Journal of Clinical Endocrinology and Metabolism
Volume 81 • Number 5 • May 1, 1996
Copyright © 1996 The Endocrine Society

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Antiprogestin and/or Gonadotropin-Releasing Hormone Agonist for Endometriosis Treatment and Bone Maintenance: A 1-Year Primate Study*

Dr.sinan DOĞANTÜRK

Ankara

DANIEL R. GROW

ROBERT F. WILLIAMS

J. G. HSIU

GARY D. HODGEN

Department of Obstetrics and Gynecology, Baystate Medical Center, Tufts University School of Medicine (D.R.G.),
Springfield, Massachusetts 00000; The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School (D.R.G., R.F.W., G.D.H.),
Norfolk, Virginia 23507; and the Department of Pathology, DePaul Hospital (J.G.H.),
Norfolk, Virginia

 

Received August 23, 1995. Revision received November 29, 1995. Accepted December 14, 1995.

Address all correspondence and requests for reprints to: Dr. Gary D. Hodgen, The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia 23507. * Presented at the 41st Annual Meeting of the Society for Gynecologic Investigation, Chicago, IL, March 22-26, 1994.

ABSTRACT

The fact that RU 486 curtailed estrogen-induced endometrial proliferation in primates and relieved pelvic pain in women with endometriosis is the reason for continuing research on antiprogestins. Thirty-two adult female cynomolgus monkeys demonstrating menstrual regularity had surgery for the induction of endometriosis. After lesion staging, four treatment groups (n = 8), each of 1-yr duration, were made. Group I received combination/sequential therapy with depot GnRH agonist (GnRH-a) for 3 months, followed by weekly RU 486 for 9 months. Group II received weekly RU 486, group III received monthly GnRH-a, and group IV served as a vehicle control. A staging laparotomy was performed every 3 months to assess the area of peritoneal endometriosis (square centimeters) and the thickness of in situ endometrium. Bone density was measured serially by dual x-ray absorptiometry. Serum was collection weekly.

Mean (± SE) serum estradiol levels were lower after GnRH-a (77.1 ± 2.6 pmol/L) than after RU 486 (231 ± 12 pmol/L) treatment and lower than those in untreated cycling controls (231 ± 13 pmol/L). GnRH-a produced significant atrophy of endometriotic plaques within 3 months of therapy; this lesion reduction was sustained with RU 486. Both GnRH-a and RU 486 alone produced profound thinning of ectopic and eutopic endometrium throughout 1 yr of continuous therapy. Bone density decreased significantly after 6 months of GnRH-a alone (P < 0.05), without significant changes in the other groups. After RU 486 treatment, there were no significant changes in testosterone, androstenedione, sex hormone-binding globulin, or cortisol. Like GnRH-a, long term antiprogestin therapy produced a reduction in the volume of pelvic endometriotic lesions as well as atrophy of in situ endometrium; however, RU 486 allowed maintenance of tonic ovarian estradiol secretion, suggesting that efficacious endometriosis therapy can be sustained long term without the sequelae of hypoestrogenism, specifically bone density loss. (J Clin Endocrinol Metab 81: 1933-1939, 1996)

ENDOMETRIOSIS implies the presence of endometrial-like glands and stroma at ectopic sites beyond the confines of the uterus. Monthly proliferation of these lesions, secretory gland formation, and hemorrhage in synchronization with the ovarian/menstrual cycle cause debilitating pain in millions of women worldwide. For many, this problem persists throughout their reproductive lives. Endometriosis can be medically and/or surgically treated; however, effective current endocrine therapies result in either suppression of the secretion of ovarian steroids to castrate levels or inhibition of endometrial cells via androgenic/progestogenic medications ^[1] , ^[2] .

One of the most efficacious treatments to date involves administration of continuous GnRH agoinst (GnRH-a) to suppress pituitary gonadotropins, inducing a state of pseudomenopause due to profound inhibition of ovarian steroid hormone secretion. Whereas endometriotic symptoms, especially pelvic pain, quickly regress, pain frequently returns when active endometriotic lesions reappear within several weeks to a few months after normal ovarian/menstrual cycles return. The U.S. FDA has warned that extended severe estrogen deprivation may not be safe for intervals greater than 6 months due to potentially adverse sequelae, including osteoporosis ^[3] , ^[4] . The heightened incidence of acute side-effects, such as headaches, and reversible menopausal-like symptoms (hot flashes, dry vagina, etc .) is expected during GnRH-a treatment ^[1] . Superior therapies that can enhance patient tolerance are needed for the potential treatment of this chronic condition. The antiprogestins may offer such promise. The antiprogestins bind with high affinity to the progesterone receptors, thereby accomplishing blockade of receptor-ligand activity almost completely. Antiprogestins also possess noncompetitive antiestrogenic (antiproliferative and antivascular) activities that have been shown in laboratory primates to curtail estradiol-induced endometrial growth and menstrual bleeding ^[5] . At appropriate doses, RU 486 causes anovulation and/or amenorrhea during chronic therapy ^[6] . Preliminary studies with RU-486 have shown this antiprogestin to ameliorate pelvic pain promptly in women with persistent endometriosis ^[6] , ^[7] . Similar regimens shrink leiomyomata ^[8] . However, unlike GnRH-a, the antiprogestins allow continuation of tonic (basal) ovarian estrogen secretion, which may be sufficient to protect against rapid estrogen-dependent bone density loss among women of reproductive age.

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Historically, the surgical induction of endometriosis using laboratory primate models has proven useful in designing clinically relevant investigations ^{[9] [10] [11]} . Also insightful in these monkey models is the wealth of data available on the primate climacteric and the hypothalamic-pituitary-ovarian-uterine response to GnRH and its analogs ^{[12] [13] [14]} as well as inhibition of endometrial proliferation by RU 486 ^[5] , ^[15] . Here, we asked whether long term antiprogestin treatment, either in conjunction with initial GnRH-a therapy or alone, provides any apparent advantage over the common GnRH-a regimens currently used in the clinical management of endometriosis patients. Of particular interest was whether the bone density loss known to accompany protracted GnRH-a therapy can be avoided during a 1-yr treatment interval using antiprogestins.

Materials and Methods

Primate husbandry

Adult female cynomolgus monkeys (Macaca fasicularis ) were housed individually in a controlled light and temperature environment. They were fed a commercial primate diet and had unrestricted access to water. Experiments were undertaken with the approval of the institutional animal care and use committee in compliance with the regulations of the USDA (Animal Welfare Act) and the guidelines of the NIH Guide for the Care and Use of Laboratory Animals. The experiments were undertaken at Eastern Virginia Medical School facilities, accredited by the American Association for the Accreditation of Laboratory Animal Care.

Surgical induction of endometriosis

Thirty-two adult female cynomolgus monkeys (Macaca fascicularis) demonstrating menstrual regularity had surgery for the induction of endometriosis ^[9] . Using anesthesia (20 mg/kg ketamine, im; 1 mg/kg xylazine), on day 3 of the menstrual cycle, a diagnostic laparoscopy was performed to rule out preexisting pelvic adhesive disease. Monkeys with obvious pelvic adhesions were excluded. Blood was collected on menstrual cycle days 8-14 via venipuncture (10 mg/kg ketamine, im), and serum was assayed for 17beta-estradiol by RIA. Laparotomy (20 mg/kg ketamine; 1 mg/kg xylazine) was performed 3-5 days after a clearly defined preovulatory estradiol peak. A 2-cm fundal hysterotomy was performed; approximately 100 mg endometrium were removed by curettage and minced in sterile 0.9% saline. The uterine incision was closed with 4-0 polyglactin suture. The minced endometrial tissue was injected subperitoneally into five sites: the left and right vesicouterine folds, the right and left broad ligaments, and the cul-de-sac. Procedures were performed with aseptic technique and careful tissue handling to help prevent adhesive disease due to peritoneal trauma. The abdominal incision was then closed in layers. Postsurgical pain control was provided (1 mg/kg Nubain).

During the subsequent menstrual cycle, laparotomy was again performed 3-5 days after the preovulatory rise in estradiol. The presence of ectopic endometrial tissue and the extent of adhesions were noted, with peritoneal implants carefully measured. Photographs of all lesions were taken to accumulate a pictorial record. The area of each pelvic endometriotic lesion was measured across its longest and its shortest axis. Biopsies were taken from representative lesions to assess glandular and stromal tissue status after tissue fixation in 10% formalin and hematoxylin and eosin staining for histological examination.

Monkeys were divided into four treatment groups (n = 8 each). Group I was given monthly depot im injections of GnRH-a (leuprolide, TAP Pharmaceuticals, Chicago, IL; 80 mug/kg, im) beginning on menstrual day 21 of the second laparotomy cycle. This regimen was continued at 28-day intervals for a total of three injections, at which time GnRH-a therapy was discontinued, and weekly injections of the antiprogestin RU 486 began (initially 5 mg/kg, im, in oil, then 2 mg/kg·week). RU 486 was continued for a period of 40 weeks to complete 1 yr of therapy. Group II was given weekly im injections of RU 486 (5 mg/kg initially, then 2 mg/kg·week) beginning on menstrual day 1 of the cycle after viable endometriotic lesions were confirmed. This was continued for a treatment interval of 52 weeks. Group III was given monthly depot doses of leuprolide only through 1 yr. Group IV were controls and received only vehicle (0.5 mL normal saline, im, weekly) for 52 weeks.

Assessment of endometriosis

After daily injections began, a staging laparotomy was performed at 3, 6, and 9 months to assess the status (progression, maintenance, or regression) of the pelvic disease.

To enhance consistency, all surgical procedures were performed by the first author. Areas of peritoneal involvement with endometriotic lesions were again measured in two dimensions and recorded. Biopsies were taken of the intraabdominal lesions on an intermittent basis for histological assessment of disease; photographs of in situ plaques were also taken and catalogued.

Assessment of hormonal milieu

Using ketamine anesthesia, blood was collected on alternate days in all groups for 28 days after initiation of treatment after confirmation of endometriosis. The serum was frozen for later hormonal analysis. Thereafter, blood was collected weekly. Blood collection continued until after the conclusion of injections (1 yr), and serum was frozen. Estradiol and progesterone levels were determined on all collected samples. Testosterone, androstenedione, sex hormone-binding globulin (SHBG), and cortisol were determined at 4-week intervals.

Serum assays for estradiol, progesterone, testosterone, androstenedione, and cortisol were performed by RIA with kits from ICN Biomedicals (Costa Mesa, CA). Multiple assay runs were required for estradiol and progesterone. The estradiol assay had a sensitivity of 59 pmol/L, with interassay and intraassay coefficients of variation (CVs) of less than 16% and less than 10%, respectively. The progesterone assay had a sensitivity of 0.64 pmol/L, with interassay and intraassay CVs of less than 13% and less than 10%, respectively.

The sensitivity, interassay CV, and intraassay CV for androstenedione were 0.35 nmol/L, less than 14%, and less than 7%, respectively; those for testosterone were 0.35 nmol/L, less than 12%, and less than 8%, respectively; and those for cortisol were 1 mug/dL, less than 16%, and less than 11%, respectively.

A saturation analysis, adapted from previously reported methodologies ^[16] , ^[17] , was performed to determine the concentration of SHBG in serum samples. The specimens (100 muL) were stripped of endogenous steroids by incubation for 10 min at 37 C with a charcoal (0.8%) solution, followed by centrifugation at 2500 × g for 10 min at 4 C. Four 10-muL aliquots of the stripped specimen were diluted with 190 muL assay buffer (phosphate-buffered saline). Two of these four aliquots were heated for 30 min at 60 C to inactivate the SHBG; these duplicates were used for the determination of nonspecific binding. [ ³ H]dihydrotestosterone ([³ H]DHT; 500 pg in 50 muL buffer) was added to each tube and incubated for 30 min at 37 C before being chilled at 4 C for 15 min. The assay was terminated by the addition of 500 muL dextran (0.05%)-charcoal (0.5%) solution and incubation at 4 C for 15 min, followed by centrifugation at 2500 × g for 10 min. The radioactivity in the supernatant was determined by liquid scintillation spectrometry. The number of nanomoles of [³ H]DHT bound was calculated. As each SHBG molecule is assumed to bind one molecule of [³ H]DHT, the concentration of SHBG per mL equals the nanomoles of [³ H]DHT bound per mL serum. The intra- and interassay coefficients of variation were 8% and 15%, respectively.

Assessment of endometrium and vaginal mucosa

Full thickness endometrial biopsies were repeated at the time of laparotomies (3-month intervals). Upon completion of the 1-yr medical therapies, a hysterectomy was performed. All tissues were fixed with 10% formalin and stained with hematoxylin and eosin. A gynecological pathologist, blinded to the treatment groups, assessed the histology of the glandular and stromal elements. The depth of the endometrium from the mucosal surface to the endometrial/myometrial interface was measured in a line parallel to the long axis of the glands using a micrometer at low power magnification. Vaginal epithelium was obtained at the time of each surgery, using a Kevorkian biopsy forceps taken from the lateral

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vaginal side of the upper two thirds of the vagina. The same pathologist measured the thickness of the vaginal epithelium, the keratin layer, and the total epithelium to the base of the rete pegs.

Assessment of general well-being

Primates were observed daily on morning rounds. Changes in behavior were noted. Skin was examined for signs of rash or inflammatory changes at the injection site. The external genitalia were examined for signs of menses. Body weight was determined monthly, and any changes in appetite were noted.

Assessment of bone metabolism

Dual x-ray absorptiometry (DXA; Norland, Fort Atkinson, WI) was used to measure the bone mineral density of the lumbar spine (L2-L4). Monkeys were anesthetized with ketamine and restrained to minimize motion artifact. DXA was performed before medical therapies began, as well as at intervals of 3, 6, and 12 months in treatment groups I-IV.

Statistical methods

Data were analyzed using repeated measures ANOVA to assess group differences in the area of pretreatment endometriotic plaques and to assess treatment effects within each group. Hormonal data were analyzed using ANOVA, with multiple comparisons made using least significant differences. Bone densitometry data were analyzed using repeated measures ANOVA to detect changes within a treatment group over time. Histological data were analyzed using ANOVA and least significant differences.

Results

As shown in Table 1 , serum estradiol was severely suppressed during the 3 months of treatment with GnRH-a (group I); basal ovarian estradiol secretion returned soon after GnRH-a was discontinued at the time RU 486 injections began. Group II (RU 486 only) had a mean ± SEM serum estradiol level of 231 ± 12 pmol/L, which was significantly higher than that in the GnRH-a only monkeys (group III; 77.1 ± 2.6 pmol/L; P < 0.05); this estrogen level (during RU 486 treatment) was similar to that in the early follicular phase, vehicle-treated (normally menstruating) controls (group IV; 231 ± 13 pmol/L). Both antiprogestin and GnRH-a reliably suppressed ovulation, as shown by the low mean serum progesterone values. In each treatment group, there were progesterone levels indicative of recent ovulation. Assuming ovulation for a serum progesterone of greater than 3.2 pmol/L (1 ng/mL), there were three ovulations detected

a P < 0.05 vs. vehicle and RU 486.
b P < 0.05 vs. GnRH-a and RU 486.

during 120 months of GnRH-a treatment, 11 ovulations during 168 months of RU 486 treatment, and 100 ovulations during 96 months of saline treatment. There is not a statistical difference in frequency of ovulation between the RU 486- and GnRH-a-treated primates ( P > 0.1, by X² test), although both groups significantly suppressed ovulation compared to controls.

The area (square centimeters) of ectopic endometrium visualized on peritoneal surfaces is shown in Fig. 1 . The pretreatment areas in all groups were similar (by ANOVA, P > 0.1). The posttreatment lesioned areas (square centimeters ± SEM) for the GnRH-a and antiprogestin groups were significantly different from the respective pretreatment areaa as well as from the posttreatment endometriotic area in the control group (P < 0.05). This shows clearly that both GnRH-a and RU 486 diminished the size of the endometriotic lesions. Group I was switched to RU 486 after completing the initial 3-month course of GnRH-a therapy. The mean area of peritoneal disease remained suppressed during subsequent RU 486 therapy. That is, endometriosis remained under control (static) in the face of antiprogestin therapy as well as in the GnRH-a only group (group III). The vehicle-treated (control) monkeys had no statistically significant change in the area of peritoneal endometriosis throughout the 1-yr study. No significant differences in the area of endometriotic plaques were detected during or after treatment in groups I, II, and III.

The DXA bone density determinations are shown in Fig. 2 . Baseline bone density was similar in all groups ( P > 0.1). Group I, receiving sequential GnRH-a then RU 486 therapy, showed no statistical change in bone density from baseline after 3 months of GnRH-a treatment or during the following 9 months of the antiprogestin regimen. There was no significant increase in bone density after 3 months of RU 486 treatment (P = 0.08; group II, continuous RU 486); likewise,

Figure 1. Bar graphs showing the area of peritoneal endometriosis before initiating therapy and at 3-month intervals of treatment. Group I, Combination sequential therapy with GnRH-a/RU 486; group II, chronic RU 486; group III, chronic GnRH-a; group IV, vehicle control. The areas of the endometriotic plaques for groups I, II, and III decreased after 3, 6, and 9 months compared to baseline and to the respective time points in the control group IV ( P < 0.05). There were no statistical differences detected among groups I, II, and III.

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the data showed no significant changes from baseline through 1 yr of treatment. In contrast, the chronic GnRH-a group III revealed a reduction in bone density from baseline after 6 months (P < 0.02), a change that persisted at 1 yr (P < 0.02). No bone density changes were detectable in the vehicle-treated group IV. A tabular representation of the bone density measurements (in grams per square centimeter ± SE) is summarized in Table 2 .

The histological assessment of endometrial and vaginal epithelia by hematoxylin and eosin staining is summarized in Table 3 . Endometrium in group I became thin and atrophic (static) after 3 months of GnRH-a treatment; it remained thin and quiescent during 9 months of RU 486 therapy. The antiprogestin only endometrium group (group II) consistently appeared to be either atrophic as well as thin at the early secretory stage, such as cycle days 15-17, in agreement with previous reports ^[5] , ^[15] , ^[18] . Both the GnRH-a-treated and RU 486-exposed endometrial tissues were statistically thinner (<1.0 mm) than the control monkeys (group IV), whose biopsies were taken randomly during the menstrual cycle (2.79 ± 0.26 mm; P < 0.05). Regarding the thickness of the vaginal epithelium, tissue was statistically thicker from the surface of the keratin layer to the base of the rete pegs in the antiprogestin group and the control group than in the GnRH-a groups.

Figure 2. Graph showing the change in DXA bone density determinations of the lumbar spine compared to baseline measurements after 3, 6, and 12 months of treatment (grams per cm² ± SEM). Group I, Combination/sequential therapy; group II, chronic RU 486; group III, chronic GnRH-a; group IV, vehicle control. Chronic GnRH-a resulted in a significant decrease in bone density at 6 and 12 months compared to the pretreatment baseline (P < 0.05).

Hysterectomy specimens were obtained after the completion of 1 yr of medical therapy (all groups) and showed endometrial histology similar to that in the previous quarterly endometrial biopsies. There was occasional cystic dilation of the endometrial glandular elements in the RU 486 only monkeys of group II, but completely without hyperplasia. The endometrial thickness from the surface to the endometrial-myometrial junction to the epithelium remained thin (~5-fold less than controls) and similar to the minimal endometrium of chronically hypoestrogenic monkeys given GnRH-a for only 1 yr Fig. 3 . The endometrium from monkeys (group I) given the combination/sequential therapy was the thinnest of that in any group (P < 0.05). Not surprisingly, the endometrium from the control females was much thicker than that in any of the groups receiving medical therapy (P < 0.05).

Taking diurnalism into account, serum collection in the morning (~0800 h) showed no change in cortisol levels from baseline among primates receiving RU 486, nor was there any significant change in testosterone, androstenedione, or SHBG throughout the study.

Daily assessment of overall well-being revealed no note-worthy changes in behavior, skin coloration, or dietary habits. Body weights were unaffected in any of the groups.

Discussion

GnRH-a are widely used clinically for the treatment of endometriosis, leiomyomata, and other sex steroid hormone-dependent conditions ^[19] . The mechanism of action is understood, in as much as suppression of pituitary gonadotropin secretion inhibits folliculogenesis and, in turn, the production of estradiol as well as progesterone. A reversible pseudocastration condition supervenes. The resulting hypoestrogenism causes generalized atrophy of organ systems and tissues that depend on estrogenic stimulation, including gonads, the reproductive tract, bone, and certain central neuroendocrine functions. Others have provided evidence that in women receiving extended GnRH-a therapy, a menopausal-like hormone replacement regimen ("addback" therapy) of estrogen plus progestin can counter the frank symptoms of the pharmacologically induced pseudocastration and possibly avert the long term side-effects of estrogen deprivation ^[20] , ^[21] . However, the practicality of overlapping three concurrent medications (GnRH-a, estrogen, and progestin) is questionable.

Chronic GnRH-a therapy, although it can be quite effective in reducing pelvic pain from endometriosis, is unacceptable for long term (>6 months) use in patients because of associated health risks, including osteoporosis and cardiovascular

a P < 0.02 vs. pretreatment.

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a P < 0.05 vs. GnRH-a and RU 486.
b P < 0.05 vs. RU 486 and control.

diseases ^[4] , ^[22] , ^[23] , as well as the unpleasant symptoms of vasomotor flushes, vaginal dryness, depression, etc., experienced by some patients ^[1] . All of these side-effects are potential sequelae of severe estrogen deprivation. A more desirable therapeutic route would be one that allows modest (tonic) circulating estradiol to preserve basal estrogen-dependent functions. Simultaneously, efficacy must be sustained by negating the potentially proliferative actions of basal estrogen on the ectopic endometrium.

RU 486 has been shown to block the proliferative action of estradiol on endometrium in monkeys by a mechanism termed the noncompetitive antiestrogen effect ^[5] , ^[15] , ^[24] . Concurrent administration of estradiol and RU 486 has demonstrated a dose-dependent inhibition of glandular development and endometrial growth ^[15] . Unlike the actions of a progestin, this antiproliferative effect of RU 486 in the endometrium occurs despite a substantial increase in the estradiol receptor concentrations ^[24] . Chronic RU 486 treatment commonly produces a state of extended amenorrhea or oligomenorrhea and anovulation even though early follicular phase or higher levels of circulating estradiol are present throughout ^[15] . Full follicular maturation, the preovulatory LH surge, and ovulation are blocked at the appropriate doses of antiprogestin ^[15] , ^{[25] [26] [27] [28]} .

In concert with the findings reported previously ^[1] , ^[29] , we observed that lesions of endometriosis regress in size within 3 months after initiation of GnRH-a therapy. In addition, we established that regression of this pelvic disease was maintained after discontinuation of GnRH-a by the addition of chronic antiprogestin (RU 486) therapy. This combination of sequential therapy is expected to bring prompt relief of symptomatology (pain reduction) through castration-like hypoestrogenism. Then, basal (tonic) ovarian estradiol production is restored during antiprogestin treatment without forfeiting the desired noncompetitive antiestrogen effect that

Figure 3. Bar graph showing 1 yr data for the endometrial thickness after hysterectomy for each of the treatment groups (mean ± SEM). Group I endometrium was significantly thinner than that in each of the other groups (P < 0.05). Group IV endometrium was significantly thicker than that in each of the other groups (P < 0.05). The endometrial thickness in group II vs. that in group III was not statistically different.

inhibits ectopic endometrial proliferation long term. Concurrently, bone density loss is avoided via a sustained endogenous tonic estrogen supply. Although not specifically tested here, it is possible that mifepristone (RU 486) has some intrinsic bone-sparing affect, an action previously described for certain progestational compounds ^[30] . Moreover, mifepristone is known to have some mixed function agonistic/antagonist properties in the endometrium of monkeys and women ^[31] , ^[32] . In this primate model, the response of uterine endometrium and experimental endometriosis to chronic RU 486 alone (group II) suggests that the initial GnRH-a-induced hypoestrogenism (group I), albeit potentially facilitatory in the sense of accelerated efficacy, may not be necessary. Significant reductions in the area of peritoneal endometriotic lesions were observed within 3 months, an effect that continued for the duration of therapy (1 yr). Our vehicle control group IV served to validate the experimental model, showing no discernible change in the lesioned areas over time; that is, an active process of tissue proliferation and sloughing persisted in the absence of medical treatment.

Our original observations that basal ovarian estrogen production persisted in the face of ovulation inhibition and amenorrhea in primates receiving RU 486 ^[33] were extended by Kettel et al. ^[6] , who found that all six cycling women with endometriosis enjoyed clinically significant pain relief despite sustained basal ovarian estrogen secretion while receiving RU 486 for 3 months. Of the five who had follow-up surgery for staging their pelvic endometriosis plaques, only one had complete resolution of the disease. In a follow-up study with low dose RU 486, overall improvement was revealed by a reduction in the endometriosis score, evaluated according to the American Society for Reproductive Medicine (formerly American Fertility Society) classification system ^[7] , ^[34] . Likewise, RU 486-induced regression of myomas was accompanied by ongoing tonic estrogen secretion by the ovaries ^[8] . However, in a more recent clinical trial ^[35] ,

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antiestrogenic actions of RU 486 on endometrium were ambiguous when a regimen of 50 mg/day over 6 months was used. However, like the prior observations, stromal compaction was evident ^[15] , ^[35] . Whether dosage, tissue receptor differences, or other variables are the cause of these changes between studies is unknown. However, no cytological atypia were seen ^[35] .

Here, full thickness endometrial biopsies were used to evaluate the volume of glands and stroma at the time of each laparotomy; those served as an objective measure of the endometrial suppressive effects of the medicinal regimens. Our gynecological pathologist, who was blinded to the hormonal treatment groups, found endometrial thickness to be significantly reduced in both GnRH-a- and RU 486-treated monkeys within 3 months compared to that in cycling controls. These antiproliferative effects of RU 486 were confirmed by histological evaluation posthysterectomy after 1 yr of continuous treatment. Although the endometrial thickness after GnRH-a alone was slightly less than that after RU 486 alone, the difference was not statistically different.

Bone density conservation must be a hallmark of an acceptable long term (multiyear) therapy for hormonally responsive endocrine-based diseases, including endometriosis and uterine myomas. Osteoporosis is responsible for bone fractures in over 40% of women before they reach the age of 70 yr ^[36] . Iatrogenic estrogen deficiency, early menopause, or conditions that adversely effect estrogen production chronically are among the main causes of early and rapid bone density loss ^[37] , ^[38] .

Chronic GnRH-a therapy frequently causes accelerated bone loss in young women during 6 months of estrogen depletion therapy ^[4] , ^[29] . Here, we were able to demonstrate in our primate model a similar bone density decrease using DXA measurements of the lumbar spine in monkeys receiving GnRH-a treatment for 1 yr. No such decrease in bone density was observed in the antiprogestin only group (II). This may be explained by the sustained early follicular phase levels of serum estradiol during chronic RU 486 therapy. Tonic circulating estrogen levels may affect bone directly via its receptor system ^[39] , indirectly through greater availability of calcium ^[40] , or both. Nonetheless, the noncompetitive antiestrogen effect of RU 486 on endometrium appears to be tissue selective and/or dose dependent ^[5] , ^[15] , ^[24] . This concept of selective tissue antagonism is not newly introduced here. Clomiphene citrate, although a competitive antagonist of estradiol in the female reproductive tract, is capable of preventing bone loss despite relative hypoestrogenism in rats ^[41] and laboratory primates ^[42] . Likewise, women receiving the antiestrogen tamoxifen for the proposed suppression of estrogen receptor-positive breast cancer growth have been shown to respond with increased bone density, an estrogenic impact ^[43] .

Similarly, vaginal atrophy, a frequent problem during GnRH-a therapy, may not occur with antiprogestin regimens. The thicknesses of the vaginal epithelium and keratin layer during chronic RU 486 treatment (groups I and II) were similar to those observed in regularly cycling primates and statistically thicker than those in hypoestrogenic primates receiving only GnRH-a (group III; P < 0.05). That vaginal mucosa resists thinning even when exposed to the same RU 486 doses that produce profound retardation of endometrial growth suggests a certain hierarchy of tissue specificity within the female reproductive tract. Notice that monkey oviductal tissues still respond normally to estrogen in the face of RU 486 treatments that simultaneously inhibit endometrial proliferation ^[44] . Similarly, in rabbits and rats, the selective antiestrogenic impact of another antiprogestin, onapristone, has been reported ^[45] , ^[46] . Interestingly, onapristone is said to be a pure antagonist of progesterone, lacking agonistic properties such as those of mifepristone; still, it exerts profound antiestrogenic impact on uterine tissues. Others have reported that the A forms of the progesterone receptor modulate estrogen receptor functions where both A and B form are present. In turn, RU 486 exerts antiestrogenic activity through a novel progesterone receptor A form-mediated mechanism ^[47] that results in tissue-selective expressions, differential not only to normal cells but possibly also to neoplasia such as breast cancer ^[48] . It seems that RU 486 has several desirable and intriguing properties for the treatment of certain gynecological conditions. It causes regression of endometriosis, in situ endometrium, and leiomyomata ^[8] while preserving bone density, vaginal epithelial thickness, and circulating SHBG levels, as shown here.

Despite RU 486 having well known antiglucocorticoid effects and its use to successfully treat the symptoms of Cushing's syndrome in doses nearly 10-fold higher than those used here ^[49] , we saw no such impact in this study. High dose therapy (10 mg/kg·day) results in excessive diurnal elevations of ACTH and cortisol ^[50] . Here, monthly determinations of early morning cortisol did not reveal any significant elevations from baseline during the 1-yr course of therapy, as had been observed in women ^[7] . The low dose used here (2 mg/kg·week) is far lower than those used in previous studies and approximately one sixth (on an equal body weight basis) of the dose used to suppress endometriosis in women with few adverse effects ^[6] . Thus, the antiglucocorticoid potential of RU 486 may be negligible at low doses while achieving control over ectopic endometrial tissue.

The findings presented here support the premise that long term antiprogestin regimens, administered alone or after completion of a course of ovarian suppression via GnRH-a, may provide symptomatic relief of endometriosis or induce atrophy of uterine fibroids without the generalized side-effects of extended severe estrogen deprivation. Although these results are highly encouraging, a number of issues deserve cautions interpretation: 1) subjective complaints of side-effects, i.e. vasomotor flush, dizziness, emotional changes, etc., are not adequately assessed in this primate model and will need elevated attention when human subjects are studied further; 2) although highly informative, 1 yr of antiprogestin therapy is insufficient to predict the sequelae of even longer therapeutic regimens; 3) despite the comparatively low doses employed here, the minimal effective dose of mifepristone to achieve the noncompetitive antiestrogenic affect has yet to be determined for these laboratory primates or women; and 4) the degree to which these actions of mifepristone are generalizable to other antiprogestins, especially those that are more purely antagonistic, is unknown.

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Acknowledgments

We thnak the members of the Primate Core Husbandry Lab, led by Mr. Bruce Lamar, for their attention to detail in the careful execution of this study. We thank the Hormone Assay Laboratory, led by Ms. Marybeth Southern and Ms. Barbara Atkinson, for their dedication in performing the assays, and Dr. Michael Sutherland for his assistance with statistical analysis. We also thank Ms. Margarita Fuentes-Negron and especially Ms. Dara Willett-Leary for manuscript preparation.

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