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Effects of different estrogen and progestin regimens on the mechanical properties of rat femur
Abstract
The purpose of this study was to examine the effects of estrogen replacement, in concert with three different progestin regimens, on the mechanical properties of rat femoral cortical bone. Ninety-two 11-month-old female Sprague-Dawley rats were randomly divided into six groups and were treated for a duration of 6 months. Group-1 rats were intact controls, group-2 rats were ovariectomized controls, and groups 3-6 were ovariectomized and given continuous doses of estrogen with 5% estradiol 17B silicone-rubber implants. Groups 4, 5, and 6 were also given different doses of progestin (norethindrone): group 4 received a continuous dose of 3 micrograms per animal per day, group 5 received a cyclic dose of 6 micrograms per animal per day for 14 days of a 28-day cycle, and group 6 received an interrupted dose of 3 micrograms per animal per day for 3 days of a 6-day cycle. Femurs from each group were mechanically tested. Bending stiffness was measured by nondestructive three-point bending tests and maximum torque capacity, by destructive torsion tests. Geometrical properties and apparent density of cortical bone were also measured. The significant differences were: the increases in elastic modulus (measured from the three-point bending stiffness) of group 5 (cyclic norethindrone) compared with those of group 2 (ovariectomized controls) and group 3 (estrogen only); the increases in the size represented by the moment of inertia, the moment of the area, and medial-lateral width of group 2 compared with those of group 5; and the increases in apparent density and decreases in moment of inertia of group 6 (interrupted norethindrone) compared with those of group 2. Cyclic or interrupted treatment of progestin along with continuous treatment of estrogen after ovariectomy likely improves material properties of cortical bone, increases its density, and reduces the size of the bone compared with ovariectomized rats.
... Serum alkaline phosphatase (ALP, bone specific) levels and osteocalcin (OCN) are frequently used as bone formation markers to monitor drug actions [24][25][26]. Bone turnover markers were measured using commercially available kits as specified by the manufacturers. ...
... axis of a Bionix 858 materials testing system (MTS Systems, Eden Prairie, Minnesota), with its proximal and distal ends potted in bone cement (polymethylmethacrylate) at a constant gauge length of 15 mm 20,21 . A 1.4-N-m reaction torque transducer (Futek, Irvine, California) measured applied torque. ...
Background:
Lithium, a treatment for bipolar disorder, is not clinically indicated for use in fracture management but has been reported to positively influence bone biology. It is hypothesized that lithium dosing for beneficial effects on bone health may be much lower than the dosing required for psychotropic benefits in patients with bipolar disorder. A preclinical study with a rodent fracture model was utilized to best define the lowest effective dose, best timing of treatment onset, and optimal treatment duration for the use of lithium as a new treatment in fracture care.
Methods:
A design-of-experiments approach was used to assess the parameters of dose, timing of treatment onset, and treatment duration. Closed femoral shaft fractures were generated and analyzed with use of destructive torsional mechanical testing and microcomputed tomography-based image analysis. Eleven different outcome measures were quantified, with maximum yield torque as the primary study outcome, to assess the quality of long-bone fracture-healing.
Results:
Fracture-healing was maximized with a lithium treatment combination of a low dose (twenty milligrams per kilogram of body weight per day), later onset of lithium treatment (seven days after fracture), and longer treatment duration (two weeks), with maximum yield torque displaying a 46% increase compared with nontreated and sham-treated controls (481.1 ± 104.0 N-mm compared with 329.9 ± 135.8 N-mm; p = 0.04). Design-of-experiments analysis determined the timing of treatment onset to be the most influential parameter for improving fracture-healing, with femora treated at a later onset (seven days after fracture) showing a significant (21%) increase in maximum yield torque compared with those treated at an earlier onset (three days after fracture) (p = 0.01).
Conclusions:
A later onset of lithium administration significantly improved femoral fracture-healing. Trends indicated that a lower dose and longer treatment duration also had a positive effect on fracture repair.
Clinical relevance:
Orally administered low-dose lithium therapy with a large postfracture administration window has the potential to yield a safe, reliable, and cost-effective treatment to enhance bone-healing and restore earlier function and mobility pending appropriate large-animal proof-of-concept models, safety data, and U.S. Food and Drug Administration clinical trials approval.
... Fig. 5 indicates that the elastic modulus of the rat bone beam in high vacuum is 4.98 ± 0.25 GPa, low vacuum is 5.24 ± 0.11 GPa and wet, in air is 5.22 ± 0.15 GPa and does not vary greatly over the time period examined. Previous mechanical testing on fully hydrated whole rat bone femur using 3-point bending configuration gives an elastic modulus of 5.12 ± 0.77 GPa (Kasra et al., 1997), 8.0 ± 0.4 GPa (Barengolts et al., 1993), 6.88 ± 0.31 GPa (Jorgensen et al., 1991), and 4.9 ± 0.4 GPa (Ejersted et al., 1993) which is similar to the calculated elastic modulus values in our work and indicates that the vacuum of the SEM chamber does not have an effect on elastic modulus of the samples over the time period investigated. Interestingly, the similarity between the elastic modulus of our relatively small bone volumes and whole bone testing suggests an effective transfer of stresses throughout the bone material, indicating that all bone components are not failing or slipping but are tightly bound to one another. ...
The lamellar unit is a critical component in defining the overall mechanical properties of bone. In this paper, micro-beams of bone with dimensions comparable to the lamellar unit were fabricated using focused ion beam (FIB) microscopy and mechanically tested in bending to failure using atomic force microscopy (AFM). A variation in the mechanical properties, including elastic modulus, strength and work to fracture of the micro-beams was observed and related to the collagen fibril orientation inferred from back-scattered scanning electron microscopy (SEM) imaging. Established mechanical models were further applied to describe the relationship between collagen fibril orientation and mechanical behaviour of the lamellar unit. Our results highlight the ability to measure mechanical properties of discrete bone volumes directly and correlate with structural orientation of collagen fibrils.
Copyright © 2015 Elsevier Ltd. All rights reserved.
... 5 indicates that the elastic modulus of the rat bone beam in high vacuum is 4.98 ± 0.25 GPa, low vacuum is 5.24 ± 0.11 GPa and wet, in air is 5.22 ± 0.15 GPa and does not vary greatly over the time period examined. Previous mechanical testing on fully hydrated whole rat bone femur using 3-point bending configuration gives an elastic modulus of 5.12 ± 0.77 GPa (Kasra et al., 1997), 8.0 ± 0.4 GPa (Barengolts et al., 1993), 6.88 ± 0.31 GPa (Jorgensen et al., 1991), and 4.9 ± 0.4 GPa (Ejersted et al., 1993) which is similar to the calculated elastic modulus values in our work and indicates that the vacuum of the SEM chamber does not have an effect on elastic modulus of the samples over the time period investigated. Interestingly, the similarity between the elastic modulus of our relatively small bone volumes and whole bone testing suggests an effective transfer of stresses throughout the bone material, indicating that all bone components are not failing or slipping but are tightly bound to one another. ...
The mechanical properties of rat bone at micron length scales have been evaluated as a function of environmental conditions using an in situ atomic force microscope (AFM) setup while observing using scanning electron microscopy (SEM). Focused ion beam fabricated rat bone cantilever samples were tested in both low and high vacuum conditions in the SEM as well as wet in air using the AFM to measure their elastic modulus. The elastic modulus of rat bone at micron length scales is shown to be independent of the environmental testing conditions and indicates water is bound to bone material even under relatively high vacuum conditions. Our work therefore shows how in situ mechanical testing of bone while observing using high resolution SEM can provide results similar to testing wet in air.
... The diaphysis of the right femur was tested for mechanical failure using three-point bending according to a procedure previously described [21]. In brief, samples were subjected to a pre-load of 1 N and then deformed at a rate of 1 mm/min until failure. ...
VN/14-1 [4-(±)-(1H-Imidazol-1-yl)-(E)-retinoic acid], a novel retinoic acid metabolism blocking agent (RAMBA), works by inhibiting the breakdown of all-trans-retinoic acid. The purpose of this study was to evaluate the anti-tumor effects of VN/14-1 on the N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinoma model, and peripheral organ effects on the uteri of immature ovariectomized (OVX) rats. In tumor burden experiments, after 56 days of administration of VN/14-1 5, 10, and 20 mg/kg/day, significant tumor reductions in mean tumor weight of 19.1, 34.4, and 44.3%, compared to tumors in control animals occurred. Cumulative tumor growth was also significantly slower in a dose-dependent manner in groups receiving 5, 10, and 20 mg/kg/day of VN/14-1 compared to growth rates in the control group. Tumor apoptosis was significant increases in animals treated with 5, 10, and 20 mg/kg/day of VN/14-1. In uterotrophic experiments, immature OVX rats given VN/14-1 significantly reduced uterine weight and blocked endometrial stimulation induced by unopposed β-estradiol (E2). In both rat models, adverse toxicities included weakness, anorexia, and reduction in body weight in the groups given the highest dose of 20 mg/kg/day. In summary, VN/14-1 inhibited tumor growth in the MNU-induced estrogen receptor (ER)-positive rat mammary tumor model, and antagonized the stimulatory effect of estrogens on the uterus. The studies suggest that VN/14-1 may be a useful novel therapy for ER-positive breast cancer.
... Three-point-bending, load-deformation, and stress-strain data were determined according to procedures described previously. (45) The diaphysis of each right femur was tested in three-point bending. Briefly, each femur was positioned posterior side down with a gauge length of 15.6 mm. ...
We have bred a strain of rats to maximize urine (u) calcium (Ca) excretion and model hypercalciuric nephrolithiasis. These genetic hypercalciuric stone-forming (GHS) rats excrete more uCa than control Sprague-Dawley rats, uniformly form kidney stones, and similar to patients, demonstrate lower bone mineral density. Clinically, thiazide diuretics reduce uCa and prevent stone formation; however, whether they benefit bone is not clear. We used GHS rats to test the hypothesis that the thiazide diuretic chlorthalidone (CTD) would have a favorable effect on bone density and quality. Twenty GHS rats received a fixed amount of a 1.2% Ca diet, and half also were fed CTD (4 to 5 mg/kg/d). Rats fed CTD had a marked reduction in uCa. The axial and appendicular skeletons were studied. An increase in trabecular mineralization was observed with CTD compared with controls. CTD also improved the architecture of trabecular bone. Using micro-computed tomography (µCT), trabecular bone volume (BV/TV), trabecular thickness, and trabecular number were increased with CTD. A significant increase in trabecular thickness with CTD was confirmed by static histomorphometry. CTD also improved the connectivity of trabecular bone. Significant improvements in vertebral strength and stiffness were measured by vertebral compression. Conversely, a slight loss of bending strength was detected in the femoral diaphysis with CTD. Thus results obtained in hypercalciuric rats suggest that CTD can favorably influence vertebral fracture risk. CTD did not alter formation parameters, suggesting that the improved vertebral bone strength was due to decreased bone resorption and retention of bone structure.
... The diaphysis of the right humeri was tested in threepoint bending according to the procedure described previously. (46) Briefly, samples were subjected to a preload of 2 N and deformed at a rate of 2 mm/min until failure. The point of failure was defined as a successive drop in load >10%. ...
Kidney stone patients often have a decrease in BMD. It is unclear if reduced BMD is caused by a primary disorder of bone or dietary factors. To study the independent effects of hypercalciuria on bone, we used genetic hypercalciuric stone-forming (GHS) rats. GHS and control (Ctl) rats were fed a low Ca (0.02% Ca, LCD) or a high Ca (1.2% Ca, HCD) diet for 6 wk in metabolic cages. All comparisons are to Ctl rats. Urine Ca was greater in the GHS rats on both diets. GHS fed HCD had reduced cortical (humerus) and trabecular (L(1)-L(5) vertebrae) BMD, whereas GHS rats fed LCD had a reduction in BMD similar to Ctl. GHS rats fed HCD had a decrease in trabecular volume and thickness, whereas LCD led to a approximately 20-fold increase in both osteoid surface and volume. GHS rats fed HCD had no change in vertebral strength (failure stress), ductibility (failure strain), stiffness (modulus), or toughness, whereas in the humerus, there was reduced ductibility and toughness and an increase in modulus, indicating that the defect in mechanical properties is mainly manifested in cortical, rather than trabecular, bone. GHS rat cortical bone is more mineralized than trabecular bone and LCD led to a decrease in the mineralization profile. Thus, the GHS rats, fed an ample Ca diet, have reduced BMD with reduced trabecular volume, mineralized volume, and thickness, and their bones are more brittle and fracture prone, indicating that GHS rats have an intrinsic disorder of bone that is not secondary to diet.
... The diaphysis of the right femur was tested to failure in three-point bending according to a procedure previously described (13). Briefly, samples were subjected to a preload of 2 N and then deformed at a rate of 2 mm/min until failure. ...
Exemestane (EXE) and letrozole (LET) are third-generation aromatase inhibitors currently prescribed for postmenopausal hormone-dependent breast cancer. The impact on end organs of estrogen depletion in menopausal women is of significant clinical importance. We studied the effects of EXE, its principal metabolite, 17-hydroexemestane (17-H-EXE), and LET on bone and lipid metabolism in ovariectomized (OVX) rats.
OVX rats were treated by weekly intramuscular injection for 16 weeks with 20, 50, and 100 mg/kg EXE, 20 mg/kg 17-H-EXE, and daily oral gavage of 1 mg/kg LET. At the end of the treatment period, bone mineral density (BMD), the bone resorption marker serum pyridinoline, the bone formation marker serum osteocalcin, bone mechanical properties, histomorphometry, and serum lipid concentrations were determined.
Lumbar vertebral and femoral BMD, bending strength of the femur, compressive strength of the fifth lumbar vertebra, and trabecular bone volume were significantly higher in OVX animals given EXE and 17-H-EXE than in OVX controls. EXE and 17-H-EXE significantly reduced an ovariectomy-induced increase in serum pyridinoline and serum osteocalcin. EXE and 17-H-EXE given to OVX rats caused significant reductions of serum cholesterol and low-density lipoprotein cholesterol. In contrast, OVX rats treated with LET had BMD, bone biomarkers, mechanical failure properties, and lipid levels similar to those of OVX controls.
EXE and 17-H-EXE significantly prevent bone loss, enhance bone mechanical strength, and lower serum cholesterol and low-density lipoprotein levels in OVX rats. These protective effects on end-organ function are not seen with the nonsteroidal inhibitor LET.
... The diaphysis of the right femur was tested to failure in a three-point bending test according to a procedure described previously [14]. Briefly, samples were subjected to a preload of 1 N at a rate of 1 mm/min until failure. ...
Our objective was to determine the effects of SCH 57068 alone and with 17 beta-estradiol (E(2)) on bone, lipids and uteri in ovariectomized (OVX) rats. In OVX animals lumbar vertebral and femoral bone mineral density (BMD) were significantly higher after 12 weeks of treatment with SCH 57068 than in untreated OVX controls. Similarly BMD was superior in OVX + E(2) + SCH 57068 treated animals than in OVX + E(2) controls. SCH 57068 also significantly reduced the increase in bone turnover markers, serum pyridinoline and serum osteocalcin levels, induced by OVX, and increased mechanical bone strength. SCH 57068 also significantly reduced the rise in serum cholesterol and low-density lipoprotein cholesterol induced by OVX. SCH 57068 had no stimulatory effect on uterine epithelium when given alone in OVX rats. SCH 57068 (1 and 2.5 mg/kg) reduced uterine weight and blocked endometrial stimulation induced by E(2). In summary, SCH 57068 adds to the positive effects of E(2) on bone and lipid metabolism but blocks the stimulatory effects of E(2) on the uterus. Potentially, E(2) + SCH 57068 could be combined for the treatment and prevention of breast cancer or as a novel hormone replacement therapy.
... The diaphysis of the left femur was tested to failure in three-point bending according to the procedure described previously [28]. Briefly, samples were subjected to a preload of 1 N and then deformed at a rate of 1 mm/min until failure. ...
Vanadium-based drugs lower glucose by enhancing the effects of insulin. Oral vanadium drugs are being tested for the treatment of diabetes. Vanadium accumulates in bone, though it is not known if incorporated vanadium affects bone quality. Nine- to 12-month-old control and streptozotocin-induced diabetic female Wistar rats were given bis(ethylmaltolato)oxovanadium(IV) (BEOV), a vanadium-based anti-diabetic drug, in drinking water for 12 weeks. Non-diabetic rats received 0, 0.25 or 0.75 mg/ml BEOV. Groups of diabetic rats were either untreated or treated with 0.25-0.75 mg/ml BEOV as necessary to lower blood glucose in each rat. In diabetic rats, this resulted in a Controlled Glucose group, simulating relatively well-managed diabetes, and an Uncontrolled Glucose group, simulating poorly managed diabetes. Plasma insulin, glucose and triglyceride assays assessed the diabetic state. Bone mineral density (BMD), mechanical testing, mineral assessment and histomorphometry measured the effects of diabetes on bone and the effects of BEOV on non-diabetic and diabetic bone. Diabetes decreased plasma insulin and increased plasma glucose and triglycerides. In bone, diabetes decreased BMD, strength, mineralization, bone crystal length, and bone volume and connectivity. Treatment was effective in incorporating vanadium into bone. In all treated groups, BEOV increased osteoid volume. In non-diabetic bone, BEOV increased cortical bone toughness, mineralization and bone formation. In controlled glucose rats, BEOV lowered plasma glucose and improved BMD, mechanical strength, mineralization, bone crystal length and bone formation rate. In poorly controlled rats, BEOV treatment slightly lowered plasma glucose but did not improve bone properties. These results suggest that BEOV improves diabetes-related bone dysfunction primarily by improving the diabetic state. BEOV also appeared to increase bone formation. Our study found no negative effects of vanadium accumulation in bone in either diabetic or non-diabetic rats at the dose given.
... In both experiments, force and deformation data were collected at a rate of 25 Hz using a 12-bit data acquisition card (National Instruments, USA), Labview 5.0 data acquisition software (National Instruments), and a Pentiumk II computer (Compaq Canada). The diaphysis of the right femur was tested to failure in three-point bending according to a procedure previously described [17]. Briefly, samples were subjected to a pre-load of 2 N and then deformed at a rate of 2 mm/min until failure. ...
The irreversible steroidal aromatase inhibitor exemestane (EXE) is one of three third generation aromatase inhibitors currently prescribed for advanced breast cancer in postmenopausal women. Its principal mechanism of action is to reduce estrogen by inhibiting its synthesis. In addition to its efficacy against breast cancer, its effects on other organs are important, especially when given to women with good-prognosis breast cancer or potentially to healthy women at increased risk of developing breast cancer. The purpose of this study was to evaluate the effects of EXE on bone and lipid metabolism in ovariectomized (OVX) rats. Ten-month-old Sprague-Dawley female rats were sorted into intact controls, intact + EXE, OVX controls, and OVX + EXE groups, and treated by weekly intramuscular injection with vehicle or 100 mg/kg EXE for 16 weeks. The bone mineral density (BMD), mechanical testing, histomorphometry, bone resorption marker-serum pyridinoline (PYD), and bone formation marker-serum osteocalcin (OC) were used to determine the effects of treatment on bone. In addition, total serum cholesterol, triglyceride, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were determined. BMD of the lumbar spine and femur were 11% and 7%, respectively, higher in OVX animals given EXE than in OVX controls (all Ps<0.001). Significant increases in the bending strength and toughness of the femora as well as the compressive strength and elastic modulus of the vertebrae were observed in OVX rats given EXE (all Ps<0.02 vs. OVX controls). Trabecular bone volume (BV) was significantly higher in OVX rats treated with EXE than in OVX controls (P<0.0001). In OVX animals, EXE reduced the OVX-induced increase of serum PYD by 96% (P<0.0001), and the OVX-induced increase of serum OC was completely prevented by treatment with EXE. In OVX animals, EXE resulted in a 28% reduction of serum cholesterol (P<0.0001) and reduced LDL by 64% compared with OVX controls (P<0.0001). The positive results of EXE on bone and lipid metabolism in the OVX rat model merit further investigation of the effects of EXE in postmenopausal women.
The purpose of this study was to evaluate the effects of withdrawal of minodronic acid (MIN) for 3 months after 12 months of treatment in ovariectomized (OVX) rat. OVX rats were orally treated with MIN (6, 30, and 150 µg/kg/day) for 12 months and necropsied on the day after the last dosing or following 3 months of withdrawal. Lumbar and femoral BMD were decreased in OVX controls. MIN dose-dependently increased BMD. Withdrawal eliminated the effect of MIN on BMD loss after treatment at 6 µg/kg, but not after treatment at 30 and 150 µg/kg. In MIN-treated rats, trabecular thinning occurred during withdrawal after treatment at 6 µg/kg, but the trabecular microstructure was maintained at 30 and 150 µg/kg. In a mechanical test of the femoral diaphysis, stiffness of in OVX controls was decreased but ultimate load was similar to that in sham after withdrawal. MIN increased ultimate load and stiffness, but endosteal length decreased after withdrawal. Suppression of bone turnover by MIN based on bone turnover markers and histomorphometric indices was attenuated by withdrawal after treatment at 6 and 30 µg/kg and partially at 150 µg/kg. The MIN concentration in the humerus decreased during withdrawal, and half-life at 30 µg/kg was shorter than that at 150 µg/kg. These results show that the antiresorptive action of MIN was dose-dependently attenuated by 3-month withdrawal in a rat OVX model. An absence of BMD increase was only observed at a low dose but decreases in antiresorptive activity occurred over a wide dose range.
Osteoporosis is a disease known to promote bone fragility but the effect on the mechanical properties of bone material, which is independent of geometric effects, is particularly unclear. To address this problem, micro-beams of osteoporotic bone were prepared using focused ion beam microscopy and mechanically tested in compression using an atomic force microscope while observing them using in situ electron microscopy. This experimental approach was shown to be effective for measuring the subtle changes in the mechanical properties of bone material required to evaluate the effects of osteoporosis. Osteoporotic bone material was found to have lower elastic modulus and increased strain to failure when compared to healthy bone material, while the strength of osteoporotic and healthy bone was similar. Surprisingly, the increased strain to failure for osteoporotic bone material provided enhanced toughness relative to the control samples, suggesting that lowering of bone fragility due to osteoporosis is not defined by material performance. A mechanism is suggested based on these results and previous literature that indicates degradation of the organic material in osteoporosis bone is responsible for resultant mechanical properties.
The focus of this review is hormone replacement therapy (HRT) with continuous administration of micronised, oral 17β-estradiol 1 mg/day (herein referred to as continuous estradiol) plus micronised, oral norgestimate 90 μg/day administered for 3 days then withdrawn for 3 days in a 6-day repeating sequence (herein referred to as intermittent norgestimate).
According to data from randomised, comparative trials of 1 year’s duration, continuous estradiol 1 mg/day plus intermittent norgestimate 90 μg/day relieves climacteric symptoms (vasomotor symptoms and vulvovaginal atrophy) in postmenopausal women. Continuous estradiol 1 mg/day plus intermittent norgestimate 90 μg/day appeared as effective as estradiol 1 mg/day alone or continuous estradiol 2 mg/day plus continuous norethisterone acetate 1 mg/day in the treatment of postmenopausal women with vasomotor symptoms. Continuous estradiol 1 mg/day plus intermittent norgestimate 90 μg/day was as effective as continuous estradiol 1 mg/day in causing the maturation of vaginal epithelial cells.
In a randomised, double-blind study, bone mineral density (BMD) increased to a significantly greater extent and the rate of bone turnover was slower in postmenopausal women treated with continuous oral estradiol 1 mg/day plus intermittent norgestimate 90 μg/day than in placebo-treated patients.
Two randomised, double-blind studies indicated that the addition of norgestimate 90 μg/day to continuous estradiol 1 mg/day did not attenuate the beneficial effects of estradiol on lipid parameters. Continuous estradiol 1 mg/day plus intermittent norgestimate 90 μg/day was associated with increases in mean serum high density lipoprotein (HDL)-cholesterol levels and decreases in total cholesterol, low density lipoprotein (LDL)-cholesterol and lipoprotein (a) levels, compared with baseline. There was no statistically significant increase in triglyceride levels.
In comparative trials, continuous oral estradiol 1 mg/day plus intermittent oral norgestimate 90 μg/day was well tolerated. Headache, breast pain or discomfort, abdominal pain or discomfort, uterine bleeding, dysmenorrhoea, oedema, nausea and depression were the most commonly reported adverse events. Continuous estradiol 1 mg/day plus intermittent oral norgestimate 90 μg/day was associated with a favourable uterine bleeding profile that improved over time. In a randomised trial, 80% of women were free from bleeding (irrespective of spotting) during month 12 of treatment. Norgestimate 90 μg/day was effective in protecting postmenopausal women against induction of endometrial hyperplasia by continuous estradiol 1 mg/day.
In conclusion, data from a limited number of randomised studies indicate that HRT with continuous estradiol 1 mg/day plus intermittent norgestimate 90 μg/day is effective in relieving climacteric symptoms, increasing BMD and slowing the rate of bone turnover in postmenopausal women. This HRT regimen is well tolerated and is associated with a similar incidence of adverse events to that reported in recipients of continuous estradiol 1 mg/day. The norgestimate component of the regimen provides good endometrial protection and is associated with a favourable bleeding profile. Long-term studies investigating the associated risk of breast cancer and thromboembolic events in recipients of continuous estradiol plus intermittent norgestimate are needed. In the meantime, continuous oral estradiol plus intermittent oral norgestimate can be regarded as an effective new option for HRT in postmenopausal women.
Pharmacodynamic Profile
This review concerns hormone replacement therapy (HRT) in the form of continuous administration of micronised, oral 17β-estradiol 1 mg/day (herein referred to as continuous estradiol) plus micronised, oral norgestimate 90 μg/day administered for 3 days then withdrawn for 3 days in a 6-day repeating sequence (herein referred to as intermittent norgestimate).
Animal studies have shown that progestogens inhibit the synthesis of their own receptors as well as estrogen receptors in the endometrium. By administering norgestimate in an intermittent manner, this inhibition is periodically removed. When norgestimate is withdrawn, the unopposed estrogen increases the number of estrogen and progestogen receptors. The target cells are thus resensitised to both these hormones, allowing a lower total dosage of norgestimate to be administered.
In two randomised trials in postmenopausal women, the addition of intermittent norgestimate 90 μg/day did not negate the beneficial effects of estradiol 1 mg/day on lipid and lipoprotein levels in postmenopausal women. Treatment with continuous estradiol 1 mg/day plus intermittent norgestimate 90 μg/day was associated with increases in mean serum high density lipoprotein (HDL)- (5 and 10%) and HDL2- (24 and 29%) cholesterol and apolipoprotein A1 levels (7%) and decreases in total cholesterol (2 and 9%), low density lipoprotein (LDL)-cholesterol (5 and 15%) and lipoprotein (a) [11 and 31%], compared with baseline. Although triglyceride levels increased by 4 to 9%, this increase was not statistically significant compared with baseline.
Continuous estradiol 1 mg/day plus intermittent norgestimate 90 μg/day had a more beneficial effect on the lipid/lipoprotein profile than continuous oral estradiol 2 mg/day plus norethisterone acetate 1 mg/day in a randomised, doubleblind study.
Pharmacokinetic Properties
Following multiple doses of continuous estradiol plus intermittent norgestimate, estradiol reached a peak serum concentration (Cmax) of 46.2 ng/L after approximately 7 hours. The plasma elimination half-life (t½β) of estradiol is approximately 16 hours. Estradiol is metabolised in the liver to estrone, estriol and their conjugates. The metabolites are mainly excreted as sulphate and glucuronide conjugates in the urine, although some enterohepatic recirculation may occur. Norgestimate is extensively metabolised by first-pass mechanisms in the gastrointestinal tract and/or liver to 17-deacetylnorgestimate (norelgestromin). The Cmaxof 17-deacetylnorgestimate of 643 ng/L was reached after 2 hours. The mean t½β of 17-deacetylnorgestimate was 37 hours. Norgestimate metabolites are eliminated in the urine or faeces.
Therapeutic Use
In three randomised, 1-year studies, treatment with continuous estradiol 1 mg/ day plus intermittent norgestimate 90 μg/day was effective in eliminating climacteric symptoms. Continuous estradiol 1 mg/day plus intermittent norgestimate 90 μg/day appeared as effective as continuous unopposed estradiol 1 mg/day or continuous estradiol 2 mg/day plus continuous norethisterone acetate 1 mg/day in the treatment of postmenopausal women with vasomotor symptoms, with ≥70% of women free from symptoms at 3 to 4 months. Continuous estradiol 1 mg/day plus intermittent norgestimate 90 μg/day was as effective as continuous unopposed estradiol 1 mg/day in causing the maturation of vaginal epithelial cells.
In a randomised, double-blind study, bone mineral density increased to a significantly greater extent and the rate of bone turnover was slower in postmenopausal women treated with continuous oral estradiol 1 mg/day plus intermittent norgestimate 90 μg/day than in placebo-treated patients.
Tolerability
In comparative 1-year trials, continuous estradiol 1 mg/day plus intermittent norgestimate 90 μg/day was well tolerated. Headache, breast discomfort, abdominal discomfort, uterine bleeding, dysmenorrhoea, oedema, nausea and depression were the most commonly reported adverse events. The incidence of adverse events associated with continuous estradiol 1 mg/day plus intermittent norgestimate 90 μg/day was similar to that in recipients of continuous estradiol 1 mg/day, with the exception of breast pain, which tended to be more commonly reported in this group. However, slightly fewer recipients of continuous estradiol plus intermittent norgestimate than recipients of continuous estradiol alone withdrew because of adverse events (7 vs 16%).
In a 12-month, randomised study, the incidence of breast discomfort, abdominal pain, oedema and dysmenorrhoea was lower in postmenopausal women treated with continuous estradiol 1 mg/day plus intermittent norgestimate 90 μg/day than in women treated with continuous estradiol 2 mg/day plus continuous norethisterone acetate 1 mg/day.
Randomised trials indicated that continuous estradiol 1 mg/day plus intermittent norgestimate 90 μg/day was associated with a uterine bleeding profile that improved over time. In a combined analysis of two randomised, double-blind 1-year trials, 69, 71 and 80% of women were free from bleeding (irrespective of spotting) at 1, 6 and 12 months, respectively. In a randomised 1-year trial, continuous estradiol 1 mg/day plus intermittent norgestimate 90 μg/day had a bleeding profile that was similar to that of continuous oral estradiol 2 mg/day plus continuous norethisterone acetate 1 mg/day.
In a comparative trial in postmenopausal women, 90 μg/day was the minimal dosage of norgestimate that was required to protect postmenopausal women against induction of endometrial hyperplasia in a regimen of intermittent norgestimate and continuous estradiol 1 mg/day. Endometrial hyperplasia developed in 28% of women treated with continuous estradiol 1 mg/day alone, and in 6, 0 and 0% of women after the addition of intermittent norgestimate 30, 90 and 180 μg/day, respectively.
Dosage and Administration
In the continuous estradiol plus intermittent norgestimate regimen, a single oral tablet containing estradiol 1mg is taken once daily for the first 3 days of therapy, and a different tablet containing estradiol 1mg plus norgestimate 90μg is taken once daily on days 3 to 6. This 6-day sequence is then repeated continuously.
Estradiol 1 mg/day plus intermittent norgestimate 90 μg/day is contraindicated in women with known or suspected pregnancy, undiagnosed abnormal genital bleeding, known or suspected cancer of the breast, known or suspected estrogen-dependent neoplasia, or active or past history of thrombophlebitis or thromboembolic disorders.
Postmenopausal osteoporosis leads to a significant increase in bone fragility. In this study we used the rat tibia plateau fracture model to investigate the efficiency of estrogen replacement therapy (ERT) to mitigate the post-ovariectomy decrease in fracture load.
A total of 73 virgin Sprague Dawley rats had been ovariectomized and 26 animals underwent sham operation. The ovariectomized animals were either untreated (n=35) or treated with estrogen injections (10 μg/kg per day 3 days a week until sacrifice), starting treatment at either 0, 5, 8, or 13 days post surgery.
Before starting ERT and at 50 days post surgery, the trabecular structure of the right proximal tibial metaphysis of each animal was imaged non-invasively using high resolution X-ray topography. The animals were then sacrificed and the right knee from each animal was harvested and mounted into a servo-hydraulic materials testing system so that the distal femoral condyle could be forced into the proximal tibial plateau until fracture occurred. The failure load (F) of the ovariectomized group without estrogen administration was significantly less than that for the sham group. The mean stiffness (K) of the ovariectomized group was 22 percent less than that of the sham group, though this difference did not reach statistical significance.
Across all groups, the failure load and stiffness were significantly correlated with the trabecular bone volume. Our data suggest that prompt ERT can increase the fracture load and stiffness of trabecular bone by allowing bone formation to continue in previously activated bone remodeling units while suppressing the production of new remodeling units. This may be the mechanism by which estrogen and other antiresorptive agents increase bone mass, and thereby reduce the risk of osteoporotic fractures in postmenopausal women.
The benefit of glucosamine (GlcN) in bone and joint disorders remains controversial. N-acetylation and other N-acylations of GlcN alter its biological properties fundamentally. We have shown previously that N-butyryl glucosamine (GlcNBu) preserved strikingly the subchondral bone structure in a destructive arthritis rat model. Here, we examine whether GlcNBu preserves bone in the ovariectomized (OVX) rat, a model for postmenopausal osteoporosis. Rats were randomized into 4 groups: group 1, sham OVX glucose (Glc) fed; group 2, sham OVX GlcNBu fed; group 3, OVX Glc fed; and group 4, OVX GlcNBu fed. A single, oral, 200-mg/kg dose of GlcNBu or Glc was administered daily for 6 months. Bone mineral content (BMC) and bone mineral density, and biomechanical properties of the femurs and spines were determined by standardized techniques. Two-way analysis of variance with a Bonferroni posttest was used for statistical analysis. Ovariectomy in group 3 resulted either in significant or highly significant effects in a number of the tests. For spinal BMCs the interaction between GlcNBu and OVX was significant. For the femurs, this interaction was also seen in energy to failure, and ultimate displacement and ultimate strain tests. In general, ovariectomy was necessary to show significant preventive effects of GlcNBu on mineral content and some biomechanical properties. We conclude that GlcNBu feeding in the OVX rat preserves bone mineral and some biomechanical properties. Translationally, GlcNBu can be positioned between nutriceuticals and pharmaceuticals for the prevention and treatment of osteoporosis. Advantages include low production costs and a favorable safety profile.
The use of animal models is a very powerful tool for the preclinical assessment of potential therapies for osteoporosis. However, the effective use of animal models has two prerequisites. The first is the use of appropriate techniques to assess the overall effects of therapy on bone. As spontaneous fractures do not occur in any species other than humans, the efficacy of a therapy cannot be assessed by its impact on fracture incidence. Instead, a suite of parameters (collectively referred to as ‘bone quality’), including bone architecture, mineralization and mechanical properties, is examined. While techniques such as histomorphometry and dual-energy x-ray absorptiometry are useful for elucidating specific effects of therapies on bone, mechanical testing is crucial to integrate all the changes into parameters which can serve as a proxy for fracture risk. The second prerequisite is an understanding of the similarities and differences between various animal models of osteoporosis and the human case. Two important animal models of post-menopausal osteoporosis are discussed here: the ovariectomized (OVX) rat and the non-human primate (NHP) model. The OVX rat is convenient, inexpensive and responds rapidly to ovariectomy and to therapy at a number of cancellous sites, which is congruent to the human case. However, the OVX-induced changes are largely confined to cancellous bone. Non-human primates, however, are biologically much more similar, particularly in terms of their reproductive physiology, to humans. The response of cortical bone of skeletally mature NHPs to ovariectomy and other stimuli closely parallels that in humans, but this is not mirrored in the cancellous bone in younger animals. This suggests that these two animal models complement each other as models for osteoporosis in humans. Drug Dev. Res. 49:146–158, 2000. © 2000 Wiley-Liss, Inc.
Rosiglitazone (RSG) is an insulin-sensitizing drug used to treat type 2 diabetes mellitus. The A Diabetes Outcome Progression Trial (ADOPT) shows that women taking RSG experienced more fractures than patients taking other type 2 diabetes drugs. These were not osteoporotic vertebral fractures but, rather, occurred in the limbs. The purpose of this study was to investigate how RSG treatment alters bone quality, which leads to fracture risk, using the Zucker fatty rat as a model.
A total of 61 female 4-month-old rats were divided into six groups. One Sham group was a control and another was administered oral RSG 10 mg/kg/day. Four ovariectomized (OVX) groups were dosed as follows: controls, RSG 10 mg/kg, alendronate (ALN, injected at 0.7 mg/kg/week), and RSG 10 mg/kg plus ALN. After 12 weeks of treatment, bone quality was evaluated by mechanical testing. Microarchitecture, bone mineral density (BMD), cortical bone porosity, and bone remodeling were also measured.
OVX RSG 10 mg/kg rats had lower vertebral BMD and compromised trabecular architecture versus OVX controls. Increased cortical bone porosity and decreased mechanical properties occurred in these rats. ALN treatment prevented decreased BMD and architectural and mechanical properties in the OVX model. Reduced bone formation, increased marrow adiposity, and excess bone resorption were observed in RSG-treated rats.
RSG decreases bone quality. An unusual finding was an increase in cortical bone porosity induced by RSG, consistent with its effect on long bones of women. ALN, an inhibitor of bone resorption, enhanced mechanical strength and may provide an approach to partially counter the deleterious skeletal effects of RSG.
Complete estrogen blockade remains under investigation as a means to optimize anti-estrogen therapy in breast cancer thus both the efficacy and end-organ toxicities are of interest with combinations. We hypothesized that a steroidal aromatase inhibitor (AI) atamestane (ATA) alone, and in combination with the anti-estrogens tamoxifen (TAM) or toremifene (TOR) would have beneficial effects in ovariectomized (OVX) rats on key end-organ functions including bone and lipid metabolism and on the endometrium. Significant positive effects on bone were noted with ATA, TOR, TAM, ATA+TOR, or ATA+TAM. TOR, TAM, ATA+TOR, or ATA+TAM caused significant decreases in serum cholesterol and low-density lipoprotein cholesterol whereas ATA had no effect. Uterine weight and epithelium lining height were not increased by ATA but were by TOR and TAM. No significant differences were found in the key parameters outlined above between OVX rats given TOR and ATA+TOR, or TAM and ATA+TAM. Our data show that ATA in combination with TOR or TAM is equivalent to TOR or TAM alone in terms of end-organ effects within a range of clinically relevant doses. Further studies of combinations of AIs with anti-estrogens on end-organ function are merited.
To find a more potent alternative with less oestrogen-related side effects for hormone replacement therapy (HRT) in postmenopausal osteoporosis, we designed and synthesized a NO-releasing prodrug of genistein (NO-G) according to the structure of NONSAIDs. The purpose of this study was to evaluate the effects of the prodrug on bone in ovariectomised (OVX) rats.
Forty-eight adult Sprague-Dawley female rats were ovariectomised and treated with vehicle, 9 mg/kg genistein and 4.5, 9 or 18 mg/kg NO-G by oral administration daily. The bioassays were performed in terms of bone mineral density (BMD), mechanical testing, bone formation markers, bone alkaline phosphatase (b-ALP) and osteocalcin (OCN) and bone resorption marker urine deoxypyridinoline (DPD). In addition, the effects of the drugs on uterus and body weight were examined.
After treatment for 12 weeks, the BMD of whole femur and tibia in the NO-G (9 and 18 mg/kg) groups were 12.1% and 12.2% higher than that of OVX group (P<0.01); the bending strength of the femur was 11.2% and 12.2% higher than OVX group (P<0.01). The OVX-induced increase of serum b-ALP, OCN and urinary DPD were markedly attenuated. The prodrug showed no side effects on uterus and body weight.
The NO-releasing prodrug of genistein improves the bone loss in OVX rats without stimulatory effects on the uterus, which suggests that the product could potentially be used for the prevention and treatment of postmenopausal osteoporosis.
The purpose of this study was to examine the effects of estrogen replacement, in concert with three different progestin regimens, on the mechanical properties of rat lumbar vertebrae. Ninety-two Sprague-Dawley rats (11 months old) were divided into six groups for treatment. The first group was an intact control, the second group (OVX) was ovariectomized only, and the third group (estrogen-only) was ovariectomized and received continuous estrogen through a 17β-estradiol implant. The remaining groups were ovariectomized and received estrogen and progestin (norethindrone, NET) therapy; 3 μg of NET was injected daily (estrogen plus continuous NET), or 6 μg of NET was injected for 14 consecutive days of a 28-day cycle (estrogen plus cyclic NET), or for 3 consecutive days of a 6-day cycle estrogen plus interrupted NET). The animals were sacrificed after 6 months, and the vertebrae were dissected out. The vertebral processes of the fourth lumbar vertebrae were removed, and the density of the vertebral bodies was determined. They were then subjected to compression testing.
We found that all three estrogen/progestin regimens maintain bone density and all mechanical properties at a level indistinguishable from the control. However, the cyclic and continuous NET treatment results were, with the exception of density, also indistinguishable from those of the ovariectomized group. The estrogen plus interrupted NET group on the other hand, has a significantly greater compressive modulus and density than the ovariectomized group. In conclusion, with respect to the ovariectomized group, the estrogen plus interrupted NET treatment resulted in a superior density and compressive modulus. The remaining mechanical properties were equivalent to those resulting from the continuous or cyclic progestin regimens.
This review outlines the basic principles of a novel interrupted progestin HRT regimen in which estrogen is administered continuously, and progestin is given in a 3-days on, 3-days off pulsed fashion. The rationale for this regimen is to prevent receptor down-regulation and allow increased estrogen and progestin sensitivity during the progestin-free periods. Background information is provided including the reasons for poor patient acceptance of HRT, and the concerns of the potential association of HRT with breast and endometrial cancer. Experimental studies in the rat are described which provide evidence in support of the rationale for the interrupted progestin regimen. Clinically, two pilot studies examining symptom control, bleeding rates and safety of the interrupted progestin regimen, as well as preliminary results of a third study examining the usefulness of this regimen for addback therapy in GnRH agonist treated patients, are outlined. The preliminary results of phase III trials are presented. These clinical studies all demonstrated good symptom control, low bleeding rates, endometrial protection, and excellent patient acceptance. The combination of continuous estrogen with interrupted progestin appears to result in increased sensitivity to estrogen and progestin in estrogen responsive tissues. As a result, lower doses of estrogen and progestin may be used for HRT with good biological effects. Further clinical studies, preferably in prospective randomized trials, are required to demonstrate an advantage of this new regimen compared to continuous combined HRT.
The goals of this study were to develop a protocol to induce a compressive fracture at the tibial plateau of the rat knee in vitro and to determine if the biomechanical parameters provided a sensitive assessment of the early skeletal changes induced by estrogen deficiency. Sixty-one rats underwent an ovariectomy (n = 36) or sham operation (n = 25) and were maintained for 50 days after the procedure. Just before death, the proximal tibia of each animal was scanned with high-resolution x-ray tomography. From the three-dimensional images, the mean trabecular bone volume, thickness, and separation and the number of trabeculae were calculated. The knees were then harvested and mounted into a servohydraulic materials testing system so that the distal femoral condyle could be forced into the proximal tibial plateau until fracture. The fracture load of the ovariectomized rats was 24% less than that of the rats that had the sham operation. Similarly, the structural stiffness of the ovariectomized knees was decreased by 22%. Both of these differences were statistically significant (p < 0.01) and were explained by differences in trabecular bone volume (r = 0.56, p < 0.0001 and r = 0.42, p < 0.005, respectively). The other measures of trabecular bone structure were correlated with the volume and did not improve the prediction by the biomechanical parameters. These data demonstrate that biomechanical testing of the tibial plateau in rats can quantify the structural consequences of estrogen deficiency at an early time point before they become apparent at other bone sites, such as the lumbar spine.
The purpose of this study was to evaluate the effects of ovariectomy on the mechanical properties of bone in the aged Sprague-Dawley rat model of osteoporosis. Eight female rats were sacrificed at the start of the study, at the age of four months. Twenty-four remaining rats were then bilaterally ovariectomized (OVX), and another twenty-four served as controls. Eight rats from each group were sacrificed at five, ten or fifteen weeks. The mean density of L1 vertebral bodies from OVX rats was lower than in their control counterparts, as was the compressive modulus, the ultimate compressive stress and the toughness. The second lumbar vertebra from each rat was cored to remove the bulk of the cancellous bone and tested in compression to failure. Ovariectomy did not affect the mechanical properties of the cored vertebrae, suggesting that the mechanically relevant changes take place in the cancellous bone. The gravimetric density and bone mineral density (measured by dual-energy x-ray absorptiometry) of whole femora were lower in OVX rats than control rats. However, the gravimetric density of the cortical bone was unchanged, and when femora were tested to fracture in three-point bending, the mechanical properties of the midshaft were found to be unaffected. This study suggests some limitations to the mature ovariectomized rat model of osteoporosis: mechanically significant loss of cancellous bone occurs in vertebrae much more rapidly than suggested by other studies. Further, there is little evidence of mechanically important bone loss in cortical bone within four months of ovariectomy.
The focus of this review is hormone replacement therapy (HRT) with continuous administration of micronised, oral 17beta-estradiol 1 mg/day (herein referred to as continuous estradiol) plus micronised, oral norgestimate 90 microg/day administered for 3 days then withdrawn for 3 days in a 6-day repeating sequence (herein referred to as intermittent norgestimate). According to data from randomised, comparative trials of 1 year's duration, continuous estradiol 1 mg/day plus intermittent norgestimate 90 microg/day relieves climacteric symptoms (vasomotor symptoms and vulvovaginal atrophy) in postmenopausal women. Continuous estradiol 1 mg/day plus intermittent norgestimate 90 microg/day appeared as effective as estradiol 1 mg/day alone or continuous estradiol 2 mg/day plus continuous norethisterone acetate 1 mg/day in the treatment of postmenopausal women with vasomotor symptoms. Continuous estradiol 1 mg/day plus intermittent norgestimate 90 microg/day was as effective as continuous estradiol 1 mg/day in causing the maturation of vaginal epithelial cells. In a randomised, double-blind study, bone mineral density (BMD) increased to a significantly greater extent and the rate of bone turnover was slower in postmenopausal women treated with continuous oral estradiol 1 mg/day plus intermittent norgestimate 90 microg/day than in placebo-treated patients. Two randomised, double-blind studies indicated that the addition of norgestimate 90 microg/day to continuous estradiol 1 mg/day did not attenuate the beneficial effects of estradiol on lipid parameters. Continuous estradiol 1 mg/day plus intermittent norgestimate 90 microg/day was associated with increases in mean serum high density lipoprotein (HDL)-cholesterol levels and decreases in total cholesterol, low density lipoprotein (LDL)-cholesterol and lipoprotein (a) levels, compared with baseline. There was no statistically significant increase in triglyceride levels. In comparative trials, continuous oral estradiol 1 mg/day plus intermittent oral norgestimate 90 microg/day was well tolerated. Headache, breast pain or discomfort, abdominal pain or discomfort, uterine bleeding, dysmenorrhoea, oedema, nausea and depression were the most commonly reported adverse events. Continuous estradiol 1 mg/day plus intermittent oral norgestimate 90 microg/day was associated with a favourable uterine bleeding profile that improved over time. In a randomised trial, 80% of women were free from bleeding (irrespective of spotting) during month 12 of treatment. Norgestimate 90 microg/day was effective in protecting postmenopausal women against induction of endometrial hyperplasia by continuous estradiol 1 mg/day. In conclusion, data from a limited number of randomised studies indicate that HRT with continuous estradiol 1 mg/day plus intermittent norgestimate 90 microg/day is effective in relieving climacteric symptoms, increasing BMD and slowing the rate of bone turnover in postmenopausal women. This HRT regimen is well tolerated and is associated with a similar incidence of adverse events to that reported in recipients of continuous estradiol 1 mg/day. The norgestimate component of the regimen provides good endometrial protection and is associated with a favourable bleeding profile. Long-term studies investigating the associated risk of breast cancer and thromboembolic events in recipients of continuous estradiol plus intermittent norgestimate are needed. In the meantime, continuous oral estradiol plus intermittent oral norgestimate can be regarded as an effective new option for HRT in postmenopausal women.
A number of epidemiological studies have suggested that cigarette smoking is a risk factor for osteoporosis. Benzo(a)pyrene (BaP) and 7,12-dimethylbenz(a)anthracene (DMBA) are polycyclic aromatic hydrocarbons (PAHs) found in the tar fraction of cigarette smoke, as well as in car exhaust and furnace gases. We hypothesized that BaP and DMBA are responsible, through interaction with the aryl hydrocarbon receptor (AhR), for the bone loss and fragility seen in smoking-related osteoporosis. In this study four groups of 9-month-old Sprague-Dawley rats were examined. An intact group served as controls. A second control was the ovariectomized (ovx) group. The third group (ovx + E(2)) were ovariectomized and also given a continuous basal dose of estrogen by implanted estrogen pellet (0.085 mg of 17beta-estradiol per rat). The fourth group (ovx + E(2) + BaP/DMBA) was ovariectomized with an estradiol pellet, and received subcutaneous injections of 250 microg/kg of BaP/DMBA weekly for 15 weeks. The loss of ovarian function allowed the study of a direct effect of BaP/DMBA on bone while the concomitant estrogen repletion prevented ovx-related bone loss. Dual-energy X-ray absorptiometry (DEXA), histomorphometry, image analysis, and mechanical testing were used to determine the effect of the treatments on bone. The DEXA results showed a significant (p < 0.05) decrease in bone mineral density compared with intact controls with both ovx alone and with ovx + E(2) + BaP/DMBA treatment. The ovx + E(2) rats were similar to the intact controls. The osteoid parameters showed a significant increase (p < 0.05) with BaP/DMBA addition vs. intact controls, mimicking the ovx rats. The ovx + E(2) rats had osteoid parameters comparable to those of intact rats. Bone connectivity was decreased in the ovx and ovx + E(2) + BaP/DMBA animals. Connectivity of the ovx + E(2) rats was comparable to that of intact animals. A decrease in failure force was seen in three-point bending for the ovx + E(2) + BaP/DMBA group and in vertebral compression in both the ovx and ovx + E(2) + BaP/DMBA groups vs. intact controls. The mechanical properties of the ovx + E(2) rats were similar to those of intact rats. These results demonstrate that BaP/DMBA causes a loss of bone mass and bone strength, possibly through an increase in bone turnover. This is the first in vivo study linking environmental toxicants, found in the tar fraction of cigarette smoke and in urban air pollution, to loss of bone mass and strength in estrogen-replete ovx rats.
To examine the utility of a low-dose estrogen and pulsed progestogen hormone replacement therapy (HRT) regimen for add-back during long-term gonadotropin-releasing hormone-agonist (GnRH-agonist) therapy.
A pilot clinical trial conducted at a tertiary referral, academic, reproductive sciences center. The study included 15 patients with endometriosis and 5 patients with severe premenstrual syndrome (PMS). Patients with endometriosis received leuprolide acetate depot 3.75 mg IM monthly until their symptoms had resolved (2-3 months), at which time HRT was initiated along with the GnRH-agonist. Patients with severe PMS received the same treatment with the addition of HRT after 1 month. The HRT regimen consisted of 1 mg oral micronized estradiol daily and 0.35 mg norethindrone daily for 2 days alternating with 2 days without norethindrone. The main outcome measure included bone density assessment in the lumbar spine and femoral neck by dual-energy x-ray absorptiometry at 6- to 12-month intervals. The mean follow-up duration +/- SD while on GnRH-agonist treatment was 31.2 +/- 17 months (for endometriosis patients) and 37.7 +/- 8.4 months (for patients with severe PMS).
Bone mineral density was stable after initiation of HRT for the entire follow-up period. No patient had return of pelvic pain or resumption of mood swings after HRT add-back. After the first 3 months of HRT, all women remained amenorrheic.
Long-term GnRH-agonist down-regulation is safe and effective when combined with HRT add-back. Furthermore, on the basis of this small study, the low-dose pulsed progestogen, continuous estrogen HRT regimen seems to be safe for use as add-back therapy in terms of bone health.
Experimental, epidemiological, and clinical data indicate that progesterone is active in bone metabolism. Progesterone appears to act directly on bone by engaging an osteoblast receptor or indirectly through competition for a glucocorticoid osteoblast receptor. Progesterone seems to promote bone formation and/or increase bone turnover. It is possible, through estrogen-stimulated increased progesterone binding to the osteoblast receptor, that progesterone plays a role in the coupling of bone resorption with bone formation. A model of the interdependent actions of progesterone and estrogen on appropriately-"ready" cells in each bone multicellular unit can be tied into the integrated secretions of these hormones within the ovulatory cycle. Figure 5 is an illustration of this concept. It shows the phases of the bone remodeling cycle in parallel with temporal changes in gonadal steroids across a stylized ovulatory cycle. Increasing estrogen production before ovulation may reverse the resorption occurring in a "sensitive" bone multicellular unit while gonadal steroid levels are low at the time of menstrual flow. The bone remodeling unit would then be ready to begin a phase of formation as progesterone levels peaked in the midluteal phase. From this perspective, the normal ovulatory cycle looks like a natural bone-activating, coherence cycle. Critical analysis of the reviewed data indicate that progesterone meets the necessary criteria to play a causal role in mineral metabolism. This review provides the preliminary basis for further molecular, genetic, experimental, and clinical investigation of the role(s) of progesterone in bone remodeling. Much further data are needed about the interrelationships between gonadal steroids and the "life cycle" of bone. Feldman et al., however, may have been prophetic when he commented; "If this anti-glucocorticoid effect of progesterone also holds true in bone, then postmenopausal osteoporosis may be, in part, a progesterone deficiency disease."
The study comprised 12 groups of female rats: 6 groups of intact rats killed at 2, 6, 9, 12, 15, and 24 months of age, 4 groups of rats ovariectomized at 6 months and killed together with the intact rats at 9, 12, 15, and 24 months of age, and 2 groups of rats (one intact and one ovariectomized) treated with estrogen (2 micrograms estradiol valerate/rat/week s.c.) for 8 months before they were killed at 24 months of age. The composition, dimensions, and mechanical strength of intact bone and bone collagen from femoral diaphyses were investigated in relation to age, ovariectomy, and estrogen administration. Up to 6-9 months of age, the axial length, percentage ash, density, and compressive mechanical stress increased, whereas percentage collagen decreased. An age-related increase in bone mass, cross-sectional area, and wall thickness and a decrease in mechanical quality of bone collagen were apparent from 2 to 24 months of age. An age-related periosteal bone formation and the absence of endosteal bone resorption were demonstrated in intact rats. Compared with intact rats, ovariectomy was followed by an increase in body weight, a tendency to reduced percentage ash and a depressed bone mass, cross-sectional area, and wall thickness of femoral diaphyses. The compressive mechanical stress of intact bone and the mechanical quality of bone collagen were unaffected by ovariectomy. Ovariectomy did not influence the periosteal bone formation but induced an endosteal bone resorption not present in the intact rats.(ABSTRACT TRUNCATED AT 250 WORDS)
Since bone reacts to imposed loads by formation and resorption of tissue, analysis of tissue distribution within a bone provides evidence of the adaptation of that bone to a given mechanical function. Definition of these structure-function relationships permits the physical anthropologist to clarify the wide variety of behavioral/morphological adaptations to specific ecological niches in extant primates. From this information, behavior and locomotor function can ultimately be inferred in fossil primates. This paper reviews research which shows the relationships between the physical, geometrical and mechanical properties of bone, so that researchers who are investigating the properties of bone are aware of the numerous interpretations which may be made about structure and function from basic data. In addition, this paper is an attempt to apprise investigators working with primates that comparative data on the properties of primate bone are available, though sparse.
A 10-year, double-blind prospective study was undertaken to evaluate the effects of estrogen replacement therapy. The sample population consisted of 84 pairs of randomly chosen postmenopausal patients who were matched for age and diagnosis. One half of the patients received conjugated estrogens and cyclic progesterone, while the other half received placebo. Estrogen-treated patients whose therapy started within 3 years of menopause showed improvement or no increase in osteoporosis. Control patients demonstrate an increase in their osteoporosis.
Department of Medicine, Manchester Royal Infirmary, Manchester, M13 9WL
(Received 17 October 1977)
Recently Feldman, Dziak, Koehler & Stern (1975) have detected high-affinity gluco corticoid receptors in cytosol from isolated foetal rat calvarial bone cells. They estimated the binding constant (Kd) to be 7×10⁻⁹ mol/l for [³H]dexame[unk]hasone; however, their estimates were based on a maximum bound: free ratio of only 1%. Their data from cell cultures suggest that such binding may nevertheless be of physiological importance (Chen, Aronow & Feldman, 1977). The very small amount of binding might be due to a low initial concentration of binding sites, to their destruction during or after the trypsin–collagenase digestion, to their high dilution or to a combination of all three factors. We report here a much simpler method whereby we have greatly increased the percentage binding by setting up the incubation in intact calvaria.
Calvaría were dissected from foetal rats (19–21 days
We have previously identified glucocorticoid binding proteins in cytosol of cells dispersed from fetal rat calvaria by collagenase digestion. The present study, employing primary culture of these cells, provides further evidence that these binding proteins represent glucocorticoid receptors. [3H]Dexamethasone bound to cytoplasmic extracts of cultured cells with an apparent Kdiss of 6.8 nM and exhibited approximately 8500 binding sites/cell. Nuclear translocation of [3H]dexamethasone was demonstrated with approximately 50% of bound steroid extractable from the nuclear pellet after incubation at 37 C; little nuclear transfer occurred at 0 C. The specificity of these binding site was characterized by competition studies with other steroids in whole cells, the order of affinities being: triamcinolone acetonide greater than dexamethasone greater than progesterone greater than cortisol greater than corticosterone = cortexolone. Non-glucocorticoids except progesterone competed only poorly. Sedimentation analysis of [3H]dexamethasone-protein complexes on sucrose gradients revealed a cytoplasmic peak of 6.5 S in salt-free gradients and 3.8 S in 0.3 M KCl gradients. Dexamethasone addition to the culture medium resulted in a dose-dependent inhibition of cell growth with approximately 40% reduction in cell number at 13 nM. That this inhibition was receptor mediated was substantiated by the partial blockade of the dexamethasone effect in the presence 1.3 microM progesterone. Functionally, dexamethasone inhibits the growth of these cells. These data provide evidence for receptor mediated inhibitory effects of glucocorticoids directly at the level of the bone cell.
Bone mass, calcium and lipid metabolism, climacteric symptoms, bleeding, blood pressure, and weight changes were studied in 62 healthy postmenopausal women at 3-month intervals throughout 2 years of treatment with continuous estradiol valerate (2 mg) plus cyproterone acetate (1 mg), sequential estradiol valerate (2 mg) plus levonorgestrel (75 micrograms), or placebo. During the 2 years of the study, bone mineral content of the distal and ultradistal regions of the forearm (measured by single-photon absorptiometry) remained unchanged in the hormone groups, whereas bone mineral content at these sites decreased by 5 and 6%, respectively, in the placebo group. Bone mineral density in the spine (measured by dual-photon absorptiometry and dual-energy x-ray absorptiometry) increased by 3-4% in the hormone groups and decreased by 2% in the placebo group. Biochemical estimates of bone turnover (serum alkaline phosphatase and fasting urinary calcium/creatinine) decreased significantly to premenopausal levels in the hormone groups, but remained unchanged in the placebo group. Serum concentrations of total and low-density lipoprotein cholesterol were significantly reduced by 5-10% (P less than .05-.01) in the estradiol + cyproterone acetate group and by 10-15% (P less than .001) in the estradiol valerate + levonorgestrel group. There were no significant changes in high-density lipoprotein cholesterol in the hormone groups. Virtually no changes were observed in the placebo group. Climacteric symptoms and hot flushes were significantly reduced in both hormone groups compared with the placebo group.(ABSTRACT TRUNCATED AT 250 WORDS)
Hormone replacement therapy (HRT) comprises oestrogen with or without progestogen.
Benefits—HRT is the most effective treatment for the relief of menopausal symptoms and for the primary prevention of postmenopausal osteoporosis. There are also possible benefits for coronary heart disease, colorectal cancer, and neurocognitive function, but these are yet to be established....
An animal model of postmenopausal bone loss can be defined as a living animal in which spontaneous or induced bone loss due to ovarian hormone deficiency can be studied, and in which the characteristics of the bone loss and its sequalae resemble those found in postmenopausal women in one or more respects. Although in comparison to humans, the skeletal mass of rats remains stable for a protracted period during their lifespan, rats can be ovariectomized to make them sex-hormone deficient, and to stimulate the accelerated loss of bone that occurs in women following menopause. Ovariectomy induced bone loss in the rat and postmenopausal bone loss share many similar characteristics. These include: increased rate of bone turnover with resorption exceeding formation; and initial rapid phase of bone loss followed by a much slower phase; greater loss of cancellous than cortical bone; decreased intestinal absorption of calcium; some protection against bone loss by obesity; and similar skeletal response to therapy with estrogen, tamoxifen, bisphosphonates, parathyroid hormone, calcitonin and exercise. These wide-ranging similarities are strong evidence that the ovariectomized rat bone loss model is suitable for studying problems that are relevant to postmenopausal bone loss.
The influence of biosynthetic human growth hormone (b-hGH) on female rat cortical femur and tibia was studied after administration of hormone doses of 0.16, 1.10, or 8.33 mg/kg body weight/day for 90 days. The mechanical properties, dimensions, real density, ash weight, and the mineral and collagen concentrations of the bones were measured. In both femur and tibia a positive linear relation was found between the dose of hormone and ultimate load, ultimate stiffness, energy absorption at ultimate load, load at failure, energy absorption at failure, and deflection at failure. In the femur a positive correlation between dose and deflection at ultimate load was also found. After normalizing the mechanical data for the dimensions of the bones, no differences were found in the hormone treated groups compared to placebo, except for the elastic modulus (Young's modulus), which was decreased in the femur in the group given 8.33 mg b-hGH. The mineral and collagen concentration were unaffected in both femur and tibia, whereas the real density was decreased in the femur. The growth-hormone-induced changes in the mechanical properties seem to be caused mainly by increased dimensions of the bones.
Bleeding and climacteric symptoms were recorded in two groups of postmenopausal women receiving either continuous combined estradiol and norethisterone acetate or estradiol and cyproterone acetate. Out of a sample of 99 postmenopausal women aged 45 to 54 years, 86 completed a 2-year, double-blind, placebo-controlled study. Comparison of the bleeding patterns in the two groups revealed a statistically significant difference: More women in the estradiol-cyproterone acetate group experienced bleeding and for a longer duration. Thirteen women in the estradiol-norethisterone acetate group were amenorrheic, compared with two in the other group. The Kupperman index score in both groups declined to about 30% to 40% of initial values (p less than 0.001). The hot flushes in both treatment groups decreased to a highly significant degree (p less than 0.001), to a value below 20% of baseline values. We conclude that a continuous combination of estrogen and progestogen can produce amenorrhea and symptomatic relief. However, the progestogen components seem to differ in their ability to control bleeding.
The effects of progesterone on oophorectomy-induced bone loss in aged rats were evaluated. Female rats aged 12 months were divided into three groups: (1) sham-operated controls (SHAM); (2) oophorectomized (OVX); (3) OVX rats treated with progesterone (OVX + PROG). After 20 weeks the dry weight, bone ash, and calcium content of femur, tibia, and fourth lumbar vertebra were significantly lower in OVX than in sham rats. These reductions did not occur in OVX rats treated with PROG. There was no difference in the bone composition between the control and progesterone-treated rats. Vertebral bone histomorphometry showed increased bone resorption as well as increased bone formation parameters in OVX rats. Progesterone treatment inhibited the increased resorption indices, but the bone formation remained elevated. The results indicate that progesterone therapy prevents the postovariectomy bone loss in aged rats. The protective effect of progesterone is mediated by inhibition of bone resorption while maintaining the increased bone formation. These findings suggest that progesterone alone may be a valuable agent for management of postmenopausal osteoporosis.
The elastic moduli of human subchondral, trabecular, and cortical bone tissue from a proximal tibia were experimentally determined using three-point bending tests on a microstructural level. The mean modulus of subchondral specimens was 1.15 GPa, and those of trabecular and cortical specimens was 4.59 GPa and 5.44 GPa respectively. Significant differences were found in the modulus values between bone tissues, which may have mainly resulted from the differences in the microstructures of each bone tissue rather than in the mineral density. Furthermore, the size-dependency of the modulus was examined using eight different sizes of cortical specimens (heights h = 100-1000 microns). While the modulus values for relatively large specimens (h greater than 500 microns) remained fairly constant (approximately 15 GPa), the values decreased as the specimens became smaller. A significant correlation was found between the modulus and specimen size. The surface area to volume ratio proved to be a key variable to explain the size-dependency.
The effect of oophorectomy and hormone replacement therapy on cortical and trabecular bone mass was assessed in Sprague-Dawley rats. Bone mineral density (BMD) of the femur and the lumbar spine was determined by dual photon absorptiometry 4 months after surgery. Femoral mineral content was also determined. A significant decrease in bone density and in calcium content was observed after surgical castration. Bone mineral loss was prevented by either progesterone or estrogen, while the combination of progesterone and estrogen had no effect on the bone mineral content. The present study suggests the possibility that estrogen-progestin treatment may be less effective than a therapy with estrogen alone, and that further study on the effect of progesterone alone would be worthwhile.
10 healthy early postmenopausal women were treated with oestrogen and progestagen for two cycles of 28 days. Serum alkaline phosphatase and bone Gla protein increased during progestagen administration, whereas urinary excretion of calcium and hydroxyproline fell significantly during treatment, independently of progestagen intake. Thus, bone formation increases when progestagen is added to oestrogen treatment, whereas bone resorption may be kept constantly low during oestrogen plus progestagen treatment, leading to a positive calcium balance. This makes possible effective treatment of postmenopausal osteoporosis--treatment of elderly postmenopausal women with substantial bone loss before their bones have fractured or when they have just started to fracture. This study design can be used for easy and rapid screening of potential drugs for the prevention and treatment of postmenopausal bone loss.
We present the results of the static and dynamic histomorphometric analyses of parameter measurements of trabecular bone remodeling activity in nonoophorectomized, untreated oophorectomized, 17 beta-estradiol-supplemented oophorectomized, as well as progestagen-supplemented oophorectomized beagle dams. If the spayed beagle dam can be considered a model in which one may equivalate bone changes occurring in the ovarian hormone-deficient human female, we reach the following conclusions: The presence or absence of quasi-physiological levels of single ovarian hormones do affect the status of trabecular bone. The evidence indicates that the presence or absence of individual ovarian hormones do not have a simple effect on trabecular bone remodeling. Apart from affecting the numbers of new bone remodeling units (BMUs) that are initiated in unit time, their effect is seen to be extended to the working efficiency of existing osteoclasts and osteoblasts. The effect is also manifest on the total amount of work performed by the collections of osteoclasts and osteoblasts that provide the respective resorptive and formation phases of the BMU. Finally, we are led to believe that there may be a close interdependence both in physiological activity and therapeutic response between the four active bone envelopes. Such protein effects have hitherto not received their due consideration from researchers in this field.
Resorptive and formative characteristics on the periosteal and corticoendosteal bone envelopes were assessed on the ribs of intact, spayed and medroxyprogesterone acetate (MPA)-supplemented Beagle dams. Histomorphometric analyses of the alterations on surface activities of these two envelopes were carried out using tetracycline labeling. It was found that continuous administration of progestogen decreased the extent of resorptive surfaces on the corticoendosteal surfaces of progestogen-treated animals vs. spayed animals. On the periosteal surfaces the progestogen-treated dogs showed a marked increase in formation when compared to spayed and intact dogs. As a result of continuous progestogen treatment, mineralization lag time was shortened on the corticoendosteum and periosteum of progestogen-treated animals when compared to the same surfaces on the bones of intact and spayed animals.
Histomorphometric analyses of resorptive and formative parameters of the periosteal and corticoendosteal bone envelopes were performed on ribs from intact, spayed and spayed 17 beta-estradiol-supplemented Beagle dams. Dynamic formative features were evaluated after 2 pulses of in vivo tetracycline administration. The results indicated that following oophorectomy there was an increase in resorptive surfaces on the periosteum and corticoendosteum, and a decline in formative activity on these same envelopes. With continuous 17 beta-estradiol supplementation, the resorptive surfaces on both the periosteum and corticoendosteum of the spayed dams decreased. The formative activity on the periosteum seemed to decline further after the administration of 17 beta-estradiol, while on the corticoendosteum it tended to be restored to the level obtained for the intact dams. The increase in mineralization lag-time detected on the periosteum following oophorectomy appears not to be corrected by continuous 17 beta-estradiol supplementation in the spayed dams.
Estradiol (E2) plays a major role in maintaining women's skeletal integrity. Loss of bone mass is a regular occurrence with E2 deficiency, regardless of etiology. Estrogen-dependent bone loss follows a predictable pattern. Initially, it is quite rapid, preferentially affecting trabecular bone, which may decrease by 5-8% annually, whereas compact or cortical bone decreases by 1-3% annually. After 10-15 years of E2 deficiency, the rate of loss each year decreases; however, by that time, skeletal mass may be one-third to one-half of its youthful level. Because of the resultant skeletal fragility, even minimal trauma can produce fractures of the spine and wrist. After an additional 10-15 years of bone loss, hip fractures occur with alarming frequency. Timely restoration of E2 levels can prevent estrogen-dependent bone loss and can reduce significantly the risk of fracture. Studies show that 2 mg of E2 administered orally is an adequate dose; 1 mg will suffice if it is combined with a high dietary calcium intake. High calcium intake without estrogen is not effective in preventing the accelerated loss of bone that occurs in the years immediately after menopause. Long-term studies confirm that postmenopausal women who regularly use estrogen have greater bone mass and fewer osteoporotic fractures. Physicians should be encouraged to treat all estrogen-deficient women, particularly those who appear to be at higher risk for osteoporosis.
An estrogen-progestogen regimen of hormone replacement therapy has become widely used in recent years, primarily as a means to protect the endometrium from the carcinogenic effects of unopposed estrogen therapy (ERT). In this article, we evaluate the probable effects of this regimen on mortality from endometrial cancer as well as mortality from other chronic diseases. We conclude from this analysis that ERT is to be preferred to combination therapy for postmenopausal women without a uterus, primarily because it is predicted that ERT confers a significantly greater benefit on heart disease risk. In women with a uterus, if progestogens are to be prescribed, they should be given in the lowest possible dose needed to achieve the desired histologic changes in the endometrium, since the predicted loss in heart disease benefit from adding the progestogen is substantial.
Geometric, elastic, and structural properties of growing rat femora were determined from bending and torsion tests followed by bone sectioning and measurement of areal properties. Rosette strain gages bonded to the bone surface measured the strain during testing. A computer generated elliptical cross-sectional representation of the cross section geometry was used for calculation of material and structural properties. All structural and material properties increased with increasing age, exhibiting age-related changes that were best represented by an allometric or "heterauxic" growth pattern (y = axb) up to maturity. The femoral axial, flexural, and torsional rigidity increased 5.7, 10.1, and 14.8 fold, respectively, during maturation from 21 to 119 days of age. The increase in whole bone rigidity during maturation was caused primarily by changes in geometry. The bone tissue tensile longitudinal elastic modulus and shear modulus approximately doubled, and the shear strength increased approximately fourfold over this same period. Following maturity, a much slower increase in bending and torsional properties was noted. The results suggest that bone structural properties are regulated by changes in both geometric and material properties.
Fifty-two mature female rats on a controlled diet were studied to compare the effects of oophorectomy, and hormone replacement therapy after oophorectomy, on femoral morphology and mineral content. Oophorectomy was followed by the development of osteoporosis after 11 months of observation. This was characterized by a reduction in ash per unit length of bone and a diminution of mid-shaft femoral cortical width. The administration of a progestogen (9 μg ethynodiol diacetate/rat/day) for 10 months after oophorectomy prevented the reduction in ash per unit length from occurring, whereas an oestrogen (0·9 μg mestranol/rat/day) had no significant effect on either parameter of osteoporosis. The progestogen appeared to produce this effect by a relative increase in periosteal new bone formation at the expense of increased loss of bone from the endosteal surface.
The influence of ovarian steroids on food intake (FI), water intake (WI) and body weight (BWt) was measured under various conditions. Ovariectomy results in an increase in FI and BWt, which plateaued around one month after surgery. Daily injection of 1.5 μg oestradiol benzoate (OB) initiated at this time significantly reduced both FI and BWt. This effect of daily OB treatment on FI is only transitory since the FI returns to normal during OB treatment although the effect on BWt is maintained throughout and beyond OB treatment. Following ovariectomy, WI gradually falls, but is returned to normal by daily OB treatment. When oestrogen treatment is initiated at the time of ovariectomy, the increase in FI and BWt is prevented. In additional ovariectomized rats, 3 μg OB was injected every fifth day with either progesterone or oil administered on the intervening days. Although no influence of progesterone injection (either with OB or alone) was detected, the intermittent injection of OB induced cyclic suppression of FI, and the pattern of FI approached that of the intact cycling female. Adaptation to the intermittent injection of OB was not observed. Finally, OB treatment was found to decrease the increased FI seen during pseudopregnancy by a proportion similar to the effect of oestrogen in the long-term ovariectomized animal. These results suggest that oestrogen, but not progesterone, is the ovarian hormone active in the regulation of intake parameters and body weight in the female rat.
The value of oestrogen therapy in the prevention of osteoporosis after oophorectomy was assessed in 114 middle-aged women who participated in a double-blind controlled trial of mestranol in an average daily dose of 23 mug. The skeletal response to treatment was measured by a photon absorption technique. Where treatment was started within two months of operation subsequent bone mineral loss was prevented. Treatment started three years after oophorectomy caused a highly significant increase in bone mineral content. When treatment was delayed for six years mestranol failed to prevent subsequent bone mineral loss with age. These effects occurred independently of the associated humoral changes in calcium and phosphorus homoeostasis. Mestranol in this dosage appeared to be relatively safe, but it is too early to evaluate the long-term hazards of such therapy.
Cell nuclear estrogen receptors and cytosol progestin receptors were measured in the pituitary gland, preoptic area and hypothalamus throughout the estrous cycle of the rat. Cell nuclear estrogen receptor levels paralleled changes in serum estradiol concentrations with highest values on proestrus and lowest on diestrus. Proestrous values were 50-60% of capacity for each tissue. Cytosol progestin receptor number in these tissues was also highest on proestrus and lowest on diestrus. With these data as a guide, Silastic capsules filled with estradiol-cholesterol mixtures were used to generate physiologic levels of estrogen receptor occupation within the brain-pituitary complex of ovariectomized rats and to examine the kinetics of estradiol stimulation of lordosis behavior and cyclic gonadotropin release. Our results indicate that the effectiveness of an estradiol stimulus to elicit lordosis or luteinizing hormone release depends on at least three factors: the magnitude of the increment in serum estradiol and brain and pituitary cell nuclear estradiol receptor levels; the duration over which these increments area maintained; and the interval from previous exposure to estrogen.
The effect of combined oestrogen/progestogen and calcium therapy on vitamin D metabolites was studied in a double-blind, controlled clinical study over a period of 12 mth. Seventeen healthy post-menopausal women were randomly allocated for treatment with either oestrogen/progestogen or placebo. All the participants received a daily calcium supplement of 0.5 g throughout the study. The oestrogen-treated group showed the well-known changes in calcium metabolic variables, i.e. decreases in serum phosphate (P less than 0.001), serum alkaline phosphatase (P less than 0.001) and 24-h urinary calcium excretion rate (P less than 0.01). However, the serum calcium and serum 1,25-dihydroxycholecalciferol concentrations were unchanged. In the placebo group all the measured values remained virtually unchanged throughout the study. The findings indicate that calcium seems to potentiate the bone-preserving effect of oestrogen treatment in early post-menopausal women by keeping the serum calcium level unchanged and thus maintaining an unchanged vitamin D metabolism.
Medroxyprogesterone acetate (MPA) is the progestin used most commonly in postmenopausal women to attempt to reduce the risk of endometrial cancer associated with estrogen replacement and, more recently, as the sole agent for the treatment of hot flashes. The effects of this progestin were examined on the fasting urinary ratios of calcium (Ca) and hydroxyproline (OHPr) to creatinine (Cr) as indices of bone resorption. MPA, 20 mg, significantly reduced Ca/Cr and OHPr/Cr to levels similar to those in premenopausal women. The effects of MPA were eightfold greater than those of megestrol acetate (MA) as previously reported. The effects of ethinyl estradiol were greater than those of both MPA and MA. Long-term studies of bone density will be required to confirm these apparent, benficial effects of MPA on bone metabolism in postmenopausal women.
The purpose of this study was to examine the effects of estrogen replacement, in concert with three different progestin regimens, on the mechanical properties of rat lumbar vertebrae. Ninety-two Sprague-Dawley rats (11 months old) were divided into six groups for treatment. The first group was an intact control, the second group (OVX) was ovariectomized only, and the third group (estrogen-only) was ovariectomized and received continuous estrogen through a 17β-estradiol implant. The remaining groups were ovariectomized and received estrogen and progestin (norethindrone, NET) therapy; 3 μg of NET was injected daily (estrogen plus continuous NET), or 6 μg of NET was injected for 14 consecutive days of a 28-day cycle (estrogen plus cyclic NET), or for 3 consecutive days of a 6-day cycle estrogen plus interrupted NET). The animals were sacrificed after 6 months, and the vertebrae were dissected out. The vertebral processes of the fourth lumbar vertebrae were removed, and the density of the vertebral bodies was determined. They were then subjected to compression testing.
We found that all three estrogen/progestin regimens maintain bone density and all mechanical properties at a level indistinguishable from the control. However, the cyclic and continuous NET treatment results were, with the exception of density, also indistinguishable from those of the ovariectomized group. The estrogen plus interrupted NET group on the other hand, has a significantly greater compressive modulus and density than the ovariectomized group. In conclusion, with respect to the ovariectomized group, the estrogen plus interrupted NET treatment resulted in a superior density and compressive modulus. The remaining mechanical properties were equivalent to those resulting from the continuous or cyclic progestin regimens.
To investigate the effects of estrogen (E) alone or with cyclic, continuous, or interrupted P regimens on bone density in an aged rat model.
Randomized controlled trial.
Twenty 12-month-old female Sprague-Dawley rats were sham operated (intact control) and 100 were ovariectomized (OVX).
Four groups of 20 OVX rats were implanted with silastic capsules containing 5% E2 (wt/wt) in cholesterol. All rats in the intact control (group 1), OVX (group 2), and the first of the OVX plus E groups (group 3) were injected subcutaneously daily for 6 months with corn oil (vehicle). Three other groups of rats with E capsules received daily injections of norethindrone in corn oil, according to the following dosage schedules: 6 micrograms norethindrone/rat per day for 2 of every 4 weeks (cyclic; group 4); 3 micrograms norethindrone/rat per day every day (continuous; group 5); or 3 micrograms norethindrone/rat per day for 3 of every 6 days (interrupted; group 6).
Bone mineral content (BMC) and bone mineral density (BMD) in the femur and vertebrae were measured by dual-energy roentgenogram absorptiometry.
The OVX rats (group 2) receiving corn oil alone had the lowest BMD. Intact controls (group 1), E plus cyclic P (group 4), and E and interrupted P (group 6) were all similar and had significantly greater bone density in the vertebrae than the OVX controls. In contrast, vertebral BMD with E alone (group 3) and continuous E and P (group 5) was not significantly different from the OVX group. Femur BMD was significantly lower in the OVX group compared with the other five groups, which did not differ significantly from each other.
In this experimental model, compared with OVX controls, combined hormone replacement therapy with E and cyclic or interrupted P resulted in the best vertebral BMD whereas continuous E and P resulted in the worst BMD. In the femurs, E alone and E plus P had equal effects on BMD.
Feral adult female cynomolgus monkeys were divided into two groups: normal controls and ovariectomized. Tibiae and trabecular bones from the femoral head, from each group, were tested using a materials testing machine. The bending stiffness of the tibiae was measured by nondestructive three-point bending tests and their maximum torque capacity by destructive torsion tests. The compressive strength of the trabecular bones was measured by compression tests. Ovariectomy caused significant decreases in elastic modulus of the tibiae (p < 0.008), measured by three-point bending tests, and in shear modulus (p < 0.015), failure shear stress (p < 0.01), and failure torque (p < 0.001) of the tibiae, measured by torsion tests. It caused a significant decrease in cortical bone density (p < 0.005), but no significant changes in tibial cross-sectional area and in cortical shaft external and internal diameters. The differences in elastic modulus, maximum compressive strength, and density of femoral trabecular bone samples between the two groups were not significant.
Exercise may play a role in the prevention of bone fractures in postmenopausal osteoporosis. The effects of endurance exercise on bone properties were assessed in 9-month-old sham-operated (SH) and ovariectomized (OVX) rats. The rats were either kept sedentary (SED) or were exercised (EX) on a rodent treadmill at 21 m/minute, 7% grade, 40 minutes/day, 4 days/week for 3 months. Bone mineral (by ash weight), morphometry, and biomechanical properties (by three-point bending) were evaluated after excision of bones at sacrifice. Ovariectomy resulted in a loss of bone mineral in femur, tibia, and fourth lumbar vertebra (L4), but biomechanical (force, deformation, stress, strain, and modulus of elasticity) and morphometric (length, cortical and medullary area, and moment of inertia) properties of femur were maintained. The ash weight of femur and tibia, but not L4, as well as femur yield and maximum force and moment of inertia, were improved in OVX-EX rats compared to OVX-SED animals. In SH rats exercise had no influence on ash weight of any of the three bones or femur morphometric properties, yet femur maximum force and plastic deformation were significantly enhanced compared to SH-SED rats. The results of the present study suggest that endurance exercise has beneficial effects on the bone mineral as well as biomechanical properties (femur yield and maximum force) during early stages after ovariectomy and improves the bending strength of the intact femur without an effect on bone mineral in sham-operated rats.
We tested a new hormone replacement formulation based on the hypothesis that interrupted administration of progestin in the presence of continuous estrogen would result in receptor up-regulation and resensitization of target tissues to both estrogen and progestin. As a result, symptom control might be possible with lower doses of steroids and in the absence of withdrawal bleeding.
Forty postmenopausal women were entered in a 6-month pilot study, including an 18-month extension. They received piperazine estrone sulfate 0.75 mg daily. Norethindrone 0.35 mg daily was added in 3-day phases, alternating with progestin-free phases of 3 days. There was no steroid-free withdrawal period. We examined symptom control, bleeding patterns, endometrial protection, and lipid profiles in the women over the 24 months of the study.
Hot flushes were completely eliminated in 76% of women, and 80% had no bleeding by 6 months. There were three dropouts. Thirty-three women elected to continue after the first 6 months and completed 24 months on therapy for a compliance rate of 82.5%. No endometrial hyperplasia was seen on serial biopsies, and no changes occurred in lipids except for a small but statistically significant decrease in high-density lipoproteins and triglycerides at 24 months.
Our preliminary results of low bleeding rates, good symptom control, and endometrial protection suggest that hormone replacement with low-dose estrogen and interrupted progestin is effective and may lead to improved compliance in menopausal women.
Detection of high-affinity glu-cocorticoid binding in rat bone Bleeding patterns during 457-67, 1986 continuous combined estrogen-progestogen therapy
- Manolagas Sc
- Anderson
- Riis U Bj Marslew
- Christiansen
Manolagas SC, Anderson DC: Detection of high-affinity glu-cocorticoid binding in rat bone. J Endocrinol 76:379-380, 24. Marslew U, Riis BJ, Christiansen C: Bleeding patterns during 457-67, 1986 continuous combined estrogen-progestogen therapy. A m J Obsrer Gynecol 164:1163-1168, 1991
- Aitken
The effects of ovarian steroids on food and water intake and body weight in the female rat
- Tarttelin MF