL was added in all four culture media. The degree of suppression was not significantly different between these two concentrations, however. Results obtained with 10 ngjmL, a subtherapeutic concentration, were less consistent with suppression achieved only in group A. Overall, no consistent dose-dependent effect was noted. In an attempt to assess the direct effects of the various media supplements, the mean change in [3H]-thymidine incorporation compared with control during the 72-hour harvest was compared for each supplement at each concentration of MPA and danazol employed (Fig. 4). A greater degree of suppression was noted for cells cultured in medium supplemented by 2.5% CS alone (group B) at each concentration of both MPA and danazol. This difference reached a level of statistical significance (P < 0.01) with MPA 3 and 300 ngjmL as well as LEUPROLIDE ACETATE

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The effects on stromal cell proliferation of the addition of the three concentrations of MPA are displayed in Figure 1. Note that after 72 hours, cumulative [3H]-thymidine incorporation was significantly lower than control (P < 0.01) for all concentrations of the progestin in all four media employed. The degree of suppression was greatest with MPA 30 ngjmL in groups A, C, D, and with 300 ngjmL in group B. However, these differences did not achieve statistical significance among the three concentrations of MPA in any group. Overall, there was no clear evidence of a dose-dependent effect. Cumulative [3H]-thymidine incorporation of stromal cells exposed to three concentrations of the GnRH -a LA in each of the four supplemented Vol. 58, No.2, August 1992

C

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DANAZOL 10'" PRE-OVULATORY SERUM 3200

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with danazoll00 and 1,000 ngjmL. There were no significant differences among the other three media.

gestins alone are somewhat conflicting. Employing mixed endometrial cultures, investigators have demonstrated that P decreased endometrial proliferation in both short-term culture and cell lines (8, 14). In contrast, others have reported that P and MP A stimulated endometrial stromal cell growth in culture (15, 16). The use of secretory as opposed to proliferative phase cells in the latter experiments may explain these varying results. Danazol has also been demonstrated to be a highly effective agent in the management of symptomatic endometriosis (1, 2). Concentrations employed in this investigation that resulted in an antiproliferative effect were well within the range for plasma levels achieved after acute and chronic oral administration of this agent in a clinical setting (9, 10). It has been assumed that danazol acts primarily as an antigonadotropin with additional direct inhibitory effects on steroidogenic enzymes as well as binding capabilities to androgen and Preceptors (17). However, evidence of direct receptor binding has not been demonstrated in endometrium or endometriotic implants. The data presented in the current investigation provide strong evidence to support a direct antiproliferative effect on isolated endometrial stromal cells in short-term culture. Employing mixed endometrial cell cultures obtained at varying points in the menstrual cycle, Rose et al. (18) have previously suggested that danazol and its metabo-

DISCUSSION

In this investigation, we have assessed the direct tissue effects of three chemotherapeutic agents that have been successfully used in the treatment of endometriosis. We have demonstrated that therapeutic levels of MPA (Fig. 1) and danazol (Fig. 3) suppressed proliferation of isolated proliferative phase endometrial stromal cells to a greater degree than cells grown in nutrient medium alone as measured by [3H]-thymidine incorporation. These effects were noted under all conditions tested. In contrast, LA had no consistent direct effect on stromal cell proliferation (Fig. 2). Oral MP A has been demonstrated previously to be effective in improving symptoms and diminishing extent of implants in patients with endometriosis (5). Oral daily doses of 30 to 50 mg, which produce serum levels well within the range of concentrations employed in this investigation, have been used to achieve these effects (11). The current data would confirm that MPA has a direct effect on ectopic endometrial tissue. Progestogens have been shown to have antimitotic effects on estrogen-primed endometrium (13), but data on the effects of P and pro276

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l!. CONTROL AT 72 HOURS MEDROXYPROGESTERONE ACETATE 3ng/mL

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Endometrial stromal cells and chemotherapy

Fertility and Sterility

lites acted directly to inhibit cell growth by employing direct cell-counting techniques. Others have shown this effect in long-term mixed endometrial cell cultures (14). The effect of varying the estrogenic environment on the antiproliferative actions of these agents was assessed by employing four different supplements to the basic nutrient medium. We have previously demonstrated that in this model, the 24 hours before the 72-hour cell harvest is the time interval of maximal cell proliferation (7). Varying E2 concentrations were used to reflect early and late follicular phase levels in vivo (groups C and D, respectively). A positive control consisted of pooled preovulatory serum from women receiving exogenous gonadotropin stimulation (group A). After dilution, E 2 levels were similar to those in group D. A negative control consisted of unsupplemented medium (group B). The suppressive effects of MPA and danazol in this model were maximal after 72 hours of culture in the complete absence of E2 as well as other mitogens present in human serum (Fig. 4). Absence of the previously described consistent independent doseresponse effects of exogenous E2 on cell proliferation (19) may be explained by the extremely low physiological concentrations employed in our present investigation. A broader concentration range was employed in our earlier study (19). A slight degree of inherent estrogenicity in the culture medium employed may have further masked amplification of cell proliferation by this combination (20). One could hypothesize that the clinical effectiveness of these agents could be enhanced by prior induction of a hypoestrogenic state with GnRH -a. Various highly potent GnRH-a have been demonstrated to be effective agents in the medical management of endometriosis with a mode of action felt to be secondary to induction of a hypoestrogenic state by interaction at the level of the hypothalamicpituitary axis (3, 4). This investigation provides evidence for lack of any direct effect of these agents on endometrial stromal cell proliferation (Fig. 2). Weare aware of no data to support the presence of direct endometrial effects of either GnRH or GnRH -a in the human. Although the cells employed in this investigation were not derived from endometriotic implants and their in vitro responses may not be identical, the rationale for the use of late-proliferative phase endometrial stromal cells in short-term culture as a model for the stromal components of endometriotic implants has been previously discussed (7). In brief, endometrial stromal cells survive more consistently than epithelial cells in tissue culture (21). Although Vol. 58, No.2, August 1992

the degree of synchrony between uterine endometrium and endometriotic implants is controversial (22, 23), only proliferative phase endometrial stromal cells have been shown to display a similar number of nuclear estrogen receptor sites as foci of endometriosis (24). It is important to recognize that the interactive effects of the local peritoneal environment on proliferation of endometriotic implants have not been assessed in the current investigation. Using a similar model, we have previously demonstrated that cellfree peritoneal fluid fractions obtained from women with endometriosis stimulated endometrial stromal cell proliferation to a significantly greater degree than fractions obtained from women without the disease (7). This effect was also achieved with the addition of a macrophage secretory product, plateletderived growth factor, in a similar model (19). The presence of minimal amounts of such growth factors in the serum supplemented medium employed in this investigation cannot be excluded. Assessment ofthe direct antiproliferative effects of these chemotherapeutic agents in the presence of mitogenic peritoneal fluid fractions would further add to the understanding of their mechanism of action. Nevertheless, the data presented in the current investigation provide further evidence to support a direct effect on endometriotic implants of MPA and danazol, but not LA, in preventing proliferation of endometriosis. Acknowledgments. We express appreciation to TAP Pharmaceuticals (North Chicago, IL) for their generous donation of LA, Elisa Obnial, B.S., and Eufemia Tsui, B.S., for technical assistance, and Ms. Hazel Myers and Ms. Laurie Levine for typographical skills in preparing this manuscript. REFERENCES 1. Buttram VC Jr, Reiter RC, Ward S. Treatment of endometriosis with danazol: report of a 6-year prospective study. Fertil Steril 1980;43:353-60. 2. Dmowski WP, Kapetanakis E, Scommegna A. Variable effects of danazol on endometriosis at 4 low-dose levels. Obstet GynecoI1982;59:408-15. 3. Steingold KA, Cedars M, Lu JKH, Randle D, Judd HL, Meldrum DR. Treatment of endometriosis with a long-acting gonadotropin-releasing hormone agonist. Obstet Gynecol 1987;69:403-11. 4. Dlugi AM, Miller JD, Knittle J, Lupron Study Group. Lupron depot (leuprolide acetate for depot suspension) in the treatment of endometriosis: a randomized, placebo-controlled, double-blind study. Fertil Steril 1990;54:419-27. 5. Luciano AA, Turksoy RN, Carleo J. Evaluation of oral medroxyprogesterone acetate in the treatment of endometriosis. Obstet Gynecol 1988;72:323-6. 6. diZerega GS, Barber DL, Hodgen GD. Endometriosis: role of ovarian steroids in initiation, maintenance, and suppression. Fertil Steril 1980;33:649-53.

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7. Surrey ES, Halme J. Effect of peritoneal fluid from endometriosis patients on endometrial stromal cell proliferation in vitro. Obstet GynecoI1990;76:792-7. 8. Fleming H, Gurpide E. Growth characteristics of primary cultures of stromal cells from human endometrium. J Steroid Biochem 1982;16:717-20. 9. Lloyd-Jones JC, Labross A, Williams-Ross T, Ross RW, Edelson J, Davison C. Danazol plasma concentration in man. J Int Med Res 1977; SuppI5:18-24. 10. Williams TA, Edelson J, Ross RW Jr. A radioimmunoassay for danazo1. Steroids 1978;31:205-17. 11. Sall S, DiSaia P, Morrow CP, Mortel R, Prem K, Thigpen T, et a1. A comparison of medroxyprogesterone serum concentrations by the oral or intramuscular route in patients with persistent or recurrent endometrial carcinoma. Am J Obstet GynecoI1979;135:647-50. 12. deVane GW, Czekala NM, Judd HL, Yen SSC. Circulating gonadotropins, estrogens, and androgens in polycystic ovarian disease. Am J Obstet Gynecol 1975;121:496-500. 13. Whitehead MI, Townsend PT, Pryse-Davis J. Progestins: effects of various types and dosages on the postmenopausal endometrium. J Reprod Med 1982;27:539-48. 14. Neulen J, Wagner B, Runge M, Breckwoldt M. Effect of progestins, androgens, estrogens and anti-estrogens on 3H-thymidine uptake by human endometrial and endosalpinx cells in vitro. Arch Gynecol 1987;240:225-32. 15. Irwin JC, Kirk D, King RJB, Quigley MM, Gwatkin RBL. Hormonal regulation of human endometrial stromal cells in culture: an in vitro model for decidualization. Fertil Steril 1989;52:761-8. 16. Zhu HH, Huang JR, Mazella J, Rosenberg M, Tseng L. Differential effects of progestin and relaxin on the synthesis and

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secretion of immunoreactive prolactin in long term culture of human endometrial stromal cells. J Clin Endocrinol Metab 1990;71:889-99. 17. Barbieri RL, Ryan KJ. Danazol: endocrine pharmacology and therapeutic applications. Am J Obstet GynecoI1981;141:45363. 18. Rose GL, Dowsett M, Mudge JE, White JO, Jeffcoate SL. The inhibitory effects of danazol, danazol metabolites, gestrinone, and testosterone on the growth of human endometrial cells in vitro. Fertil Steril 1988;49:224-8. 19. Surrey ES, Halme J. Effect of platelet-derived growth factor on endometrial stromal cell proliferation in vitro: a model for endometriosis? Fertil Steril 1991;56:672-9. 20. Berthois Y, Katzenellenbogen JA, Katzenellenbogen BS. Phenol red in tissue culture media is a weak oestrogen: implications concerning the study of oestrogen-responsive cells in culture. Proc Natl Acad Sci USA 1986;83:2496-500. 21. Kirk D, King RJB, Heyes J, Peachey L, Hirsch PJ, Taylor RWT. Normal human endometrium in cell culture. I. Separation and characterization of epithelial stromal components in vitro. In Vitro 1978;14:651-62. 22. Berquist A, Ljungberg 0, Myhre E. Human endometrium and endometriotic tissue obtained simultaneously: a comparative histologic study. Int J Gynecol Pathol 1984;3:13345. 23. Lessey BA, Metzger DA, Haney AF, McCarthy KS Jr. Immunohistochemical analysis of estrogen and progesterone receptors in endometriosis: comparison with normal endometrium during the menstrual cycle and the effect of medical therapy. Fertil Steril1989;51:409-15. 24. Gould SF, Shannon JM, Cunha GR. Nuclear estrogen binding site in human endometriosis. Fertil Steril 1983;39:520-4.

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