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Gonadotropin responses to luteinizing releasing factor in boys treated with cyclophosphamide for nephrotic syndrome
December 1976 The Journal o f P E D I A T R I C S
941
Gonadotropin responses to luteinizing releasing factor in boys treated with cyclophosphamide for nephrotic syndrome Cyclophosphamide therapy of the nephrotic syndrome has been associated with oligo- and azooapermia and with abnormalities of testicular histology in adults and pubertal boys. In 15 prepubertal boys, no abnormalities of basal serum levels of LH, FSH, or T were found when they were studied 8 months to 7 years after eyelophosphamide therapy. Five boys who were pubertal du~qng therapy were found to have elevated mean basal values of gonadotropins with normal testosterone levels and elevated LH responses to LRF; the FSH responses to L R F were elevated in Jbur patients. One of four boys who were prepubertal during therapy but pubertal at the time of testing had an elevated basal LH and L H response to LRF. Three boys who were prepubertal at the times of therapy and testing had normal LH responses to LRF. The L R F test may provide a means of identifying the patient who has sustained testicular injury and who may require testieular biopsy.
Rebecca T. Kirkland, M.D.,* Alfred M. Bongiovanni, M.D., David Cornfeld, M.D., Joseph B. McCormick, M.D., John S. Parks, M.D., and Alfred Tenore, M.D.,
Philadelphia, Pa.
CYCLOPHOSPHAMIDE has been utilized in the therapy of steroid-dependent and steroid-resistant minimalchange nephrotic syndrome in childhood since 1963.1 This alkylating agent has been shown to produce prolonged remissions when used comcomitantly with corticosteroid therapy in patients with the nephrotic syndrome Who have had multiple relapses. ~ The immediate side effects of chemical cystitis and alopecia have occurred in approximately 7 to 33% of patients,-' and the long-term hazards of gonadal injury in the prepubertal and pubertal child are From the Department of Pediatrics, University of Pennsylvania, Children's Hospital of Philadelphia. Supported by Clinical Research Center Grant FRO0240, the National Institutes of Health Public Health Service Grants HDO0371, AM05197, and AMO0004-O1, and in part by a grant from Eli Lilly Pharmaceutical Company. Presented in part at the Society for Pediatric Researeh, Washington, D.C., Spring, 1974. *Reprint address: Department of Pedialrics, Division of Endocrinology, Baylor College of Medicine, 1200 Moursand A re., Houslon, TX 77030.
still under investigation. In adults and pubertal boys complications of cyclophosphamide therapy have included diminished sperm counts and testicular atrophy with absence of germinal cells in biopsied testicular tissue.:' ~ Penso and colleagues" have indicated that the germinal cells in the testes of prepubertal and pubertal Abbreviations used LH: luteinizing hormone FSH: follicle-stimulating hormone LRF: luteinizing hormone releasing factor T: testosterone mlU/ml: milli-international units per milliliter boys may be susceptible to damage by the drug. Since an abnormal elevation of serum luteinizing hormone and follicle-stimulating hormone levels usually occurs by puberty as a result of widespread gonadal injury or castration, ~ the present study was undertaken to further evaluate gonadotropin release in prepubertal and pubertal boys treated with cyclophosphamide, who may have sustained testicular injury.
Vol. 89, No. 6, pp. 941-944
942
Kirkland et al.
The Journal of Pediatrics December 1976
Table I. Cyclophosphamide-treated patients
Case No.
Months of therapy
Age (yr) at onset of therapy
Total dose (gm)
Daily dose (mg/kg)
Interval since drug use(yr)
Age at time ofstudy(yr)
3.8 0.75. 5.0
7.7,2.8 5.2 7.2
3.8 5.5 1.4-2.8
4.0 5.7 5.2
7.8 6.5 10;2
0.95-2.9 2.7 2.3 2.96
3.8 6.0 3.8 4.8
14.8 15.9 14.8 15.8
2.5 2.6 2.5 1.3-2.6 1.5
1.5 3.0 4.5 4.0 5.3
16.5 20.0 19 17 19.9
Group A* 1
2 3 Group Bt 4
5 6 7 Group C$ 8
9 10 11 12
5, 1.5 3.5 5.6 5.0 4, 2.5 3.0 3.0
11 9.5 t1 11
6.7 12, 7.2 9 1.6
3.5 8.5 3.0 10.0 2.4
15 17 15.5 13 14.7
10.2 25.5 14.3 20.0 7.2
*Cases l-3: prepubertal at time of therapy and testing. tCases 4-7: prepubertaI at time of therapy and pubertal at time of testing. $Cases 8-12: pubertal at time of therapy and testing. SUBJECTS Fifteen prepubertal boys, aged 1 to 13.5, were studied 8 months to 7 years following cyclophosphamide therapy for steroid-dependent or steroid-resistant nephrotic syndrome. The total daily oral dose of cyclophosphamide was 0.5 to 18.5 gm. The somatic ages were assessed by the multiple center method s and by the method of Greulich and PyleY Basal serum levels of LH, FSH, and T were obtained. Three prepubertal boys, Group A (Cases 1 to 3, Table l), received luteinizing releasing factor to assess their pituitary-gonadal status. Cases 1. and 2 were receiving alternate-day prednisone therapy at the time of administration of LRF. Four boys, Group B (Cases 4 to 7, Table 1), who were prepubertal at the time of administration of cyclophosphamide, 9.5 to 11 years of age, were studied after the onset of puberty from 14.8 to 15.9 years. They were tested with LRF 3.8 to 6 years following therapy. The sexual development at the time of the study of cases 4, 5, and 7 was Tanner Stage VI~ Case 6 was Tanner Stage III. Cases 4 and 5 had received azathioprine 5 years prior to study. Case 5 was receiving alternate-day prednisone therapy at the time of administration of LRF. Five boys, Group C (Cases 8 to 12, Table I), who were pubertal during cyclophosphamide therapy were 16.5 to 20 years of age at the time of the study and were evaluated 1.5 to 5.3 years following therapy. The pubertal boys were Tanner Stage IV and V; with the exception of case 11 who had unilateral cryptorchidism, they had normal-sized testes bilaterally. Cases 11 and 12 had received azathiprine, and all had received glucocorticoids 3 to 4 years prior to the study.
A second L R F test was performed in Cases 4, 8, 10, and 11 after a 9-month interval. Assessment of renal function included normal creatinine clearances with no evidence of edema or proteinuria in Cases 1 through 12. METHODS
The L R F used in this investigation was synthesized and kindly provided by Dr. Roger Guilliman. After two basal serum samples were obtained, a bolus of 5 0 o r 100/~g of L R F was injected intravenously with serum collected for measurement of L H and F S H levels and testosterone levels (in 5 patients) at intervals up to 4 hours. Serum LH H and F S H ...... expressed in m I U / m l were measured by radioimmunoassay with the double antibody method employing LER 907 as standard. Normal basal levels of LH in our laboratory were 6.8 • 2.0 m I U / m l for prepubertal and 14.1 • 3.8 m l U / m l for pubertal boys, respectively; these corresponded with data of Johanson. TM Normal F S H values were 5.3 -- 2 m I U / m l and 9.2 • 4 m I U / m l for prepubertal and pubertal boys, respectively. The range of response of pubertal boys to L R F was 20 to 100 m I U / m l for LH and 10 to 20 m I U / m l for F S H occurring at 20 minutes and 90 minutes, respectively. Plasma testosterone ':':~ levels were determined by a modification of a radioimmunoassay utilizing rabbit antiserum supplied by Calbiochem, La Jolla, California. The normal levels were < 3 0 0 n g / d l and 300 to 900 n g / d l for prepubertal and pubertal boys, respectively. RESULTS The 15 prepubertal boys, average age 9.7 years, were studied at an average of 2.4 years following treatment
Volume 89 Number 6
Gonadotropin responses to L R F
943
Table II. Serum LH and F S H levels ( m l U / m l ) basal and in response to L R F , increment of elevation of LH and F S H following LRF, and medication received by the patients at the time of testing Case No.
Basal L H
Peak L H
A LH
Basal FSH
Peak FSH
A FSH
Medications
Group A 1
2.4
2 3 Group B 4 5 6 7 Group C 8 9 10 11 12
6.9 5.5
9.5 9.5 17.8
7.1 2.6 12.3
2.0 2.6 5.7
* * *
* * *
Prednisone same day Prednisone day before Nolle
22.4 9.0 10.0 51.75
41.8 64 100.8 133
19.4 55 90.8 81.25
20.2 9.0 4.0 19.8
18.8 14.5 8.0 26.5
- 1.4 5.5 4.0 6.7
None Prednisone None None
22.3 41.6 51.5 44.4 48.25
139.8 119 153 121.8 174
117.5 77.4 101.5 77.4 125.75
36.9 15.0 29.3 19.8 45.2
58.8 15.5 47.5 37.5 72.0
21.9 0.5 18.2 17.7 26.8
None None None None None
*No change from basal levels. with cyclophosphamide. The mean LH was 9.6 _+ 5.34 (SD) m l U / m l and F S H was 5.9 _+ 3.44 (SD) m I U / m l . The mean serum T was 251 ng/dl. The mean somatic age (10/15 patients with an average chronologic age of 9.5 years) was 8.75 years. Three boys who were prepubertal during therapy and at the time of testing (Group A, Table II) had normal basal L H levels (mean 4.95 m I U / m l ) and elevation of LH in response to L R F (mean 12.3 m I U / m l ) . Mean basal F S H was 3.6 m l U / m l with no increase in response to L R F stimulation. O f the four boys of Group B (Table II) who were prepubertal during cyclophosphamide therapy and pubertal at the time o f testing, Case 7 had elevation of the basal L H level, 51.75 m l U / m l , and L H response to LRF, 133.0 m l U / m l . Cases 4 to 6 had normal basal LH levels (mean 13.7 m I U / m l ) and peak LH levels (mean 68.9 m I U / m l ) . Basal FSH levels were slightly elevated in Cases 4 and 7, 20.2 and 19.8 m l U / m l , respectively, with no increase of FSH to L R F in Case 4. The five boys of Group C had a m e a n basal LH of 41.6 m I U / m l and mean basal F S H of 29.2 m l U / m l . These values represented a significant elevation from normal control values (p < 0.001). The L R F stimulation test in these five revealed that all had a peak response of LH greater than 100 m I U / m l (108 to 174 m I U / m l ; mean 141.5 m l U / m l ) . Case 9 had a normal basal F S H and no rise of FSH in response to L R F ; and Cases 8, 10, 11, and 12 had peaks of F S H from 37.5 to 72 m I U / m l with LRF.
Four patients (Cases 4, 8, 10, and 11) had a second L R F test after a 9-month interval. The rise in LH with the initial L R F test (100/,g) in Cases 8 and 10 and with the repeat test (50 #g) revealed that the LH increments in response to the second test with the lower dose of L R F
were higher. With the first test, the increments of rise of L H were 50.2 and 62.1 m I U / m l in Cases 8 and 10, respectively, and with the second test the increments were 117.5 and 101.5 m l U / m l , The F S H increments with the first test were 19.1 and 9.6 m I U / m l in cases 8 and 10, respectively, and 21.9 and 18.2 m I U / m l with the second test. Repeat testing in Cases 4 and 11 demonstrated no significant change from the initial results in LH and F S H responses to LRF. The testosterone levels following L R F administration were measured in Cases 1 to 8 and 10 to 12. The mean basal testosterone of all pubertal boys (Cases 4 to 8 and 10 to 12) was 541.2 ng/dl with a peak (mean 793.4 ng/dl) occurring between 120 and 180 minutes after the LRF dose was administered. This was 90 to 120 minutes after the LH peak was observed. The mean increment of elevation was 252.2 ng/dl. The mean basal testosterone of the three prepuhertaI boys (Cases 1 to 3) was 23.1 ng/dI with no significant elevation following L R F (mean 31.6 ng/dl). DISCUSSION In pubertal boys and adults, reported complications of the cyclophosphamide therapy have included testicular atrophy and oligo- and azoospermia? " These effects may have been related to duration of therapy and to total drug dose.'- ~ Correlation of testicular damage with dose and length of time of administration of cyclophosphamJde has not been conclusive. ' ': The total close of 7.2 and 25.5 gm of cyclophosphamide administered to the pubertal boys over a 3.5 to 10-month period in this study compares favorably with that of Penso and associates." In prepubertal boys, the long-term effect of cyclophosphamide on the testis has not been adequately assessed.':
944
Kirkland et al.
In one study, prepubertal boys who had received the alkylating agent progressed into puberty normally. TM DeGroot and associates 1' reported normal basal gonadotropins. In the present study the prepubertal boys of Group A have not demonstrated any abnormalities of LH, FSH, and testosterone levels or of response of LH, FSH, and testosterone to LRF. However, Case 7 of Group B demonstrated abnormalities of basal L H and peak LH levels of L R F . He was prepubertal at the time of treatment and was studied when he was Tanner Stage V. The finding of the abnormal level of LH is this boy suggests that before the possibility of testicular damage can be excluded, the prepubertal patients in Group A should be restudied at a pubertal age. Studies of gonadotropin levels in pubertal'subjects have revealed elevation of F S H and LH in azoospermic boys,~.._,0 with normal testosterone levels. -'~ Thus the pubertal patients of Group C in this study who received cyclophosphamide may have sustained gonadal injury which was reflected in the elevated basal LH and F S H levels and peak response of LH and F S H to LRF. in addition, the injury may be progressive. The increased response to the second test after a 9-month interval in 2 of the 3 patients in Group C who were retested may represent a continuing inability to sustain a normal hormonal relationship with the gonadostat. Testosterone response following L R F has been found to occur after 4 to 8 hours in pubertal boys or 30 to 100 minutes after the LH peak in 7 of 9 adult males. -'1 The rise in testosterone occurred 20 to 80 minutes following the LH rise during sleep in pubertal boys. = The boys in this study demonstrated a substantial rise in testosterone 90 to 120 minutes after the LH peak occurred. This study as well as others '~, ._,o have shown that the serum T m a y be normal in cyclophosphamide-treated patients. Thus, the gonadotropins and more specifically the LH and F S H responses to L R F may be o f value in providing a noninvasive method of following the recovery phase of the cyclophosphamide-treated patient and in identifying those patients who may require additional study by sperm counts and testicular biopsies. We are grateful to Miss Jean Marino for her technical assistance. REFERENCES
1. Coldbeck JI-t: Experience with alkylating agents in the treatment of children with the nephrotic syndrome, Med J Aust 2:987, 1963. 2. Worrall PM, Elliott DL, and Harris JR: Retrospective study of 481 patients with minimal change nephrotic syndrome treated with cyclophosphamide, Third International Symposium of Pediatric Nephrology Abstract, Washington, DC, 1974, p 26.
The Journal of Pediatrics December 1976
3. Fairley KF, Barrie JU, and Johnson W: Sterility and testicular atrophy related to cyclophosphamide therapy. Lancet 1:568, 1972. 4. Qureshi MSA, Goldsmith H J, Pennington JH, and Cox PE: Cyclophosphamide therapy and sterility, Lancet 2:1290, 1972. 5. Rapola J, Koskimies O, Huttunen NP, Floman P, Vilska J. and Hallman N: Cyclophosphamide and the pubertal testis. Lancet 1:98, 1973. 6. Penso J, Lippe B, Ehrlich R, and Smith FG: Testicular function in prepubertal and pubertal male patients treated with cyclophosphamide for nephrotic syndrome, J PEDIAIa 84:831, 1974. 7. Winter JSD, and Faiman C: Serum gonadotropin secretion in agonadal children and adults, J Clin Endocrinol Metab 35:561, 1972. 8. Wilkins L: The diagnosis and treatment of endocrine disorders in childhood and adolescence, ed 3 in Blizzard RM, and Migeon CJ, editors: Springfield, Ill, 1965, Charles C Thomas, Publisher, pp 37-39. 9. Greulich WW, and Pyle SI: Radiographic atlas of skeletal development of the hand and wrist, ed 2, Stanford, Calif, 1959, Stanford University Press. 10. Tanner JM: Growth and endocrinology of the adolescent, in Gardner LI, editor: Endocrine and genetic diseases of childhood, Philadelphia, 1969, WB Saunders Company, pp 19-60. 11. Odell WP, Rayford PL, and Ross GT: Simple, partially automated method for radioimmunoassay of human thyroid stimulating, growth, luteinizing and follicle stimulating hormone, J Lab Clin Med 70:973, 1967. 12. Midgley AR: Radioimmunoassay for human follicle-stimulating hormone, J Clin Endocrinol Metab 27:295, 1967. 13. Faiman C, and Ryan RH: Radioimmunoassay of human FSH, J Clin Endocrinol Metab 27:444, 1967. 14. Johanson AJ: FSH and LH in the serum and urine of normal children and adults and in endocrine disorders, Recent advances in endocrinology, Proc Seventh Pan-Am Congress of Endocrinol, Sao Paulo, Brazil, 1970, p 182. 15. Coyotupa JA, Parlow AF, and Abraham GE: Simultaneous radioimmunoassay of plasma for testosterone and dihydrotestosterone, Anal Lett 5:329, 1972. 16. Furuyama S, Mayes DM, and Nugent CA: A radioimmunoassay for plasma testosterone, Steroids 16:415, 1970. 17. Buchanan JD, Fairley KF, and Barrie JU: Return of spermatogenesis after stopping cyclophosphamide therapy, Lancet 1:156, 1975. 18. Arneil GC: Cyclophosphamide and the prepubertal testis, Lancet 2:1259, t972. 19. DeC-root GW, Faiman C, and Winter JSD: Cyclophosphamide and the prepubertal gonad: a negative report, J PEDIATR 84:123, 1974. 20. Ettledorf JN; West C, Pitcock J, and Williams D: Gonadal function, testicular histology, and meiosis following cyclophosphamide therapy in patients with nephrotic syndrome, J PEDIATR 88:206, 1976. 21. Roth JC, Grumbach MM, and Kaplan SL: Effect of synthetic luteinizing hormone-releasing factor on serum testosterone and gonadotropins in prepubertal, pubertal and adult males, J Ctin Endocrinol Metab 37:680, 1973. 22. Judd HL, Parker DC, and Yen SSC: The physiological role of the sleep related rise during puberty in boys, Endocrine Society, Atlanta, G-a, June, 1974, Abstract, 2, A-56.
941
Gonadotropin responses to luteinizing releasing factor in boys treated with cyclophosphamide for nephrotic syndrome Cyclophosphamide therapy of the nephrotic syndrome has been associated with oligo- and azooapermia and with abnormalities of testicular histology in adults and pubertal boys. In 15 prepubertal boys, no abnormalities of basal serum levels of LH, FSH, or T were found when they were studied 8 months to 7 years after eyelophosphamide therapy. Five boys who were pubertal du~qng therapy were found to have elevated mean basal values of gonadotropins with normal testosterone levels and elevated LH responses to LRF; the FSH responses to L R F were elevated in Jbur patients. One of four boys who were prepubertal during therapy but pubertal at the time of testing had an elevated basal LH and L H response to LRF. Three boys who were prepubertal at the times of therapy and testing had normal LH responses to LRF. The L R F test may provide a means of identifying the patient who has sustained testicular injury and who may require testieular biopsy.
Rebecca T. Kirkland, M.D.,* Alfred M. Bongiovanni, M.D., David Cornfeld, M.D., Joseph B. McCormick, M.D., John S. Parks, M.D., and Alfred Tenore, M.D.,
Philadelphia, Pa.
CYCLOPHOSPHAMIDE has been utilized in the therapy of steroid-dependent and steroid-resistant minimalchange nephrotic syndrome in childhood since 1963.1 This alkylating agent has been shown to produce prolonged remissions when used comcomitantly with corticosteroid therapy in patients with the nephrotic syndrome Who have had multiple relapses. ~ The immediate side effects of chemical cystitis and alopecia have occurred in approximately 7 to 33% of patients,-' and the long-term hazards of gonadal injury in the prepubertal and pubertal child are From the Department of Pediatrics, University of Pennsylvania, Children's Hospital of Philadelphia. Supported by Clinical Research Center Grant FRO0240, the National Institutes of Health Public Health Service Grants HDO0371, AM05197, and AMO0004-O1, and in part by a grant from Eli Lilly Pharmaceutical Company. Presented in part at the Society for Pediatric Researeh, Washington, D.C., Spring, 1974. *Reprint address: Department of Pedialrics, Division of Endocrinology, Baylor College of Medicine, 1200 Moursand A re., Houslon, TX 77030.
still under investigation. In adults and pubertal boys complications of cyclophosphamide therapy have included diminished sperm counts and testicular atrophy with absence of germinal cells in biopsied testicular tissue.:' ~ Penso and colleagues" have indicated that the germinal cells in the testes of prepubertal and pubertal Abbreviations used LH: luteinizing hormone FSH: follicle-stimulating hormone LRF: luteinizing hormone releasing factor T: testosterone mlU/ml: milli-international units per milliliter boys may be susceptible to damage by the drug. Since an abnormal elevation of serum luteinizing hormone and follicle-stimulating hormone levels usually occurs by puberty as a result of widespread gonadal injury or castration, ~ the present study was undertaken to further evaluate gonadotropin release in prepubertal and pubertal boys treated with cyclophosphamide, who may have sustained testicular injury.
Vol. 89, No. 6, pp. 941-944
942
Kirkland et al.
The Journal of Pediatrics December 1976
Table I. Cyclophosphamide-treated patients
Case No.
Months of therapy
Age (yr) at onset of therapy
Total dose (gm)
Daily dose (mg/kg)
Interval since drug use(yr)
Age at time ofstudy(yr)
3.8 0.75. 5.0
7.7,2.8 5.2 7.2
3.8 5.5 1.4-2.8
4.0 5.7 5.2
7.8 6.5 10;2
0.95-2.9 2.7 2.3 2.96
3.8 6.0 3.8 4.8
14.8 15.9 14.8 15.8
2.5 2.6 2.5 1.3-2.6 1.5
1.5 3.0 4.5 4.0 5.3
16.5 20.0 19 17 19.9
Group A* 1
2 3 Group Bt 4
5 6 7 Group C$ 8
9 10 11 12
5, 1.5 3.5 5.6 5.0 4, 2.5 3.0 3.0
11 9.5 t1 11
6.7 12, 7.2 9 1.6
3.5 8.5 3.0 10.0 2.4
15 17 15.5 13 14.7
10.2 25.5 14.3 20.0 7.2
*Cases l-3: prepubertal at time of therapy and testing. tCases 4-7: prepubertaI at time of therapy and pubertal at time of testing. $Cases 8-12: pubertal at time of therapy and testing. SUBJECTS Fifteen prepubertal boys, aged 1 to 13.5, were studied 8 months to 7 years following cyclophosphamide therapy for steroid-dependent or steroid-resistant nephrotic syndrome. The total daily oral dose of cyclophosphamide was 0.5 to 18.5 gm. The somatic ages were assessed by the multiple center method s and by the method of Greulich and PyleY Basal serum levels of LH, FSH, and T were obtained. Three prepubertal boys, Group A (Cases 1 to 3, Table l), received luteinizing releasing factor to assess their pituitary-gonadal status. Cases 1. and 2 were receiving alternate-day prednisone therapy at the time of administration of LRF. Four boys, Group B (Cases 4 to 7, Table 1), who were prepubertal at the time of administration of cyclophosphamide, 9.5 to 11 years of age, were studied after the onset of puberty from 14.8 to 15.9 years. They were tested with LRF 3.8 to 6 years following therapy. The sexual development at the time of the study of cases 4, 5, and 7 was Tanner Stage VI~ Case 6 was Tanner Stage III. Cases 4 and 5 had received azathioprine 5 years prior to study. Case 5 was receiving alternate-day prednisone therapy at the time of administration of LRF. Five boys, Group C (Cases 8 to 12, Table I), who were pubertal during cyclophosphamide therapy were 16.5 to 20 years of age at the time of the study and were evaluated 1.5 to 5.3 years following therapy. The pubertal boys were Tanner Stage IV and V; with the exception of case 11 who had unilateral cryptorchidism, they had normal-sized testes bilaterally. Cases 11 and 12 had received azathiprine, and all had received glucocorticoids 3 to 4 years prior to the study.
A second L R F test was performed in Cases 4, 8, 10, and 11 after a 9-month interval. Assessment of renal function included normal creatinine clearances with no evidence of edema or proteinuria in Cases 1 through 12. METHODS
The L R F used in this investigation was synthesized and kindly provided by Dr. Roger Guilliman. After two basal serum samples were obtained, a bolus of 5 0 o r 100/~g of L R F was injected intravenously with serum collected for measurement of L H and F S H levels and testosterone levels (in 5 patients) at intervals up to 4 hours. Serum LH H and F S H ...... expressed in m I U / m l were measured by radioimmunoassay with the double antibody method employing LER 907 as standard. Normal basal levels of LH in our laboratory were 6.8 • 2.0 m I U / m l for prepubertal and 14.1 • 3.8 m l U / m l for pubertal boys, respectively; these corresponded with data of Johanson. TM Normal F S H values were 5.3 -- 2 m I U / m l and 9.2 • 4 m I U / m l for prepubertal and pubertal boys, respectively. The range of response of pubertal boys to L R F was 20 to 100 m I U / m l for LH and 10 to 20 m I U / m l for F S H occurring at 20 minutes and 90 minutes, respectively. Plasma testosterone ':':~ levels were determined by a modification of a radioimmunoassay utilizing rabbit antiserum supplied by Calbiochem, La Jolla, California. The normal levels were < 3 0 0 n g / d l and 300 to 900 n g / d l for prepubertal and pubertal boys, respectively. RESULTS The 15 prepubertal boys, average age 9.7 years, were studied at an average of 2.4 years following treatment
Volume 89 Number 6
Gonadotropin responses to L R F
943
Table II. Serum LH and F S H levels ( m l U / m l ) basal and in response to L R F , increment of elevation of LH and F S H following LRF, and medication received by the patients at the time of testing Case No.
Basal L H
Peak L H
A LH
Basal FSH
Peak FSH
A FSH
Medications
Group A 1
2.4
2 3 Group B 4 5 6 7 Group C 8 9 10 11 12
6.9 5.5
9.5 9.5 17.8
7.1 2.6 12.3
2.0 2.6 5.7
* * *
* * *
Prednisone same day Prednisone day before Nolle
22.4 9.0 10.0 51.75
41.8 64 100.8 133
19.4 55 90.8 81.25
20.2 9.0 4.0 19.8
18.8 14.5 8.0 26.5
- 1.4 5.5 4.0 6.7
None Prednisone None None
22.3 41.6 51.5 44.4 48.25
139.8 119 153 121.8 174
117.5 77.4 101.5 77.4 125.75
36.9 15.0 29.3 19.8 45.2
58.8 15.5 47.5 37.5 72.0
21.9 0.5 18.2 17.7 26.8
None None None None None
*No change from basal levels. with cyclophosphamide. The mean LH was 9.6 _+ 5.34 (SD) m l U / m l and F S H was 5.9 _+ 3.44 (SD) m I U / m l . The mean serum T was 251 ng/dl. The mean somatic age (10/15 patients with an average chronologic age of 9.5 years) was 8.75 years. Three boys who were prepubertal during therapy and at the time of testing (Group A, Table II) had normal basal L H levels (mean 4.95 m I U / m l ) and elevation of LH in response to L R F (mean 12.3 m I U / m l ) . Mean basal F S H was 3.6 m l U / m l with no increase in response to L R F stimulation. O f the four boys of Group B (Table II) who were prepubertal during cyclophosphamide therapy and pubertal at the time o f testing, Case 7 had elevation of the basal L H level, 51.75 m l U / m l , and L H response to LRF, 133.0 m l U / m l . Cases 4 to 6 had normal basal LH levels (mean 13.7 m I U / m l ) and peak LH levels (mean 68.9 m I U / m l ) . Basal FSH levels were slightly elevated in Cases 4 and 7, 20.2 and 19.8 m l U / m l , respectively, with no increase of FSH to L R F in Case 4. The five boys of Group C had a m e a n basal LH of 41.6 m I U / m l and mean basal F S H of 29.2 m l U / m l . These values represented a significant elevation from normal control values (p < 0.001). The L R F stimulation test in these five revealed that all had a peak response of LH greater than 100 m I U / m l (108 to 174 m I U / m l ; mean 141.5 m l U / m l ) . Case 9 had a normal basal F S H and no rise of FSH in response to L R F ; and Cases 8, 10, 11, and 12 had peaks of F S H from 37.5 to 72 m I U / m l with LRF.
Four patients (Cases 4, 8, 10, and 11) had a second L R F test after a 9-month interval. The rise in LH with the initial L R F test (100/,g) in Cases 8 and 10 and with the repeat test (50 #g) revealed that the LH increments in response to the second test with the lower dose of L R F
were higher. With the first test, the increments of rise of L H were 50.2 and 62.1 m I U / m l in Cases 8 and 10, respectively, and with the second test the increments were 117.5 and 101.5 m l U / m l , The F S H increments with the first test were 19.1 and 9.6 m I U / m l in cases 8 and 10, respectively, and 21.9 and 18.2 m I U / m l with the second test. Repeat testing in Cases 4 and 11 demonstrated no significant change from the initial results in LH and F S H responses to LRF. The testosterone levels following L R F administration were measured in Cases 1 to 8 and 10 to 12. The mean basal testosterone of all pubertal boys (Cases 4 to 8 and 10 to 12) was 541.2 ng/dl with a peak (mean 793.4 ng/dl) occurring between 120 and 180 minutes after the LRF dose was administered. This was 90 to 120 minutes after the LH peak was observed. The mean increment of elevation was 252.2 ng/dl. The mean basal testosterone of the three prepuhertaI boys (Cases 1 to 3) was 23.1 ng/dI with no significant elevation following L R F (mean 31.6 ng/dl). DISCUSSION In pubertal boys and adults, reported complications of the cyclophosphamide therapy have included testicular atrophy and oligo- and azoospermia? " These effects may have been related to duration of therapy and to total drug dose.'- ~ Correlation of testicular damage with dose and length of time of administration of cyclophosphamJde has not been conclusive. ' ': The total close of 7.2 and 25.5 gm of cyclophosphamide administered to the pubertal boys over a 3.5 to 10-month period in this study compares favorably with that of Penso and associates." In prepubertal boys, the long-term effect of cyclophosphamide on the testis has not been adequately assessed.':
944
Kirkland et al.
In one study, prepubertal boys who had received the alkylating agent progressed into puberty normally. TM DeGroot and associates 1' reported normal basal gonadotropins. In the present study the prepubertal boys of Group A have not demonstrated any abnormalities of LH, FSH, and testosterone levels or of response of LH, FSH, and testosterone to LRF. However, Case 7 of Group B demonstrated abnormalities of basal L H and peak LH levels of L R F . He was prepubertal at the time of treatment and was studied when he was Tanner Stage V. The finding of the abnormal level of LH is this boy suggests that before the possibility of testicular damage can be excluded, the prepubertal patients in Group A should be restudied at a pubertal age. Studies of gonadotropin levels in pubertal'subjects have revealed elevation of F S H and LH in azoospermic boys,~.._,0 with normal testosterone levels. -'~ Thus the pubertal patients of Group C in this study who received cyclophosphamide may have sustained gonadal injury which was reflected in the elevated basal LH and F S H levels and peak response of LH and F S H to LRF. in addition, the injury may be progressive. The increased response to the second test after a 9-month interval in 2 of the 3 patients in Group C who were retested may represent a continuing inability to sustain a normal hormonal relationship with the gonadostat. Testosterone response following L R F has been found to occur after 4 to 8 hours in pubertal boys or 30 to 100 minutes after the LH peak in 7 of 9 adult males. -'1 The rise in testosterone occurred 20 to 80 minutes following the LH rise during sleep in pubertal boys. = The boys in this study demonstrated a substantial rise in testosterone 90 to 120 minutes after the LH peak occurred. This study as well as others '~, ._,o have shown that the serum T m a y be normal in cyclophosphamide-treated patients. Thus, the gonadotropins and more specifically the LH and F S H responses to L R F may be o f value in providing a noninvasive method of following the recovery phase of the cyclophosphamide-treated patient and in identifying those patients who may require additional study by sperm counts and testicular biopsies. We are grateful to Miss Jean Marino for her technical assistance. REFERENCES
1. Coldbeck JI-t: Experience with alkylating agents in the treatment of children with the nephrotic syndrome, Med J Aust 2:987, 1963. 2. Worrall PM, Elliott DL, and Harris JR: Retrospective study of 481 patients with minimal change nephrotic syndrome treated with cyclophosphamide, Third International Symposium of Pediatric Nephrology Abstract, Washington, DC, 1974, p 26.
The Journal of Pediatrics December 1976
3. Fairley KF, Barrie JU, and Johnson W: Sterility and testicular atrophy related to cyclophosphamide therapy. Lancet 1:568, 1972. 4. Qureshi MSA, Goldsmith H J, Pennington JH, and Cox PE: Cyclophosphamide therapy and sterility, Lancet 2:1290, 1972. 5. Rapola J, Koskimies O, Huttunen NP, Floman P, Vilska J. and Hallman N: Cyclophosphamide and the pubertal testis. Lancet 1:98, 1973. 6. Penso J, Lippe B, Ehrlich R, and Smith FG: Testicular function in prepubertal and pubertal male patients treated with cyclophosphamide for nephrotic syndrome, J PEDIAIa 84:831, 1974. 7. Winter JSD, and Faiman C: Serum gonadotropin secretion in agonadal children and adults, J Clin Endocrinol Metab 35:561, 1972. 8. Wilkins L: The diagnosis and treatment of endocrine disorders in childhood and adolescence, ed 3 in Blizzard RM, and Migeon CJ, editors: Springfield, Ill, 1965, Charles C Thomas, Publisher, pp 37-39. 9. Greulich WW, and Pyle SI: Radiographic atlas of skeletal development of the hand and wrist, ed 2, Stanford, Calif, 1959, Stanford University Press. 10. Tanner JM: Growth and endocrinology of the adolescent, in Gardner LI, editor: Endocrine and genetic diseases of childhood, Philadelphia, 1969, WB Saunders Company, pp 19-60. 11. Odell WP, Rayford PL, and Ross GT: Simple, partially automated method for radioimmunoassay of human thyroid stimulating, growth, luteinizing and follicle stimulating hormone, J Lab Clin Med 70:973, 1967. 12. Midgley AR: Radioimmunoassay for human follicle-stimulating hormone, J Clin Endocrinol Metab 27:295, 1967. 13. Faiman C, and Ryan RH: Radioimmunoassay of human FSH, J Clin Endocrinol Metab 27:444, 1967. 14. Johanson AJ: FSH and LH in the serum and urine of normal children and adults and in endocrine disorders, Recent advances in endocrinology, Proc Seventh Pan-Am Congress of Endocrinol, Sao Paulo, Brazil, 1970, p 182. 15. Coyotupa JA, Parlow AF, and Abraham GE: Simultaneous radioimmunoassay of plasma for testosterone and dihydrotestosterone, Anal Lett 5:329, 1972. 16. Furuyama S, Mayes DM, and Nugent CA: A radioimmunoassay for plasma testosterone, Steroids 16:415, 1970. 17. Buchanan JD, Fairley KF, and Barrie JU: Return of spermatogenesis after stopping cyclophosphamide therapy, Lancet 1:156, 1975. 18. Arneil GC: Cyclophosphamide and the prepubertal testis, Lancet 2:1259, t972. 19. DeC-root GW, Faiman C, and Winter JSD: Cyclophosphamide and the prepubertal gonad: a negative report, J PEDIATR 84:123, 1974. 20. Ettledorf JN; West C, Pitcock J, and Williams D: Gonadal function, testicular histology, and meiosis following cyclophosphamide therapy in patients with nephrotic syndrome, J PEDIATR 88:206, 1976. 21. Roth JC, Grumbach MM, and Kaplan SL: Effect of synthetic luteinizing hormone-releasing factor on serum testosterone and gonadotropins in prepubertal, pubertal and adult males, J Ctin Endocrinol Metab 37:680, 1973. 22. Judd HL, Parker DC, and Yen SSC: The physiological role of the sleep related rise during puberty in boys, Endocrine Society, Atlanta, G-a, June, 1974, Abstract, 2, A-56.