Clinical Endocrinology (1979) 11,523-532.
SLEEP-WAKE PATTERNS AND INTEGRATED VALUES O F LUTEINIZING HORMONE, FOLLICLE STIMULATING HORMONE, PROLACTIN, GROWTH H O R M O N E A N D THYROID STIMULATING H O R M O N E IN NORMAL A N D CRYPTORCHID PUBERTAL PATIENTS M. G I U S T I , F. B O L O G N E S I , R . M O R T A R A , D . M I G N O N E , P. G R I M A L D I A N D G . G I O R D A N O Department of Endocrinology. University of Genoa, Genoa, Italy (Receiiied I I July 1978: reimed 2 April 1979: arccpred 20 May 1979)
SUMMARY
The sleep-wake behaviour of LH, FSH, PRL, GH and TSH was studied in seven cryptorchid patients (four unilateral and three bilateral cryptorchids) average age 12 years and in nine normal pubertal boys of 13 years (mean age). Blood samples were collected by a continuous withdrawal pump, every hour, for 24 h. The hormonal concentration for every fraction of time was measured and related to the sleep (SS.),wake (WE) and total 24 h period (DS.). The integrated concentrations of the corresponding periods (IS, IW, ID) were calculated as well as their ratios (IS/IW; IS/ID%). For GH and TSH, the data obtained demonstrated no differences between cryptorchid and pubertal subjects. The PRL secretion in cryptorchid patients was moderately increased during the hours of nocturnal sleep. A normal pubertal sleepwake rhythm was found for gonadotrophins in both groups of subjects. More marked levels of LH secretion were observed in cryptorchid boys compared to normal pubertals. The presence of a sleep-wake rhythm was also found in the cryptorchid patients and normal pubertal subjects in the P 1 stage. These data suggest that the CNS ‘programme’ which controls the onset of puberty may be normal in cryptorchid patients. The pathogenesis of testicular maldescent is only partially understood. Besides the role of mechanical factors, the hypothalamo-pituitary axis may be important (Cacciari et a!., 1976, 1977; Canlorbe, 1974; Canlorbeetal., 1974b; Job etal., 1974, 1977a, b; Sizonenko et al., 1973). Some authors found in some cases a reduced basal gondotrophin secretion (Canlorbe et al., 1974a; Job et al., 1974, 1977a, b) and a decreased LH response to gonadotrophin releasing hormone (LHRH) (Canlorbe et al.; 1974a; Gendrel et al., 1977; Correspondence: Dr. M. Giusti, Cattedra di Endocrinologia, Universiti di Genova, viale Benedelto XV. 16100 Genoa, Italy. .
0300-0664/79/1100-0523$02.00 101979 Blackwell Scientific Publications
523
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M . Giusti et al.
Job el al., 1974, 1977a, b) with no difference between subjects with unilateral or bilateral cryptorchidism (Gendrel et al., 1977; Job et a/., 1977b). Normalization of the gonadotrophin response to LHRH is observed at pubertal stage 3 (Canlorbe et al., 1974b; Gendrel et al, 1977). Others (Bulgarelli, 1976; Canlorbe et al., 1974b; Gendrel et al., 1977), on the other hand, suggest that the leydig cells of cryptorchid patients may progress spontaneously to normal. Additional information on the behaviour of the hypothalamo-pituitary-gonadotrophin axis in cryptorchid patients during puberty may be obtained from the study of the sleep-wake rhythm of gonadotrophin secretion. The 24 h secretion in pubertal patients is clearly different compared to the normal adult subjects. In particular, a marked increase in LH secretory episodes is demonstrated during sleep in males and in females (Boyar et a/., 1972; Lee et al., 1976). By contrast, prepubertal subjects show no definite circadian variations in LH secretion (Lee et al., 1976; Weitzman et al., 1975). Thus it seems likely that puberty occurs with a progressive maturation of gonadotrophin secretion with secretory episodes seeming first during sleep, and then in the wake period (Weitzman el al., 1975). On the basis of these data we studied the circadian pituitary secretion in a group of cryptorchid patients and in a group of normal pubertal subjects.
MATERIALS A N D METHODS Seven patients (mean age 12 years, range 10-14) with undescended unilateral (4) or bilateral (3) testes were studied (Table I). The studies were also performed on a group of nine pubertal males (mean age 13 years, range 12-1 5) (Table I ) . Informed consent for the investigation was obtained from the patients and their legal guardians. No particular patient had received any medication for at least 1 month prior to the investigations. They were sexually staged according to the pubic hair and penile size criteria of Tanner (1962). Boys with retractile testis were excluded from the study. Bilateral cryptorchidism was diagnosed on the basis of a positive testosterone response to HCG (1000 i.u./day for 5 days) (Vandershueren-Lodeweycky & Laurance, 1977; Waaler, 1976) and on the absence of elevated basal LH values (Lee el al., 1974). The study was performed after acclimatization to hospital life. During the test the subjects were free to carry on normal activity and were not confined to bed during the hours of waking. Each of them slept in a separate room under the constant supervision of the medical personnel. The onset of sleep began at 22.40 h ( 2 48 min, SD) for cryptochid boys and at 23.10 h ( ? 42 min) for the normal pubertal boys while waking took place at 06.56 h ( ? 42 min) in the former and at 06.30 h (f56 min) in the latter. EEG monitoring of sleep was not undertaken since the objective of this investigation, as that of others (Alford ef al., 1973a; Kowarski et al., 1971; Lee et al., 1976), was not to study the direct correlation between EEG stages and secretory episodes during sleep, but the whole secretory phenomenon during this period. Meals of balanced content were consumed at uniform hours (breakfast 08.00 h, lunch 12.00 h, dinner 19.00 h). The collection of specimens was carried out by means of a constant withdrawal pump
L H , FSH, PRL, G H and TSH in cryprorchidpubertalpatients
525
Table I . Patients studied Case Age (years) Height (cm) Weight (kg) Cryptorchid subjects
Normal pubertal subjects
LV GL TM PF AA MG BM
14
NA ME RS SA GF AA
15 14
0s
CA CF
13 14
13 10 10
10
13 15 12
13 14 12 12
I58 149 I49 157 I32 I50 1 50
55 41 39 46 32 53 3u
141 145 I34 157 I32 I30 143 I34 153
33 42 30 48 30 2u 34 31 53
Pubertal Stage’ 3 2 4 4 1 1 I
B** U U U U
B B
*According to Tanner (1962). **Unilateral (u) and bilateral (B) cryptorchid subjects
(Kowarski et al., 1971). A 2.5 ml volume of blood was collected hourly. The samples were immediately centrifuged and stored at - 20°C until assayed. LH, FSH, PRL and TSH were measured by double antibody radioimmunoassay using reagents obtained from Biodata (Rome); the G H was determined by a similar method (CEA-IRE-Sorin, Saluggia). The concentrations of G H and PRL were expressed as ng/ml, the sensitivity of the methods was 0. I ng/ml and 1 ng/ml, respectively. For LH and FSH concentrations were expressed as ml.U./ml(2nd IRP-HMG), the sensitivity of the method was 0.5 mI.U./ml; for TSH a s pU/ml (reference preparation WHO 63/38), the sensitivity of the method was 0.3 pU/ml. The possibility of interference by hemolysis, as occasionally caused by the pump’s rotor, was excluded (Giusti et al., 1978). Constant withdrawal sampling permitted the measurement of the mean concentrations related to single fractions of time, generally hourly, and the average of the concentrations related to single periods of the day: the wake (We), sleep (SC) or entire 24 h period (DE) (Giordano & Giusti, 1979; Giusti et al., 1978). In addition the secretory area (I) were evaluated according to the formula: I = E x time, for the corresponding periods of time (IW, IS, ID) (Giordano & Giusti, 1979; Giusti et al., 1978). The study of the ratios SC/WC and IS/ID% can indirectly give information on the secretory rate of the hormones in a single time period (Giordano & Giusti, 1979; Giusti et al., 1978). Statistical significance was determined by the use of the Mann-Whitney U test and Wilcoxon’s signed-rank test (Sokal & Rohlf, 1969) RESULTS The experiment was of minimal discomfort to all the patients and subjects who experi-
M .Giusti et al.
526
enced regular sleep averaging 8 h 6 min f I h 12 min - SD) in cryptorchid patients and 7 h ( & I h 1 1 min -SD) in controls. LH concentrations (Fig. I ) were higher in cryptorchid boys compared to controls with a statistically significant difference a t 13.00-14.00 h (P < 0.05; Mann-Whitney test). In both groups of subjects an increase in LH was demonstrable during the sleep period with a zenith in the hours 02.00-03.00 h for the cryptorchids and 06.00-07.00 h in the normals (Fig. I ) . Case by case analysis has demonstrated the presence of a sleep-wake rhythm in four out of seven cryptorchid boys (Fig. 2) and six out of nine normal pubertal subjects (Fig. 2). This phenomenon was absent in two cryptorchid subjects in the P 1 stage (MG, BM) and in one in the P 4 stage (PF), in the latter probably attributable to the conclusion of the pubertal period (Weitzman et al., 1975). this phenomenon was present even in an unilateral cryptorchid subject (AA) in the P 1 stage despite the absence of clinical signs indicating the onset of puberty (Fig. 2). With regard to normal subjects the absence of an LH sleep-wake rhythm was found only in subjects in the PI stage (Fig. 2). The LH DE was higher in cryptorchid patients (8.2 f-2.3 ml.U./rnl - SEM) than in controls (4. I 5 1.0 ml.U./ml) (ns) (Table 2). The LH SC/WC ratio was found to be positive in both groups (cryptorchids: 1.5kO.4; pubertals: 1.9kO.4) (Table 2). The IS of LH represented 38.4+5.6% of ID in cryptorchids (compared to 33.9% for the same time period of sleep with hormone secretion rate constant) and 42.4 f4.9% of ID in normal pubertal subjects (theoretical value: 30.1 (Table 2). The 24 hour profile of FSH did not demonstrate a significant difference between the two groups of subjects examined (Fig. I ) . A sleep-wake rhythm for the hormone was not found in the majority of the cryptorchid boys with the exception of case A A (Fig. 2). This phenomenon, with the exception of two cases (OS,CA), also appeared absent in normal pubertal subjects (Fig. 2).
x)
l5
M+ SE *P
SLEEP-WAKE PATTERNS AND INTEGRATED VALUES O F LUTEINIZING HORMONE, FOLLICLE STIMULATING HORMONE, PROLACTIN, GROWTH H O R M O N E A N D THYROID STIMULATING H O R M O N E IN NORMAL A N D CRYPTORCHID PUBERTAL PATIENTS M. G I U S T I , F. B O L O G N E S I , R . M O R T A R A , D . M I G N O N E , P. G R I M A L D I A N D G . G I O R D A N O Department of Endocrinology. University of Genoa, Genoa, Italy (Receiiied I I July 1978: reimed 2 April 1979: arccpred 20 May 1979)
SUMMARY
The sleep-wake behaviour of LH, FSH, PRL, GH and TSH was studied in seven cryptorchid patients (four unilateral and three bilateral cryptorchids) average age 12 years and in nine normal pubertal boys of 13 years (mean age). Blood samples were collected by a continuous withdrawal pump, every hour, for 24 h. The hormonal concentration for every fraction of time was measured and related to the sleep (SS.),wake (WE) and total 24 h period (DS.). The integrated concentrations of the corresponding periods (IS, IW, ID) were calculated as well as their ratios (IS/IW; IS/ID%). For GH and TSH, the data obtained demonstrated no differences between cryptorchid and pubertal subjects. The PRL secretion in cryptorchid patients was moderately increased during the hours of nocturnal sleep. A normal pubertal sleepwake rhythm was found for gonadotrophins in both groups of subjects. More marked levels of LH secretion were observed in cryptorchid boys compared to normal pubertals. The presence of a sleep-wake rhythm was also found in the cryptorchid patients and normal pubertal subjects in the P 1 stage. These data suggest that the CNS ‘programme’ which controls the onset of puberty may be normal in cryptorchid patients. The pathogenesis of testicular maldescent is only partially understood. Besides the role of mechanical factors, the hypothalamo-pituitary axis may be important (Cacciari et a!., 1976, 1977; Canlorbe, 1974; Canlorbeetal., 1974b; Job etal., 1974, 1977a, b; Sizonenko et al., 1973). Some authors found in some cases a reduced basal gondotrophin secretion (Canlorbe et al., 1974a; Job et al., 1974, 1977a, b) and a decreased LH response to gonadotrophin releasing hormone (LHRH) (Canlorbe et al.; 1974a; Gendrel et al., 1977; Correspondence: Dr. M. Giusti, Cattedra di Endocrinologia, Universiti di Genova, viale Benedelto XV. 16100 Genoa, Italy. .
0300-0664/79/1100-0523$02.00 101979 Blackwell Scientific Publications
523
524
M . Giusti et al.
Job el al., 1974, 1977a, b) with no difference between subjects with unilateral or bilateral cryptorchidism (Gendrel et al., 1977; Job et a/., 1977b). Normalization of the gonadotrophin response to LHRH is observed at pubertal stage 3 (Canlorbe et al., 1974b; Gendrel et al, 1977). Others (Bulgarelli, 1976; Canlorbe et al., 1974b; Gendrel et al., 1977), on the other hand, suggest that the leydig cells of cryptorchid patients may progress spontaneously to normal. Additional information on the behaviour of the hypothalamo-pituitary-gonadotrophin axis in cryptorchid patients during puberty may be obtained from the study of the sleep-wake rhythm of gonadotrophin secretion. The 24 h secretion in pubertal patients is clearly different compared to the normal adult subjects. In particular, a marked increase in LH secretory episodes is demonstrated during sleep in males and in females (Boyar et a/., 1972; Lee et al., 1976). By contrast, prepubertal subjects show no definite circadian variations in LH secretion (Lee et al., 1976; Weitzman et al., 1975). Thus it seems likely that puberty occurs with a progressive maturation of gonadotrophin secretion with secretory episodes seeming first during sleep, and then in the wake period (Weitzman el al., 1975). On the basis of these data we studied the circadian pituitary secretion in a group of cryptorchid patients and in a group of normal pubertal subjects.
MATERIALS A N D METHODS Seven patients (mean age 12 years, range 10-14) with undescended unilateral (4) or bilateral (3) testes were studied (Table I). The studies were also performed on a group of nine pubertal males (mean age 13 years, range 12-1 5) (Table I ) . Informed consent for the investigation was obtained from the patients and their legal guardians. No particular patient had received any medication for at least 1 month prior to the investigations. They were sexually staged according to the pubic hair and penile size criteria of Tanner (1962). Boys with retractile testis were excluded from the study. Bilateral cryptorchidism was diagnosed on the basis of a positive testosterone response to HCG (1000 i.u./day for 5 days) (Vandershueren-Lodeweycky & Laurance, 1977; Waaler, 1976) and on the absence of elevated basal LH values (Lee el al., 1974). The study was performed after acclimatization to hospital life. During the test the subjects were free to carry on normal activity and were not confined to bed during the hours of waking. Each of them slept in a separate room under the constant supervision of the medical personnel. The onset of sleep began at 22.40 h ( 2 48 min, SD) for cryptochid boys and at 23.10 h ( ? 42 min) for the normal pubertal boys while waking took place at 06.56 h ( ? 42 min) in the former and at 06.30 h (f56 min) in the latter. EEG monitoring of sleep was not undertaken since the objective of this investigation, as that of others (Alford ef al., 1973a; Kowarski et al., 1971; Lee et al., 1976), was not to study the direct correlation between EEG stages and secretory episodes during sleep, but the whole secretory phenomenon during this period. Meals of balanced content were consumed at uniform hours (breakfast 08.00 h, lunch 12.00 h, dinner 19.00 h). The collection of specimens was carried out by means of a constant withdrawal pump
L H , FSH, PRL, G H and TSH in cryprorchidpubertalpatients
525
Table I . Patients studied Case Age (years) Height (cm) Weight (kg) Cryptorchid subjects
Normal pubertal subjects
LV GL TM PF AA MG BM
14
NA ME RS SA GF AA
15 14
0s
CA CF
13 14
13 10 10
10
13 15 12
13 14 12 12
I58 149 I49 157 I32 I50 1 50
55 41 39 46 32 53 3u
141 145 I34 157 I32 I30 143 I34 153
33 42 30 48 30 2u 34 31 53
Pubertal Stage’ 3 2 4 4 1 1 I
B** U U U U
B B
*According to Tanner (1962). **Unilateral (u) and bilateral (B) cryptorchid subjects
(Kowarski et al., 1971). A 2.5 ml volume of blood was collected hourly. The samples were immediately centrifuged and stored at - 20°C until assayed. LH, FSH, PRL and TSH were measured by double antibody radioimmunoassay using reagents obtained from Biodata (Rome); the G H was determined by a similar method (CEA-IRE-Sorin, Saluggia). The concentrations of G H and PRL were expressed as ng/ml, the sensitivity of the methods was 0. I ng/ml and 1 ng/ml, respectively. For LH and FSH concentrations were expressed as ml.U./ml(2nd IRP-HMG), the sensitivity of the method was 0.5 mI.U./ml; for TSH a s pU/ml (reference preparation WHO 63/38), the sensitivity of the method was 0.3 pU/ml. The possibility of interference by hemolysis, as occasionally caused by the pump’s rotor, was excluded (Giusti et al., 1978). Constant withdrawal sampling permitted the measurement of the mean concentrations related to single fractions of time, generally hourly, and the average of the concentrations related to single periods of the day: the wake (We), sleep (SC) or entire 24 h period (DE) (Giordano & Giusti, 1979; Giusti et al., 1978). In addition the secretory area (I) were evaluated according to the formula: I = E x time, for the corresponding periods of time (IW, IS, ID) (Giordano & Giusti, 1979; Giusti et al., 1978). The study of the ratios SC/WC and IS/ID% can indirectly give information on the secretory rate of the hormones in a single time period (Giordano & Giusti, 1979; Giusti et al., 1978). Statistical significance was determined by the use of the Mann-Whitney U test and Wilcoxon’s signed-rank test (Sokal & Rohlf, 1969) RESULTS The experiment was of minimal discomfort to all the patients and subjects who experi-
M .Giusti et al.
526
enced regular sleep averaging 8 h 6 min f I h 12 min - SD) in cryptorchid patients and 7 h ( & I h 1 1 min -SD) in controls. LH concentrations (Fig. I ) were higher in cryptorchid boys compared to controls with a statistically significant difference a t 13.00-14.00 h (P < 0.05; Mann-Whitney test). In both groups of subjects an increase in LH was demonstrable during the sleep period with a zenith in the hours 02.00-03.00 h for the cryptorchids and 06.00-07.00 h in the normals (Fig. I ) . Case by case analysis has demonstrated the presence of a sleep-wake rhythm in four out of seven cryptorchid boys (Fig. 2) and six out of nine normal pubertal subjects (Fig. 2). This phenomenon was absent in two cryptorchid subjects in the P 1 stage (MG, BM) and in one in the P 4 stage (PF), in the latter probably attributable to the conclusion of the pubertal period (Weitzman et al., 1975). this phenomenon was present even in an unilateral cryptorchid subject (AA) in the P 1 stage despite the absence of clinical signs indicating the onset of puberty (Fig. 2). With regard to normal subjects the absence of an LH sleep-wake rhythm was found only in subjects in the PI stage (Fig. 2). The LH DE was higher in cryptorchid patients (8.2 f-2.3 ml.U./rnl - SEM) than in controls (4. I 5 1.0 ml.U./ml) (ns) (Table 2). The LH SC/WC ratio was found to be positive in both groups (cryptorchids: 1.5kO.4; pubertals: 1.9kO.4) (Table 2). The IS of LH represented 38.4+5.6% of ID in cryptorchids (compared to 33.9% for the same time period of sleep with hormone secretion rate constant) and 42.4 f4.9% of ID in normal pubertal subjects (theoretical value: 30.1 (Table 2). The 24 hour profile of FSH did not demonstrate a significant difference between the two groups of subjects examined (Fig. I ) . A sleep-wake rhythm for the hormone was not found in the majority of the cryptorchid boys with the exception of case A A (Fig. 2). This phenomenon, with the exception of two cases (OS,CA), also appeared absent in normal pubertal subjects (Fig. 2).
x)
l5
M+ SE *P
