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Original Studies |
Departments of Medicine (S.M.O.) and Epidemiology and Biostatistics (D.S., A.Z.L., W.E.B.), University of Washington, and the Center for Health Studies, Group Health Cooperative of Puget Sound (L.E.I., C.K.Y.), Seattle, Washington 98195-6426
Address correspondence and requests for reprints to: Susan M. Ott, M.D., Division of Metabolism, Box 356426, University of Washington, Seattle, Washington 98195-6426.
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Abstract |
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Introduction |
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Several biochemical markers that measure bone resorption and formation rates have been developed over the last decade. Collagen contains cross-linking molecules that are covalently bonded between three collagen fibrils and released by bone resorption. The N-telopeptide (NTX) assay measures the urine excretion of one of these collagen fragments that is located at the site of the cross-linking and is specific to bone collagen. Osteocalcin is a protein that is secreted by mature osteoblasts, and it has been shown to correlate with the bone formation rate in normal persons (20) as long as there are no pathological alterations in the metabolism of the protein or in osteoid mineralization. In young healthy females aged 11–32 yr, NTX and osteocalcin showed good correlation with calcium isotope kinetic studies of bone resorption and formation, respectively (21).
The purpose of this analysis was to compare biochemical markers of bone resorption and formation in young women who were using DMPA, OCs, or no hormonal contraception. PTH and calcium were also measured to determine the effect of hormonal contraceptives on these aspects of bone metabolism. A second purpose of this study was to examine the relationship between the biochemical markers and bone density in this same group of premenopausal women.
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Materials and Methods |
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The subjects in this study are a subset of those included in a recent report (1). Women aged 18–39 yr who had recently received DMPA were identified using the administrative computerized databases of Group Health Cooperative of Puget Sound, a large health maintenance organization. A random sample of comparison women was also selected from the Group Health enrollment database and frequency matched on the basis of age and primary care clinic. The comparison subjects were enrolled without regard to their contraceptive method, so the prevalence of OC use reflected that of the population. OCs containing progestin without estrogen and progesterone implants were not used by this group of women. Although OC content was not recorded for this study, the computerized pharmacy database provided the distribution of OC prescriptions for all 18–39-yr-old women enrollees at the time of study recruitment. Pills containing ethinyl estradiol (35 µg) and norethindrone (0.5–1 mg) were used by 53.6% of women; ethinyl estradiol (35 µg) and either levonorgestinel (1 mg) or ethynodiol diacetate (1 mg) were used by 18%; ethinyl estradiol (30 µg) and norethindrone (1.5 mg) by 13.7%; and ethinyl estradiol (20 µg) with levonorgestrel or norethindrone by 9.7% of women. Subjects were excluded if they were pregnant or trying to become pregnant, lactating, or had diseases known to cause secondary osteoporosis. The protocol was approved by the Human Subjects Committees of the University of Washington and Group Health. All subjects gave written informed consent.
The study group consisted of 183 women using DMPA and 274 comparison women. A random subset of 227 women was asked to provide blood and urine samples; of these, 116 were using DMPA, 39 were using OCs containing estrogen and progestin, and 72 were not currently using hormonal contraceptives. One subject was excluded from analysis because she was found to have secondary hyperparathyroidism with a PTH of 120.
Bone densitometry
Bone density was measured with a Hologic 2000 bone densitometer (Hologic, Inc., Waltham, MA). The study was concurrent with the Fracture Intervention Trial, whose quality control methods have been reported (22). The sites measured were the whole body, the lumbar spine (L1–L4) and the proximal femur (total hip). To avoid the risk of radiation exposure in a woman who could possibly be pregnant, the clinic visits were scheduled during the early follicular phase in those women who had menstrual cycles.
Increase in bone size results in increases in the areal bone density as measured by dual-energy x-ray absorptiometry, even if there is no change in the density of the bone (mass per volume). We did not make adjustments for the projectional method of measuring bone mass in this study because after age 18 the increases in bone size are very small.
Bone biochemistry
Serum samples (nonfasting) were frozen at -70 C for batch analysis. Urine samples were obtained during the daytime clinical visit. Osteocalcin was measured using a two-site immunoradiometric assay with rabbit antibodies and human standards (23). The reported intra-assay coefficient of variation was 6%. Reagents were obtained from Immunotopics, Inc. (San Clemente, CA). Intact PTH was measured with a chemiluminescence assay (24) (Nichols Laboratory, San Juan Capistrano, CA). The intra-assay coefficient of variation for PTH was less than 6%. Urine NTX was measured using a RIA (Ostex International, Inc. Seattle, WA) (25). The reported intra-assay variation was 7%, with a circadian variation of 22% from the mean 24-h value (26).
Clinical data
Weight and height were measured at the clinic visit, the latter with a Harpenden stadiometer (Holtain, Pembrokeshire, UK). Other variables were obtained from a self-administered questionnaire that was reviewed at the clinic visit. Calcium and protein intakes were assessed by the Fred Hutchinson Cancer Research Center Dietary Intake Questionnaire (27).
Statistical analysis
Programs used for statistics were SAS and Statview 5.0 (both from SAS Institute, Inc., Cary, NC). Basic descriptive statistics for the biochemical tests, risk factors, and bone density measurements were calculated and compared among contraceptive groups using ANOVA.
The association of selected baseline variables with biochemical markers was examined univariately after adjusting for age. The variables examined were age, body mass index (BMI), calcium intake, protein intake, physical activity score, smoking, alcohol use, age at menarche, ethnicity, fracture in a female relative, and age at first pregnancy. These same variables were examined for their association with contraceptive method. Variables associated with contraceptive use or at least one of the biochemical markers were entered in a multivariate model. Only those factors that remained significant or had a strong association with the biochemical markers were retained in the final multivariate model. This model used the general linear models procedure.
The least squares mean values for each biochemical marker, after adjustment for the other factors, were then compared among the contraceptive groups using pairwise comparisons.
Pearson correlation coefficients between the bone density measurements and the biochemical markers were calculated with and without adjustment for age.
The mean NTX and osteocalcin levels were calculated for five age categories and plotted to show the effects of age on these levels within each contraceptive group.
Within the groups using contraceptives, the partial correlation coefficients between bone density or biochemcial markers and duration of contraceptive use were calculated with adjustment for age.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows the unadjusted mean
values for NTX, osteocalcin, PTH, calcium, bone density, and the
clinical risk factors in the three contraceptive exposure groups. There
were no significant differences among groups in the clinical factors,
except that a greater percentage of women who used DMPA had been
pregnant. Amenorrhea, defined as no period within the last 6 months,
was reported in 49.6% of women in the DMPA group, 2.6% in the OC
group, and 1.4% in the nonusers. Using one-way ANOVA, the significance
for differences in bone density at the spine was 0.051. Pairwise
comparisons showed that the DMPA group was lower (P =
0.02) than the OC group, but a Bonferroni adjustment for multiple
comparisons resulted in a significance level of 0.06. The nonusers had
intermediate values that were not significantly different from either
hormonally exposed group. The bone density values at the hip and whole
body were not significantly different among groups.
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shows
the results from the final multivariate model with NTX as the dependent
variable.
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shows the values for the
biochemical markers using adjusted means from the final multivariate
model. The NTX levels were highest in the DMPA group and lowest in the
OC group; differences were significant in all pairwise comparisons.
Osteocalcin levels showed the same pattern, except there was no
statistically significant difference between the DMPA group and
nonusers. There were no differences among groups in their PTH levels.
The serum calcium was slightly lower in the OC group than in the DMPA
group (P = 0.05).
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shows the
results of NTX in different age ranges for each of the exposure groups.
In all age ranges, NTX was higher in the DMPA group than in the OC
group.
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As reported previously (1), duration of use of DMPA was associated with decreased bone density in younger women, with lower bone density in women who had used DMPA the longest. Among the 95 OC users in the entire cohort, the correlation coefficient between duration of use and bone mineral density (BMD) was 0.02 (hip) and 0.10 (spine); these remained nonsignificant when adjusted for age. Within each 4-yr age group the correlation coefficients between hip BMD and duration of OC use ranged from 0.04–0.29 (not significant). Similar correlations were seen for spine BMD, except in the youngest age group, 18–21 yr, where the correlation coefficient between BMD and duration of OC use was 0.47 (P = 0.01). The value was similar when adjusted for age (partial correlation, 0.51).
The NTX and osteocalcin levels were both negatively correlated with the
bone density. Figure 2 shows the scatter
plots of the spine BMD and the biochemical values. Including all
subjects, the correlation coefficient between NTX and spine bone
density was -0.33 (P < 0.001), which was not changed
after adjustment for age (-0.34); for osteocalcin the coefficients
were -0.27 (P < 0.001) unadjusted and -0.31 after
age adjustment. Within each contraceptive group, the correlation
coefficient between NTX and spine bone density was -0.36 in the
nonusers, -0.25 in the oral contraceptive group, and -0.27 in the
DMPA group. For osteocalcin the values were -0.18, -0.09, and -0.32,
respectively.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The main purpose of this analysis was to compare biochemical markers among groups of women using different methods of contraception. To interpret our findings, we first discuss the relationship between markers and bone physiology in this age range. We then discuss the results seen in DMPA users, followed by the findings in the OC users. Finally, we consider the second aspect of this analysis, to examine the relationship between biochemical markers and bone density in these premenopausal women.
Biochemical markers and bone physiology in young women
Both NTX and osteocalcin were lower in subjects who were older. This was seen in all the exposure groups of this study. The relationship with age is most valid in the nonuser group, because hormonal therapy could have altered normal physiologic changes. Because peak bone mass is not attained until approximately age 25 (7, 28, 29), the decrease in markers in the nonuser group probably reflects the maturing of the skeleton. Osteocalcin and NTX are frequently referred to as markers of "bone turnover," but this is not always accurate, especially during growth. In immature skeletons bone modeling contributes to the levels of these markers. Thus, teenagers who are rapidly growing have NTX levels that are an order of magnitude higher than those in middle aged persons (30). Osteocalcin levels during adolescence are also increased by skeletal growth (31).
The bone remodeling process occurs in both immature and mature skeletons. The net effect of remodeling on the bone mass depends on the overall balance of resorption to formation. During normal skeletal consolidation in young adults, both formation and resorption rates are higher than in older adults, but bone formation is greater than bone resorption, and bone density increases. However, if the eroded cavities are not completely filled (resorption exceeds formation at the individual bone remodeling unit), then high levels of NTX will be associated with loss of bone mass.
There are some limitations to the use of markers in the evaluation of bone remodeling. One source of error is the diurnal variation of NTX, which is 22% higher in the morning than the 24-h mean (26). In this study, the markers were obtained during the day, but were not strictly done at the same time.
Also, in young women, some markers vary with the menstrual cycle. Osteocalcin does not vary with the cycle (32, 33), but deoxypyridinoline, another collagen cross-link that reflects bone resorption, is higher in the follicular phase (32) and NTX is highest at mid-cycle and lower again during the luteal phase (34). The subjects in this study were generally measured during the follicular phase; if they had been measured during the luteal phase the difference between users and nonusers of DMPA would probably have been enhanced.
Effects of DMPA use on markers
The women using DMPA had higher values of NTX and lower bone density than nonusers, which suggests that bone resorption exceeded bone formation. The bone density values in the subset of subjects who also had biochemical markers did not show the same level of significance as seen in the entire cohort because the number was smaller. Our findings of low bone density are consistent with other reports (2, 3, 4, 5, 6). Regarding osteocalcin, a prospective study of 11 subjects showed an increase in the osteocalcin with DMPA use (35); in our study, osteocalcin was higher in DMPA users, but this was not statistically significant.
Estrogen levels were not measured in this study, but others have shown
that estradiol levels are low with DMPA use (4, 5) and
amenorrhea occurs in 
45% of women (5). Estrogen
deficiency is consistently associated with rapid bone loss, especially
in young women (36). Bone resorption is increased,
osteoclasts have longer life spans and erode deeper cavities, and NTX
levels increase. The abnormalities seen in the DMPA users in this study
are very similar to the changes reported in women with estrogen
deficiency. Therefore, it is a plausible hypothesis that DMPA causes
abnormal bone physiology due to low estrogen levels.
A direct effect of DMPA on bone cells is also possible. A biopsy study in beagles showed an increased bone formation with medroxyprogesterone acetate (MPA) therapy, but the bone porosity and Haversian canal diameters were also significantly increased, so that there was no improvement in the bone volume (37). This would be consistent with the high bone resorption seen in the women in this study. A recent dose-response study of MPA in women taking estrone showed a smaller increase in bone density in those with the higher (10 mg) dose of MPA than with lower doses (38). In postmenopausal women who are taking estrogen, the addition of MPA or micronized progesterone does not improve bone density (39, 40). In premenopausal women, MPA does not protect bone from effects of GnRH (41), ovariectomy (42), or amenorrhea (43). In contrast to the above studies, one controlled trial in physically active women who had ovulatory disturbances found an improvement in bone density with MPA treatment (44). Additional studies in situations with normal estrogen levels are needed to define the independent effects on MPA on bone physiology.
The long-term effect of DMPA use on bone remains unknown. Women with high bone formation and resorption have the potential to increase bone density when bone resorption is decreased, which could potentially occur when DMPA is discontinued and estrogen levels return to normal. This is seen in postpartum women after weaning, when bone density and bone biochemical markers return to the prepregnancy levels (45), and in postmenopausal women who are treated with estrogen (40). Cundy et al. (46) followed 13 women for 8 months after discontinuation of DMPA and noted an increase of 3.4% per year in spine bone density. Additional longitudinal studies are needed to determine the effect of DMPA discontinuation on bone density and bone biochemical markers.
Effects of OC use on bone markers
The group using OCs had lower levels of NTX and osteocalcin than nonusers, but their bone density was not different. Even in the complete study group, the bone density for all anatomic sites was not different in the 95 women who were currently using OCs compared with the 180 who were not using hormonal contraception. This suggests that, although the values of NTX and osteocalcin were both depressed, the net balance between bone resorption and bone formation was similar to that in women not using hormones, so there was no increase in bone density. Other studies of biochemical markers have shown decreased levels of osteocalcin (12, 14, 47) and NTX (14) in women using OCs, results consistent with those in this study.
Suppression of bone resorption usually results in an initial gain of bone mass while the active bone metabolic units continue to fill in the previously resorbed cavities. Thereafter the bone formation rate is suppressed as well. Register et al. (48) performed a randomized trial in a young primate model and found that the final bone density was lower in those on the OCs than in the nonusers. They suggested that oral estrogen-progestin contraceptives currently in use can suppress endogenous androgen secretion, which would independently lower the bone formation. If this were true, then bone formation would be depressed to a further extent than it would from suppression of bone resorption alone, and bone density would not increase. Consistent with this hypothesis were results from Polatti et al. (11), who studied 200 young women and found no increase in bone density after 5 yr in those using OCs, whereas the controls gained 7.8%. Recker et al. (7), however, observed that young college-aged women who were using OCs had a greater increase in bone density than nonusers. In our study, the youngest women, aged 18–21 yr, showed a positive correlation between duration of use of OCs and bone density at the spine, but not at the hip. Although statistical adjustment for age did not alter the results, the findings should be interpreted with caution, because age and duration of use are colinear in this population and this is a cross-sectional study.
Most cross-sectional studies of premenopausal women have not found a difference in bone density between users and nonusers of OCs (12, 13, 14, 15, 16). Some studies in postmenopausal or perimenopausal women found a higher bone density in women who had previously used OCs (8, 9), but others failed to find a difference in bone density with past use (17, 18). Two large cohort studies of fracture end points in women who had used OCs came to opposite conclusions: one found a 25% reduction in hip fractures (10), the other a relative risk for fractures of 1.2 (19). These different results may be due to variable amounts of estrogen and progestin in the different OC preparations.
Relationship between biochemical markers and bone density
The bone biochemical markers provide valuable information about bone physiology in groups of subjects, but there is debate about the clinical use in individual women. In this study, the bone density was inversely correlated with both NTX and osteocalcin. When all subjects were included in the analysis, the NTX values predicted only 11% of the variation in bone density. When the relationship between bone density and NTX or osteocalcin was examined within the treatment groups, similar correlation coefficients were seen regardless of contraceptive choice. Garnero et al. (49) found no correlation between osteocalcin and bone density in premenopausal women, but the NTX did show significant correlations (r = -0.21 at lumbar spine and -0.31 at total hip), similar to those in this study. Thus, the biochemical markers show significant correlations to bone density and to bone resorption and formation rates in groups of women, but for individual women the errors are large. The markers are useful in studies of bone physiology, but they would perform poorly as a screening test for low bone density.
Summary and conclusions
Women using DMPA contraception have bone resorption rates that are higher than formation rates, with a negative bone balance and decreased bone density. Those women who use OCs have lower bone formation and resorption rates than women not using hormonal contraception, but this does not result in higher bone density.
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Acknowledgments |
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Footnotes |
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Received January 19, 2000.
Revised September 11, 2000.
Accepted September 15, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, Women using DMPA;
, women using OCs; 
, nonusers. Error bars are ±SE.