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Endocrine Care |
Departments of Obstetrics and Gynecology, Medicine and Orthopedics, St. Luke’s-Roosevelt Hospital Center and the Columbia College of Physicians and Surgeons, New York, New York 10032
Address all correspondence and requests for reprints to: Michelle P. Warren, M.D., Department of Obstetrics and Gynecology, PH 16-127, Columbia University, 622 West 168th Street, New York, New York 10032. E-mail: . mpw1{at}columbia.edu
Abstract
Few longitudinal studies have investigated the effects of amenorrhea and amenorrhea plus exercise on bone mineral density (BMD) of young women. We carried out a 2-yr comparison of dancers and nondancers, both amenorrheic and normal, that investigated the role of hypothalamic amenorrhea on bone in this context. We studied 111 subjects (mean age, 22.4 ± 4.6 yr; age of menarche, 14.1 ± 2.2 yr), including 54 dancers, 22 with hypothalamic amenorrhea, and 57 nondancers, 22 with hypothalamic amenorrhea. Detailed hormonal and nutritional data were obtained in all groups to determine possible causal relationship to osteoporosis. The amenorrheic groups, dancers and nondancers, both showed reduced BMD in the spine, wrist, and foot, which remained below controls throughout the 2 yr. Only amenorrheic dancers showed significant changes in spine BMD (12.1%; P < 0.05) but still remained below controls, and within this subgroup, only those with delayed menarche showed a significant increase. The seven amenorrheic subjects (three dancers and four nondancers) who resumed menses during the study showed an increase in spine and wrist BMD (17%; P < 0.001) without achieving normalization. Delayed menarche was the only variable that predicted stress fractures (P < 0.005), which we used as a measure of bone functional strength. Analysis of dieting and nutritional patterns showed higher incidence of dieting behavior in this group, as manifested by higher Eating Attitudes Test scores (16.3 ± 2.00 vs. 11.5 ± 1.45; P < 0.05) and higher fiber intakes (30.7 ± 3.00 vs. 17.5 ± 2.01 g/24 h; P < 0.001). We concluded that low bone mass occurs in young women with amenorrhea and delayed menarche, both exercisers and nonexercisers. Crucial bone mass accretion may be compromised by their reproductive and nutritional health.
HYPOTHALAMIC AMENORRHEA IN young women is associated with reduced bone accretion or premature bone loss during adolescence (1, 2, 3, 4, 5, 6, 7, 8, 9, 10), which places women at high risk for fractures, significant osteopenia, and severe osteoporosis at menopause. The potential for reversibility of this problem warrants further investigation, especially due to the current lack of longitudinal studies.
Research has suggested that exercise, particularly weight-bearing exercise, might increase bone accretion in women with hypothalamic amenorrhea (11) and in young adults and adolescents (12). However, hypothalamic amenorrhea has also been increasingly linked to nutritional insults, particularly caloric deprivation, which suggests that the metabolic axis might be involved with the amenorrhea and osteopenia in these cases (13, 14, 15).
This study investigates the roles of hypothalamic amenorrhea and exercise in the accretion of bone mass during adolescence and young adulthood, a time frame during which bone mass is known to increase significantly. We examined the effects of prolonged amenorrhea on bone mass in exercising and nonexercising young women. We investigated whether exercise affected the rate of bone accretion and the functional strength of bones, as measured by the frequency of stress fractures.
Materials and Methods
A group of 111 subjects was followed for 2 yr without intervention. This study compared an exercising group to a group of controls and was designed to examine the effects of prolonged hypoestrogenism with and without exercise. Ballet dancers were chosen for the exercising group because they begin to train before adolescence and are well known to have both delayed menarche and a high incidence of amenorrhea during adolescence (7).
Subjects
We followed 111 subjects. The mean age of this group was 22.4 ± 4.6 yr, and the mean age of menarche was 14.1 ± 2.2 yr. The exercising group consisted of 54 ballet dancers, 22 of whom were amenorrheic. A control group consisted of 57 nonexercising subjects, 22 of whom had nonexercise-associated hypothalamic amenorrhea. Of these 44 exercising and nonexercising amenorrheics, 26 (59.1%) had delayed menarche. Volunteers were solicited from national and regional schools and dance companies, advertisements in college publications, and physicians’ referrals for hormonal problems and interest in bone density measurements. These subjects were part of a cross-sectional study previously reported (5), and 30 subjects were part of a previous report (1). All subjects were white and from middle to upper class families. Information was obtained by interview, questionnaire, and medical examination. Informed consent was obtained by physicians or nurse practitioners. The procedures followed were approved by the Institutional Review Board at the St. Luke’s-Roosevelt Hospital Center. Secondary amenorrhea was defined as amenorrhea of 5 or more months immediately preceding the study and delayed menarche age as 14 or older. Subjects taking hormones or oral contraceptives for 6 months before the study were eliminated. All subjects were seen yearly, and all measures were repeated. The educational levels of the subjects were determined by the seven-point scale developed by Hollingshead and Redlich (16).
Medical evaluation
A nurse practitioner took a menstrual and hormonal history, including weight fluctuations, dieting behavior, hours spent dancing per week, and a history of past and present illness. Women with chronic illness were eliminated from the study. Weight and height measurements and a brief physical examination were performed by a physician or a nurse practitioner. Blood for estradiol levels was drawn at random in amenorrheic subjects and in the early follicular phase (d 3–7) in menstruating subjects. Amenorrheic subjects also had FSH, LH, prolactin (PRL), dehydroepiandrosterone sulfate (DHEAS), and testosterone levels measured. All stress fractures were diagnosed by an orthopedic surgeon on the basis of symptoms and were confirmed by an x-ray or bone scan.
Nutritional evaluation
Food intake was determined using a 2-d dietary history as well as the Walter Willet semiquantitative food frequency questionnaire (17). The food frequency measure is designed to target frequently consumed food items that contain relatively high values for those nutrient groups known to either contain or influence calcium intake or its absorption, i.e. calcium, vitamin D, fiber, and caffeine (18). The 24-h recall food intake diaries were based on a modified version of that described by Frank et al. (19). The food intake records and food frequency records were coded separately, using the established coding protocol and the Nutri-calc software package (CADME Corp., Rochester, NY).
Activity level
Activity level was determined on the basis of the number of calories expended per day according to the method of Bouchard et al. (20). Subjects were asked to fill out a 3-d questionnaire over 2 weekdays and 1 weekend day. Each day of the record was divided into 96 periods of 15 min each. The participant selected a code number from nine categories of listed activities that best represented her level of activity during each period. Activities were grouped according to similar energy costs in kilocalories per kilogram per minute, and a median value for each category was used to compute energy expenditure. Mean kilocalorie expenditure for 3 d is highly reliable, with a coefficient of variation of 0.96 (P < 0.01).
Psychometrics and eating disorders
Eating problems were assessed through questionnaires and subject interviews, including Garner and Garfinkel’s Eating Attitudes Test (EAT)-26, an abbreviated version of their EAT-40 (5). During a semistructured interview, each subject was also asked to indicate how typical for her was each of the thoughts or behaviors required by Diagnostic and Statistical Manual III for a diagnosis of anorexia, bulimia, and atypical eating disorder. Results were also rescored according to Diagnostic and Statistical Manual III-R criteria (5).
Measurements of bone density
Bone density was measured at the spine, wrist, and foot. Studies have shown that spinal BMD is particularly affected in amenorrheic athletes (21) because high-impact activity appears to have a beneficial effect on BMD primarily at the hip (22). Bone mass analyses of the spine and radius were carried out using a DP3 Dual Photon Spine/Femur Scanner and a SP2 Single Photon Scanner (Lunar Corp., Madison, WI) (5). The bone density measurement of the foot, specifically the first metatarsal, was done with the Dual Photon Absorptiometer with a method described previously (5). Dual energy x-ray absorptiometers became available in the middle of the study but were not used for the sake of longitudinal consistency. The decision to continue with the same equipment was advised by our osteoporosis consultant.
The precision of BMD testing was verified in 16 healthy, normal women. BMD was measured 10 times with repositioning. Coefficients of variation (CV) were 1.9%, 1.6%, and 4.0% for spine, wrist, and metatarsal, respectively. To correct for source decay and source change, calibrations were made daily using a standard that consisted of a block of tissue-equivalent material with three bone-simulating chambers (small, medium, large) of known bone mineral content. Mean measurements, SD, and CV were made during the period of study. Percentage CV for photon standard measurements on DP3 was less than 1%, and SP2 was less than 2% over the 2 yr for the three chambers. These values remained constant before and after a source change with a percentage CV of less than 2% on the DP3 and less than 2% on the SP2. The same machine was used during the entire study. Subjects were seen at baseline and yearly for 2 yr, for a total of three visits. All measurements, including bone density, were done at each visit.
Statistical analysis
We used ANOVA to evaluate differences within and between four groups to look at the following variables: amenorrhea dancer, normal cycles dancer, amenorrhea control (nondancer), and normal cycles control (nondancer). We examined these groups across the years of study while controlling for possible differences between individual subjects. Data were analyzed using one-way ANOVA at each point in time for the four groups for all valid data points. We also compared controls (both dancers and nondancers) to amenorrheic groups (both dancer and nondancer) using independent t tests. For the longitudinal analyses, repeated measures ANOVA were used for the data for subjects with valid data at all three visits. In addition, percentage increase in BMD at 12 and 24 months was measured by subtracting BMD at baseline from the value at the later time point and dividing by the baseline value. Multiple comparisons were made using the Bonferroni method, available in SPSS software (SPSS, Inc., Chicago, IL), and correlations between variables and bone mass using Pearson linear correlation. Multiple regressions were performed to determine the change in BMD from baseline to 24 months controlling for the following variables: changes in weight, height, caloric intake, hormonal levels, body mass index (BMI), percentage body fat, and kilocalories expended per 24 h. ANOVA was used to compare differences in the spine, wrist, and foot bone mineral density (BMD); weight; caloric intake; and other variables in the four groups. We also examined bone density changes by comparing the slopes of the regression lines after examining the correlation between BMD and time.
Results
One hundred eleven subjects entered, and 62 completed the study (22 normal controls, 21 normal dancers, 9 amenorrheic controls, and 10 amenorrheic dancers); 50 subjects who finished the study had all three bone density measurements at the three time intervals; 7 amenorrheic subjects resumed menses during the study (3 amenorrheic dancers and 4 amenorrheic controls). The age of menarche for amenorrheic subjects was significantly older than controls (14.7 ± 2.3 vs. 13.7 ± 1.9 yr; P < 0.05) (Table 1
). Amenorrheic dancers were younger than normal controls, and normal controls were heavier and nearer their normal weight (Table 1). Behavior and nutrition patterns are shown in Table 2. Significantly higher EAT scores for dancers and the amenorrheic nondancers indicate that dieting behavior was more frequent among these groups, although this was significant only for the latter. These groups also showed a higher incidence of anorexia in the past (significant for control amenorrheics and normal dancers) and higher fiber intake (significant in both exercising and nonexercising amenorrheic groups). All amenorrheic subjects had normal PRL, testosterone, and DHEAS levels, normal to low LH and FSH levels, and low estradiol levels so that they fit the hypothalamic amenorrhea profile (Table 3).
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reports data analyzed using ANOVA at each point in time for all valid data points. BMD was significantly lower at baseline in both amenorrheic groups, and BMD remained lower in the spine during the 2 yr of observation (Fig. 1), independent of weight or age and despite the return or onset of menses in seven subjects. The normal exercising group (dancers) showed significantly higher bone density in the weight-dependent sites (i.e. spine and foot) at baseline when compared with amenorrheics of both groups (Fig. 1); this effect persisted over the 2 yr. These findings were weight-dependent but were not affected by controlling for age. The wrist BMD was significantly higher only at baseline in the normal group, and a significant rise in wrist BMD was noted in the amenorrheic dancers over the 2 yr. None of the groups studied showed a significant decline in BMD.
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), and they were significantly younger and had an older age of menarche than the other groups. The increase in spine BMD was related to amenorrhea, delayed menarche, and exercise (kilocalories expended per 24 h; P < 0.005). When controlling for age and weight, a significant increase in BMD occurred only in the amenorrheic dancers (the exercising group) with delayed menarche.
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Another interesting issue is the discrepancy between energy input and output in all of the groups. As several investigators have reported, groups who exercise (particularly amenorrheic groups) may develop energy efficiency (23, 24, 25). However, an underestimation of energy intake may also occur, a problem that has been noted in nutritional surveys.
Despite the increase in dancers’ spine BMD, the BMD of all amenorrheics (dancer and nondancer) remained below normal. The seven subjects who resumed menses had a significant increase in BMD (17% in wrist and spine; P < 0.001) but did not achieve normalization (Table 4). Multiple regression correlating the change in BMD from baseline to 24 months and controlling for the changes in weight, height, caloric intake, hormonal levels, BMI, percentage body fat, and kilocalories expended per 24 showed only an effect of kilocalories expended at 24 months on spine BMD. Because the significant changes occurred only in amenorrheic dancers, this was not surprising.
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. Overall, estradiol levels were higher in the normal groups (dancer and nondancer) vs. amenorrheic groups (dancer and nondancer) throughout the study, and testosterone and PRL levels were higher in normal controls at baseline (testosterone significant vs. amenorrheic dancers only). The amenorrheic dancers were the only group to show a significant increase in estradiol and testosterone levels over time. A total of 108 stress fractures were diagnosed, with 25 occurring at baseline and 83 during the 2 yr of the study. Delayed menarche was the only variable that predicted stress fractures. An increased frequency of stress fractures (P < 0.05) was associated with lower BMDs in the spine. Subjects with stress fractures showed an older age of menarche (15.2 ± 2.3 vs. 13.5 ± 1.5 yr; P = 0.002), more dieting behavior (EAT scores, 16.3 ± 2.00 vs. 11.5 ± 1.43; P < 0.05), and higher amounts of fiber in the diets (30.7 ± 3.3 vs. 17.5 ± 2.01 g; P < 0.001). There was no difference in calcium or fat intake in these groups.
The dropout rate in this study was 45%. Our analyses showed no difference in age, BMD, weight, change in BMD, level of exercise, fractures, or menstrual status for the dropouts vs. compliant subjects. A review of the subjects found that 72% dropped out because of geographic relocation, 8% due to lack of time, 15% for undetermined reasons, and 5% due to lack of interest.
To further eliminate the effects of noncompliant subjects on the longitudinal observations, we examined the subjects (n = 50) who had all BMD measurements at 0, 12, and 24 months. These data are presented in Table 5. When examining this compliant group, the amenorrheic group (both dancer and nondancer) showed slightly lower spine BMD at baseline when compared with normals (both dancer and nondancer). Findings were similar to previous analyses with the following exceptions: an increase in spine BMD was significant only from 12–24 months for amenorrheic dancers and not in the first year. This group also showed a significant decline in calcium intake in the first year (833.3 ± 337.9 vs. 679.5 ± 173.9 mg/24 h; P < 0.01) and significant increases in PRL (5.6 ± 1.2 vs. 6.8 ± 1.3 ng/ml; P < 0.01) as well as estradiol (111.3 ± 120.5 vs. 184.8 ± 125.6 pmol/liter; P < 0.05) during this year. Again, multiple regression demonstrated an effect of kilocalories expended per 24 h at 24 months, which would be expected because the dancers were the only group to show an increase in spine BMD. There was no effect of caloric or calcium intake, hormonal levels, change in weight, height, BMI, or percentage body fat.
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This is the first longitudinal study of this magnitude using a homogenous group of exercising subjects from one athletic discipline. Our results show that women with hypothalamic amenorrhea also have lower bone density that remains below normal menstruating counterparts over 2 yr. These results were independent of weight and age and occurred despite the continuous exercise in one group. The findings are tempered by the large dropout rate but are consistent whether compliant or noncompliant subjects were included.
Only one group, the amenorrheic dancers, showed a significant change in spinal BMD, and within this group it was only those with delayed menarche who showed an increase in spinal BMD. This subgroup with delayed menarche also showed the most stress fractures and the most distorted pattern of eating behavior. Despite the increase, their BMD remained below that of their normal counterparts.
All amenorrheic subjects were deficient in BMD at baseline, which suggests that the bone mass deficiency was present before the start of this study. The loss of bone or lack of accretion, therefore, may have occurred at a crucial time not fully addressed by this study. An increase in spinal BMD was significant when controlled for age and weight only in the amenorrheic dancers. Thus, exercise-associated amenorrhea (seen in our dancers) is associated with delayed sexual development and possibly delayed bone accretion; this increase in BMD may represent a catch-up phase. This is also suggested by the significant increase in estradiol in the compliant subgroup.
In the study, seven women (three dancers and four controls) resumed menses and showed a significant increase in their spine and wrist BMD (both 17%; Fig. 3) without normalization. Past studies on athletes (6%; Refs. 1 and 26) and subjects with anorexia nervosa (0–19%; Refs. 10 , 27 , and 28) have also associated returned menstrual cyclicity with increased bone mass. Significantly, none of the women who resumed menses achieved normalization of BMD. The lack of normalization suggests that reduced bone accretion might result in a permanent failure to achieve peak bone mass, which underscores the importance of intervention, either preventative or therapeutic.
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Insufficient caloric intake for the level of activity has been proposed as a factor in the genesis of exercise-associated reproductive dysfunction (24, 31, 32, 33), and the osteopenia may also be an adaptive response to chronic low energy intake. Metabolic factors in response to nutritional insults might mediate reproductive and bone growth adaptations. Our observations are consistent with the findings of others (34) and suggest that poor nutrition or an energy deficit with an adaptation to large caloric needs is fundamentally linked with the prolonged amenorrheic state and the osteopenia (34, 35, 36).
Other recent data also suggest that nutritionally regulated processes may underlie the osteopenia. Hypercortisolemia (37, 38, 39), IGF-I deficiency (34), or other metabolic adaptations seen in these athletes may contribute to the osteopenia. Lowered bone formation markers, T3 and IGF-I, are seen in amenorrheic runners but not in controls (34, 35). Because our original studies on ballet dancers showed normal vitamin D and parathyroid levels, we suggest that these are not the causal factors in the osteopenia (7). Two nutritionally dependent hormones, IGF-I and T3, predicted change in trabecular bone mass with estrogen-progestin therapy in past studies on anorexia nervosa (28). Low leptin levels have been associated with amenorrhea (31, 40) and with disordered eating (31), a behavior that our amenorrheic group demonstrated. Recently, leptin receptors have been found on bone (41), and it has been suggested that leptin may be involved in the skeletal regulation and may possibly be centrally controlled, most likely in the hypothalamus (42, 43, 44). This provides a possible mechanism for an interaction between the nutritional and metabolic bone axes and suggests that leptin may function as a physiological regulator of bone mass. Therefore, a mechanism other than hypoestrogenism may also account for the low bone density and the alarming stress fracture rate seen in women whose amenorrhea is associated with caloric deficiency, nutritional insults, and exercise (29, 31, 45, 46).
Another surprising finding is the lack of further bone loss in the amenorrheic subjects. This may be due to factors such as the low bone turnover reported in this state (35, 36, 47) which in turn may be induced by a central mechanism protecting against further loss and perhaps involving a metabolic signal such as leptin.
Recent studies suggest that accretion of peak bone mass during adolescence and young adulthood is essential because at least 40% of bone mass is formed at this time, and it may be difficult for a woman to accrue additional bone mass later in life (13, 14, 15, 48). Maximizing total bone mass accretion for young women at risk cannot be implemented without further understanding of the relationship between hypothalamic amenorrhea and bone density. More studies on this unique form of bone loss are needed to improve the efficacy of prevention and treatment of osteopenia.
Footnotes
This research was supported by NIH/National Institute of Child Health and Human Development Grants R01-HD22171-01 through R01-HD22171-07.
Abbreviations: BMD, Bone mineral density; BMI, body mass index; CV, coefficient(s) of variation; DHEAS, dehydroepiandrosterone sulfate; EAT, Eating Attitudes Test; PRL, prolactin.
Received October 6, 2000.
Accepted March 21, 2002.
References
This article has been cited by other articles:
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  M. P. WARREN and A. T. CHUA Exercise-Induced Amenorrhea and Bone Health in the Adolescent Athlete Ann. N.Y. Acad. Sci., June 1, 2008; 1135(1): 244 - 252. [Abstract] [Full Text] [PDF]
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 S. L. Lo, A. C. Zoga, I. Elias, J. R. Peterson, W. Chao, J. Green, and W. B. Morrison Stress Fracture of the Distal Phalanx of the Great Toe in a Professional Ballet Dancer: A Case Report Am. J. Sports Med., September 1, 2007; 35(9): 1564 - 1566. [Full Text] [PDF]
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 M. J. De Souza and N. I. Williams Physiological aspects and clinical sequelae of energy deficiency and hypoestrogenism in exercising women Hum. Reprod. Update, September 1, 2004; 10(5): 433 - 448. [Abstract] [Full Text] [PDF]
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 J. Z. Kasa-Vubu, M. Sowers, W. Ye, N. E. Carlson, and T. Meckmongkol Differences in Endocrine Function With Varying Fitness Capacity in Postpubertal Females Across the Weight Spectrum Arch Pediatr Adolesc Med, April 1, 2004; 158(4): 333 - 340. [Abstract] [Full Text] [PDF]
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 M. L. Gourlay and S. A. Brown Clinical Considerations in Premenopausal Osteoporosis Arch Intern Med, March 22, 2004; 164(6): 603 - 614. [Abstract] [Full Text] [PDF]
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, normal dancers (n = 32, 22, and 21 in yr 1, 2, and 3, respectively); 
, amenorrheic controls (n = 22, 14, and 9 in yr 1, 2, and 3, respectively); 
, normal controls (n = 35, 26, and 22 in yr 1, 2, and 3, respectively).
