0021-972X/90/7101-0105$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society
Vol. 71, No. 1 Printed in U.S.A.
A Potential Primate Model for Bone Loss Resulting from Medical Oophorectomy or Menopause* DAVID R. MANN, KENNETH G. GOULD, AND DELWOOD C. COLLINS Department of Physiology, Morehouse School of Medicine, Yerkes Regional Primate Research Center, and the Medical Research Service, Veterans Administration Medical Center/Emory University School of Medicine, Atlanta, Georgia 30310
ABSTRACT. This study examined the potential use of the GnRH agonist-treated female monkey as a model for bone loss after medical oophorectomy or the onset of menopause in women. Three female rhesus monkeys (13-16 yr of age) were treated continuously for 10 months with 25 /xg/day GnRH agonist using osmostic minipumps. All three animals exhibited normal menstrual cycles before treatment. Within 5 weeks of the beginning of GnRH agonist treatment, serum progesterone and estradiol concentrations had fallen to low values and did not rise significantly during the remaining treatment period. The decline in ovarian steroidogenesis was correlated with a reduction in bone mineral density (BMD; bone mineral content/ bone width) of the caudal vertebrae and humerus. The reduction of BMD of the caudal vertebrae occurred gradually. The downward trend was evident during the first 3 treatment months, but did not fall significantly below pretreatment levels until 9 months of GnRN agonist treatment. The overall decline in BMD for the caudal vertebrae was approximately 14% after 9 months
of GnRH agonist treatment. The measured decline in BMD of the humerus was 11%. Serum osteocalcin levels rose more than 10-fold above pretreatment values between 4 and 7 months of GnRH agonist treatment before declining to levels that approached pretreatment concentrations between 8 and 10 months of treatment. Menstrual cycles were reinitiated within 4 weeks after the termination of treatment, as shown by luteal phase increases in serum progesterone concentrations. BMD of the humerus and caudal vertebrae remained subnormal 2 months posttreatment, but by 5 months had recovered to near-pretreatment values. These data suggest that ovarian hormone deprivation induced by GnRH agonist administration is associated with a decline in BMD in female monkeys, and that this animal model may be an excellent model for postmenopausal bone loss or bone reduction resulting from medical oophorectomy. The GnRH agonist-treated monkey also has the potential to be developed as a model for type I postmenopausal osteoporosis. (J Clin Endocrinol Metab 7 1 : 105-110,1990)
A
density than younger animals (10), and ovariectomy is associated with a significant loss of trabecular bone in Old World monkeys (11). All of these data suggest that this nonhuman primate may be a valuable model for the loss of bone associated with the onset of menopause. We have shown that continuous GnRH agonist treatment of female monkeys causes a cessation of menstrual cycles and a decline of circulating ovarian hormone levels to near-castrate values (12, 13). This effect is fully reversible, with menstrual cycles resuming within 4-5 weeks of termination of agonist treatment (12). Thus, agonist treatment induces a reversible medical ovariectomy and ovarian hormone deprivation similar to those in the early postmenopausal period or the period after surgical ovariectomy. These data suggested that the agonist-treated monkey might be a useful model for the bone loss that occurs as a result of ovarian hormone deprivation following menopause or surgical ovariectomy. The objective of this study was to examine the effect of administration of a GnRH agonist on bone density in adult female monkeys and to correlate any changes with
N APPROPRIATE animal model has not been developed for postmenopausal bone loss in women (1). A variety of animal models have been used to study the effect of age and ovarian hormone deprivation on bone mass. These models include the aged ovariectomized rat (1-5) and the aged ovariectomized dog (6, 7). Each of these models has the distinct disadvantage of not being a primate model. Female macaque monkeys exhibit menstrual cycles of similar length and hormonal patterns to those in the human. Moreover, these monkeys show irregular cycles or amenorrhea, a sustained rise in serum gonadotropins, and low circulating levels of serum estradiol and progesterone (characteristic of the onset of human menopause) between 22 and 30 yr of age (8, 9). Female monkeys over 30 yr of age have lower humerus and vertebrae bone Received August 30,1989. Address all correspondence and requests for reprints to: David R. Mann, Ph.D., Department of Physiology, Morehouse School of Medicine, 720 Westview Drive SW, Atlanta, Georgia 30310-1495. * This work was supported in part by a grant from Genentech, Inc., NIH Grants HD-23295, RR-08248 (via cofunding from NIMH), and RR-00165; and V.A. Grant 1513-081.
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circulating levels of ovarian hormones and osteocalcin, a marker of bone turnover.
followed by the Dunnett test for multiple comparisons between treatment times (16).
Materials and Methods
Results
Three female rhesus monkeys (Macaca mulatto), ranging in age from 13-16 yr, were used in this study. Animals were maintained in a temperature (22-24 C)- and light (12 h of light, 12 h of darkness)-controlled room throughout the study. Monkeys were treated continuously for 10 months with a GnRH agonist (Wyeth-Ayerst Laboratories, Princeton, NJ; DTrp6-AT-Me-Leu7-des-Glylo-Pro9-NHEt-GnRH) using osmotic minipumps (2 mL 4; Alza Corp. Palo Alto, CA). Pumps were inserted sc and replaced every 4 weeks under sterile conditions and ketamine anesthesia (15-20 mg/kg BW). Blood samples were taken from conscious animals three times a week during one control menstrual cycle and then twice a week during the treatment and the posttreatment periods. Serum was stored at -20 C until assayed for hormones. Every 4 weeks, animals were anesthetized with ketamine, and the bone mineral density [BMD; bone mineral content/ bone width (BMC/BW); grams per cm2) of the midshaft humerus and eighth caudal vertebrae was measured using the Norland model 2780 single photon absorptiometer. In all cases, five scans were taken of each bone from each subject, and the mean of the five scans was used as the measurement for that bone. The in vivo precision for the humerus was 1.2%, and that for the caudal vertebrae was 3.8%. In an earlier study we compared the BMD of hydrated isolated humeri, eighth caudal vertebrae, and third lumbar vertebrae obtained at autopsy using the single photon unit (10). Using isolated lumbar vertebrae, it is possible to measure BMD using the single photon unit. There was good correlation between the readings for lumbar vertebrae and caudal vertebrae (correlation coefficient = 0.92; P < 0.01; n = 33) and between lumbar vertebrae and the humerus (correlation coefficient = 0.92; P < 0.01; n = 31) in these necropsy specimens. There was also good correlation for BMC/BW between live us. necropsy specimens for the humerus (correlation coefficient = 0.98; P < 0.01; n = 7) and a lesser but significant correlation for the caudal vertebrae (correlation coefficient = 0.76; P < 0.05; n = 7). These data indicate that the single photon unit provides reliable data on the humerus and caudal vertebrae of live tissue that is highly correlated with measurements on isolated bone specimens. Serum samples were analyzed for progesterone (14) and estradiol (15) by RIA. Serum osteocalcin concentrations were assayed by RIA, using a kit purchased from Incstar Corp. (Stillwater, MN). This assay uses bovine osteocalcin for a radioactive tracer (125I) and standard. Increasing aliquots of a serum pool from female monkeys ran parallel to the bovine standard in the RIA. Intra- and interassay coefficients of variation for the progesterone, estradiol, and osteocalcin assays were 6.0%, 6.8%, and 6.8% and 9.2%, 9.8%, and 5.1%, respectively. The limits for detection of progesterone, estradiol, and osteocalcin were 0.8 nmol/L, 88 pmol/L, and 0.8 ng/mL, respectively. Hormonal and BMD measurements were analyzed initially with a one-way analysis of variance with repeated measures,
All three monkeys exhibited normal menstrual cycle patterns of serum estradiol and progesterone concentrations before treatment (Fig. 1). By the second month of GnRH agonist treatment, serum levels of estradiol and progesterone had fallen to low values. The three animals were apparently anovulatory throughout the GnRH agonist treatment, as assessed by the lack of a luteal phase rise in serum progesterone (Fig. 2). The suppression of ovarian steroidogenesis by the GnRH agonist was associated with an increase in serum osteocalcin concentrations (Fig. 3). Serum osteocalcin levels began to rise during the fifth month of GnRH agonist treatment and were more than 8-fold {P < 0.01) and 12-fold (P < 0.01) greater than pretreatment levels during the sixth and seventh treatment months. After peak osteocalcin levels were achieved at 7 months of GnRH agonist treatment, serum osteocalcin levels declined to values that were slightly but not significantly greater than pretreatment values. GnRH agonist treatment caused an overall decrease in the BMD of both the caudal vertebrae (P < 0.005) and humerus (P < 0.001; Fig. 4). BMD values of the caudal vertebrae showed a gradual decline throughout treatment. By the ninth month of GnRH agonist administration, BMD reached levels that were significantly below (P < 0.05) the pretreatment value (Fig. 4, top). The overall decline in the BMD of the caudal vertebrae was approximately 14%. The overall decline in the BMD of the humerus was 11%, but the decrease in the humerus reached levels of significance earlier (6 months; P < 0.05; Fig. 4, bottom) than the caudal vertebrae (9 months). This was probably not the result of a more rapid loss of humeral bone, but was caused by the lower variability in humeri vs. caudal vertebrae readings. Humeral BMD readings remained significantly lower (P < 0.05) at 9 months of GnRH agonist treatment, but did not differ from the values at 6 months of treatment. Menstrual cycles resumed within 4 weeks of the termination of GnRH agonist treatment in all three monkeys. The pattern of serum estradiol and progesterone levels during the first posttreatment cycle did not differ from that during the pretreatment cycle (data not shown). Serum osteocalcin concentrations during the posttreatment period did not differ from those in the late treatment period (Fig. 3). Two months posttreatment, bone density values for the humerus (P < 0.05) and caudal vertebrae (P < 0.05) were still subnormal, but had recovered to near-pretreatment values by 5 months posttreatment (Fig. 4).
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BONE MASS IN GnRH AGONIST-TREATED MONKEYS
107 -•12.0
500>
400-
-9.0
LJ
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300FIG. 1. Mean (±SE) serum estradiol and progesterone concentrations in three female monkeys during a pretreatment menstrual cycle.
o E ^^
i
200-
o o
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-12
-4
0
4
12
16
PRETREATMENT CYCLE DAYS
FlG. 2. Mean (±SE) serum estradiol and progesterone concentrations in three female monkeys during GnRH agonist treatment.
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Discussion Treatment of female monkeys with a GnRH agonist induced an amenorrheic anovulatory condition that resembles the ovarian hormone milieu of postmenopausal women and patients after ovariectomy (12, 13). In this study we have shown that the decline in ovarian steroidogenesis in these monkeys is associated with a reduction in bone mass, suggesting that the GnRH agonist-treated monkey may be useful as a model for postmenopausal bone loss or the decline in bone mass after medical oophorectomy in women. Upon further definition (bone histomorphometry), the GnRH agonist-treated monkey has potential as a model for type I postmenopausal osteoporosis. We envision the use of this model for testing the efficacy of therapeutic approaches for decelerating bone loss as a result of ovarian hormone deprivation and defining the mechanisms (which may involve invasive approaches) by which ovarian steroids or pos-
4 5 6 7 MONTHS OF TREATMENT
8
9
10
11
sible therapeutic agents preserve bone mass in ovarian hormone-deficient women. In the present study, the effect of GnRH agonist treatment on bone metabolism in female monkeys was comparable to changes reported for women in which a different analog was used to treat endometriosis (17,18). After 9 months of GnRH agonist treatment in monkeys, bone density (single photon absorptiometry) of the caudal vertebrae and the humerus had declined 14% and 7%, respectively. This compares to a decline of more than 7% in BMC of the spinal trabecular bone (quantitative computed topography) (17) and 6% in bone mass of the distal radius (single photon absorptiometry) and spinal vertebrae (dual photon absorptiometry) (18) in women treated for 6 months with a GnRH agonist. The greater overall decline in bone density of monkeys in the present study may be related to the 50% longer period of GnRH agonist administration. The pattern of circulating osteocalcin is interesting
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16.0
Poattroatment Treatment
Posttreatment
14.0-
FIG. 3. Mean (±SE) serum osteocalcin concentrations in female monkeys before (zero value), during, and after GnRH agonist treatment. *, Significantly different from the pretreatment value at the P < 0.05 level or better.
and provides new information about the longitudinal changes that occur in osteocalcin during treatment with a GnRH agonist. Osteocalcin levels remained near pretreatment levels for the first 5 months, rose about 10fold during months 6 and 7 (8.2 and 12.1 ng/mL) of treatment, followed by a drop to approximately 3 ng/mL by 9 months of treatment. Comparison of these data with results in the human is not possible at the present time because sufficient longitudinal data are not available in the human. Gudmundsson et al. (19) reported the effect of nafarelin on osteocalcin in women and showed increased osteocalcin at 6 months. Unfortunately, they measured osteocalcin only during pretreatment and after 6 months of treatment. Johansen et al. (18) measured serum osteocalcin levels in women with endometriosis treated with nafarelin for 6 months and found an elevation of osteocalcin after 6 months of treatment and 3 months posttreatment. Osteocalcin levels were only measured at 3-month intervals, and treatment was terminated at 6 months. Both of these studies suggest, however, that GnRH agonist treatment is associated with a rise in serum osteocalcin, as it was in the present study in GnRH agonist-treated monkeys. In two cross-sectional studies, the mean postmenopausal osteocalcin values were not different from premenopausal values (20, 21). Yasumura et al. (21) reported elevated levels of osteocalcin levels in the perimenopausal period that later returned to normal. Brown et al. (20) found that most osteophoretic postmenopausal women had osteocalcin levels within the normal range. However, a small percentage of patients with high turnover osteoporosis based on bone histomorphometry had high osteocalcin levels, similar to those reported in our animals after 6-7 months of treatment with GnRH agonist. This period may represent a resorptive or osteoclastic phenomenon that drives coupled osteoblastic ac-
Poittreatmont
6
9 MONTHS
FIG. 4. Mean bone ^density measurements (±SE) of the caudal vertebrae (top) and humerus (bottom), expressed as percentage of the pretreatment values, in female monkeys before, during, and after GnRH agonist treatment. *, Significantly different from the pretreatment value at the P < 0.05 level or better.
tivity. Our results suggest that the monkey may serve as a model reflecting the high turnover period of activity at 6-7 months of treatment, followed by a return toward more normal turnover rates. Menstrual cycles were initiated in the monkeys within 5 weeks of terminating GnRH agonist treatment. The recovery of bone mass in monkeys after GnRH agonist treatment appears to be similar to that reported for women after GnRH agonist therapy (17, 19). The BMD measurements remained subnormal during the first 2 months posttreatment in both the humerus and caudal vertebrae. In both cases, BMD measurements were increased to near pretreatment values within 5 months of termination of GnRH agonist treatment. Osteopenia is an established characteristic of human aging, but little comparative data are available for other primate species. Twenty pigtail and two longtail macaques showed age-related changes in metacarpal cortical thickness similar to those observed in humans (22). Age-
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BONE MASS IN GnRH AGONIST-TREATED MONKEYS related osteopenia was also observed in the skeletons of female, but not male, rhesus monkeys from the Cayo Santiago collection (23). These results suggest that similar processes are involved in skeletal aging of human and macaque monkeys. However, the aged monkey model is of limited usefulness for the study of osteoporosis because of the protracted maintenance period (several decades) necessary to obtain aged animals (1). The cost to provide sufficient animals for such studies would be prohibitive. Ovarian hormone deprivation resulting from ovariectomy of monkeys is associated with bone loss similar to that occurring during the first decade after the onset of menopause or after surgical ovariectomy in women (2428). Twenty-two months after ovariectomy, longtailed macaques had less trabecular bone than intact monkeys (11, 29). In the present study we have shown that reversible medical ovariectomy in female monkeys with GnRH agonist treatment is also associated with bone loss. Thus, GnRH agonist administration induces a hypogonadal postmenopausal-like state that is associated with a measurable loss of bone mass, but, unlike surgical castration, is fully reversible. There are three advantages of this model over the ovariectomized monkey model for bone loss associated with ovarian hormone deprivation in women. 1) The reversibility of the model allows examination of both the effect of ovarian hormone deprivation on bone mass and the effect of reinitiation of menstrual cycles on bone structure and mass. 2) The reversibility of the treatment does not sacrifice the reproductive potential of the animal. 3) Using this model, it is theoretically possible to use animals more than once to study the effect of acute ovarian hormone deprivation on bone mass. The aged ovariectomized rodent has been used as a model for bone loss. Female, but not male, rats castrated at 12 months of age exhibited decreased intestinal absorption of calcium, and greater loss of trabecular than cortical bone as the animals aged (2, 5). These changes are similar to those seen in postmenopausal women (3032). Conversely, intact mice and rats fed an adequate diet do not show a decline in bone mass between 1 and 2 yr of age (33, 34). Furthermore, fractures do not result from bone loss in ovariectomized rats (5). The cortical bone in rats lacks Haversian canals (5), rodents do not have lamellar bone, and rodents have only a limited capacity for bone remodeling (1). Thus, rodent models, despite their cost effectiveness, have limited value as models for bone loss in humans because of these and other characteristics dissimilar to human bone and bone changes associated with human aging and ovarian deficiency. Ovariectomy is also associated with a loss of trabecu-
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lar, but not cortical, bone loss in dogs (7), and bone morphology in dogs resembles that in humans (1). However, bone turnover is much greater and fracture rate lower in dogs than humans (1), limiting the usefulness of this species as a model for bone loss in humans. In summary, these data suggest that the GnRH agonist-treated female monkey warrants further study as a model for bone loss associated with medical oophorectomy or menopause and for examining the efficacy of therapeutic approaches for treating these conditions. Clearly, GnRH agonist-treated monkeys show decreases in serum estradiol and progesterone and a rise in osteocalcin characteristic of women during the early stages of menopause. Further studies are necessary to determine its usefulness as a model for the study of type I postmenopausal osteoporosis. Acknowledgments We thank Wyeth-Ayerst Laboratories for providing the GnRH agonist used in this study. We also thank Mr. Thomas Buckner, Ms. Toni Duffey, Ms. Robin King, Ms. Sheres Caines, and Ms. Janice Tittle for their expert technical assistance. All experiments were performed according to principles and procedures of the NIH Guidelines for the Care and Use of Laboratory Animals. The Yerkes Regional Primate Research Center is fully accredited by the American Association for Accreditation of Laboratory Animal Care.
References 1. Draper HH. Osteoporosis: animal models for the study of nutrition and disease. In: Bird RP, Mercer NJH, Draper HH, eds. Advances in nutrition research. New York: Plenum Press; 1985; 172-86.
2. Gurkan L, Ekeland A, Gautvik KM, Langeland N, Ronningen H, Solheim LF. Bone changes after castration in rats: a model for osteoporosis. Orthrop Scand. 1986;57:67-70. 3. Safadi M, Shapira D, Leichter I, Reznick A, Silbermann M. Ability of different techniques of measuring bone mass to determine vertebral bone loss in aging female rats. Calcif Tissue Int. 1988;42:37582. 4. Okumura H, Yamamuro T, Kasai R, Hayashi T, Tada K, Nishii Y. Effect of la-hydroxyvitamin D3 on osteoporosis induced by immobilization combined with ovariectomy in rats. Bone. 1988;8:351-5. 5. Kalu DN, Liu C-C, Hardin RR, Hollis BW. The aged rat model of ovarian hormone deficiency bone loss. Endcrinology. 1989; 124:716. 6. Jee WSS, Kimmel DB, Hashimoto EG, Dell RB, Woodbury LA. Quantitative studies of Beagle vertebral bodies. In: Jaworski ZFG, ed. Bone morphometry. Ottawa: University of Ottawa Press; 1976:110-7. 7. Martin RB, Butcher RL, Sherwood LL, et al. Effects of ovariectomy in beagle dogs. Bone. 1987;8:23-31. 8. Hodgen GD, Goodman AL, O'Connor A, Johnson DK. Menopause in rhesus monkeys: model for study of disorders in the human climacteric. Am J Obstet Gynecol. 1977;127:581-4. 9. Van Wagenen G. Menopause in a subhuman primate. Anat Rec. 1970;166:392. 10. Pope NS, Gould KG, Anderson DC, Mann DR. Effects of age and sex on bone density in the rhesus monkey. Bone. 1989;10:109-12. 11. Miller LC, Weaver DS, McAlister JA, Koritnik DR. Effects of ovariectomy on vertebral trabecular bone in the cynomolgus monkey (Macaca fascicularis). Calcif Tissue Int. 1986;38:62-5. 12. Mann DR, Collins DC, Smith MM, Kessler MT, Gould KG. Treatment of endometriosis in monkeys: effectiveness of continu-
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20. 21.
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ous infusion of a gonadotropin-releasing hormone agonist compared to treatment with a progestational steroid. J Clin Endocrinol Metab. 1986;63:1277-83. Fraser HM, Sandow J. Suppression of follicular maturation by infusion of a luteinizing hormone-releasing hormone agonist starting during the late luteal phase in stumptailed macaque monkey. J Clin Endocrinol Metab. 1985;60:579-84. Graham CE, Collins DC, Robinson H, Preedy JRK. Urinary levels of estrogens and pregnanediol and plasma levels of progesterone during the menstrual cycle of the chimpanzee. Endocrinology. 1972;91:13-24. Wright K, Collins DC, Preedy JRK. The use of specific radioimmunoassays to determine the renal clearance rates of estrone and 17/3-estradiol during the menstrual cycle. J Clin Endocrinol Metab. 1978;47:1084-91. Keppel G. Design and analysis: a researcher's handbook. Englewood Cliffs: Prentice-Hall; 1973. Cann CE, Henzl M, Burry K, et al. Reversible bone loss is induced by GnRH agonists. Proc of the 68th Annual Meet of The Endocrine Soc. 1986;37. Johansen JS, Riis BJ, Hassager C, Moen M, Jacobson J, Christiansen C. The effect of a gonadotropin-releasing hormone agonist analog (Nafarelin) on bone metabolism. J Clin Endocrinol Metab. 1988;67:701-6. Gudmundsson JA, Ljunghall S, Bergquist C, Wide L, Nillius SJ. Increased bone turnover during gonadotropin-releasing hormone superagonist-induced ovulation inhibition. J Clin Endocrinol Metab. 1987;65:159-63. Brown JP, Delmas PD, Malaval L, Edouard C, Chapuy MC, Meunier PJ. Serum bone Gla-protein: a specific marker for bone formation in postmenopausal osteoporosis. Lancet. 1984;l:1091-3. Yasumura S, Aloia JF, Gundberg CM, et al. Serum osteocalcin and total body calcium in normal pre-and postmenopausal women and postmenopausal osteoporotic patients. J Clin Endocrinol Metab.
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1987;64:681-5. 22. Bowden DM, Teets C, Witkin J, Young DM. Long bone calcification and morphology. In: Bowden DM, ed. Aging in nonhuman primates. New York: Van Nostrand Reinhold; 1979:335-47. 23. DeRousseau CJ. Aging in the musculoskeletal system of rhesus monkeys. II. Bone loss. Am J Physical Anthropol. 1985;68:157-67. 24. Newton-John HF, Morgan DB. The loss of bone with age, osteoporosis, and fractures. Clin Orthop. 1970;71:229-52. 25. Mazess RB. On aging bone loss. Clin Orthop. 1982;165:239-52. 26. Krolner B, Nielsen SP. Bone mineral content of the lumbar spine in normal and osteoporotic women: cross-sectional and longitudinal studies. Clin Sci. 1982;62:329-36. 27. Cann CE, Genant HK, Ettinger B, et al. Spinal mineral loss in oophorectomized women. JAMA. 1980;244:2056-9. 28. Christiansen C, Christiansen MS, McNair P, et al. I. Prevention of early postmenopausal bone loss: controlled 2-year study in 315 normal females. Eur J Clin Invest 1980; 10:273-9. 29. Bowles EA, Weaver DS, Telewski FW, Wakefield AH, Jaffe MJ, Miller LC. Bone measurement by enhanced contrast image analysis: ovariectomized and intact Macaca fascicularis as a model for human postmenopausal osteoporosis. Am J Physical Anthropol. 1985;67:99-103. 30. Gallagher JC, Riggs BL, Eisman J, Hanstra A, Arnaud SB, DeLuca HF. Intestinal calcium absorption and serum vitamin D metabolites in normal subjects and osteoporotic patients. Effect of age and dietary calcium. J Clin Invest. 1979;64:729-36. 31. Riggs BL, Melton HI LJ. Evidence for two distinct syndromes of involutional osteoporosis. Am J Med. 1983;75:899-901. 32. Riggs BL, Melton III LJ. Involutional osteoporosis. N Engl J Med. 1986;314:1676-86. 33. Hansard SL, Crowder HM. The physiological behavior of calcium in the rat. J Nutr. 1957;62:325-39. 34. Shah BG, Krishnarao GVG, Draper HH. The relationship of Ca and P nutrition during adult life and osteoporosis in adult mice. J Nutr. 1967;92:30-42.
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Vol. 71, No. 1 Printed in U.S.A.
A Potential Primate Model for Bone Loss Resulting from Medical Oophorectomy or Menopause* DAVID R. MANN, KENNETH G. GOULD, AND DELWOOD C. COLLINS Department of Physiology, Morehouse School of Medicine, Yerkes Regional Primate Research Center, and the Medical Research Service, Veterans Administration Medical Center/Emory University School of Medicine, Atlanta, Georgia 30310
ABSTRACT. This study examined the potential use of the GnRH agonist-treated female monkey as a model for bone loss after medical oophorectomy or the onset of menopause in women. Three female rhesus monkeys (13-16 yr of age) were treated continuously for 10 months with 25 /xg/day GnRH agonist using osmostic minipumps. All three animals exhibited normal menstrual cycles before treatment. Within 5 weeks of the beginning of GnRH agonist treatment, serum progesterone and estradiol concentrations had fallen to low values and did not rise significantly during the remaining treatment period. The decline in ovarian steroidogenesis was correlated with a reduction in bone mineral density (BMD; bone mineral content/ bone width) of the caudal vertebrae and humerus. The reduction of BMD of the caudal vertebrae occurred gradually. The downward trend was evident during the first 3 treatment months, but did not fall significantly below pretreatment levels until 9 months of GnRN agonist treatment. The overall decline in BMD for the caudal vertebrae was approximately 14% after 9 months
of GnRH agonist treatment. The measured decline in BMD of the humerus was 11%. Serum osteocalcin levels rose more than 10-fold above pretreatment values between 4 and 7 months of GnRH agonist treatment before declining to levels that approached pretreatment concentrations between 8 and 10 months of treatment. Menstrual cycles were reinitiated within 4 weeks after the termination of treatment, as shown by luteal phase increases in serum progesterone concentrations. BMD of the humerus and caudal vertebrae remained subnormal 2 months posttreatment, but by 5 months had recovered to near-pretreatment values. These data suggest that ovarian hormone deprivation induced by GnRH agonist administration is associated with a decline in BMD in female monkeys, and that this animal model may be an excellent model for postmenopausal bone loss or bone reduction resulting from medical oophorectomy. The GnRH agonist-treated monkey also has the potential to be developed as a model for type I postmenopausal osteoporosis. (J Clin Endocrinol Metab 7 1 : 105-110,1990)
A
density than younger animals (10), and ovariectomy is associated with a significant loss of trabecular bone in Old World monkeys (11). All of these data suggest that this nonhuman primate may be a valuable model for the loss of bone associated with the onset of menopause. We have shown that continuous GnRH agonist treatment of female monkeys causes a cessation of menstrual cycles and a decline of circulating ovarian hormone levels to near-castrate values (12, 13). This effect is fully reversible, with menstrual cycles resuming within 4-5 weeks of termination of agonist treatment (12). Thus, agonist treatment induces a reversible medical ovariectomy and ovarian hormone deprivation similar to those in the early postmenopausal period or the period after surgical ovariectomy. These data suggested that the agonist-treated monkey might be a useful model for the bone loss that occurs as a result of ovarian hormone deprivation following menopause or surgical ovariectomy. The objective of this study was to examine the effect of administration of a GnRH agonist on bone density in adult female monkeys and to correlate any changes with
N APPROPRIATE animal model has not been developed for postmenopausal bone loss in women (1). A variety of animal models have been used to study the effect of age and ovarian hormone deprivation on bone mass. These models include the aged ovariectomized rat (1-5) and the aged ovariectomized dog (6, 7). Each of these models has the distinct disadvantage of not being a primate model. Female macaque monkeys exhibit menstrual cycles of similar length and hormonal patterns to those in the human. Moreover, these monkeys show irregular cycles or amenorrhea, a sustained rise in serum gonadotropins, and low circulating levels of serum estradiol and progesterone (characteristic of the onset of human menopause) between 22 and 30 yr of age (8, 9). Female monkeys over 30 yr of age have lower humerus and vertebrae bone Received August 30,1989. Address all correspondence and requests for reprints to: David R. Mann, Ph.D., Department of Physiology, Morehouse School of Medicine, 720 Westview Drive SW, Atlanta, Georgia 30310-1495. * This work was supported in part by a grant from Genentech, Inc., NIH Grants HD-23295, RR-08248 (via cofunding from NIMH), and RR-00165; and V.A. Grant 1513-081.
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circulating levels of ovarian hormones and osteocalcin, a marker of bone turnover.
followed by the Dunnett test for multiple comparisons between treatment times (16).
Materials and Methods
Results
Three female rhesus monkeys (Macaca mulatto), ranging in age from 13-16 yr, were used in this study. Animals were maintained in a temperature (22-24 C)- and light (12 h of light, 12 h of darkness)-controlled room throughout the study. Monkeys were treated continuously for 10 months with a GnRH agonist (Wyeth-Ayerst Laboratories, Princeton, NJ; DTrp6-AT-Me-Leu7-des-Glylo-Pro9-NHEt-GnRH) using osmotic minipumps (2 mL 4; Alza Corp. Palo Alto, CA). Pumps were inserted sc and replaced every 4 weeks under sterile conditions and ketamine anesthesia (15-20 mg/kg BW). Blood samples were taken from conscious animals three times a week during one control menstrual cycle and then twice a week during the treatment and the posttreatment periods. Serum was stored at -20 C until assayed for hormones. Every 4 weeks, animals were anesthetized with ketamine, and the bone mineral density [BMD; bone mineral content/ bone width (BMC/BW); grams per cm2) of the midshaft humerus and eighth caudal vertebrae was measured using the Norland model 2780 single photon absorptiometer. In all cases, five scans were taken of each bone from each subject, and the mean of the five scans was used as the measurement for that bone. The in vivo precision for the humerus was 1.2%, and that for the caudal vertebrae was 3.8%. In an earlier study we compared the BMD of hydrated isolated humeri, eighth caudal vertebrae, and third lumbar vertebrae obtained at autopsy using the single photon unit (10). Using isolated lumbar vertebrae, it is possible to measure BMD using the single photon unit. There was good correlation between the readings for lumbar vertebrae and caudal vertebrae (correlation coefficient = 0.92; P < 0.01; n = 33) and between lumbar vertebrae and the humerus (correlation coefficient = 0.92; P < 0.01; n = 31) in these necropsy specimens. There was also good correlation for BMC/BW between live us. necropsy specimens for the humerus (correlation coefficient = 0.98; P < 0.01; n = 7) and a lesser but significant correlation for the caudal vertebrae (correlation coefficient = 0.76; P < 0.05; n = 7). These data indicate that the single photon unit provides reliable data on the humerus and caudal vertebrae of live tissue that is highly correlated with measurements on isolated bone specimens. Serum samples were analyzed for progesterone (14) and estradiol (15) by RIA. Serum osteocalcin concentrations were assayed by RIA, using a kit purchased from Incstar Corp. (Stillwater, MN). This assay uses bovine osteocalcin for a radioactive tracer (125I) and standard. Increasing aliquots of a serum pool from female monkeys ran parallel to the bovine standard in the RIA. Intra- and interassay coefficients of variation for the progesterone, estradiol, and osteocalcin assays were 6.0%, 6.8%, and 6.8% and 9.2%, 9.8%, and 5.1%, respectively. The limits for detection of progesterone, estradiol, and osteocalcin were 0.8 nmol/L, 88 pmol/L, and 0.8 ng/mL, respectively. Hormonal and BMD measurements were analyzed initially with a one-way analysis of variance with repeated measures,
All three monkeys exhibited normal menstrual cycle patterns of serum estradiol and progesterone concentrations before treatment (Fig. 1). By the second month of GnRH agonist treatment, serum levels of estradiol and progesterone had fallen to low values. The three animals were apparently anovulatory throughout the GnRH agonist treatment, as assessed by the lack of a luteal phase rise in serum progesterone (Fig. 2). The suppression of ovarian steroidogenesis by the GnRH agonist was associated with an increase in serum osteocalcin concentrations (Fig. 3). Serum osteocalcin levels began to rise during the fifth month of GnRH agonist treatment and were more than 8-fold {P < 0.01) and 12-fold (P < 0.01) greater than pretreatment levels during the sixth and seventh treatment months. After peak osteocalcin levels were achieved at 7 months of GnRH agonist treatment, serum osteocalcin levels declined to values that were slightly but not significantly greater than pretreatment values. GnRH agonist treatment caused an overall decrease in the BMD of both the caudal vertebrae (P < 0.005) and humerus (P < 0.001; Fig. 4). BMD values of the caudal vertebrae showed a gradual decline throughout treatment. By the ninth month of GnRH agonist administration, BMD reached levels that were significantly below (P < 0.05) the pretreatment value (Fig. 4, top). The overall decline in the BMD of the caudal vertebrae was approximately 14%. The overall decline in the BMD of the humerus was 11%, but the decrease in the humerus reached levels of significance earlier (6 months; P < 0.05; Fig. 4, bottom) than the caudal vertebrae (9 months). This was probably not the result of a more rapid loss of humeral bone, but was caused by the lower variability in humeri vs. caudal vertebrae readings. Humeral BMD readings remained significantly lower (P < 0.05) at 9 months of GnRH agonist treatment, but did not differ from the values at 6 months of treatment. Menstrual cycles resumed within 4 weeks of the termination of GnRH agonist treatment in all three monkeys. The pattern of serum estradiol and progesterone levels during the first posttreatment cycle did not differ from that during the pretreatment cycle (data not shown). Serum osteocalcin concentrations during the posttreatment period did not differ from those in the late treatment period (Fig. 3). Two months posttreatment, bone density values for the humerus (P < 0.05) and caudal vertebrae (P < 0.05) were still subnormal, but had recovered to near-pretreatment values by 5 months posttreatment (Fig. 4).
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BONE MASS IN GnRH AGONIST-TREATED MONKEYS
107 -•12.0
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300FIG. 1. Mean (±SE) serum estradiol and progesterone concentrations in three female monkeys during a pretreatment menstrual cycle.
o E ^^
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FlG. 2. Mean (±SE) serum estradiol and progesterone concentrations in three female monkeys during GnRH agonist treatment.
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Discussion Treatment of female monkeys with a GnRH agonist induced an amenorrheic anovulatory condition that resembles the ovarian hormone milieu of postmenopausal women and patients after ovariectomy (12, 13). In this study we have shown that the decline in ovarian steroidogenesis in these monkeys is associated with a reduction in bone mass, suggesting that the GnRH agonist-treated monkey may be useful as a model for postmenopausal bone loss or the decline in bone mass after medical oophorectomy in women. Upon further definition (bone histomorphometry), the GnRH agonist-treated monkey has potential as a model for type I postmenopausal osteoporosis. We envision the use of this model for testing the efficacy of therapeutic approaches for decelerating bone loss as a result of ovarian hormone deprivation and defining the mechanisms (which may involve invasive approaches) by which ovarian steroids or pos-
4 5 6 7 MONTHS OF TREATMENT
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sible therapeutic agents preserve bone mass in ovarian hormone-deficient women. In the present study, the effect of GnRH agonist treatment on bone metabolism in female monkeys was comparable to changes reported for women in which a different analog was used to treat endometriosis (17,18). After 9 months of GnRH agonist treatment in monkeys, bone density (single photon absorptiometry) of the caudal vertebrae and the humerus had declined 14% and 7%, respectively. This compares to a decline of more than 7% in BMC of the spinal trabecular bone (quantitative computed topography) (17) and 6% in bone mass of the distal radius (single photon absorptiometry) and spinal vertebrae (dual photon absorptiometry) (18) in women treated for 6 months with a GnRH agonist. The greater overall decline in bone density of monkeys in the present study may be related to the 50% longer period of GnRH agonist administration. The pattern of circulating osteocalcin is interesting
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MANN, GOULD, AND COLLINS
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FIG. 3. Mean (±SE) serum osteocalcin concentrations in female monkeys before (zero value), during, and after GnRH agonist treatment. *, Significantly different from the pretreatment value at the P < 0.05 level or better.
and provides new information about the longitudinal changes that occur in osteocalcin during treatment with a GnRH agonist. Osteocalcin levels remained near pretreatment levels for the first 5 months, rose about 10fold during months 6 and 7 (8.2 and 12.1 ng/mL) of treatment, followed by a drop to approximately 3 ng/mL by 9 months of treatment. Comparison of these data with results in the human is not possible at the present time because sufficient longitudinal data are not available in the human. Gudmundsson et al. (19) reported the effect of nafarelin on osteocalcin in women and showed increased osteocalcin at 6 months. Unfortunately, they measured osteocalcin only during pretreatment and after 6 months of treatment. Johansen et al. (18) measured serum osteocalcin levels in women with endometriosis treated with nafarelin for 6 months and found an elevation of osteocalcin after 6 months of treatment and 3 months posttreatment. Osteocalcin levels were only measured at 3-month intervals, and treatment was terminated at 6 months. Both of these studies suggest, however, that GnRH agonist treatment is associated with a rise in serum osteocalcin, as it was in the present study in GnRH agonist-treated monkeys. In two cross-sectional studies, the mean postmenopausal osteocalcin values were not different from premenopausal values (20, 21). Yasumura et al. (21) reported elevated levels of osteocalcin levels in the perimenopausal period that later returned to normal. Brown et al. (20) found that most osteophoretic postmenopausal women had osteocalcin levels within the normal range. However, a small percentage of patients with high turnover osteoporosis based on bone histomorphometry had high osteocalcin levels, similar to those reported in our animals after 6-7 months of treatment with GnRH agonist. This period may represent a resorptive or osteoclastic phenomenon that drives coupled osteoblastic ac-
Poittreatmont
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FIG. 4. Mean bone ^density measurements (±SE) of the caudal vertebrae (top) and humerus (bottom), expressed as percentage of the pretreatment values, in female monkeys before, during, and after GnRH agonist treatment. *, Significantly different from the pretreatment value at the P < 0.05 level or better.
tivity. Our results suggest that the monkey may serve as a model reflecting the high turnover period of activity at 6-7 months of treatment, followed by a return toward more normal turnover rates. Menstrual cycles were initiated in the monkeys within 5 weeks of terminating GnRH agonist treatment. The recovery of bone mass in monkeys after GnRH agonist treatment appears to be similar to that reported for women after GnRH agonist therapy (17, 19). The BMD measurements remained subnormal during the first 2 months posttreatment in both the humerus and caudal vertebrae. In both cases, BMD measurements were increased to near pretreatment values within 5 months of termination of GnRH agonist treatment. Osteopenia is an established characteristic of human aging, but little comparative data are available for other primate species. Twenty pigtail and two longtail macaques showed age-related changes in metacarpal cortical thickness similar to those observed in humans (22). Age-
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BONE MASS IN GnRH AGONIST-TREATED MONKEYS related osteopenia was also observed in the skeletons of female, but not male, rhesus monkeys from the Cayo Santiago collection (23). These results suggest that similar processes are involved in skeletal aging of human and macaque monkeys. However, the aged monkey model is of limited usefulness for the study of osteoporosis because of the protracted maintenance period (several decades) necessary to obtain aged animals (1). The cost to provide sufficient animals for such studies would be prohibitive. Ovarian hormone deprivation resulting from ovariectomy of monkeys is associated with bone loss similar to that occurring during the first decade after the onset of menopause or after surgical ovariectomy in women (2428). Twenty-two months after ovariectomy, longtailed macaques had less trabecular bone than intact monkeys (11, 29). In the present study we have shown that reversible medical ovariectomy in female monkeys with GnRH agonist treatment is also associated with bone loss. Thus, GnRH agonist administration induces a hypogonadal postmenopausal-like state that is associated with a measurable loss of bone mass, but, unlike surgical castration, is fully reversible. There are three advantages of this model over the ovariectomized monkey model for bone loss associated with ovarian hormone deprivation in women. 1) The reversibility of the model allows examination of both the effect of ovarian hormone deprivation on bone mass and the effect of reinitiation of menstrual cycles on bone structure and mass. 2) The reversibility of the treatment does not sacrifice the reproductive potential of the animal. 3) Using this model, it is theoretically possible to use animals more than once to study the effect of acute ovarian hormone deprivation on bone mass. The aged ovariectomized rodent has been used as a model for bone loss. Female, but not male, rats castrated at 12 months of age exhibited decreased intestinal absorption of calcium, and greater loss of trabecular than cortical bone as the animals aged (2, 5). These changes are similar to those seen in postmenopausal women (3032). Conversely, intact mice and rats fed an adequate diet do not show a decline in bone mass between 1 and 2 yr of age (33, 34). Furthermore, fractures do not result from bone loss in ovariectomized rats (5). The cortical bone in rats lacks Haversian canals (5), rodents do not have lamellar bone, and rodents have only a limited capacity for bone remodeling (1). Thus, rodent models, despite their cost effectiveness, have limited value as models for bone loss in humans because of these and other characteristics dissimilar to human bone and bone changes associated with human aging and ovarian deficiency. Ovariectomy is also associated with a loss of trabecu-
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lar, but not cortical, bone loss in dogs (7), and bone morphology in dogs resembles that in humans (1). However, bone turnover is much greater and fracture rate lower in dogs than humans (1), limiting the usefulness of this species as a model for bone loss in humans. In summary, these data suggest that the GnRH agonist-treated female monkey warrants further study as a model for bone loss associated with medical oophorectomy or menopause and for examining the efficacy of therapeutic approaches for treating these conditions. Clearly, GnRH agonist-treated monkeys show decreases in serum estradiol and progesterone and a rise in osteocalcin characteristic of women during the early stages of menopause. Further studies are necessary to determine its usefulness as a model for the study of type I postmenopausal osteoporosis. Acknowledgments We thank Wyeth-Ayerst Laboratories for providing the GnRH agonist used in this study. We also thank Mr. Thomas Buckner, Ms. Toni Duffey, Ms. Robin King, Ms. Sheres Caines, and Ms. Janice Tittle for their expert technical assistance. All experiments were performed according to principles and procedures of the NIH Guidelines for the Care and Use of Laboratory Animals. The Yerkes Regional Primate Research Center is fully accredited by the American Association for Accreditation of Laboratory Animal Care.
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2. Gurkan L, Ekeland A, Gautvik KM, Langeland N, Ronningen H, Solheim LF. Bone changes after castration in rats: a model for osteoporosis. Orthrop Scand. 1986;57:67-70. 3. Safadi M, Shapira D, Leichter I, Reznick A, Silbermann M. Ability of different techniques of measuring bone mass to determine vertebral bone loss in aging female rats. Calcif Tissue Int. 1988;42:37582. 4. Okumura H, Yamamuro T, Kasai R, Hayashi T, Tada K, Nishii Y. Effect of la-hydroxyvitamin D3 on osteoporosis induced by immobilization combined with ovariectomy in rats. Bone. 1988;8:351-5. 5. Kalu DN, Liu C-C, Hardin RR, Hollis BW. The aged rat model of ovarian hormone deficiency bone loss. Endcrinology. 1989; 124:716. 6. Jee WSS, Kimmel DB, Hashimoto EG, Dell RB, Woodbury LA. Quantitative studies of Beagle vertebral bodies. In: Jaworski ZFG, ed. Bone morphometry. Ottawa: University of Ottawa Press; 1976:110-7. 7. Martin RB, Butcher RL, Sherwood LL, et al. Effects of ovariectomy in beagle dogs. Bone. 1987;8:23-31. 8. Hodgen GD, Goodman AL, O'Connor A, Johnson DK. Menopause in rhesus monkeys: model for study of disorders in the human climacteric. Am J Obstet Gynecol. 1977;127:581-4. 9. Van Wagenen G. Menopause in a subhuman primate. Anat Rec. 1970;166:392. 10. Pope NS, Gould KG, Anderson DC, Mann DR. Effects of age and sex on bone density in the rhesus monkey. Bone. 1989;10:109-12. 11. Miller LC, Weaver DS, McAlister JA, Koritnik DR. Effects of ovariectomy on vertebral trabecular bone in the cynomolgus monkey (Macaca fascicularis). Calcif Tissue Int. 1986;38:62-5. 12. Mann DR, Collins DC, Smith MM, Kessler MT, Gould KG. Treatment of endometriosis in monkeys: effectiveness of continu-
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1987;64:681-5. 22. Bowden DM, Teets C, Witkin J, Young DM. Long bone calcification and morphology. In: Bowden DM, ed. Aging in nonhuman primates. New York: Van Nostrand Reinhold; 1979:335-47. 23. DeRousseau CJ. Aging in the musculoskeletal system of rhesus monkeys. II. Bone loss. Am J Physical Anthropol. 1985;68:157-67. 24. Newton-John HF, Morgan DB. The loss of bone with age, osteoporosis, and fractures. Clin Orthop. 1970;71:229-52. 25. Mazess RB. On aging bone loss. Clin Orthop. 1982;165:239-52. 26. Krolner B, Nielsen SP. Bone mineral content of the lumbar spine in normal and osteoporotic women: cross-sectional and longitudinal studies. Clin Sci. 1982;62:329-36. 27. Cann CE, Genant HK, Ettinger B, et al. Spinal mineral loss in oophorectomized women. JAMA. 1980;244:2056-9. 28. Christiansen C, Christiansen MS, McNair P, et al. I. Prevention of early postmenopausal bone loss: controlled 2-year study in 315 normal females. Eur J Clin Invest 1980; 10:273-9. 29. Bowles EA, Weaver DS, Telewski FW, Wakefield AH, Jaffe MJ, Miller LC. Bone measurement by enhanced contrast image analysis: ovariectomized and intact Macaca fascicularis as a model for human postmenopausal osteoporosis. Am J Physical Anthropol. 1985;67:99-103. 30. Gallagher JC, Riggs BL, Eisman J, Hanstra A, Arnaud SB, DeLuca HF. Intestinal calcium absorption and serum vitamin D metabolites in normal subjects and osteoporotic patients. Effect of age and dietary calcium. J Clin Invest. 1979;64:729-36. 31. Riggs BL, Melton HI LJ. Evidence for two distinct syndromes of involutional osteoporosis. Am J Med. 1983;75:899-901. 32. Riggs BL, Melton III LJ. Involutional osteoporosis. N Engl J Med. 1986;314:1676-86. 33. Hansard SL, Crowder HM. The physiological behavior of calcium in the rat. J Nutr. 1957;62:325-39. 34. Shah BG, Krishnarao GVG, Draper HH. The relationship of Ca and P nutrition during adult life and osteoporosis in adult mice. J Nutr. 1967;92:30-42.
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