Gonadal function, testicular histology, and meiosis following cyclophosphamide therapy in patients with nephrotic syndrome

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February 1976 The Journal o f P E D I A T R I C S

Gonadal function, testicular histology, and meiosis following cyclophosphamide therapy in patients with nephrotic syndrome Gonadal function, histology, and meiosis were studied in patients after puberty who had received cyclophosphamide in the treatment of nephrotic syndrome. Four males with minimal lesion nephrotic syndrome received cyclophosphamide, 2 to 4 mg/kg/24 hr, during periods ranging from 49 to 60 days and had normal semen at 15 3/12 to 22 3/12 years of age. Four males who received cyclophosphamide, 2 to 5 mg/kg/24 hr, during 89 to 489 days were azoospermic at 15 6/12 to 21 11/12years of age. Plasma testosterone levels were normal in both groups. L H and F S H levels were elevated in the azoospermicgroup. Light and electron microscopy and meiotic analysis o f testicular tissue were normal in two normospermic patients tested. Germinal cells were absent but Leydig and Sertoli cells were present in three azoospermic patients tested. No neoplasia was observed. Two preadolescent and two postadolescent males received 64 to 129 mg/kg of cyclophosphamide during 14 to 45 days and have since fathered normal children. One preadolescent female received 182 mg/kg o f cyclophosphamide during 49 days and later bore a normal child. Two preadolescent females who died o f complications o f their nephrotic syndrome during childhood after courses o f cyclophosphamide had normal preadolescent ovaries.

James N. Etteldorf, M.D.,* Clark D. West, M.D., James A. Pitcock, M.D., and Dorothy L. Williams, M.D., Memphis, Tenn., and Cincinnati, Ohio

THE THERAPEUTIC VALUE of cyclophosphamide in frequently relapsing nephrotic syndrome, as well as in other nonfatal and fatal conditions, is well established. 1-5 The potential gonadal dysfunction, which may be associated with its use, has caused an international study From the Departments of Pediatrics and Pathology, The University o f Tennessee Center for the Health Sciences, Baptist, Memorial Hospital, and the Department of Pediatrics, College o f Medicine, University of CincinnatL Supported in part by United States Public Health Service Grants HE-00454 and HL-05285, and by a grant (RR-211) from the General Clinical Research Centers Program o f the Division of Research Resources, National Institutes o f Health. Presented at the Third International Symposium of Pediatric Nephrology, Washington, D. C., September 28-October 1, 1974. *Reprint address: Dobbs Research Institute, The University of Tennessee Centerfor the Health Sciences, 951 Court A re. Room 544D, Memphis, Tenn. 38163.

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group to recommend abandonment of its use in the treatment of nephrotic syndrome pending further toxicity studies despite a demonstrated therapeutic benefit in a controlled study. +-+ Abbreviations used NS: nephrotic syndrome MLNS: minimal lesion nephrotic syndrome Experimental evidence, as well as observations in human beings, leaves no doubt that cyclophosphamide is capable of producing permanent gonadal dysfunction2 -1+ Reversible side effects such as leukopenia, alopecia, and hematuria have been seen in human beings. Following an international conference in New York City in 1972, TM to evaluate the risk/benefit aspects associated with the use of cyclophosphamide in the treatment of NS, systemic lupus erythematosus, and rheumatoid arthritis, it was recommended that the use o f this agent be limited to life-threatening conditions. We presented

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Effects of cyclophosphamide in nephrotic syndrome

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Table I. Clinical data

Patient (diagnosis)

Age at onset of nephritis (yo

Normospermic patients Group A G.K. (MLNS) 5 1/12 J.M.t (MLNS) 6 6/12 M.W.t (MLNS) 2 9/12 D.O.t (MLNS) 4 6/12 Azoospermic patients C.A. (MLNS) 7 11/12 C.P.t (MLNS) 5 N.S.t (MLNS) 6 5/12

J.P.$ (MLNS)

8 6/12

Period since eyclophosDose cyclophosphamide phamide Age at Cumulative I 24hr I Total evaluation treatment (mg/kg) (mg/kg) (gin) (yr) (yr)

Age at onset of cyclophosphamide treatment (yr)

Duration of cyclophosphamide treatment (days)

12 4/12 9 10 9/12 13

60 50 52 49

180 200 104 196

11 9/12 7 7/12 7 7/12

134 265 406

12 1/12

83 489 89

8 9/12

3 4 2 4

10.5 6.2 8.3 14.3

20 15 18 22

5/12 3/12 7/12 3/12

536 1325 1015

4 5 2.5

13.4 39.3 23.1

21 11/12 18 4/12 19 2/12

166 1181 312

2.0 av2.4 3.5

6.8 29.9 11.75

Side effects*

7 11/12 6 2/12 7 8/12 9 1/12

A L L,A L,A,C

9 10/12 10 10 6/12 #1

L,A,C L,A L,A

6 10/12 #2 15 6/12

6 6/12

A,C

MLNS = minimal lesion nephrotic syndrome. *A = alopeeia, L = leukopenia, C = h e m o r r h a g i c cystitis. tTesticular biopsy. $Oligospermia.

preliminary data at this conference that sterility in males who received cyclophosphamide during preadolescence appeared to be dose related and that beneficial therapeutic effects in children with NS are possible with doses which will not result in azoospermia later in life. We wish to record our observations of gonadal function, histology, and meiosis during postadolescence in ten males and three females who received cyclophosphamide during preadolescence, and in two males who received the drug during early adolescence. All patients were being treated for nephrotic syndrome. Our observations include sperm counts, determinations of plasma levels of testosterone, luteinizing hormone, and follicle-stimulating hormone, and reproductive histories. Testicular biopsies were studied by light and electron microscopy, and for meiosis.

PATIENT GROUPS Group A. Eight males with frequently relapsing minimal lesion nephrotic syndrome were treated with a combination of cyclophosphaniide and prednisone at ages 7 7/12 to 13 years (Table I). The cumulative dose of cyclophosphamide during single courses of therapy ranged from 104 to 1,325 mg/kg over periods of 49 to 406 days. One patient (N.S.) received two courses for 406 and 83 days, respectively. The period from discontinuation of cyclophosphamide therapy to the time of this study ranged from 6 2/12 years to 10 6/12 years. Sperm counts

and plasma levels of testosterone, LH, and FSH were determined when the patients were between 15 3/12 and 22 3/12 years of age (Table II). Testicular biopsies obtained in five of these patients at the time of hormonal investigations were examined by light and electron microscopy and studied for meiotic chromosomal phases. Group B. Two preadolescent (Patients T.D. and M. Wes) and two postadolescent (Patients T.P. and T.K.) males and one preadolescent female (Patient T.L.) with nephrotic syndrome due to a variety of conditions received 64 to 182 mg/kg of cyclophosphamide in addition to prednisone for 14 to 49 days, and their reproductive histories were evaluated at ages 18 11/12 to 24 10/12 years, which is 6 4/12 to 10 8/12 years later (Table III). Group C. Ovarian histology was studied in two children who died with pulmonary thrombosis and aspiration pneumonia at 5 and 6 years of age, respectively, after receiving cumulative doses of 269 mg/kg and 171 mg/kg of cyclophosphamide during multiple courses of therapy.

METHODS Plasma testosterone levels were measured by radioimmunoassay including preassay chromatography on alumina columns as described by Furuyama and associates? 7 Plasma FSH and LH levels were assayed by the two-stage, double-antibody radioimmunoassay of Morgan and colleaguesTM as applied by Wiser and associates? 9

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Table IL Testicular functions

Patients (diagnosis)

Testosterone (ng%)

Group A Normospermia G.K. J.M. M.W. D.O. Azoospermia C.A. C.P.

FSH (mlU/m 0

S p e rcount m

LH (mlU/ml)

Testicular biopsy

• 1061mO

1,021 535 782 720

5 5 7 7

11 10 12 16

50* 50* 87* 60*

650 808

40 14

64 22

0 0

677 1,068

54 7

41 29

0 5 (sluggish)

450-1,000

6.3-15.8

5.5-22

50-60 m i n i m u m for fertility

N.S. J.P.

GK, MW, DO: Normal

CP, NS: Absence of meiotic figures; absence of spermatogenesis; tubules lined by Sertoli cells; Leydig cells prominent

Normal adults

*Highly motile/normal morphology.

T a b l e HI.

Clinical data

Patient (diagnosis)

Age at onset of nephritis (yr)

Males fathering children Group B T.P. (MPG) 3 7/12 T.K.(memb neph)

15 9/12

Age at onset of cyclophosphamide treatment

(yr)

Duration of cyclophosphamide treatment (days)

15 5/12

30

129

16 10/12 17 11/12

29 16 45 22 14

58 45 103 66 64IV

T.D.(MLNS) 1 6/12 9 10/12 M.Wes. (FGN) 11 11/12 12 9/12 Female bearing a child T.L. (MLNS) 11 4/12 11 8/12 49 Group C Females with "normal preadolescent ovaries" at autopsy J.R.~ (MLNS) 11/12 3 10/12 46 5 5/12 24 6 4/12 15 85 D.B.$ (FGS) 5 1/12 5 2/12 29 5 7/12 14 43

Dose cyclophosphamide Cumulative I 24 hr [ Total (mg/kg) (mg/kg) (gin)

Age birth of child or autopsy (yr)

Period since cyclophosphamide treatment

(yr)

Side effects*

21 10/12 23 10/12 24 10/12

6 4/12 8 4/12 7

A,L

20 7/12 22 4/12

10 8/12 10 4/12

A,L L

8.4

18 11/12

7 1/12

L,C

3.55 2.3 1.35 7.20 2.7 .7 3.4

6 5/12

1 7/12 8/12 6 days

A,L A,L

5 10/12

7/12 3/12

A,L

4.3

6.8

2.0 2.8 av2.3 3.0 4.6

3.1 2.6 5.7 2.2 2.2

182

3.7

143 91 35 269 136 35 171

3.1 3.8 2.3 av3.2 4.7 2.5 av4.0

A

Abbreviations used: MLNS = minimal lesion nephrotic syndrome, memb neph = membranous nephropathy, FGS = focal glomerulosclerosis, FGN = focal glornerulonephritis; MPG = mesangial proliferative glomerulonephrifis. *A = alopecia, L = leukopenia, C = hemorrhagic cystitis. tDied of pulmonary artery thrombosis. SDied of aspiration.

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Fig. 1. Biopsy reveals normal spermatogenesis (SP) in patient with short-term therapy. Leydig (L) and Sertoli (St) cells are normal. (H & E. x 250.)

Fig. 2. Spermatogenesis is absent in patient with prolonged therapy leaving only Sertoli cells (St). The tubules are slightly smaller. Leydig cells (L) are not remarkable. (H & E. x 250.)

Semen was collected at 9 A.M., and a 1:20 dilution in formalinized 5% sodium bicarbonate solution was prepared and placed in a hemocytometer for counting. Morphology and motility were studied in undiluted semen. Smears of semen were also stained by the Giemsa method. Testicular tissue was obtained by open surgical biopsy following informed consent. Tissue was processed for light and electron microscopy by standard techniques. Meiotic chromosomes were prepared using a modification of a previously published procedure. 2~ A phosphate buffer with a pH of 7.3 was used for preparing 0.9% sodium chloride and 0.7% sodium citrate. The fixative was methyl alcohol-glacial acetic acid (3:1 v/v). The tissue was cut into 2 to 3 mm cubes and placed in buffered 0.9% sodium chloride at 35~ for 20 minutes. It was then incubated in buffered 0.7% sodium citrate at 35~ for 60 minutes. The cubed tissue was fixed for three minutes and then minced in fi afive, obtaining a fine cell suspension. The slides were prepared by the air drying technique, fixed for an additional ten minutes (total 30 minutes), and stained with Giemsa.

The cumulative dose ranged from 104 to 200 mg/kg (mean 145 mg/kg). Plasma levels of testosterone, FSH, and LH were normal when measured at ages 15 3/12 to 22 3/12 years (Table II). The time following therapy ranged from 6 2/12 to 9 1/12 years. It is of interest that these quantifies of cyclophosphamide, when combined with prednisone therapy, resulted in permanent remissions of nephrotic syndrome in these patients as in the azoospermic patients of this group. In contrast, azoospermia occurred in Patients C.A., C.P., and N.S. and oligospermia in Patient J.P. with alterations in hormonal secretion when the daily doses o f cyclophosphamide ranged from 2.4 to 5 mg/kg, average 3.7 mg/kg, during 89 to 489 days; the cumulative dose ranged from 312 to 1,325 mg/kg. Plasma LH levels were elevated or in the upper limit of our adult normal range in all four patients. FSH levels were definitely elevated in Patients C.A. and N.S., at the upper limit of normal in Patient C.P., and normal in Patient J.P. Plasma testosterone levels were normal in the azoospermic and normospermic patients (Table II). Testicular biopsies from Patients J.M., M.W., D.O., C.P., and N.S., H&E stains and electromicrographs in Patients G.K., M.W., and D.O., who had normal sperm counts, revealed active spermatogenesis and normalappearing Leydig and Sertoli cells. No evidence of neoplasia was observed (Figs. 1, 2, and 3). Major meiotic stages also were normal (Fig. 4). In Patients C.P. and N.S., who were azoospermic, H&E stains and electron micros-

RESULTS Data in Tables I and II, Group A, clearly indicate that cyclophosphamide in daily doses of 2 to 4 mg/kg (average 3.3 mg/kg) given to males (Patients G.K., J.M., M.W., and D.O.) with MLNS during preadolescence over periods of 49 to 60 days did not influence pituitary gonadal function.

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The Journal of Pediatrics February 1976

F SPERMATOGONIAL MITOSIS

DIAKINESIS

Fig. 3. Electron microscopy confirms absence of spermatogenesls and preservation of Sertoli cells in patient with prolonged therapy. (• 2,200.) copy revealed complete absence of spermatogonia with normal-appearing Leydig and Sertoli cells (Fig. 3). No evidence of neoplasia was detected. Unfortunately a testicular biopsy was not obtainable in Patient J.P. who had oligospermia and normal plasma FSH levels. Fertility histories were available for evaluation in two males who received cyclophosphamide during preadolescence and two who received the drug during postadolescence (Table III, Group B). The cumulative dose o f cyclophosphamide in these patients ranged from 64 to 129 mg/kg, or 2.3 to 4.6 m g / k g / d a y during 14 to 45 days. All four patients fathered one or more normal children, 6 4/ i2 to 10 8/12 years following therapy. In addition, one

SECOND MEIOTIC METAPHASE Fig. 4. Examples of normai chromosome phases as observed m Patients J.M., M.W., and D.O. These are representative of the major meiotic stages found to be present in all three patients: (1) spermatogonial mitosis (stem cell division), (2) diakinesis (first meiotic division), (3) secondmeiotic metaphase (second meiotic division).

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preadolescent female (Patient T.L.) received 182 mg/kg during 49 days and bore a normal child 7 1/ 12 years after treatment (Table III, Group C). Karyotypes on Patient T.L. and her offspring revealed normal chromosomes. Two females died of complications of their nephrotic syndrome during the three months following therapy. Each had received two or three courses of therapy between the ages of 3 and 6 years with cumulative doses of 269 and 171 mg/kg. The average daily doses ranged from 3.2 to 4.0 mg/kg (Table III, Group C). Histologic studies in these children at the time of autopsy revealed normal preadolescent ovaries. DISCUSSION These results give evidence that abnormal hormonal levels and histologic alterations following single courses of cyctophosphamide with the daily dose in the usual therapeutic range are related to the duration of therapy. With doses of cyclophosphamide ranging from 2 to 4.6 m g / k g / day (mean 3.5 mg/kg) for periods of 60 days or less, gonadal function and histology remained normal in eight males and three females. However, azoospermia and disappearance of germinal epithelium with preservation of Leydig and Sertoli cells occurred in two patients and azoospermia or oligospermia in two others when the dose of cyclophosphamide ranged from 2.4 to 5.0 mg/kg (mean 3.7 mg/kg) for 89 days or longer. In these four patients plasma levels of FSH and LH tended to be elevated and levels of plasma testosterone were normal. The normal plasma FSH level with an elevated LH in Patient J.P. may be related. Perhaps the relatively shorter duration of therapy, i.e., 89 days, represents an intermediate stage progressing to aspermia while permitting normal FSH values and a pulsatile increase in secretion of LH. 21 Unfortunately we were unable to obtain permission for a testicular biopsy in this patient. Another potential risk with cyclophosphamide therapy is future malignant degeneration of reproductive tissue. No evidence of malignancy was observed in the testicular biopsy material of any of the five patients in whom biopsied tissue was examined 6 2/12 to 10 years following cessation of therapy. Single karyotypes in Patient T.L. (Table III) and her normal offspring suggest that cyclophosphamide in doses which do not cause infertility will riot produce significant chromosomal aberrations. Additional observations are obviously necessary and planned. SUMMARY

These results indicate that courses of 2.0 to 4.6 mg/kg o f cyclophosphamide for not more than 60 days caused no abnormalities of the functions evaluated in the study.

Effects of cyclophosphamide in nephrotic syndrome

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Additional data, especially following repeated courses of this dosage schedule, must be obtained before this therapeutic regimen can be unequivocally recommended in treatment of nonfatal conditions. Serial follow-up observations for potential untoward effects in azoospermic patients who have elevated plasma FSH and LH levels are indicated and contemplated. Sincere appreciation is expressed to Dr. R. L. Summitt for the chromosomal analyses, to Dr. James R. Givens for the hormonal studies, and to Mrs. Susan Keslensky and Mrs. Eva Boyd for assisting in the assembling of data and typing of this manuscript. REFERENCES

1. West CD, Hong R, and Holland NH: Effect of cyclophosphamide on lipoid nephrosis in the human and on aminonucleoside nephrosis in the rat, J PEDIATR68:516, t966. 2. Etteldorf JN, Roy S, Summitt RL, et al: Cyclophosphamide in the treatment of idiopathic lipoid nephrosis, J PEmATR 70:758, 1967. 3. Drummond KN, Hillman DA, Marchessault JHY, et al: Cyclophosphamide in the nephrotic syndrome of childhood, Can Med Assoc J 98:524, 1968. 4. Moncrieff MW, White RHR, Ogg CS, et al: Cyclophosphamide therapy in the nephrotic syndrome in childhood, Br Med J 1:666, 1969. 5. Barratt TM, and Soothill JF: Controlled trial of cyclophosphamide in steroid-sensitive relapsing nephrotic syndrome of childhood, Lancet 2:479, 1970. 6. Spitzer A: The effect of cyclophosphamide in childhood nephrotic syndrome. A report for the International Study of Kidney Disease in Children, Pediatr Res 6:330, 1972. 7. Prospective controlled trim of cyclophosphamide therapy for children with the nephrotic syndrome. A report of the International Study of Kidney Disease in Children, Lancet 2:423, 1974. 8. Spitzer A: Imrnunosuppressive drugs in nephrotic syndrome, Pediatrics 49:472, 1972. (Letter.) 9. Miller DG: Alkylating agents and human spermatogenesis, JAMA 217:1662, 1971. 10. Hyman LR, and Gilbert EF: Testicular atrophy in a prepubescent male after cyclophosphamide therapy, Lancet 2:426, 1972. 11. Fairley KF, Barrie JU, and Johnson, W: Sterility and testicular atrophy related to cyclophosphamide therapy, Lancet 1:568, 1972. 12. Berry CL, Cameron JS, Ogg CS, et al: Cyclophosphamide and prepubertal testis, Lancet 2:1033, 1972. 13. Ameil GC: Cyclophosphamide and the pubertal testis, Lancet 2:1259, 1972. 14. Rapola J, Koskimies O, Huttunen NP, et al: Cyclophosphamide and the pubertal testis, Lancet 1:98, 1973. 15. Penso J, Lippe B, Ehrlich R, et al: TesticuIar function in prepubertal and pubertal male patients treated with cyclophosphamide for nephrotic syndrome, J P~mATR 84:831, 1974. 16. Greifer I, and Barnett HL: International workshop on riskbenefit assessment of cyclophosphamide in renal disease, Kidney Int 2:352, 1972.

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17. Furuyama S, Mayes DM, and Nugent CA: A radioimmunoassay for plasma testosterone, Steroids 16:415, 1970. 18. Morgan CR, Sorenson RL, and Lazarow A: Studies of an inhibitor of the two antibody immunoassay system, Diabetes 13:1, 1964. 19. Wiser WL, Givens JR, and Fish SA: Plasma gonadotropin assays in the diagnostic work-up of amenorrhea, Postgrad Med 52:198, 1972. 20. Camacho AM, Williams DL, and Montalvo JM: Alterations of testicular histology and chromosomes in patients with

The Journal of Pediatrics February 1976

constitutional sexual precocity treated with medroxyprogesterone acetate, J Clin Endocrinol Metab 34:279, 1972. 21. Reiter EO, Berent CT, Duckett GD, et al: Examination of pulsatile gonadotropin release in children and adolescents: use of constant LH-RH infusion, presented at the jointly sponsored meeting of the Southern Section, The American Federation for Clinical Research, The Southern Society for Clinical Investigation, and the Southern Society for Pediattic Research, Endocrinology and Metabolism Section, New Orleans, February 1, 1975.