OBESITY is a major health problem in the United States

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1 X/99/$03.00/0 Vol. 84, No. 3 The Journal of Clinical Endocrinology & Metabolism rinted in U.S.A. Copyright 1999 by The Endocrine Society ubertal Girls Expend Less Energy at Rest and During hysical Activity than Girls* WILLIAM W. WONG, NANCY F. BUTTE, KENNETH J. ELLIS, ALBERT C. HERGENROEDER, REBECCA B. HILL, JANICE E. STUFF, AND E. O BRIAN SMITH United States Department of Agriculture/Agricultural Research Service Children s Nutrition Research Center (W.W.W., N.F.B., K.J.E., J.E.S., E.O.S.) and Texas Children s Hospital (A.C.H., R.B.H.), Department of ediatrics, Baylor College of Medicine, Houston, Texas ABSTRACT Between 1963 and 1991, the most dramatic increases in the prevalence of overweight in the United States have been reported in girls. Lower basal energy expenditure and lack of physical activity are believed to be risk factors for excessive weight gain. We hypothesized that energy expenditure at rest and during physical activity are lower in pubertal girls than in girls. Basal metabolic rate and sleeping energy expenditure of 40 and 41 pubertal girls (matched for age, physical characteristics, body fat, and energy intake) were measured by whole-room calorimetry, energy expended for physical activity by the doubly labeled water method, sexual maturity OBESITY is a major health problem in the United States because of its association with increased risk of hypertension, coronary heart disease, diabetes, cancer, and many other medical ailments (1, 2). The National Health and Nutrition Examination Survey III (3, 4) indicated that approximately 33% of American adults and 22% of American children and adolescents are overweight. The prevalence of overweight in women is twice that in women, after adjustment for socioeconomic status (5, 6). More importantly, the most dramatic increases in the prevalence of overweight over the past 30 yr have been observed in girls, with age range-related increases of %, compared with an average increase of 35% in girls (4). Overweight children are at high risk of becoming overweight adults (7). The etiology of overweight is multifactorial. Lower energy expenditure is hypothesized to be one of the contributing factors favoring positive energy balance that leads to overweight. Three recent studies reported a lower resting metabolic rate in prepubertal children than in Received September 30, Revision received November 9, Accepted November 16, Address all correspondence and requests for reprints to: William W. Wong, h.d., United States Department of Agriculture/Agricultural Research Service Children s Nutrition Research Center, 1100 Bates Street, Houston, Texas wwong@bcm.tmc.edu. * This work was funded, in part, with federal funds from the US Department of Agriculture, Agricultural Research Service, under Cooperative Agreement No. 58 7MNI Disclaimer: The contents of this publication do not necessarily reflect the views or policies of the US Department of Agriculture, nor does mention of trade names, commercial products, or organization imply endorsement by the US Government. by physical examination, body composition by dual-energy x-ray absorptiometry, physical fitness by treadmill testing, and energy intake by 3-day food record. After adjusting for soft lean tissue mass, the basal energy expenditure ( vs kcal/day, 0.01) and energy expended for physical activity ( vs kcal/day, 0.01) were significantly lower in the girls than in the girls. The differences remained the same after controlling for differences in sexual maturity and/or physical fitness. The lower energy expenditure of the pubertal African- American girls suggests that they are at a higher risk of becoming overweight than their counterparts. (J Clin Endocrinol Metab 84: , 1999) children (8 10). The relationship between lower basal energy expenditure and excessive weight gain is controversial (11 13). Lack of physical activity, however, has been shown to be positively correlated with future weight gain in moderately obese women (13) and in children and adolescents (14 16). Because physical activity decreases through adolescence in girls (17) and fat accumulation accelerates during puberty in girls (18), lower energy expenditure during puberty may represent a greater risk for excessive weight gain in girls than in girls. We hypothesized that rates of energy expenditure at rest and during physical activity in pubertal girls are lower than those of girls. Subjects Materials and Methods Forty and 41 girls (Table 1) with greater than or equal to Tanner stage 3 of breast and pubic hair development (19) were studied. The subjects qualified for the study when both parents and grandparents were of the same ethnicity. All subjects were healthy and nondiabetic at the time of the study, based on medical history, vital signs, standard clinical blood chemistries, and physical examination. The protocol was approved by the Human Research Committee at Baylor College of Medicine. All subjects and their parents gave written informed consent. Body weight and height of each subject were measured to the nearest 0.1 kg and 1 mm, respectively, by one investigator. Body mass index (BMI) was calculated as: BMI kg/m 2 Weight Height 2 (1) Breast and pubic hair development was determined by a physician according to the Tanner stages of classification (19). 906

2 RACIAL DIFFERENCES IN ENERGY EXENDITURE 907 TABLE 1. Age, physical characteristics, sexual maturity, and energy intake of and subjects Age, yr a a 0.63 Weight, kg Height, cm BMI, kg/m Sexual maturity b 0.01 (0.07) c Tanner stage % (27.5%) 31.7% (19.5%) Tanner stage % (52.5%) 7.3% (36.6%) Tanner stage % (20.0%) 61.0% (43.9%) Energy intake, kcal/day rotein, % d Fat, % d Carbohydrate, % d a Mean SD. b ercentage of subjects within each Tanner stage of breast (pubic hair) development. c values by chi-square testing. d As percentage of total energy intake. Cardiorespiratory fitness The aerobic capacity of each girl (V O 2max ) was measured on a motorized treadmill until volitional exhaustion (20). Oxygen consumption rate (V O 2 ) and carbon dioxide production rate (V CO 2 ) were measured continuously by electronic metabolic analyzers, with the treadmill speed and elevation increased at 3-min intervals. V O 2max was achieved when V O 2 reached a plateau value and the respiratory exchange ratio exceeded 1.05 or when the heart rate was within 95% of the age-predicted maximum. Whole-room indirect calorimetry Four room respiration calorimeters were used in the study (21). Each subject entered the respiration chamber at 0800 h and ate breakfast at 0830 h, lunch at 1200 h, and dinner at 1730 h. All subjects received a standardized diet consisting of approximately 30% fat, 50% carbohydrate, and 20% protein. Their energy requirements during the calorimetric visit were estimated to be 1.5 times their predicted basal metabolic rates according to their ages, body weights, and heights, using the Schofield s equations (22). No food or drink other than water was allowed after 1900 h. All subjects remained awake until bedtime, at 2200 h. Sleeping energy expenditure was measured between 2200 h and 0650 h the next morning. At 0650 h the next morning, the subject was awakened, urinated, and returned to bed. At 0720 h, the subject was reawakened if she was asleep, and instructed to find a comfortable position in bed and to refrain from movement for the next 40 min. To eliminate the contribution of any physical activity to basal metabolic rate, only the V O 2 and V CO 2 data with activity counts 50 (Doppler microwave sensor D9/50, Microwave Sensors, Ann Arbor, MI) during the 40-min measurement period were converted to basal metabolic rate, as (23): basal metabolic rate (kcal/day) (3.941 V O 2 ) (1.106 V CO 2 ). Sleeping energy expenditure was measured to obtain a longer and more accurate estimate of basal energy expenditure, because basal metabolic rate can be affected by a child s state, i.e. anxiety, impatience, and ability to stay motionless for 40 min. The Weir equation (23) also was used to convert the V O 2 and V CO 2 measurements during sleep into sleeping energy expenditure. A 24-h urine sample was collected from each subject while she was in the calorimeter, to determine the urinary nitrogen excretion rate. Doubly labeled water method ( 2 H 2 18 O) The total energy expenditure of each subject under free-living conditions, which could not be measured by whole-room indirect calorimetry, was estimated using the 2 H 2 18 O method. After each subject exited the calorimeter, baseline plasma and saliva samples were collected. Each subject then received, by mouth, 100 mg 2 H 2 O and 125 mg 18 OasH 2 18 O (Isotec Inc., Miamisburg, OH) per kilogram body weight. The container holding the 2 H 2 18 O was rinsed three times with 50 ml of water, and the subject ingested all the rinses. The subject collected one daily saliva sample at home for the next 10 days. Immediately before her departure, a 3-h postdose plasma sample was collected. To minimize fluctuation in the basal 2 H and 18 O abundance in body water, all subjects resumed their usual diets at home and refrained from travel for the next 10 days. lasma and saliva samples were prepared for hydrogen and oxygen isotope ratio measurements by gas-isotope-ratio mass spectrometry (24, 25). The isotope dilution spaces for 2 H(N H ) and 18 O(N O ) were calculated as follows: N H or N O mol D A E E d where D is the dose of 2 H 2 OorH 2 18 O in grams; A is the amount of laboratory water, in grams, used in the dose dilution; is the amount of 2 H 2 OorH 2 18 O, in grams, added to the laboratory water in the dose dilution; E is the rise in 2 Hor 18 O abundance, per mil, in the laboratory water after the addition of the isotopic water; E d is the rise in 2 Hor 18 O abundance, per mil, in the 3-h postdose plasma sample. V CO 2 was calculated from the fractional turnover rates of 2 H(k H ) and 18 O(k O ) and the isotope dilution spaces as follows (26, 27): V CO 2 (mol/day) [(k O N O ) (k H N H )]. The V CO 2 was converted to freeliving energy expenditure as follows (23): free-living energy expenditure (kcal/day) (3.941 V O 2 ) (1.106 V CO 2 ) (2.17 U N ), where V O 2, in liters, is calculated from the 24-h respiratory quotient (RQ), measured by calorimetry, using the relationship V O 2 V CO 2 /RQ (28), and U N is the 24-h urinary nitrogen excretion in grams. Energy expended for physical activity was calculated by subtracting basal metabolic rate and diet-induced thermogenesis from free-living energy expenditure. Dietinduced thermogenesis was assumed to be 10% of free-living energy expenditure. Body composition measurement A Hologic QDR-2000 instrument (Hologic, Inc., Waltham, MA) was used to assess the body composition of each subject. The scanning software (version 5.56) is appropriate for the weight range of our study subjects, and the accuracy of the fat mass and bone mineral content measurements is independent of pubertal development (29). Fat-free mass, in kilograms, was the difference between body weight and fat mass. Because skeletal bone mass, a nonmetabolic component of fat-free mass, is higher in girls than in girls (30), soft lean tissue mass was obtained by subtracting bone mass from fat-free mass as follows (31): soft lean tissue mass (kg) (fat-free mass) (1.9 bone mineral content). Energy intake At the laboratory, each subject received instructions from a dietitian on the appropriate completion of a 3-day food record at home, including one weekend day. Each subject practiced the procedure during her stay in the respiration chamber. These instructions also were given to the parent(s). Energy intake was calculated from the food records using the (2)

3 908 WONG ET AL. JCE&M 1999 Vol 84 No 3 Minnesota Nutrition Data System (ND 2.4, Nutrition Coordinating Center, University of Minnesota, MN, 1993). Statistical analyses Because soft lean tissue mass has been shown to be the major determinant of energy expenditure in children, adolescents, and adults, whereas fat mass was only a minor contributor to energy expenditure in obese subjects (32, 33), all energy expenditure measurements were normalized to soft lean tissue mass (the mathematical ratio method) before statistical analysis. A t test was used to compare ethnic groups, with respect to age, physical characteristics, body composition, V O 2max, energy intake, and all measures of energy expenditure. Because Tanner stages of pubertal development are not continuous variables, -square was used to compare groups on sexual maturity. Because the mathematical ratio method has been shown to yield erroneous conclusions, regarding differences in energy expenditure (34), analysis of covariance also was used to determine the effect of race on all measures of energy expenditure, while controlling for soft lean tissue mass. Interactions between covariates and race were assessed. All statistical analyses were performed using standardized software (SSS for Windows, version 7.5.1, SSS, Inc., Chicago, IL). Results Age, physical characteristics, sexual maturity, and energy intake of study subjects Age, physical characteristics, sexual maturity, and energy intake of the two groups are given in Table 1. The African- American and girls were well matched for age, body weight, height, and BMI. The girls, however, were sexually more mature than the girls. Neither the total energy intake, estimated from the 3-day food records, nor the calorie intakes of the macronutrients (expressed as percentages of total energy intake) were significantly different between the two groups. V O 2max and body composition of study subjects As shown in Table 2, the V O 2max of the girls was significantly lower than those of the girls. However, the bone mineral content, bone mass, fat-free mass, and soft lean tissue mass of the girls were significantly higher than those of the girls. No difference in fat mass, expressed either in absolute kilograms or in percentage of body weight, was observed between the two groups. Differences in rates of energy expenditure, at rest, between the and girls Sleeping energy expenditure and basal metabolic rate of the and girls are summarized in Table 3. All calorimetric measures of energy expenditure, after normalization to soft lean tissue mass (by the mathematical ratio method) or after adjustment for soft lean tissue mass (by analysis of covariance), were significantly lower in the girls than in the girls. The racial differences in the calorimetric measures of energy expenditure remained significant after inclusion of sexual maturity and/or V O 2max in the analysis of covariance. Fat mass was not included in the analysis because it was not different between the two groups, one of the criteria considered as a covariate. No significant interactions were observed between race and any of the covariates in the analyses, indicating that the racial differences in basal energy expenditure were not affected by the size of soft lean tissue mass, the stages of pubertal development, or physical fitness. Differences in free-living energy expenditure and energy expended for physical activity, between the African- American and girls Total energy expenditure and energy expended for physical activity under free-living conditions (measured by the doubly labeled water method) are summarized in Table 4. The isotope dilution spaces (N H,N O ) were higher in the girls than in the girls, whereas the fractional turnover rates of 2 H and 18 O were higher among the girls than in the girls. No significant differences were observed in the 24-h RQ or U N rate between the two groups. Total energy expenditure and energy expended for physical activity under free-living conditions, after normalization to soft lean tissue mass (by the mathematical ratio method) or after adjustment for soft lean tissue mass (by analysis of covariance), were significantly lower in the girls than in the girls. The racial differences in total energy expenditure and energy expended for physical activity remained significant after inclusion of sexual maturity and/or V O 2max in the analysis. Again, no significant interactions were observed between race and any of the covariates in the analyses. Discussion Based on the weights and heights of children and adolescents reported in the National Health Examination Survey, Cycle II ( ) and the latest data recorded by the National Health and Nutrition Examination Survey ( ), the most dramatic increases in overweight have occurred in girls (4). From 1963 to 1991, a TABLE 2. V O 2 max and body composition of and subjects V O 2 max, ml/kg min a a 0.01 Body composition Bone mineral content, kg Bone mass, kg Fat mass, kg Fat mass, % Fat-free mass, kg Soft lean tissue mass, kg a Mean SD.

4 RACIAL DIFFERENCES IN ENERGY EXENDITURE 909 TABLE 3. Energy expenditure measured by whole-room indirect calorimetry of and subjects a Variables b Total sleeping energy expenditure kcal/kg sltm d kcal/d adjusted for sltm c Basal metabolic rate kcal/kg sltm d kcal/d adjusted for sltm c a Values are mean SD and value by Student t test unless indicated otherwise. b Abbreviations: sltm, soft lean tissue mass. c Mean SD and value after adjusting for soft lean tissue mass by analysis of covariance. TABLE 4. Total energy expenditure and energy expended for physical activity under free-living conditions measured by the doubly labeled water method a Variables b N H, mol N O, mol k H,d k O,d RQ U N, g/day Free-living energy expenditure kcal/kg sltm d kcal/d adjusted for sltm c Energy expended for physical activity kcal/kg sltm d kcal/d adjusted for sltm c a Values are mean SD and value by Student t test unless indicated otherwise. b Abbreviations: N H, hydrogen isotope dilution space; N O, oxygen isotope dilution space; k H, fractional turnover rate of hydrogen; k O, fractional turnover rate of oxygen; RQ, respiratory quotient; U N, urinary nitrogen excretion rate; sltm, soft lean tissue mass. c Mean SD and value after adjusting for soft lean tissue mass by analysis of covariance. 150% increase in overweight was observed in girls between 6 and 11 yr of age, and an 80% increase was reported for girls between 12 and 17 yr of age. During the same period, there was a significantly lower (35%) increase in overweight among girls 6 17 yr of age. Because overweight children are at high risk of becoming overweight adults (7), a request for proposal (HL ) was released by the National Heart, Lung, and Blood Institute on April 17, 1998 to look for ways to prevent excessive weight gain during adolescence in girls through modifications in diet and physical activity. Our data (Table 3), together with those reported recently by Kaplan et al. (8), Morrison et al. (9), and Yanovski et al. (10), clearly demonstrate the existence of a lower basal energy expenditure in prepubertal and pubertal girls, compared with girls. In this study, the sleeping energy expenditure and basal metabolic rate of the girls were 62 and 78 kcal/day, respectively, lower than those of the girls. Although Ravussin et al. (12) observed a direct relationship between a lower resting metabolic rate and later weight gain in 95 southwestern American Indian adults, over a period of 2 4 yr, the same group reported that a low resting metabolic rate was not related to later weight gain in a larger study of 130 southwestern American Indian adults (11). In a study of 24 moderately obese women, over a period of 4 yr, Weinsier et al. (13) reported that the small differences in basal energy expenditure recorded were insufficient to account for later weight gain. However, the authors (13) were able to detect a significant relationship between lower self-reported physical activity and weight gain in these women. Although soft lean tissue mass is the major determinant of energy expenditure (32, 33), Tanner stages of pubertal development were included in the analysis because the girls were sexually more mature than the girls, in spite of their similar chronological ages and physical characteristics (Table 1). The advanced sexual maturation of the girls is consistent with earlier studies showing that the mean age of onset of breast and pubic hair development is 1 2 yr earlier in African- American girls than in girls (35). More importantly, the magnitude of the differences in energy expenditure between the two ethnic groups in this study remained unchanged when sexual maturity was included in the analysis. This is in agreement with the observation by Molnar and Schutz (32) that pubertal development has no effect on energy expenditure in children and adolescents after controlling for fat-free mass. As shown in Table 2, the V O 2max of the girls was 15% lower than that of the girls. This result is similar to that reported by Trowbridge et al. (36). Inclusion of V O 2max in the analysis did not change the outcome, confirming that soft lean tissue mass is the major determinant of energy expenditure. The lower V O 2max observed in the girls might be related to a lower percentage of type I fibers in their skeletal muscle, compared with s. men have been shown to have a lower percentage of type I fibers than men (37), and their V O 2max has been shown

5 910 WONG ET AL. JCE&M 1999 Vol 84 No 3 to be highly correlated with the percentage of type I fibers in their skeletal muscle (38). However, a muscle biopsy, which was not done in this study, would be required to confirm this relationship in girls. Our results (Table 4) clearly demonstrated that energy expended for physical activity under free-living conditions was significantly lower in the girls than in the girls. Therefore, the lower level of physical activity in the African- American girls might have contributed to their lower aerobic fitness level. As shown in Table 4, the average total energy expenditure and energy expended by pubertal girls for physical activity under free-living conditions were, respectively, 410 and 462 kcal/day lower than the girls rates, after adjustment of soft lean tissue mass, by analysis of covariance. The magnitude of the difference was approximately 6-fold higher than that of the basal metabolic rate. This is in contrast to the study by Trowbridge et al. (36), showing no difference in total energy expenditure and energy expended for physical activity, under free-living conditions, between prepubertal and girls. Their inability to detect a significant difference between the two ethnic groups might have been attributable to an insufficient number of study subjects (18 and 27 girls). Because a lack of physical activity has a positive correlation with excessive weight gain (13, 39), and greater increases in adiposity have been documented in pubertal African- American girls than in girls (40), the substantially lower total energy expended for physical activity, under free-living conditions, in the pubertal girls suggests that they are at increased risk of excessive fat gain, compared with the girls. Therefore, any programs to minimize or prevent overweight in women must take into account the lower energy expenditure for physical activity in this population during childhood, as well as in adulthood. Specifically, this study suggests that greater caloric restriction and physical activity are indicated for girls than for girls. Our subjects were recruited from middle-income families. It has been shown that children of low socioeconomic status have lower levels of physical activity and higher body weight (41). Therefore, it is possible that energy expenditure for physical activity under free-living conditions is further reduced in girls in low-income families. In other words, the risk of excessive weight gain might be highest among girls in low-income families. Longitudinal studies, particularly in girls of low socioeconomic status, are needed to evaluate the efficacy of increasing physical activity to control excessive weight gain and the ability to sustain positive physical activity behavior from childhood into adulthood. Acknowledgments The authors are indebted to the volunteers; to the staff of the Metabolic Research Unit, for meeting the needs of the subjects during the study; to Dr. J. Hoyle in the ediatric Department of Kelsey-Seybold West Clinic; Dr. M. desvignes-kendrick, Director of the City of Houston Health and Human Services Department; Ms. X. Earlie, Director of Sciences of the Aldine Independent School District; Ms. S. Wooten, principal at the Teague Middle School; Dr. B. Shargey, dean of instruction, and Ms. C. C. Collins, principal at the High School for Health rofessions; and Ms. K. Wallace for subject recruitment; Dr. J. Moon, Mr. M. uyau, and Mr. F.A. Vohra, for the calorimetric measurements; Mr. R. J. Shypailo and Ms. J. ratt, for the body composition measurements; Mrs. L. L. Clarke and Mr. S. Zhang, for the isotope ratio measurements; and to Ms. L. Loddeke, for editorial assistance in the preparation of the manuscript. References 1. van Itallie TB Health implications of overweight and obesity in the United States. Ann Intern Med. 103: Bray GA Complications of obesity. Ann Intern Med. 103: Kuczmarski RJ, Flegal KM, Campbell SM, Johnson CL Increasing prevalence of overweight among US adults. The National Health and Nutrition Examination Survey, 1960 to JAMA. 272: Troiano R, Flegal KM, Kuczmarski RJ, Campbell SM, Johnson CL Overweight prevalence and trends for children and adolescents. Arch ediatr Adolesc Med. 149: Kumanyika S Obesity in black women. Epidemiol Rev. 9: Kumanyika SK Obesity in minority populations: an epidemiologic assessment. Obes Res. 2: Serdula MK, Ivery D, Coates RJ, Freedman DS, Williamson DF, Byers T Do obese children become obese adults? 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6 RACIAL DIFFERENCES IN ENERGY EXENDITURE Jensen CL, Butte NF, Wong WW, Moon JK Determining energy expenditure in preterm infants: comparison of 2 H 2 18 O method and indirect calorimetry. Am J hysiol. 263:R685 R Shypailo RJ, osada JKJ, Ellis KJ Whole-body phantoms with anthropomorphic-shaped skeletons for evaluation of dual-energy x-ray absorptiometry measurements. Appl Radiat Isot. 49: Ellis KJ, Abrams SA, Wong WW Body composition of a young, multiethnic female population. Am J Clin Nutr. 65: Snyder WS, Cook MJ, Nasset ES, Karhausen LR, Howells G, Tipton IH Weights of organs and tissues of reference man. In: Report of the Task Group on Reference Man. 1st ed. New York: ergamon ress; Molnar D, Schutz Y The effect of obesity, age, puberty and gender on resting metabolic rate in children and adolescents. Eur J ediatr. 156: Cunningham JJ Body composition as a determinant of energy expenditure: a synthetic review and a proposed general prediction equation. Am J Clin Nutr. 54: oehlman ET, Toth MJ Mathematical ratios lead to spurious conclusions regarding age- and sex-related differences in resting metabolic rate. Am J Clin Nutr. 61: Herman-Giddens ME, Slora EJ, Wasserman RC, et al Secondary sexual characteristics and menses in young girls seen in office practice: a study from the ediatric Research in Office Settings Network. ediatrics. 99: Trowbridge CA, Gower BA, Nagy TR, Hunter GR, Treuth MS, Goran MI Maximal aerobic capacity in and prepubertal children. Am J hysiol. 273:E809 E Ama FM, Simoneau JA, Boulay MR, Serresse O, Theriault G, Bouchard C Skeletal muscle characteristics in sedentary Black and males. J Appl hysiol. 61: Ivy JL, Costill DL, Maxwell BD Skeletal muscle determinants of maximum aerobic power in man. Eur J Appl hysiol. 44: Dietz WH, Gortmaker SL Factors within the physical environment associated with childhood obesity. Am J Clin Nutr. 39: Kimm SYS, Barton BA, Obarzanek E, Crawford Changes in adiposity in a biracial cohort during puberty: NHLBI growth and health study. Can J Cardiol. 13:218B. 41. Filozof C, Zuckerfeld R, Berguella AM, Saenz S, Veiga G, Gonzalez C Higher relative weight in kids of lower socioeconomic groups is associated with lower levels of physical activity and higher perceived stress. Obes Res. 5:11S. European Federation of Endocrine Societies 3rd ostgraduate Course in Molecular and Cellular Endocrinology July 7 11, 1999 Münster, Germany Topics: Introductory lectures on hormone action, selected endocrine topics, endocrinology and reproduction and development, workshops on molecular and cellular techniques in endocrinology, technology lectures. Deadline for registration: May 1, Number of registrations will be limited to 120 on a first-comefirst-serve-basis. Registration fee: 150 EURO/294 DM. The final programme and the registration form are available at the Course Secretariat: Institute of Reproductive Medicine, Domagkstr. 11 D Münster, Germany. Tel: 49/ ; Fax: 49/ ; Bbahnes@uni-muenster.de