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Hypothalamic-pituitary-gonadal function in prepubertal boys and girls with chronic renal failure
Hypothalamic-pituitary-gonadal function in prepubertal boys and girls with chronic renal failure M. C a s t e l l a n o , MD, A. Turconi, MD, E. C h a l e r , BioCh, M. A. Rivarola, MD, a n d A. Belgorosky, MD, PhD From the Endocrine Research Laboratory and Nephrology Unit, Hospital de Pediatria "Prof. Dr. J. P. Garrahan," Buenos Aires, Argentina Hypothalamic-pltultary-gonadal function was e v a l u a t e d In 24 prepubertal children with chronic renal failure (CRF). A m o n g the 47 boys, 5 were receiving conservative treatment and four long-term dialysis. Another eight boys were studied 6 months to 3.3 years after renal transplantation; their ages r a n g e d from 5 years 8 months to 45V2 years. A m o n g the girls, two patients were receiving conservative treatment and five long-term dialysis; their ages r a n g e d from 3V2 years to 44 years 2 months. In boys with CRF, but not in those after transplantation, mean serum follicle-stimulating hormone 60 minutes after administration of g o n a d o tropin releasing hormone (GnRH) was lower than in 48 control prepubertal boys (mean _ S D " 2.53 ___4.34 vs 6.25 + 2.84 IU/L, respectively; p <0.04). Testosterone s t e r o l d o g e n l c c a p a c i t y after 4 w e e k of stimulation with human chorionic gonadofropin and a n d r o g e n sensitivity ( p e r c e n t a g e of d e c r e a s e of serum sex h o r m o n e - b i n d i n g globulin 4 w e e k after intramuscular administration of testosterone e n a n t h a t e ) were normal. In girls, no difference b e t w e e n those with CRF and a control group of 49 girls was found after intravenous administration of GnRH. However, after intramuscular administration of GnRH agonlst, serum follicle-stimulating hormone concentration was lower in girls with CRF than in control girls (p <0.02); six of seven control girls had an increase of serum estradiol to more than 55 pmol/L, whereas three of seven girls with CRF had no response, and serum follicle-stimulating hormone failed to Increase after GnRH agonist therapy in two of these patients. We c o n c l u d e that hypothalamlc-pituitary function is not normal in some prepubertal boys and girls with CRF, particularly in those with low serum albumin concentrations. On the other hand, testlcular and ovarian s t e r o i d o g e n l c c a p a c i t y Is not Impaired, and the b i o l o g i c response to androgens in boys is preserved. (J PEDIArR4993;422:46-54) Pubertal development is frequently delayed and often incomplete in pubertal children with chronic renal failure. Many factors are involved) -3 but the mechanism of the disturbances in hypothalamic-pituitary-gonadal function is Supported by grants from the Consejo Nacional de Investigaciones Cientificas y T6cnicas (CONICET) of Argentina, the World Health Organization, and Programa Latinoamericano de Capacitaci6n e Investigaci6n en Reproducci6n Humana (PLACIRH). Submitted for publication Feb. 21, 1992; accepted Aug. 18, 1992. Reprint requests: Alicia Belgorosky, MD, PhD, Laboratorio de Investigaci6n, Hospital de Pediatria Garrahan, Pozos 1881, Buenos Aires (1245), Argentina. 9/20/41974 46
0022-3476/93/$01.00+OO.lO
not fully understood. In male and female adult patients with CRF, and in children during pubertal development, serum aGnRH CRF FSH GnRH hCG LH SttBG
GnRH agonist Chronic renal failure Follicle-stimulating hormone Gonadotropic releasing hormone Human chorionic gonadotropin Luteinizing hormone Sex hormone-binding globulin
gonadotropin concentrations are elevated but the gonadotropic response to gonadotropin releasing hormone is blunted. 4-6 Little information is available on the status of the hy-
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Hypothalamic-pituitary-gonadal function and chronic renal failure
pothalamic-pituitary-gonadal axis in prepubertal children with CRF. Some authors have proposed that gonadal dysfunction in children with uremia might be, at least in part, due to inadequate pituitary function. 7 We studied this issue in prepubertal children with different degrees of CRF. METHODS Procedures. Serum luteinizing hormone and folliclestimulating hormone concentrations were measured by immunoradiometric assay with the use of reagents (provided by Ares-Serono Diagnostici, Milan, Italy) containing three monoclonal antibodies. Assay sensitivity was 0.25 I U / L . The imprecision profile for the LH assay ranged from 3.8% at 0.5 I U / L to 10.0% at 100 I U / L . For FSH assay, it ranged from 12% at 0.5 I U / L to 4.7% at 75 I U / L . Interassay coefficient of variation ranged from 4.3% to 12.5% for LH and from 20% to 5.4% for FSH. The International Reference Preparation (IRP) standard (World Health Organization) used for FSH was Second IRP(78/549) and for LH was First IRP(68/40). Serum estradiol concentration was measured by radioimmunoassay with the use of reagents provided by Biodata Ltd., Milan, Italy. Assay sensitivity was 36 pmol/L (9.8 pg/ml). The imprecision profile ranged from 9.3% at 36.7 pmol/L (10 pg/ml) to 3.9% at 400 pmol/L (109 pg/ml). Serum testosterone was measured by radioimmunoassay.8 Assay sensitivity was 0.42 nmol/L (0.12 ng/ml). The imprecision profile was less than 10% for testosterone concentrations between 0.35 and 17.3 nmol/L (between 0.I0 and 5 ng/ml). Serum sex hormone-binding globulin concentration was determined by saturation analysis as previously described.9 All serum determinations were performed in duplicate. Every sample in a single patient was carried out in the same assay. The Student t test and chi-square tests were used to analyze differences among the groups. Subjects. Twenty-four prepubertal children (17 boys) with C R F were studied. Of the boys, five received conservative treatment (serum creatinine concentrations between 0.5 and 8.7 mg/dl) and four had been receiving long-term dialysis for 0.5 to 5.1 years (CRF group). Another eight boys were studied 0.5 to 3.3 years after renal transplantation. Boys' ages ranged from 5.8 to 15.6 years (mean ___SD: 8.8 ___4.2 years). Their diagnoses were as follows: obstructive uropathy (5 subjects), hemolytic uremic syndrome (1), focal glomerular sclerosis (3), nephrocystinosis (1), renal hypoplasia (5), and crescentic glomerulonephritis (2). Serum albumin concentration ranged from 33 to 47 g m / L at the time of the endocrine study, and creatinine clearances from 8.1 to 97.5 ( C R F group) and from 36 to 145 ml/min • 1.73 m 2 in those who had transplants. In the girls, two patients were receiving conservative treatment and five had been receiving long-term dialysis for 1.3 to 10.3 years. Their ages ranged from 3.1 to 11.2 years
47
(mean __. SD: 7.6 _+ 3.13 years). Their diagnoses were as follows: obstructive uropathy (2 patients), hemolytic-uremic syndrome (1), focal glomerular sclerosis (2), renal hypoplasia (I), and crescentic glomerulonephritis (1). Serum albumin concentration ranged from 12 to 45 g m / L and creatinine clearances from 7.1 to 60 ml/min x 1.73 m 2. A GnRH test was carried out in a control group of 18 prepubertal boys and 19 prepubertal girls (mean _.+ SD: 7.05 __. 2.61 and 6.5 __. 3.08 years of age, respectively). A GnRH agonist test was carried out in seven prepubertal female control subjects (mean age: 8.59 + 2.01 years). Informed consent for the studies was obtained from parents. The protocol was approved by the Hospital de Pediatria Garrahan of Buenos Aires Research Committee. Endocrine tests. The acute-phase GnRH test was carried out in both girls and boys after intravenous injection of 100 #g GnRH; blood was drawn at - 1 0 , 20, and 60 minutes for serum LH and FSH determinations. The subacute-phase aGnRH test was carried out in prepubertal girls after two subcutaneous injections of 100 ttg of an aGnRH, buserelin acetate (Suprefact; HoechstRoussel), separated by a 24-hour interval. Blood was drawn for determination of serum LH, FSH, and estradiol levels in basal conditions, 24 hours after the first injection, and 4, 6, and 24 hours after the second stimulation. The aim of this test was to evaluate both pituitary LH and FSH responsiveness and ovarian estrogenic responsiveness to endogenous gonadotropic stimulation. Maximal responses for LH ranged from 2.16 to 6.84 U / L and for FSH from 8.92 to 23.9 U / L . Serum estradiol response was considered to be positive when the value obtained was equal to or greater than 55 pmol/L (15 pg/ml) in at least one of the samples. In prepubertal boys, Leydig cell reserve and androgen sensitivity were tested successively according to a technique previously described I~ I I: after drawing blood for basal determination of serum concentrations of testosterone and of SHBG, 2500 IU of human chorionic gonadotropin was administered intramuscularly on days I and 4. On day 7, blood was drawn for measurement of the serum testosterone concentration (after administration of hCG) and 2 mg testosterone enanthate per kilogram body weight was then administered intramuscularly. Blood was drawn again on day 14 for assay of serum SHBG. Serum testosterone response to hCG was considered to be normal when the post-hCG values exceeded 3.5 nmol/L (1 ng/ml). I~ I I The androgen sensitivity was considered to be positive when serum SHBG decreased to less than 80% of the basal value on day 14. t~ I I RESULTS As shown in Table I, basal LH and FSH concentrations were normal in all male and female subjects with CRF. After 60 minutes of GnRH administration, mean serum FSH
48
Castellano et al.
The Journal of Pediatrics January 1993
T a b l e I. Basal and post-GnRH (intravenous) serum L H and F S H levels in prepubertal boys and girls with C R F Serum LH ( m U / m l )
- Serum FSH ( m U / m l )
GnRH Group
Boys CRF After transplantation Control subjects Girls Patients with CRF Control subjects
GnRH
n
Basal
At 20 mln
At 60 mln
Basal
At 20 mln
At 60 mln
9 8 18
1.61 _+ 0.97 1.18 __. 0.78 0.91 + 0.94
2.45 _+ 0.72 2.70 _ 1.13 1.65 +__ 1.05
3.39 _+ 1.80 2.76 ___2.61 2.17 +__ 1.23
1.94 _ 1.07 2.48 + 1.58 1.46 ___ 1.13
2.27 _+ 1.58 3.75 + 2.19 3.69 + 1.44
2.53 _+ 1.34" 5.58 + 4.82 6.25 + 2.84
7 19
1.14 __. 1.52 0.41 ___0.33
2.16 _ 1.64 2.41 ___ 1.94
2.62 ___2.03 2.63 __. 1.50
2.49 + 2.44 1.66 + 1.27
5.66 --- 4.05 5.05 __. 2.68
7.00 _ 3.93 8.39 +__4.66
Boys were studied during conservativetreatment, during long-termdialysis therapy, or after renal transplantation. Girls were studied during conservativetreatment or long-term dialysis only. Values are expressed as mean __.SD. *p <0.01 versus control values.
T a b l e II. Serum testosterone levels before and after acute stimulation with 5000 U I of hCG; serum S H B G levels before and after administration of 2 mg of testosterone enanthate per kilogram of body weight to prepubertal boys with C R F receiving conservative treatment or chorionic dialysis and after renal transplantation Testosterone (nmol/L)
SHBG (nmol/L)
Group
n
Basal value
After hCG
Basal v a l u e
After testosterone enanthate
% Basal v a l u e
Patients with CRF After transplantation
9 8
0.62 _+ 0.62 0.34 _+ 0.01
17.26 _ 13.59 9.49 ___8.43
92.0 ___42.8 96.4 _ 75.6
47.1 _ 22.8 49.9 _+ 24.2
51.7 __. 4.89 62.2 + 17.9
To express testosterone in nanograms per milliliter, divide by 3.467.
levels, but not mean L H levels, were significantly lower (p <0.01) in boys with C R F than in control subjects. After renal transplantation in boys, normal serum LH and F S H responses to acute G n R H were found. In girls with C R F , serum L H and FSH responses to acute G n R H stimulation were normal in comparison with their respective control subjects. In all prepubertal male patients, serum testosterone levels after hCG therapy were greater than 3.5 n m o l / L (1 n g / ml) and serum S H B G levels after testosterone enanthate treatment were equal to or less than 80% of basal value (Table II). In Fig. 1 are shown the serum LH and F S H levels in the girls after subcutaneous a G n R H administration. The mean serum L H concentration increased significantly in control ~ubjects only at 4 and 6 hours after the second a G n R H injection; the mean serum F S H concentration was already significantly greater 24 hours after the first and 4 and 6 hours after the second injection of a G n R H (p <0.01). In the girls with C R F , the serum L H level increased significantly only 6 hours after the second a G n R H injection (p <0.01); serum F S H values did not differ from basal values at any time. No significant difference in serum L H was found between control girls and the girls with C R F at any time. In contrast, the serum F S H concentration in girls with
C R F was significantly lower than in control girls, 4 and 6 hours after the second injection (p <0.02). In six of the seven control girls, serum estradiol concentration was greater than the upper limit of the basal value 24 hours after the first injection and 4 hours after the second one (positive response); one girl did not respond at any time, but her serum L H increased from 0.5 to 2.16 and her serum F S H from 0.85 to 8.92 m U / m l 4 hours after the second injection (not shown). In four of the seven patients with C R F , the serum estradiol concentration increased but in three it did not (Fig. 2). In two of these latter three patients, serum L H and F S H values did not increase after a G n R H administration. Serum estradiol response in the C R F group was significantly different from that in the control group (p <0.05, by chi-square test). Because only some of the girls with C R F had abnormal responses during the a G n R H test and others had normal responses, an attempt was made to correlate these abnormal responses with clinical and laboratory indicators of abnormal renal function. N o correlation was found with chronologic age, bone age, failure to grow, weight/height ratio, duration of C R F , creatinine clearance, or type of treatment. The only striking correlations were that (1) the two girls who had no increase in serum L H and F S H values after
Volume 122 Number !
Hypothalamic-pituitary-gonadal function attd chronic renal failure
t.
,ooo
~z
49
t.
.J o
o @
o E (x
16
**
"8 100
,,
o
o Oo 0
-6
o o
o 9
oo
9
9 9
~h h,
7 8 t,
10'
4
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BT ist
24 t 2rid
4-
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Fig.
I. Serum LH and FSH levels before (B) and after subcutaneous administration of 100 tag aGnRH buserelin (Suprefact) to girls with CRF (black bars) and to control girls (white bars). Arrows indicate first and second aGnRH injections.Numbers to the right of the second arrow indicate hours after the second injection. *p <0.05 versus corresponding B; **p <0.0I versus corresponding B; black circles, p <0.02 versus control values.
aGnRH administration had serum albumin concentrations of 12 and 15 gm/L, whereas in the other five patients the range was between 36 and 45 gm/L, and (2) these two girls were the only girls with CRF caused by focal glomerular sclerosis. DISCUSSION Many abnormalities in gonadotropin and sex steroid secretion have been described in adults with CRF. It has been proposed that uremia induces both central disturbances and alterations in the pituitary-dependent endocrine organs) 2q4 Furthermore, delayed bone age and pubertal maturation also have been reported in adolescents with CRF. t'5,15, 16 It has been suggested that abnormalities in the secretion of gonadotropins might have an impact on pubertal development} 7 In prepubertal children the available data on gonadotropin secretion are controversial. High serum LH or FSH basal levels and blunted serum gonadotropic responses to GnRH have been described in prepubertal children with CRF and during their development.5' 6, ]4-IsThese high levels of gonadotropins returned to normal after renal transplantation. In contrast to these reports, and similar to that of Giusti
e ~ ] CRF
24 t
4
6
24 hours
2 nd ol-61C
t Buserelin 1100
Hg SOl
Fig. 2. Serum estradiol levelsbefore and after subcutaneous (SC) administration of 100 tag aGnRH (buserelin) in girls with CRF (black circles) and control (C) subjects (white circles). Squares depict subjects in whom serum concentration of estradiol was never greater than 55 pmol/L (15 pg/ml) either before or after stimulation. Horizontal lines indicate the serum estradiol level of 55 pmol/L (15 pg/ml). Arrows at base of illustration indicate first and second injections of buserelin;arrows at top of illustration indicate estradiol values exceeding the scale. et al., 7 we found normal serum LH and FSH basal levels in children with uremia. Differences in assay sensitivity and specificity might have contributed to these conflicting results. We measured serum LH and FSH by an immunoradiometric assay, which utilizes a monoclonal antibody, and might have avoided c~-subunit interference in the assay. In boys the serum LH response to exogenous GnRH might be due either to chronic lesser priming of pituitary gonadotrophs by GnRH neurons or to a disturbance of pituitary cells. In men with CRF, low plasma testosterone concentrations and an inadequate rise after administration of hCG suggest that endocrine testicular function is impaired) 3, 14 No information on Leydig cell reserve in prepubertaI children with uremia has been reported. We found a normal testosterone response to hCG in our patients, suggesting that testicular steroidogenesis is conserved during prepuberty. It had been speculated that growth hormone, insulin-like growth factor-I, and dexamethasone resistance might be present in CRF.IS'2~Thus we were interested in studying the biologic response to androgens in boys with uremia. We had previously reported that the serum SHBG response to exogenous testosterone enanthate was a reliable test to eval-
50
Castellano et aL
uate androgen sensitivity,l~ tt All our boys with CRF had normal decreases in serum SHBG after receiving exogenous testosterone enanthate, suggesting that the biologic response to androgen is not disturbed. In contrast to our findings in the prepubertal boys, prepubertal girls with CRF had normal LH and FSH responses to GnRH testing. These different responses stimulated our interest in evaluating hypothalamic-pituitary-ovarian function in more detail in our patients. For this purpose, we utilized a modification of the subacute-phase aGnRH test described by Rosenfield et al. 21 Control patients had elevated levels of FSH, but not LH, 24 hours after the second injection. In girls with CRF, serum LH response was normal but that of serum FSH was low after the second injection. Indeed, serum FSH concentration did not increase after aGnRH was injected. Therefore, as speculated for boys, girls with CRF might have decreased FSH responses to GnRH because of less priming of pituitary gonadotrophs as a result of a disturbance of GnRH neurons; alternatively, the toxic effects of uremia may affect the pituitary cells themselves. The evaluation of ovarian steroidogenesis is difficult during the prepubertal period. We took advantage of the subacute-phase aGnRH test to measure serum estradiol increments as a measure of the response of the prepubertal ovary after two LH and FSH discharges. We defined the test result as positive when serum estradiol increases to greater than 55 pmol/L (15 pg/ml), because this value is never reached in prepubertal control girls in basal conditions. Positive responses were observed in more control girls than in girls with CRF. Two of the three negative estradiol responses, however, could be attributed to lack of LH and FSH responses to aGnRH stimulation. These findings suggest that prepubertal ovarian steroidogenic capacity is not impaired in CRF. The two girls in whom aGnRH failed to elicit adequate combined LH, FSH, and estradiol responses had focal glomerular sclerosis, but this failure did not seem to have any connection with hypothalamic-pituitary function. On the other hand, the girls also had low serum albumin levels. Gonadotropic function is known to be altered in severe malnutrition, which could be a factor contributing to an abnormal gonadotropin response in some patients with CRF. Delayed puberty is a common concern in patients with CRF. An abnormality in hypothalamic-pituitary function, such as the one described here, might impair the maturation of the GnRH pulse generator. However, the mechanism of the initiation of puberty is largely unknown, and other factors might be involved. We reported previously that prepubertal boys with CRF share with prepubertal boys with GH deficiency, another model of delayed puberty, a delayed increment in serum bioavailable testosterone during prepuberty. 2~ It has been suggested that patients with CRF have
The Journal of Pediatrics January 1993
peripheral resistance to growth hormone or to insulin-like growth factor 119; this resistance might play a role in their pubertal delay. We conclude that hypothalamic-pituitary function is not normal in some prepubertal boys and girls with CRF and that this abnormality, at least in girls, could be related to secondary malnutrition. In contrast, testicular and ovarian steroidogenic capacity is not impaired. Furthermore, the biologic response to androgens is also preserved. We acknowledge the contributions of P. Rossano, D. Chirico, and M. Maceiras with technical assistance. REFERENCES 1. Ferraris J, Jaenger P, LevineL, et aL Delayedpubertyin males with chronic renal failure. Kidney Int 1980;18:344-50. 2. Sharer K, Chamter C, Brunner FP, et al. Combined report on regular dialysis and transplantation of children in Europe. Proceedings of the European DialysisTransplant Association 1976;13:59-103. 3. Van Diemen-StenvoordeMD, DonckerwolckeRA, Brakel H, Wolff ED, de Gowg MCJW. Growth and sexual maturation in children after kidney transplantation. J PEDIATR 1987; 110:351-6. 4. Holdsworth S, Atkins RC, De Kretser DM. The pituitary-testicular axis in men with chronic renal failure. N Engl J Med i 977;296:1245-9. 5. Ferraris JR, Domene HM, Eseobar ME, Caletti MG, Ramirez JA, Rivarola MA. Hormonal profile in prepubertal females with chronic renal failure before and under haemodialysisand after renal transplantation. Acta Endocrinol (Copenh) 1987; I 15:289-96. 6. Oertel PJ, Liehtwall DK, Hafner S, Raueh W, Schonberg D, Shard K. Hypothalamo-pituitary-gonadal axis in children with chronic renal failure. Kidney Int 1983;115:34-9. 7. Giusti M, Perfumo F, Verrina E, et al. Biologicalactivity of luteinizing hormone in uraemic children: spontaneous nocturnal secretion and changes after administration of exogenous pulsatile luteinizing hormone releasing hormone: preliminary observations. Pediatr Nephrol 1991;5:559-65. 8. CampoS, Monteagudo S, Nieolau G, et al. Testieular function in prepubertal male pseudohermaphroditism.Clin Endocrinol (Oxf) 1981;14:11-22. 9. BelgoroskyA, Rivarola MA. Determination of sex hormone biding globulin in human serum. Medicina (Buenos Aires) 1982;42:141-8. 10. BelgoroskyA, Rivarola MA. Sex hormone binding-globulin response to human chorionicgonadotropin stimulation in children with cryptorchidism, anorchia, male pseudohermaphroditism and micropenis.J Clin Endocrinol Metab 1982;54:698704. 11. Ciaccio M, Rivarola MA, BelgoroskyA. Decreaseof serum sex hormone binding-globulinas a marker of androgen sensitivity: correlation with clinical response. Acta Endocrinol (Copenh) 1989;120:540-4. 12. Guevara A, Hallberg MC, Torn EM, Pohlman C, Wieland RG. Serum gonadotropin and testosterone levels in uremic males undergoing intermittent dialysis. Metabolism 1969; 18:1062-9. 13. Lim US, Fang VS. Gonadal dysfunction in uremic men: a
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14.
15. 16.
17.
Hypothalamic-pituitary-gonadal function and chronic renal failure
study of the hypothalamo-pituitary-testicular axis before and after renal transplantation. Am J Med 1975;58:655-62. Handelsman DJ. Hypothalamic-pituitary-gonadal dysfunction in renal failure: dialysisand renal transplantation. Endocr Rev 1985;6:151-82. Melhls O, Ritz F, Gilli EK, Renser W. Growth in renal failure. Nephrologie 1978;21:237-47. Rizzoni G, Broyer M, Guest G, Fine R, Holliday MA. Growth retardation in children with chronic renal disease: scope of the problem. Am J Kidney Dis 1986;7:256-61. ShSrer K, Shaefer F, Trott M, et al. Pubertal developmentin children with chronic renal failure. In: Laron Z, ed. Growth and endocrinechanges in children and adolescentswith chronic renal failure. Basel: Karger, 1989:151-68.
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18. Rauch KW, Ohlel PJ. Endocrine function. In: Fine CN, Gruskin A, eds. End-stage renal disease in children. Philadelphia: WB Saunders, 1984:296-307. 19. Lee PDK, Hintz RL, Sperry JB, Baxter RC, Powel DR. IGF binding proteins in growth-retarded children with chronic renal failure. Pediatr Res 1989;26:308-14. 20. BelgoroskyA, Ferraris JR, Ramirez H, Jasper H, Rivarola MA. Serum sex hormone-binding globulin and serum nonsex hormone-binding globulin-bound testosterone fractions in prepubertal boys with chronic renal failure. J Clin Endocrinol Metab 1991;73:107-10. 21. RosenfieldRL, Burstein S, Cuttler L, et al. Use of nafarelin for testing pituitary-ovarian function. J Reprod Med 1989; 34:1044-50.
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0022-3476/93/$01.00+OO.lO
not fully understood. In male and female adult patients with CRF, and in children during pubertal development, serum aGnRH CRF FSH GnRH hCG LH SttBG
GnRH agonist Chronic renal failure Follicle-stimulating hormone Gonadotropic releasing hormone Human chorionic gonadotropin Luteinizing hormone Sex hormone-binding globulin
gonadotropin concentrations are elevated but the gonadotropic response to gonadotropin releasing hormone is blunted. 4-6 Little information is available on the status of the hy-
Volume 122 Number l
Hypothalamic-pituitary-gonadal function and chronic renal failure
pothalamic-pituitary-gonadal axis in prepubertal children with CRF. Some authors have proposed that gonadal dysfunction in children with uremia might be, at least in part, due to inadequate pituitary function. 7 We studied this issue in prepubertal children with different degrees of CRF. METHODS Procedures. Serum luteinizing hormone and folliclestimulating hormone concentrations were measured by immunoradiometric assay with the use of reagents (provided by Ares-Serono Diagnostici, Milan, Italy) containing three monoclonal antibodies. Assay sensitivity was 0.25 I U / L . The imprecision profile for the LH assay ranged from 3.8% at 0.5 I U / L to 10.0% at 100 I U / L . For FSH assay, it ranged from 12% at 0.5 I U / L to 4.7% at 75 I U / L . Interassay coefficient of variation ranged from 4.3% to 12.5% for LH and from 20% to 5.4% for FSH. The International Reference Preparation (IRP) standard (World Health Organization) used for FSH was Second IRP(78/549) and for LH was First IRP(68/40). Serum estradiol concentration was measured by radioimmunoassay with the use of reagents provided by Biodata Ltd., Milan, Italy. Assay sensitivity was 36 pmol/L (9.8 pg/ml). The imprecision profile ranged from 9.3% at 36.7 pmol/L (10 pg/ml) to 3.9% at 400 pmol/L (109 pg/ml). Serum testosterone was measured by radioimmunoassay.8 Assay sensitivity was 0.42 nmol/L (0.12 ng/ml). The imprecision profile was less than 10% for testosterone concentrations between 0.35 and 17.3 nmol/L (between 0.I0 and 5 ng/ml). Serum sex hormone-binding globulin concentration was determined by saturation analysis as previously described.9 All serum determinations were performed in duplicate. Every sample in a single patient was carried out in the same assay. The Student t test and chi-square tests were used to analyze differences among the groups. Subjects. Twenty-four prepubertal children (17 boys) with C R F were studied. Of the boys, five received conservative treatment (serum creatinine concentrations between 0.5 and 8.7 mg/dl) and four had been receiving long-term dialysis for 0.5 to 5.1 years (CRF group). Another eight boys were studied 0.5 to 3.3 years after renal transplantation. Boys' ages ranged from 5.8 to 15.6 years (mean ___SD: 8.8 ___4.2 years). Their diagnoses were as follows: obstructive uropathy (5 subjects), hemolytic uremic syndrome (1), focal glomerular sclerosis (3), nephrocystinosis (1), renal hypoplasia (5), and crescentic glomerulonephritis (2). Serum albumin concentration ranged from 33 to 47 g m / L at the time of the endocrine study, and creatinine clearances from 8.1 to 97.5 ( C R F group) and from 36 to 145 ml/min • 1.73 m 2 in those who had transplants. In the girls, two patients were receiving conservative treatment and five had been receiving long-term dialysis for 1.3 to 10.3 years. Their ages ranged from 3.1 to 11.2 years
47
(mean __. SD: 7.6 _+ 3.13 years). Their diagnoses were as follows: obstructive uropathy (2 patients), hemolytic-uremic syndrome (1), focal glomerular sclerosis (2), renal hypoplasia (I), and crescentic glomerulonephritis (1). Serum albumin concentration ranged from 12 to 45 g m / L and creatinine clearances from 7.1 to 60 ml/min x 1.73 m 2. A GnRH test was carried out in a control group of 18 prepubertal boys and 19 prepubertal girls (mean _.+ SD: 7.05 __. 2.61 and 6.5 __. 3.08 years of age, respectively). A GnRH agonist test was carried out in seven prepubertal female control subjects (mean age: 8.59 + 2.01 years). Informed consent for the studies was obtained from parents. The protocol was approved by the Hospital de Pediatria Garrahan of Buenos Aires Research Committee. Endocrine tests. The acute-phase GnRH test was carried out in both girls and boys after intravenous injection of 100 #g GnRH; blood was drawn at - 1 0 , 20, and 60 minutes for serum LH and FSH determinations. The subacute-phase aGnRH test was carried out in prepubertal girls after two subcutaneous injections of 100 ttg of an aGnRH, buserelin acetate (Suprefact; HoechstRoussel), separated by a 24-hour interval. Blood was drawn for determination of serum LH, FSH, and estradiol levels in basal conditions, 24 hours after the first injection, and 4, 6, and 24 hours after the second stimulation. The aim of this test was to evaluate both pituitary LH and FSH responsiveness and ovarian estrogenic responsiveness to endogenous gonadotropic stimulation. Maximal responses for LH ranged from 2.16 to 6.84 U / L and for FSH from 8.92 to 23.9 U / L . Serum estradiol response was considered to be positive when the value obtained was equal to or greater than 55 pmol/L (15 pg/ml) in at least one of the samples. In prepubertal boys, Leydig cell reserve and androgen sensitivity were tested successively according to a technique previously described I~ I I: after drawing blood for basal determination of serum concentrations of testosterone and of SHBG, 2500 IU of human chorionic gonadotropin was administered intramuscularly on days I and 4. On day 7, blood was drawn for measurement of the serum testosterone concentration (after administration of hCG) and 2 mg testosterone enanthate per kilogram body weight was then administered intramuscularly. Blood was drawn again on day 14 for assay of serum SHBG. Serum testosterone response to hCG was considered to be normal when the post-hCG values exceeded 3.5 nmol/L (1 ng/ml). I~ I I The androgen sensitivity was considered to be positive when serum SHBG decreased to less than 80% of the basal value on day 14. t~ I I RESULTS As shown in Table I, basal LH and FSH concentrations were normal in all male and female subjects with CRF. After 60 minutes of GnRH administration, mean serum FSH
48
Castellano et al.
The Journal of Pediatrics January 1993
T a b l e I. Basal and post-GnRH (intravenous) serum L H and F S H levels in prepubertal boys and girls with C R F Serum LH ( m U / m l )
- Serum FSH ( m U / m l )
GnRH Group
Boys CRF After transplantation Control subjects Girls Patients with CRF Control subjects
GnRH
n
Basal
At 20 mln
At 60 mln
Basal
At 20 mln
At 60 mln
9 8 18
1.61 _+ 0.97 1.18 __. 0.78 0.91 + 0.94
2.45 _+ 0.72 2.70 _ 1.13 1.65 +__ 1.05
3.39 _+ 1.80 2.76 ___2.61 2.17 +__ 1.23
1.94 _ 1.07 2.48 + 1.58 1.46 ___ 1.13
2.27 _+ 1.58 3.75 + 2.19 3.69 + 1.44
2.53 _+ 1.34" 5.58 + 4.82 6.25 + 2.84
7 19
1.14 __. 1.52 0.41 ___0.33
2.16 _ 1.64 2.41 ___ 1.94
2.62 ___2.03 2.63 __. 1.50
2.49 + 2.44 1.66 + 1.27
5.66 --- 4.05 5.05 __. 2.68
7.00 _ 3.93 8.39 +__4.66
Boys were studied during conservativetreatment, during long-termdialysis therapy, or after renal transplantation. Girls were studied during conservativetreatment or long-term dialysis only. Values are expressed as mean __.SD. *p <0.01 versus control values.
T a b l e II. Serum testosterone levels before and after acute stimulation with 5000 U I of hCG; serum S H B G levels before and after administration of 2 mg of testosterone enanthate per kilogram of body weight to prepubertal boys with C R F receiving conservative treatment or chorionic dialysis and after renal transplantation Testosterone (nmol/L)
SHBG (nmol/L)
Group
n
Basal value
After hCG
Basal v a l u e
After testosterone enanthate
% Basal v a l u e
Patients with CRF After transplantation
9 8
0.62 _+ 0.62 0.34 _+ 0.01
17.26 _ 13.59 9.49 ___8.43
92.0 ___42.8 96.4 _ 75.6
47.1 _ 22.8 49.9 _+ 24.2
51.7 __. 4.89 62.2 + 17.9
To express testosterone in nanograms per milliliter, divide by 3.467.
levels, but not mean L H levels, were significantly lower (p <0.01) in boys with C R F than in control subjects. After renal transplantation in boys, normal serum LH and F S H responses to acute G n R H were found. In girls with C R F , serum L H and FSH responses to acute G n R H stimulation were normal in comparison with their respective control subjects. In all prepubertal male patients, serum testosterone levels after hCG therapy were greater than 3.5 n m o l / L (1 n g / ml) and serum S H B G levels after testosterone enanthate treatment were equal to or less than 80% of basal value (Table II). In Fig. 1 are shown the serum LH and F S H levels in the girls after subcutaneous a G n R H administration. The mean serum L H concentration increased significantly in control ~ubjects only at 4 and 6 hours after the second a G n R H injection; the mean serum F S H concentration was already significantly greater 24 hours after the first and 4 and 6 hours after the second injection of a G n R H (p <0.01). In the girls with C R F , the serum L H level increased significantly only 6 hours after the second a G n R H injection (p <0.01); serum F S H values did not differ from basal values at any time. No significant difference in serum L H was found between control girls and the girls with C R F at any time. In contrast, the serum F S H concentration in girls with
C R F was significantly lower than in control girls, 4 and 6 hours after the second injection (p <0.02). In six of the seven control girls, serum estradiol concentration was greater than the upper limit of the basal value 24 hours after the first injection and 4 hours after the second one (positive response); one girl did not respond at any time, but her serum L H increased from 0.5 to 2.16 and her serum F S H from 0.85 to 8.92 m U / m l 4 hours after the second injection (not shown). In four of the seven patients with C R F , the serum estradiol concentration increased but in three it did not (Fig. 2). In two of these latter three patients, serum L H and F S H values did not increase after a G n R H administration. Serum estradiol response in the C R F group was significantly different from that in the control group (p <0.05, by chi-square test). Because only some of the girls with C R F had abnormal responses during the a G n R H test and others had normal responses, an attempt was made to correlate these abnormal responses with clinical and laboratory indicators of abnormal renal function. N o correlation was found with chronologic age, bone age, failure to grow, weight/height ratio, duration of C R F , creatinine clearance, or type of treatment. The only striking correlations were that (1) the two girls who had no increase in serum L H and F S H values after
Volume 122 Number !
Hypothalamic-pituitary-gonadal function attd chronic renal failure
t.
,ooo
~z
49
t.
.J o
o @
o E (x
16
**
"8 100
,,
o
o Oo 0
-6
o o
o 9
oo
9
9 9
~h h,
7 8 t,
10'
4
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BT ist
24 t 2rid
4-
6
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Fig.
I. Serum LH and FSH levels before (B) and after subcutaneous administration of 100 tag aGnRH buserelin (Suprefact) to girls with CRF (black bars) and to control girls (white bars). Arrows indicate first and second aGnRH injections.Numbers to the right of the second arrow indicate hours after the second injection. *p <0.05 versus corresponding B; **p <0.0I versus corresponding B; black circles, p <0.02 versus control values.
aGnRH administration had serum albumin concentrations of 12 and 15 gm/L, whereas in the other five patients the range was between 36 and 45 gm/L, and (2) these two girls were the only girls with CRF caused by focal glomerular sclerosis. DISCUSSION Many abnormalities in gonadotropin and sex steroid secretion have been described in adults with CRF. It has been proposed that uremia induces both central disturbances and alterations in the pituitary-dependent endocrine organs) 2q4 Furthermore, delayed bone age and pubertal maturation also have been reported in adolescents with CRF. t'5,15, 16 It has been suggested that abnormalities in the secretion of gonadotropins might have an impact on pubertal development} 7 In prepubertal children the available data on gonadotropin secretion are controversial. High serum LH or FSH basal levels and blunted serum gonadotropic responses to GnRH have been described in prepubertal children with CRF and during their development.5' 6, ]4-IsThese high levels of gonadotropins returned to normal after renal transplantation. In contrast to these reports, and similar to that of Giusti
e ~ ] CRF
24 t
4
6
24 hours
2 nd ol-61C
t Buserelin 1100
Hg SOl
Fig. 2. Serum estradiol levelsbefore and after subcutaneous (SC) administration of 100 tag aGnRH (buserelin) in girls with CRF (black circles) and control (C) subjects (white circles). Squares depict subjects in whom serum concentration of estradiol was never greater than 55 pmol/L (15 pg/ml) either before or after stimulation. Horizontal lines indicate the serum estradiol level of 55 pmol/L (15 pg/ml). Arrows at base of illustration indicate first and second injections of buserelin;arrows at top of illustration indicate estradiol values exceeding the scale. et al., 7 we found normal serum LH and FSH basal levels in children with uremia. Differences in assay sensitivity and specificity might have contributed to these conflicting results. We measured serum LH and FSH by an immunoradiometric assay, which utilizes a monoclonal antibody, and might have avoided c~-subunit interference in the assay. In boys the serum LH response to exogenous GnRH might be due either to chronic lesser priming of pituitary gonadotrophs by GnRH neurons or to a disturbance of pituitary cells. In men with CRF, low plasma testosterone concentrations and an inadequate rise after administration of hCG suggest that endocrine testicular function is impaired) 3, 14 No information on Leydig cell reserve in prepubertaI children with uremia has been reported. We found a normal testosterone response to hCG in our patients, suggesting that testicular steroidogenesis is conserved during prepuberty. It had been speculated that growth hormone, insulin-like growth factor-I, and dexamethasone resistance might be present in CRF.IS'2~Thus we were interested in studying the biologic response to androgens in boys with uremia. We had previously reported that the serum SHBG response to exogenous testosterone enanthate was a reliable test to eval-
50
Castellano et aL
uate androgen sensitivity,l~ tt All our boys with CRF had normal decreases in serum SHBG after receiving exogenous testosterone enanthate, suggesting that the biologic response to androgen is not disturbed. In contrast to our findings in the prepubertal boys, prepubertal girls with CRF had normal LH and FSH responses to GnRH testing. These different responses stimulated our interest in evaluating hypothalamic-pituitary-ovarian function in more detail in our patients. For this purpose, we utilized a modification of the subacute-phase aGnRH test described by Rosenfield et al. 21 Control patients had elevated levels of FSH, but not LH, 24 hours after the second injection. In girls with CRF, serum LH response was normal but that of serum FSH was low after the second injection. Indeed, serum FSH concentration did not increase after aGnRH was injected. Therefore, as speculated for boys, girls with CRF might have decreased FSH responses to GnRH because of less priming of pituitary gonadotrophs as a result of a disturbance of GnRH neurons; alternatively, the toxic effects of uremia may affect the pituitary cells themselves. The evaluation of ovarian steroidogenesis is difficult during the prepubertal period. We took advantage of the subacute-phase aGnRH test to measure serum estradiol increments as a measure of the response of the prepubertal ovary after two LH and FSH discharges. We defined the test result as positive when serum estradiol increases to greater than 55 pmol/L (15 pg/ml), because this value is never reached in prepubertal control girls in basal conditions. Positive responses were observed in more control girls than in girls with CRF. Two of the three negative estradiol responses, however, could be attributed to lack of LH and FSH responses to aGnRH stimulation. These findings suggest that prepubertal ovarian steroidogenic capacity is not impaired in CRF. The two girls in whom aGnRH failed to elicit adequate combined LH, FSH, and estradiol responses had focal glomerular sclerosis, but this failure did not seem to have any connection with hypothalamic-pituitary function. On the other hand, the girls also had low serum albumin levels. Gonadotropic function is known to be altered in severe malnutrition, which could be a factor contributing to an abnormal gonadotropin response in some patients with CRF. Delayed puberty is a common concern in patients with CRF. An abnormality in hypothalamic-pituitary function, such as the one described here, might impair the maturation of the GnRH pulse generator. However, the mechanism of the initiation of puberty is largely unknown, and other factors might be involved. We reported previously that prepubertal boys with CRF share with prepubertal boys with GH deficiency, another model of delayed puberty, a delayed increment in serum bioavailable testosterone during prepuberty. 2~ It has been suggested that patients with CRF have
The Journal of Pediatrics January 1993
peripheral resistance to growth hormone or to insulin-like growth factor 119; this resistance might play a role in their pubertal delay. We conclude that hypothalamic-pituitary function is not normal in some prepubertal boys and girls with CRF and that this abnormality, at least in girls, could be related to secondary malnutrition. In contrast, testicular and ovarian steroidogenic capacity is not impaired. Furthermore, the biologic response to androgens is also preserved. We acknowledge the contributions of P. Rossano, D. Chirico, and M. Maceiras with technical assistance. REFERENCES 1. Ferraris J, Jaenger P, LevineL, et aL Delayedpubertyin males with chronic renal failure. Kidney Int 1980;18:344-50. 2. Sharer K, Chamter C, Brunner FP, et al. Combined report on regular dialysis and transplantation of children in Europe. Proceedings of the European DialysisTransplant Association 1976;13:59-103. 3. Van Diemen-StenvoordeMD, DonckerwolckeRA, Brakel H, Wolff ED, de Gowg MCJW. Growth and sexual maturation in children after kidney transplantation. J PEDIATR 1987; 110:351-6. 4. Holdsworth S, Atkins RC, De Kretser DM. The pituitary-testicular axis in men with chronic renal failure. N Engl J Med i 977;296:1245-9. 5. Ferraris JR, Domene HM, Eseobar ME, Caletti MG, Ramirez JA, Rivarola MA. Hormonal profile in prepubertal females with chronic renal failure before and under haemodialysisand after renal transplantation. Acta Endocrinol (Copenh) 1987; I 15:289-96. 6. Oertel PJ, Liehtwall DK, Hafner S, Raueh W, Schonberg D, Shard K. Hypothalamo-pituitary-gonadal axis in children with chronic renal failure. Kidney Int 1983;115:34-9. 7. Giusti M, Perfumo F, Verrina E, et al. Biologicalactivity of luteinizing hormone in uraemic children: spontaneous nocturnal secretion and changes after administration of exogenous pulsatile luteinizing hormone releasing hormone: preliminary observations. Pediatr Nephrol 1991;5:559-65. 8. CampoS, Monteagudo S, Nieolau G, et al. Testieular function in prepubertal male pseudohermaphroditism.Clin Endocrinol (Oxf) 1981;14:11-22. 9. BelgoroskyA, Rivarola MA. Determination of sex hormone biding globulin in human serum. Medicina (Buenos Aires) 1982;42:141-8. 10. BelgoroskyA, Rivarola MA. Sex hormone binding-globulin response to human chorionicgonadotropin stimulation in children with cryptorchidism, anorchia, male pseudohermaphroditism and micropenis.J Clin Endocrinol Metab 1982;54:698704. 11. Ciaccio M, Rivarola MA, BelgoroskyA. Decreaseof serum sex hormone binding-globulinas a marker of androgen sensitivity: correlation with clinical response. Acta Endocrinol (Copenh) 1989;120:540-4. 12. Guevara A, Hallberg MC, Torn EM, Pohlman C, Wieland RG. Serum gonadotropin and testosterone levels in uremic males undergoing intermittent dialysis. Metabolism 1969; 18:1062-9. 13. Lim US, Fang VS. Gonadal dysfunction in uremic men: a
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Hypothalamic-pituitary-gonadal function and chronic renal failure
study of the hypothalamo-pituitary-testicular axis before and after renal transplantation. Am J Med 1975;58:655-62. Handelsman DJ. Hypothalamic-pituitary-gonadal dysfunction in renal failure: dialysisand renal transplantation. Endocr Rev 1985;6:151-82. Melhls O, Ritz F, Gilli EK, Renser W. Growth in renal failure. Nephrologie 1978;21:237-47. Rizzoni G, Broyer M, Guest G, Fine R, Holliday MA. Growth retardation in children with chronic renal disease: scope of the problem. Am J Kidney Dis 1986;7:256-61. ShSrer K, Shaefer F, Trott M, et al. Pubertal developmentin children with chronic renal failure. In: Laron Z, ed. Growth and endocrinechanges in children and adolescentswith chronic renal failure. Basel: Karger, 1989:151-68.
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18. Rauch KW, Ohlel PJ. Endocrine function. In: Fine CN, Gruskin A, eds. End-stage renal disease in children. Philadelphia: WB Saunders, 1984:296-307. 19. Lee PDK, Hintz RL, Sperry JB, Baxter RC, Powel DR. IGF binding proteins in growth-retarded children with chronic renal failure. Pediatr Res 1989;26:308-14. 20. BelgoroskyA, Ferraris JR, Ramirez H, Jasper H, Rivarola MA. Serum sex hormone-binding globulin and serum nonsex hormone-binding globulin-bound testosterone fractions in prepubertal boys with chronic renal failure. J Clin Endocrinol Metab 1991;73:107-10. 21. RosenfieldRL, Burstein S, Cuttler L, et al. Use of nafarelin for testing pituitary-ovarian function. J Reprod Med 1989; 34:1044-50.
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