WOMEN WITH very low body weight and body fat are

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1 X/99/$03.00/0 Vol. 84, No. 3 The Journal of Clinical Endocrinology & Metabolism Printed in U.S.A. Copyright 1999 by The Endocrine Society Functional Hypothalamic Amenorrhea: Hypoleptinemia and Disordered Eating M. P. WARREN, F. VOUSSOUGHIAN, E. B. GEER, E. P. HYLE, C. L. ADBERG, AND R. H. RAMOS Departments of Obstetrics and Gynecology and Medicine, Columbia College of Physicians and Surgeons, New York, New York ABSTRACT Because the exact etiology of functional, or idiopathic, hypothalamic amenorrhea (FHA) is still unknown, FHA remains a diagnosis of exclusion. The disorder may be stress induced. However, mounting evidence points to a metabolic/nutritional insult that may be the primary causal factor. We explored the thyroid, hormonal, dietary, behavior, and leptin changes that occur in FHA, as they provide a clue to the etiology of this disorder. Fourteen cycling control and amenorrheic nonathletic subjects were matched for age, weight, and height. The amenorrheic subjects denied eating disorders; only after further, detailed questioning did we uncover a higher incidence of anorexia and bulimia in this group. The amenorrheic subjects demonstrated scores of abnormal eating twice those found in normal subjects (P 0.05), particularly bulimic type behavior (P 0.01). They also expended more calories in aerobic activity per day and had higher fiber intakes (P 0.05); lower body fat percentage (P 0.05); and reduced levels of free T 4 (P 0.05), free T 3 (P 0.05), and total T 4 (P 0.05), without a WOMEN WITH very low body weight and body fat are known to be at high risk for the development of hypothalamic amenorrhea. Women who are of normal weight may also experience hypothalamic amenorrhea, but the etiology is often unclear. The amenorrhea is thought to be stress induced, if exercise or dieting with weight loss is not identified. This is generally referred to as functional idiopathic hypothalamic amenorrhea (FHA). The etiology of FHA is not clearly understood, and it remains a diagnosis of exclusion. Women with FHA have been found to have neuroendocrine dysfunction, suggesting that central regulation of multiple pituitary hormones is disturbed (1). A common denominator in FHA is suppression of GnRH secretion (2, 3) although recent data from normal women and primates suggest that energy availability or overall energy deficit may be the common denominator in the suppression of GnRH secretion (4, 5). Research has uncovered a constellation of hormonal abnormalities that occur in FHA (1) and are compatible with an energy deficit despite the lack of a history of dietary insult (6, 7). Osteopenia has also been reported (8). Received August 14, Revision received December 4, Accepted December 8, Address all correspondence and requests for reprints to: Michelle P. Warren, M.D., Department of Obstetrics and Gynecology, PH 16 20, Columbia University, 622 West 168th Street, New York, New York significant change in rt 3 or TSH. Cortisol averaged higher in the amenorrheics, but not significantly, whereas leptin values were significantly lower (P 0.05). Bone mineral density was significantly lower in the wrist (P 0.05), with a trend to lower BMD in the spine (P 0.08). Scores of emotional distress and depression did not differ between groups. The alterations in eating patterns, leptin levels, and thyroid function present in subjects with FHA suggest altered nutritional status and the suppression of the hypothalamic-pituitary-thyroid axis or the alteration of feedback set-points in women with FHA. Both lower leptin and thyroid levels parallel changes seen with caloric restriction. Nutritional issues, particularly dysfunctional eating patterns and changes in thyroid metabolism, and/or leptin effects may also have a role in the metabolic signals suppressing GnRH secretion and the pathogenesis of osteopenia despite normal body weight. These findings suggest that the mechanism of amenorrhea and low leptin in these women results mainly from a metabolic/nutritional insult. (J Clin Endocrinol Metab 84: , 1999) The discovery of the hormone leptin has provided a possible mechanism by which metabolic signals may be communicated to the reproductive axis. Leptin is secreted by the adipocyte and is influenced by multiple metabolic pathways affecting appetite, energy requirements, and eating behavior (9). This may provide a link for other metabolic and hormonal pathways that are known to affect reproduction to communicate nutritional status and the degree of fat stores to the reproductive axis. Recently, low leptin levels have been reported in hypothalamic amenorrhea, although the etiology remains unclear (10). We examined a group of patients with idiopathic hypothalamic amenorrhea in depth to determine whether the metabolic abnormalities in their disorder were suggestive of abnormal eating patterns and therefore reflective of an energy deficit or dysregulation, as suggested by our previous reports (7). Materials and Methods We studied 14 subjects with amenorrhea who were matched with 14 normally menstruating controls on the basis of age, height, and weight. All were volunteers and were solicited from advertisements in college publications and by physician referral. Volunteers were told that the study examined complications of amenorrhea; the subjects were selected for amenorrhea of at least 5-month duration. All subjects denied recent weight loss, eating disorders, or athletic activity and had participated in past studies on bone density (11, 12) and hypothalamic amenorrhea (7). Subjects were within 10% of ideal body weight. The procedures followed were approved by the institutional review board of St. Luke s-roosevelt Hospital Center. The educational levels of the subjects were determined 873

2 874 WARREN ET AL. JCE&M 1999 Vol 84 No 3 by the seven-point scale developed by Hollingshead and Redlich, as described previously (13). All subjects were white and from middle to upper class families. Each subject was interviewed by a nurse practitioner, who obtained data on present and past illnesses, weight changes, fractures, and complete menstrual and medication histories. All patients with a chronic illness of any kind were eliminated from the study. The nurse practitioner also measured weight and height. Ideal weights were obtained from tables of average weight-height relations for young adults, as described previously (13), and the subjects were compared and categorized as being above or below normal weight standards. We used Sargent s tables for subjects aged 17 yr or older; these tables include six categories from underweight to obese (13). Percent body fat was determined by measuring skinfolds at four sites (biceps, triceps, subscapular, and supra-iliac) and converting the values to body fat according to the method described by Durnin and Womersley (14). No subjects had taken hormone therapy for at least 1 yr before examination, and none was receiving hormone therapy at the time the data were collected. All patients with secondary amenorrhea fit the criteria for hypothalamic amenorrhea: normal PRL, testosterone, and dehydroepiandrosterone sulfate (DHEAS) levels and low to normal FSH and LH levels. Two patients were eliminated from the study because their medical and hormonal evaluations revealed a chronic anovulatory syndrome consistent with polycystic ovary disease with normal estrogen levels. Hormonal studies were performed on a venous blood sample obtained in the midafternoon. Assays for LH, FSH, PRL, testosterone, and DHEAS were performed as previously described (15). Levels of T 3 and T 4 (both total and free), rt 3, and TSH were measured. Free T 3 was measured by 125 I RIA (Diagnostic Products Corp., Los Angeles, CA). The interassay coefficient of variation was %, the intraassay coefficient of variation was 4 11%, and the lower limit of sensitivity was 0.2 pg/ml. Free T 4 was measured by 125 I RIA (Diagnostic Products Corp.). The interassay coefficient of variation was 6 10%, the intraassay coefficient of variation was 3 10%, and the lower limit of sensitivity was 0.01 ng/dl. Total T 4 was measured by 125 I RIA (Diagnostic Products Corp.). The interassay coefficient of variation was %, the intraassay coefficient of variation was %, and the lower limit of sensitivity was 0.25 g/dl. Total T 3 was measured by 125 I RIA (Diagnostic Products Corp.). The interassay coefficient of variation was %, the intraassay coefficient of variation was %, and the lower limit of sensitivity was 7 g/dl. rt 3 was measured by RIA (Ciba-Corning/Serono Diagnostics, Medfield, MA). The interassay coefficient of variation was %, the intraassay coefficient of variation was %, and the lower limit of sensitivity was ng/ml. Serum TSH was determined by an immunoradiometric assay (Diagnostic Products Corp.). The interassay coefficient of variation was %, the intraassay coefficient of variation was %, and the lower limit of sensitivity was 0.03 IU/mL. The assay was standardized in terms of the WHO Second International Reference Preparation of TSH for immunoassay (80/558). Leptin was measured by an immunoradiometric assay (Linco Research, Inc., St. Charles, MO). The interassay coefficient of variation was %, the intraassay coefficient of variation was %, and the lower limit of sensitivity was 0.5 ng/ml. Midafternoon cortisol was measured in serum by RIA (Diagnostic Products Corp.). In this assay, the intra- and interassay coefficients of variation were % and %, respectively. The detection limit was less than 0.2 g/dl. Estradiol levels were determined at random in amenorrheic subjects and during the early follicular phase in menstruating subjects (days 3 7). Food intake was determined by use of a 2-day dietary history (two 24-h recall diaries) as well as by the Walter Willett Semiquantitative Food Frequency Questionnaire, previously described (13). 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 (16). During the nutrition interview, subjects were asked about the average frequency per week with which they ate each of the specified food items in the past year. There are nine responses, ranging from never to six or more times per day. The items are listed in quantities that represent standardized portion sizes and natural units. The 24-h recall food intake diary is an improved version of that described by Frank et al. (17). The subject filled out the Saturday diary before her scheduled visit with the nutritionist. During the visit, the subject reviewed the Saturday diary and generated a recall for the weekday diary. These established good intake information through the listing of food items consumed during six specified eating times. For each item, the type and standardized amount were recorded, and inquiry was made as to where and with whom the food was eaten. Average caloric intake was derived from the sample 24-h intake diary. Reproducibility and validity were quantified in large prospective studies among women; intraclass correlation coefficients were calculated with 1-week dietary recall measures. The overall mean of correlation coefficients comparing intakes for 18 nutrients measured on both Walter Willett Semiquantitative Food Frequency Questionnaire and the dietary recall measure was The food frequency questionnaire measured the subject s food intake over the past year. Seasonal consumption of some foods (summer fruits and hot breakfast cereals, for example) was averaged over the entire year. Activity level was determined based on the number of calories expended per day according to the method of Bouchard et al., previously described (13). The existence and severity of eating problems was assessed through both questionnaires and subject interviews. Subjects were asked to complete Garner and Garfinkel s EAT-26, an abbreviated version of their EAT-40 Eating Attitudes Test. Questions on this scale relate to three factors: dieting, bulimia and food preoccupation, and oral control, as previously described (13). During a semistructured interview with the study s research assistant, each subject was also asked to indicate whether she experienced the thoughts or behaviors required by the Diagnostic and Statistical Manual of Mental Disorders (DSM-III) for a diagnosis of anorexia, bulimia, and atypical eating disorder. The results of these interviews were also rescored according to DSM-III-R criteria for these disorders (13). Bone mineral densities of the spine, wrist and metatarsal were determined using a DP3/SP2 Lunar dual photon spine/femur scanner (Lunar Corp., Madison, WI) as previously described (13). The Hopkins Symptom Checklist (18) was used to measure the level of emotional distress and depressive symptoms currently experienced. This 64-item measure is composed of 5 reliable factors (18): somatization, obsessive-compulsive thoughts and behaviors, interpersonal sensitivity, depression, and anxiety (4-point Likert scale). Mean scores range from 1 4, with higher scores indicating more severe emotional distress. The Mann-Whitney test and t tests for paired samples were used for statistical analysis. To control for variables showing significant differences in the paired samples, variables were further assessed by analysis of covariance. Results Cycling controls and amenorrheics were matched for age, weight, and height (Table 1). The amenorrheics denied eating disorders, but our evaluation showed that they had a higher incidence of anorexia and bulimia in this small sample, based not on a history but on our in-depth testing for eating disorders. The amenorrheics also scored more than twice as high on the scale of disordered eating behavior (EAT; vs ; P 0.05) and 6 times as high on the subscale of bulimic behavior (P 0.01). They also had higher fiber intakes (23.84 vs g/day; P 0.05). Although their total expended calories per day were similar to the controls, they expended more calories in aerobic activity per day ( vs TABLE 1. Characteristics of the subjects Characteristics of the subject Amenorrheic (n 14) Matched controls (n 14) Age (yr) NS Ht (in.) NS Wt (lb) NS % of ideal wt NS Body fat (%) P

3 DISORDERED EATING AND LEPTIN IN AMENORRHEA 875 Cal/day; P 0.05; Table 2), and they tended to have a lower body fat percentage (21.55% vs %; P 0.053; Table 1). Leptin values were significantly lower in amenorrheic groups even when controlling for body fat (P 0.05). The mean scores on the Hopkins Symptom Checklist did not differ, nor did fat intake. In amenorrheic subjects, the hormonal studies revealed significantly reduced levels of free T 4 (1.19 vs ng/dl; P 0.05), free T 3 (2.46 vs pg/ml; P 0.05), total T 4 (5.45 vs g/dl; P 0.05), and leptin (5.05 vs ng/ml; P 0.05; Table 3), without a significant change in rt 3 or TSH. Consistent with past experiments, cortisol averaged higher in the amenorrheics, but not significantly. Bone mineral density was significantly lower in the wrists of amenorrheics (P 0.05). There was a trend for lower BMD in the spine (P 0.08), but values in the metatarsal did not differ (Table 3). Discussion These results indicate that alterations in leptin levels and thyroid function are present in normal weight subjects with FHA and are associated with restrictive eating patterns without obvious caloric deficits. The reproductive abnormalities in this syndrome have been the source of much research. This is the first report to link the hypoleptinemia to disordered eating and lower bone mass. Decreased leptin levels may reflect the lower body fat, restrictive eating behavior, or possibly a combination of both, but are significantly lower in amenorrheics independent of body fat. This suggests that restrictive eating behavior is more important. Although leptin is a metabolic hormone correlated with fat mass and metabolic rate (19 22) and affects appetite and eating behavior (19), it is thought to be a mediator of puberty (23), and recent research is emerging in animals showing that falling leptin seen with starvation may suppress reproduction and thyroid function while activating the hypothalamic-pituitary-adrenal axis (24). Leptin has been implicated in the neuroendocrine response to fasting and has been found to blunt the starvation-induced responses of the gonadal, thyroid, and adrenal axis when administered to male mice (25). Other studies in rats show that the hypothalamic-pituitarythyroid axis may serve as a critical locus to mediate the central actions of leptin by regulating pro-tsh gene expression (26, 27), and reports of leptin mutations in the human indicate lack of pubertal development and suppression of TSH (28). Thus, the role of leptin may be not only to control body mass, but to initiate puberty and integrate this axis with the neuroendocrine responses to starvation. This pattern of neuroendocrine changes seen in rats with energy deprivation was prevented by leptin repletion (25). The findings in our subjects suggest that chronic disordered eating may cause similar metabolic changes without an energy deficit. The diurnal rhythm of leptin is absent in amenorrheic athletes (6) and low in women with anorexia, increasing with weight gain (29). Thus, low levels of leptin are consistent with other types of hypothalamic amenorrhea that are weight or exercise induced, syndromes associated with negative energy balance. Thus, low leptin levels may be a sensitive indication of overall nutritional status and may also be associated with the metabolic changes associated with restrictive eating in women of normal weight. Low body fat may contribute to the problem in normal weight women. The potential mechanism by which leptin links the metabolic and reproductive axes remains unknown. One possible hypothesis is that a caloric deficit, even with no loss of fat mass, will trigger a cascade that will affect leptin secretion from the fat adipocytes. Leptin appears to be regulated by energy balance as well as fat stores (30), and energy deprivation has a disproportionately acute effect on lowering leptin levels even short term (9, 25, 31 33). Thus, the disordered eating, particularly the fasting-gorge behavior of bulimia, may disproportionally lower leptin levels. Leptin also regulates the basal metabolic rate, another factor that is altered in exercise-induced amenorrhea (34). Changes in the thyroid axis may also be key players in regulation of the basal metabolic rate (25, 26, 33). The sick euthyroid, or low T 3, syndrome is seen in cases of caloric deprivation and is associated with a rise in the level of rt 3 (35), which is also present in cases of anorexia nervosa (36). Although our results do not show a rise in absolute rt 3, it is possible that the conversion rate from T 4 to rt 3 is decreased in FHA, as a similar pattern with normal to decreased rt 3 levels, has been reported in exercise-induced amenorrhea and with nutritional restriction in normal individuals (34, 37, 38). Bosello et al. also found an increase in rt 3 in a dieting TABLE 2. Eating disorders and nutritional patterns Eating disorders and nutritional patterns Amenorrheic (n 14) Matched controls (n 14) P EAT 26 scores Diet Bulimia Oral control NS Anorexics 46% 0% 0.05 Bulimics 39% 9% NS Hopkin s syndrome checklist NS Cal expended/day NS Cal intake/day NS Fiber intake (g/day) Fat intake (g/day) NS Cal expended at activities/24 h High intensity Moderate intensity NS Sleep NS

4 876 WARREN ET AL. JCE&M 1999 Vol 84 No 3 TABLE 3. Endocrine studies Endocrine studies Amenorrheic (n 14) Matched controls (n 14) P FSH (TIU/mL) NS LH (TIU/mL) NS Estradiol (pmol/l) NS Testosterone (nmol/l) NS DHEA-SO 4 ( mol/l) NS PRL (ng/ml) NS Cortisol ( g/dl) NS Free T 4 (ng/dl) Free T 3 (pg/ml) Total T 4 ( g/dl) Total T 3 (ng/dl) NS rt 3 (ng/ml) NS TSH (TIU/mL) NS Leptin (ng/ml) Bone density (g/cm 2 ) Wrist Spine Metatarsal NS Values are the mean SD. group, but no change in a diet plus exercise group (39). The patients with FHA in this study showed a pattern similar to subjects in Bosello s study, with both abnormal eating behavior and high caloric expenditure. Reduced thyroid function is associated with a depressed resting metabolic rate, which results in lower levels of leptin. A recent study by Valcavi et al. (40) underscores this correlation between lower levels of leptin and hypothyroidism, and recent studies suggest that the reduced concentration of leptin may play a role in producing the decreased energy expenditure of patients with low thyroid hormone levels. The concentration of thyroid hormones may control the active function of leptin by affecting either leptin-binding proteins and/or leptin receptors (40). Thus, the role of the thyroid and leptin changes may be to preserve energy when nutritional intake is compromised. Our subjects with idiopathic FHA showed restrictive eating patterns, which could explain these changes and may be associated with changes in metabolic rate. The extreme sensitivity of the GnRH pulse generator is suggested by numerous experiments. Although stress can cause a patient to alter her food intake in either amount or type, research by Schreilhofer et al. in the primate confirm that it is mainly the energy deficit induced by short term fasting, and not stress, that causes the irregular LH secretion associated with disruption of reproductive function (5, 41). Although stress has been shown to disrupt cyclicity in monkeys, nutritional metabolic insults are more significant (42). The disordered eating in the group with FHA may be initially stress induced, but the effects on GnRH pulses are more likely due to the former. In fact, replacing low leptin restores GnRH pulses in fasted monkeys (43). In a recently published study, Berga et al. attributed FHA to stress, showing that only women with FHA had increased levels of cortisol; cycling women and women with amenorrhea of other origins (excluding eating disorders and exercise) showed no changes in cortisol (44). The characteristics of our subjects were very similar to those in Berga s study, especially in that none of the women was a confessed anorexic, bulimic, or heavy exerciser, and we too observed these increases in cortisol concentration in our subjects with FHA. Although our results were not significantly increased, our cortisol levels represent only one time sampling. The EAT scores in this study as well as the nutritional and exercise patterns and the low leptin levels suggest an association between hidden nutritional abnormalities and amenorrhea. Information was not available for Berga s subjects on leptin concentrations, EAT scores, or other nutritional data (45), and subtle nutritional differences between the two groups would not have been detected. This could explain the different conclusions drawn by the two similar studies. Low leptin levels were also detected in a new study (10), but the mechanism remained unknown. Increased cortisol levels may affect thyroid function; however, past studies have shown that glucocorticoids suppress TSH levels as well as T 3 (24, 46). Our results show normal TSH concentrations in subjects with FHA. Our results suggest that FHA may be more a function of altered metabolic state due to nutritional alteration or energy expenditure than purely stress induced. Previous studies suggest that nutritional abnormalities in FHA are subtle and need to be searched for in depth (6, 7). These results in a carefully matched sample also suggest that small or unreported nutritional insufficiencies may cause many cases of so-called idiopathic hypothalamic amenorrhea. This is important, as osteopenia is a serious complication, and intervention may need to address nutritional issues as well as hormonal replacement. Alternately, stress may induce these dietary patterns, but studies suggest that GnRH suppression rarely occurs with stress alone but requires a setting of nutritional restriction (42). The active form of thyroid hormone is a potent stimulator of bone turnover (47), and both nutritional issues and changes in thyroid metabolism may have a role in the pathogenesis of osteopenia and may be important metabolic links to the GnRH pulse generator, possibly via a leptin pathway. Interestingly, leptin has recently been found to have receptors in bone, may be a physiological regulator of bone mass (48, 49), and thus may be the link between amenorrhea and osteopenia.

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