Effect of cranial irradiation on sperm concentration of adult survivors of childhood acute lymphoblastic leukemia: a report from the St. Jude Lifetime Cohort Study

Abstract

STUDY QUESTION

Does lower dose (<26 Gy) cranial radiation therapy (CRT) used for central nervous system prophylaxis in acute lymphoblastic leukemia (ALL) adversely affect sperm concentration or morphology?

SUMMARY ANSWER

CRT doses <26 Gy had no demonstrable adverse effect on sperm concentration or morphology.

WHAT IS KNOWN ALREADY

Treatment with alkylating agents produces oligospermia and azoospermia in some patients. No prior study has been large enough to evaluate the independent effects of alkylating agents and lower dose (<26 Gy) CRT on sperm concentration or morphology.

STUDY DESIGN, SIZE, DURATION

This cross-sectional study included male adult survivors of pediatric ALL who had received alkylating agent chemotherapy with or without CRT and who enrolled in the St. Jude Lifetime Cohort Study (SJLIFE) from September 2007 to October 2013.

PARTICIPANTS/MATERIALS, SETTING, METHODS

The inclusion criteria were males, ≥18 years of age, ≥10 years after diagnosis, treated at St. Jude Children's Research Hospital for ALL, and received alkylating agent chemotherapy. Semen analyses were performed on 173 of the 241 (78.1%) adult survivors of pediatric ALL who had received alkylating agent chemotherapy with or without CRT. Cumulative alkylating agent treatment was quantified using the cyclophosphamide equivalent dose (CED). Log-binomial multivariable models were used to calculate relative risks (RRs) and 95% CI.

MAIN RESULTS AND THE ROLE OF CHANCE

Compared to those without CRT, risk of oligospermia or azoospermia was not increased for CRT <20 Gy (P = 0.95) or 20–26 Gy (P = 0.58). Participants 5–9 years of age at diagnosis compared to those 0–4 years of age (RR = 1.30, 95% CI, 1.05–.61) or those treated with 8–12 g/m² CED (RR = 2.06, 95% CI, 1.08–3.94) or ≥12 g/m² CED (RR = 2.12, 95% CI, 1.09–4.12) compared to those treated with >0 to <4 g/m² CED had an increased risk for oligospermia or azoospermia.

LIMITATIONS, REASONS FOR CAUTION

Our study relied on the results of one semen analysis. ALL survivors who did not participate in SJLIFE or who declined to submit a semen analysis may also have biased our results regarding the proportion with azoospermia or oligospermia, since those who provided a semen specimen were less likely to have previously fathered children compared to those who did not. The lower rate of previous parenthood among participants may have resulted in a higher observed frequency of azoospermia and oligospermia.

WIDER IMPLICATIONS OF THE FINDINGS

Treatment with <26 Gy CRT did not increase the risk of oligospermia or azoospermia, although a CED exceeding 8 g/m² and an age at diagnosis of 5–9 years did increase risk of oligospermia and azoospermia. These findings can be used to counsel adult survivors of pediatric ALL.

STUDY FUNDING/COMPETING INTEREST(S)

This work was supported by the National Institutes of Health (grant numbers CA 21765, CA 195547, CA00874) and the American Lebanese Syrian Associated Charities (ALSAC). The authors have no competing interests to declare.

Introduction

With modern therapy, >80% of children and adolescents with acute lymphoblastic leukemia (ALL) are expected to be 5-year survivors, with most surviving into adulthood (Pui et al., 2015). Estimates, as of January 2011, indicate a population of >62 000 survivors of childhood ALL in the USA (Phillips et al., 2015). While current therapy for most pediatric patients with ALL does not include cranial radiation therapy (CRT), many survivors treated prior to 2000 received CRT and chemotherapy (Hudson et al., 2012).

Alkylating agent chemotherapy, high-dose CRT and direct testicular irradiation can interfere with spermatogenesis. While the dose-response relationships between alkylating agent exposure (Green et al., 2014a) and direct testicular irradiation (Rowley et al., 1974) have been established, the threshold CRT dose at which pituitary production of gonadotropins is impaired, resulting in decreased or absent spermatogenesis, is poorly defined. Van Casteren et al. (2009) observed no statistically significant differences in testicular volume, testosterone level or FSH level between 55 ALL survivors who received no CRT compared to 25 treated with 15–30 Gray (Gy) (median 25 Gy), but reported no semen analysis data. Byrne et al. (2004) reported that males under age nine treated with 24 Gy CRT had reduced fertility. Limited data suggest that the doses employed for the treatment of childhood brain tumors (>40 Gy) may adversely affect FSH secretion, but the lower threshold for this effect is unknown (Merchant et al., 2009).

Previously, we examined the effect of alkylating agent treatment on sperm concentration, morphology and motility of patients from a large, well-characterized population of non-irradiated male adult survivors of childhood cancer (Green et al., 2014a). The aim of the present study was to examine the independent effect of treatment with lower dose CRT, used for prevention or treatment of central nervous system (CNS) leukemia, on sperm concentration in long-term survivors of childhood ALL. This was identified by the International Late Effects of Childhood Cancer Guideline Harmonization Group (Kremer et al., 2013, Skinner et al., 2016) as a topic for which there was no published high-quality evidence.

Materials and Methods

Patients

The current analysis uses the St. Jude Lifetime Cohort Study (SJLIFE), which consists of patients who fulfill the following criteria: (i) diagnosis of childhood malignancy treated at St. Jude Children's Research Hospital (SJCRH); (ii) survival ≥10 years from diagnosis and (iii) current age ≥18 years. The detailed methods for ascertainment, recruitment and evaluation of the members of this cohort have been reported previously (Hudson et al., 2011, 2016; Ojha et al., 2013).

The cumulative doses for 32 specific chemotherapeutic agents, surgical procedures and radiation treatment (fields, dose and energy source) were abstracted from the medical records according to a structured protocol. Alkylating agent exposure was evaluated using the cyclophosphamide equivalent dose (CED) (Green et al., 2014b).

Participants underwent an extensive clinical evaluation consisting of a core assessment battery augmented by a risk-based clinical assessment according to the Children's Oncology Group Long-Term Follow-Up Guidelines for Survivors of Childhood, Adolescent and Young Adult Cancer (COG Guidelines) (Landier et al., 2004; Children's Oncology Group, 2009). Semen analysis was offered to men who had received treatments known to be gonadotoxic, including exposure to an alkylating agent, testicular irradiation (any dose) or hypothalamic/pituitary irradiation (≥40 Gy). Those who had undergone vasectomy, those receiving exogenous androgen therapy, or those who received radiation therapy to a volume other than the whole brain or a whole brain dose exceeding 26 Gy, were excluded from the present analyses. No participant underwent bilateral orchiectomy. A flow diagram of participants is presented in Fig. 1.

Figure 1.

Flow Diagram of SJLIFE ALL cohort and SJLIFE ALL semen analysis participants. ALL, acute lymphoblastic leukemia; CED: cyclophosphamide equivalent dose; SJLIFE, St. Jude Lifetime Cohort Study.

Semen analysis

Semen samples were collected after a planned minimum of 2 days and a maximum of 7 days of sexual abstinence and were processed within 30 min of collection following the World Health Organization (WHO) Guidelines Fifth Edition (World Health Organization Department of Reproductive Health and Research, 2010). No participant had zero days of abstinence. Two non-irradiated men and one man who had received 25 Gy had only 1 day of abstinence. Five non-irradiated men, five men treated with >0–20 Gy, and four men treated with 20–26 Gy had greater than 7 days of abstinence. All were included in the current analyses. Samples were allowed to liquefy and time to liquefaction was recorded. The raw sample was microscopically evaluated. If no sperm were detected, the sample was centrifuged and concentrated. The concentrated sample was evaluated again before being classified as azoospermic. Specimens that contained >0 and <15 × 10⁶ sperm/ml were classified as oligospermic and those with ≥15 × 10⁶/ml were classified as having a normal sperm concentration. Normal sperm count was defined by the WHO (≥39 × 10⁶). Several additional characteristics, including motility (%) (World Health Organization Department of Reproductive Health and Research, 2010) and morphology (>4% Kruger Strict) (World Health Organization Department of Reproductive Health and Research, 2010) were evaluated in non-azoospermic specimens. A recent medical history was taken at the time of collection. If there was a history of recent fever over 102˚F, any medication use, or recent genitourinary tract infection or injury, a request for a repeat specimen in 1 month to confirm azoospermia was made. Repeat testing of individuals determined to be azoospermic was performed at the discretion of the patient. Semen results from the initial clinical evaluation were used in all of the analyses reported in this manuscript.

Statistical analysis

Descriptive statistics were used to summarize demographic and treatment variables between different groups. The exact χ² test was used to compare these variables between patients who did or did not undergo an on-campus evaluation, to test the differences in the percentage of those who did or did not undergo a semen analysis, to compare those who did or did not have children, and to test the differences in sperm motility and morphology between those with oligospermia and those with a normal sperm concentration, and between CRT and CED groups. To compare those with a normal sperm concentration to those with azoospermia or oligospermia, log-binomial regression models were used to evaluate associations [relative risk (RR) and 95% CI] between race, age at diagnosis, age at evaluation (semen collection), BMI and hypothalamic/pituitary radiation dose. Variables with a P-value <0.1 in univariate models were included in the multivariable model. Similar analyses were performed based on normal sperm count (≥39 × 10⁶ sperm). All analyses were conducted using SAS software (SAS 9.3, Cary, NC).

Ethical approval

This investigation was approved by the Institutional Review Board at SJCRH in accordance with an assurance filed with and approved by the Department of Health and Human Services. All participants or their guardians provided written informed consent.

Results

Of 380 eligible participants who had been treated for ALL, 244 returned for a SJLIFE on-campus clinical evaluation, 241 of whom were eligible for this analysis. The non-participants and SJLIFE participants who underwent semen analysis did not differ statistically with regard to race/ethnicity or age at diagnosis, but the non-participants were more likely to have received 4–8 g/m² and less likely to have received 8–12 g/m² CED than those who underwent a semen analysis (Table I).

Table I.

Demographic and treatment characteristics of ALL survivors eligible for SJLIFE, participated in SJLIFE, and those who provided a semen sample.

		SJLIFE on-campus evaluation with semen analysis (n = 173)		SJLIFE on-campus evaluation with no semen analysis (n = 68)		SJLIFE non-participant (n = 136)		P-valuea Pairwise comparison
		Number	%	Number	%	Number	%	SJLIFE on-campus evaluation with a semen analysis versus SJLIFE non-participants	SJIFE on-campus evaluation with no semen analysis versus SJLIFE non-participants
Race/ethnicity	Non-Hispanic white	146	84.4	62	91.2	103	75.7	0.199	0.056
	Non-Hispanic black	19	11.0	6	8.8	27	19.9
	Hispanic	4	2.3	0	0.0	3	2.2
	Other	4	2.3	0	0.0	3	2.2
Age at diagnosis (years)	0 to 4	78	45.1	24	35.3	51	37.5	0.483	0.044
	5 to 9	47	27.2	13	19.1	37	27.2
	10 to 14	35	20.2	14	20.6	34	25.0
	≥15	13	7.5	17	25.0	14	10.3
Hypothalamic/pituitary radiation dose (Gy)	none	82	47.4	25	36.8	71	52.2	0.68	0.007
	>0 to 20	62	35.8	19	27.9	43	31.6
	>20 to 26	29	16.8	24	35.3	22	16.2
Chlorambucil	Yes	0	0.0	0	0.0	0	0.0
Cyclophosphamide	Yes	173	100.0	68	100.0	136	100.0
Ifosfamide	Yes	0	0.0	0	0.0	0	0.0
Mechlorethamine (nitrogen mustard)	Yes	1	0.6	0	0.0	0	0.0	1.00
Melphalan	Yes	0	0.0	0	0.0	0	0.7	0.44	1.00
Procarbazine	Yes	0	0.0	0	0.0	0	0.0
CED (mg/m2)	>0 to <4000	17	9.8	8	11.8	14	10.3	0.043	0.369
	≥4000 to 8000	35	20.2	19	27.9	46	33.8
	≥8000 to 12 000	100	57.8	27	39.7	60	44.1
	≥12 000	21	12.1	14	20.6	16	11.8

^aExact χ² test. CED, cyclophosphamide equivalent dose; ALL, acute lymphoblastic leukemia; SJLIFE, St. Jude Lifetime Cohort Study.

Of 241 eligible participants, 173 (71.8%) underwent semen analysis. The irradiated participants had a mean age of 6.6 (SD, 4.4) years at diagnosis, a mean age of 32.9 (SD, 7.8) years at semen analysis and a mean duration of 26.3 (SD, 6.3) years after diagnosis. The non-irradiated participants had a mean age of 7.5 (SD, 5.0) years at diagnosis, a mean age of 26.2 (SD, 5.6) years at semen analysis and a mean duration of 18.7 (SD, 6.0) years after diagnosis. Of those who had a semen analysis, 82 survivors received no CRT, 62 received >0 to <20 Gy CRT and 29 received ≥20–26 Gy CRT (Table II). All were treated with cyclophosphamide. The irradiated participants were more likely to have received >12 g/m² CED (P = 0.043) (Table II). None were treated with chlorambucil, ifosfamide, melphalan or procarbazine. The irradiated patients had a longer elapsed time from diagnosis to semen analysis and a longer elapsed time from end of therapy to semen analysis than did those who had not received radiation therapy (Table II). The elapsed time from diagnosis to semen analysis was significantly different by age group at diagnosis among unirradiated men, but not among the irradiated patients (Supplementary data, Table SI). Those who underwent semen analysis were more likely to have never had children (CRT ≥20 Gy to <26 Gy, P = 0.26; CRT >0 to <20 Gy, P < 0.001; No CRT, P = 0.004) (Table III).

Table II.

Demographic and treatment characteristics of ALL survivors who underwent semen analysis by CRT status.

		No CRT (n = 82)		CRT (n = 91)		P-valuea
		Number	%	Number	%
Race/ethnicity	Non-Hispanic white	70	85.4	76	83.5	0.973
	Non-Hispanic black	8	9.8	11	12.1
	Hispanic	2	2.4	2	2.2
	Other	2	2.4	2	2.2
Age at diagnosis (years)	0 to 4	34	41.5	44	48.4	0.396
	5 to 9	23	28.0	24	26.4
	10 to 14	16	19.5	19	20.9
	≥15	9	11.0	4	4.4
	Mean (SD)	7.50 (5.00)		6.6 (4.44)		0.362b
	Median (IQR)	5.85 (2.95–11.58)		5.16 (3.14–10.21)
Age at semen analysis (years)	18 to 25	41	50.0	18	19.8	<0.001
	26 to 35	37	45.1	47	51.6
	36 to 45	3	3.7	21	23.1
	46 to 55	1	1.2	3	3.3
	>55	0	0.0	2	2.2
	Mean (SD)	26.22 (5.63)		32.91 (7.80)		<0.001b
	Median (IQR)	25.98 (22.11–28.68)		31.46 (26.74–37.86)
BMI (kg/m2)	≥15 to <18.5	0	0.0	1	1.1	<0.001
	≥18.5 to <25	36	43.9	16	17.6
	≥25 to <30	21	25.6	28	30.8
	≥30 to <35	19	23.2	24	26.4
	≥35 to <40	2	2.4	13	14.3
	≥40	4	4.9	9	9.9
Hypothalamic/pituitary radiation dose (Gy)	None	82	100.0	0	0.0	<0.001
	>0 to 20	0	0.0	62	68.1
	>20 to 26	0	0.0	29	31.9
Chlorambucil	Yes	0	0.0	0	0.0
Cyclophosphamide	Yes	82	100.0	91	100.00
Ifosfamide	Yes	0	0.0	0	0.0
Mechlorethamine (nitrogen mustard)	Yes	0	0.0	1	1.1	1.000
Melphalan	Yes	0	0.0	0	0.0
Procarbazine	Yes	0	0.0	0	0.0
CED (mg/m2)	>0 to <4000	10	12.2	7	7.7	0.043
	≥4000 to 8000	18	22.0	17	18.7
	≥8000 to 12 000	50	61.0	50	54.9
	≥12 000	4	4.9	17	18.7

CRT, cranial radiation therapy; Gy, Gray; IQR, interquartile range, %, percentage.

^aExact χ² test.

^bWilcoxon rank-sum test.

Table III.

Number of children born to ALL survivors by CRT category and semen analysis status.

	CRT dose ≥20 to 26 Gy		CRT dose >0 to <20 Gy		No CRT
Number of children	Semen analysis (N = 25)	No semen analysis (N = 24)	Semen analysis (N = 62)a	No semen analysis (N = 17)	Semen analysis (N = 82)	No semen analysis (N = 25)
0	72% (18)	58% (14)	77% (48)	24% (4)	90% (74)	60% (15)
1	20% (5)	13% (3)	11% (7)	35% (6)	7% (6)	28% (7)
2	8% (2)	13% (3)	6% (4)	24% (4)	3% (2)	8% (2)
3		3% (1)	6% (3)	12% (2)		4% (1)
4		13% (3)		5% (1)
P-value	0.26b		0.0009b		0.004b

^aTwo had vasectomies, one did not complete the questionnaires and one did not submit a semen specimen.

^bExact χ² test.

Of those who had a semen analysis, 62 survivors (35.8%) had a normal sperm concentration, 46 (26.6%) were oligospermic (sperm concentration <15 × 10⁶/ml) and 65 (37.6%) were azoospermic. There were 30 (36.6%) of the non-irradiated patients who had a normal sperm concentration compared to 32 (35.2%) of those treated with CRT. Five patients had a history of fever within the 3 months prior to semen analysis. A repeat semen analysis was obtained from one who remained azoospermic. Only five azoospermic participants submitted a repeat semen analysis, which disclosed persistent azoospermia in all cases.

In univariate analyses, risk of azoospermia or oligospermia was not statistically associated with CRT exposure at a dose of >0–20 Gy (RR = 0.99, CI, 0.70–1.28) or 20–26 Gy (RR = 1.09, CI, 0.81–1.46) or elapsed time from diagnosis to semen analysis (RR = 1.00, CI, 0.98–1.01) or from completion of therapy to semen analysis (RR = 1.00, CI, 0.98–1.02) (Table IV). However, an age at diagnosis of 5–9 years compared to 0–4 years of age, CED ≥8000 to <12 000 mg/m², and CED ≥12 000 mg/m² compared to CED >0 to <4000 mg/m², and CED (per 1000 mg/m²) satisfied the criteria for further examination in multivariable models.

Table IV.

Results of univariable analysis for RR of azoospermia or oligospermia.

Variable	Category	RR	95% CI	P-value
Race/ethnicity	Non-Hispanic black + Asian + other versus Non-Hispanic white	1.12	0.85, 1.47	0.43
Age at diagnosis (years)	5 to 9 versus <4	1.24	0.98, 1.58	0.07
	≥10 versus <4	0.91	0.67, 1.24	0.56
Age at semen analysis (years)	26 to 35 versus 18 to 25	0.82	0.60, 1.04	0.11
	≥35 versus 18 to 25	0.94	0.69, 1.26	0.67
BMI (kg/m2)	25 to 30 versus <25	1.14	0.89, 1.47	0.29
	>30 versus <25	0.82	0.62, 1.10	0.18
Hypothalamic/pituitary radiation dose (Gy)	>0 to 20 versus 0	0.99	0.70, 1.28	0.95
	≥20 to 26 versus 0	1.09	0.81, 1.46	0.58
CED	≥4000 to <8000 versus >0 to <4000	1.46	0.71, 2.99	0.31
	≥8000 to <12 000 versus >0 to <4000	1.98	1.03, 3.82	0.04
	≥12 000 versus >0 to <4000	2.29	1.17, 4.51	0.02
	CED per 1000 mg/m2	1.01	1.00, 1.02	0.05
Elapsed time from diagnosis to semen analysis	Per year increased	1.00	0.98,1.01	0.69
Elapsed time from end of therapy to semen analysis	Per year increased	1.00	0.98,1.02	0.87

RR, relative risk.

In multivariable analysis, RR for oligospermia or azoospermia was increased for those 5–9 years of age at diagnosis compared to those 0–4 years of age at diagnosis, and for CED ≥8000 to <12 000 mg/m², and CED ≥12 000 mg/m² compared to CED >0 to <4000 mg/m² (Supplementary data, Table SII).

In addition, we evaluated outcomes based on total sperm count. Only 32.3% of the 173 subjects had a normal sperm count with the remainder (67.6%) having a sperm count <39 × 10⁶, 65 of whom were azoospermic, as noted above. In univariate analyses, risk of azoospermia or oligospermia was not statistically associated with CRT exposure at a dose of >0–20 Gy (RR = 1.02, CI, 0.81–1.30) or 20–26 Gy (RR = 1.23, CI, 0.96–1.57), elapsed time from diagnosis to semen analysis (RR = 1.00, CI, 0.99–1.02), or elapsed time from completion of therapy to semen analysis (RR = 1.01, CI, 0.99–1.02) (Supplementary data, Table SIII). However, age at diagnosis 5–9 years compared to 0–4 years of age and CED ≥8000 to <12 000 mg/m² and CED ≥12 000 mg/m² compared to CED >0 to <4000 mg/m², and CED (per 1000 mg/m²) satisfied the criteria for further examination in multivariable models.

In multivariable analysis, RR for low sperm count was increased for those 5–9 years of age at diagnosis compared to those 0–4 years of age at diagnosis and for CED ≥8000 mg/m² compared to CED >0 to <4000 mg/m² (Supplementary data, Table SIV).

There was no evidence that the prevalence of very low or low motility or abnormal morphology was correlated with CED among those with oligospermia or a normal sperm concentration, whether treated with CRT or not (Table V and Supplementary data, Table SV). Patients with a normal sperm concentration who did not receive CRT were more likely to have normal sperm morphology than those with oligospermia, while those patients with a normal sperm concentration who did receive CRT were more likely to have normal sperm motility than those with oligospermia (Table VI).

Table V.

Distribution of CED by semen characteristics in non-irradiated oligospermic semen analysis participants with ALL, and those with a normal sperm concentration.

Sperm characteristic	CED (mg/m2)			Total	P-valuea
	0 to <4000	4000 to <8000	>8000
Oligospermiab
Motility (%)	N (%)	N (%)	N (%)	N
Very low or low (<40%)	0 (0.0%)	1 (20.0%)	6 (33.3%)	7	0.65
Normal (≥40%)	0 (0.0%)	4 (80.0%)	12 (66.7%)	16
Morphology (% normal)
Low (<4%)	0 (0.0%)	2 (66.7%)	5 (35.7%)	7	0.54
Normal (≥4%)	0 (0.0%)	1 (33.3%)	9 (64.3%)	10
Normal sperm concentration
Motility (%)
Very low or low (<40%)	2 (22.2%)	0 (0.0%)	2 (14.3%)	4	0.66
Normal (≥40%)	7 (77.8%)	7 (100.0%)	12 (85.7%)	26
Morphology (% normal)
Low (<4%)	2 (22.2%)	0 (0.0%)	1 (7.1%)	3	0.42
Normal (≥4%)	7 (77.8%)	7 (100.0%)	13 (92.9%)	27

N, Number of patients.

^aExact χ² test.

^bNot all oligospermic participants were evaluable for morphology and motility due to inadequate numbers of sperm.

Table VI.

Sperm characteristics among oligospermic ALL survivors, and those with a normal sperm concentrtion.

	Oligospermiaa	Normal sperm concentrtion	P-valueb
	N (%)	N (%)
No radiation therapy
Motility (%)
Very low or low (<40%)	7 (30.4%)	4 (13.3%)	0.18
Normal (≥40%)	16 (69.6%)	26 (86.7%)
Morphology (% normal)
Low (<4%)	7 (41.2%)	3 (10.0%)	0.02
Normal (≥4%)	10 (58.8%)	27 (90.0%)
CRT only
Motility (%)
Very low or low (<40%)	12 (52.2%)	3 (9.4%)	<0.001
Normal (≥40%)	11 (47.8%)	29 (90.6%)
Morphology (% normal)
Low (<4%)	7 (43.8%)	6 (18.8%)	0.09
Normal (≥4%)	9 (56.3%)	26 (81.3%)

^aNot all oligospermic participants were evaluable for morphology and motility due to inadequate numbers of sperm.

^bExact χ² test.

Six of the patients included in this analysis were reported previously as having FSH/LH deficiency (Chemaitilly et al., 2015). One who received 18 Gy underwent semen analysis and was found to be oligospermic. Five treated with 20–26 Gy underwent semen analysis, and one was azoospermic, one oligospermic and three had a normal sperm concentration.

Discussion

To the best of our knowledge, this is the largest published study of spermatogenesis among survivors of childhood ALL. We demonstrated that CRT, in the doses used for CNS prophylaxis in patients with ALL, has no demonstrable adverse effect on spermatogenesis. In contrast to previous studies, which were limited by small cohort size and the use of surrogate measures of germ cell function, we conducted a more precise evaluation of the long-term impact of low dose CRT on spermatogenesis. This evaluation was facilitated by the availability of semen samples and detailed treatment information in a relatively large cohort of men treated for ALL during childhood. Neither treatment with >0–20 Gy nor treatment with >20–26 Gy significantly increased the proportion with oligospermia or azoospermia among survivors of childhood ALL treated with alkylating agents compared to those survivors who received no CRT. In multivariable analyses, those who were ≥5 to <10 years of age at diagnosis or those who were treated with ≥8 g/m² of cyclophosphamide had an increased risk for oligospermia or azoospermia. Analyses based on normal or abnormal sperm count yielded identical results.

Previous studies evaluating germ cell function in ALL survivors described outcomes in very small (n = 12–37), clinically heterogeneous cohorts with regard to age, time from diagnosis, age at treatment and evaluation, and treatment with cyclophosphamide. In a study of 26 ALL survivors, a median of 5 years after the completion of therapy including prophylactic CRT (18–24 Gy) and no alkylating agents. Testicular volume and/or serum FSH levels suggested no significant effect on germ cell function (Sklar et al., 1990). Others reported impaired testicular germ cell function, using FSH levels, in 37 survivors of ALL treated with CRT (18–24 Gy), nine of whom also received cyclophosphamide. Follow-up evaluation at a median of 10.7 years after completion of therapy showed recovery of normal germ cell function in a substantial portion (Wallace et al., 1991). In another study describing the relationship of sperm concentration and treatment exposures in 12 survivors of childhood ALL (median, 17.1 years after diagnosis), three of four treated with CRT and cyclophosphamide (3.0–5.4 g/m²) had normal sperm concentration, compared to all of four treated without either CRT or cyclophosphamide, and both treated without CRT but with cyclophosphamide (3.0 g/m²) (Lahteenmaki et al., 2008). Although these data are generally reassuring, most utilized surrogate markers for spermatogenesis, which have poor specificity and positive predictive value (Green et al., 2013) and/or lacked a sufficient number of participants to evaluate the independent effects of treatment with CRT and with cyclophosphamide.

Our study addresses an important knowledge gap by defining a CRT dose threshold for patients also treated with an alkylating agent, below which hypothalamic–pituitary–gonadal function will likely be preserved. The effects of CRT on hypothalamic–pituitary axis functions vary by dose. Previous studies provide strong evidence of disruption of hypothalamic–pituitary–gonadal function when the CRT dose exceeds 30 Gy (Bajorunas et al., 1980; Rappaport et al., 1982). We previously observed LH/FSH deficiency in 10.8% (79/731) of adult survivors of pediatric malignancies. The prevalence was greater among those treated with 22.0–29.9 Gy (10.1%) or with ≥30 Gy (21.2%), than among those treated with ≤21.9 Gy (3.2%) (Chemaitilly et al., 2015). In the current study population, FSH/LH deficiency was previously identified by Chemaitilly et al. in five who received 20–26 Gy, three of whom had a normal sperm concentration. The diagnosis of FSH/LH deficiency was based on a single determination of FSH, LH, and testosterone. The Endocrine Society recommends two determinations performed using the same assay when the serum testosterone is borderline low (Fleseriu et al., 2016). Other factors, including known spontaneous fluctuations in testosterone levels as well as obesity may have resulted in over diagnosis of FSH/LH deficiency in our patients.

Most previous studies demonstrated no greater impairment of spermatogenesis among adult survivors of childhood cancer who were treated at a younger age (Green et al., 1981; Aubier et al., 1989; Siimes and Rautonen 1990; Lahteenmaki et al., 2008; Jahnukainen et al., 2011; Hamre et al., 2012). We do not have an explanation for the finding that oligospermia and azoospermia were statistically more likely among those who were treated at 5–9 years of age in the current study. In our prior, larger study of non-irradiated survivors, we identified no effect of age at diagnosis or age at evaluation on sperm concentration (Green et al., 2014a). The current association may reflect chance or an unrecognized association between an unevaluated parameter that is correlated with the age group 5–9 years and sperm concentration.

In addition to providing information regarding sperm concentration, clinical assessments with semen analyses also permitted evaluation of cancer treatment effects on sperm motility and morphology. In the normal population, both sperm morphology (Jouannet et al., 1988; Eggert-Kruse et al., 1996; Zinaman et al., 2000; van der Merwe et al., 2005) and sperm motility (Jouannet et al., 1988; Larsen et al., 2000; Zinaman et al., 2000; van der Merwe et al., 2005) may be associated with impairment of subsequent male fertility. In the present study, approximately one-half of those with oligospermia had normal morphology, whether treated with CRT or not. Morphology was at levels consistent with impaired fertility in a small percentage of those with normal sperm concentration, irrespective of CRT category. Thus, in addition to sperm concentration, abnormalities of motility and morphology may be contributing to the decreased fertility observed in adult male childhood cancer survivors (Green et al., 2010, 2014b) .

The strengths of this study include the assessment of semen specimens from a large, well-characterized survivor cohort that was clinically diverse with regard to alkylating agent and CRT exposure and analysis of semen specimens in a single, certified andrology laboratory. All participants were screened for recent history of genitourinary diseases, such as epididymitis and urethritis, and fever during the previous 3 months, which may affect semen analysis results. Limitations of our study include reliance on the results of one semen analysis, as the need for many participants to travel long distances to SJCRH for multiple days (average 3 days) of evaluation, precluded obtaining the two samples recommended when assessing fertility. While our study population is a selected group of long-term survivors diagnosed and treated over four decades at a single institution, we believe that the findings regarding treatment exposures can be generalized to the broader population of survivors of childhood ALL, with the caveat that ALL survivors who declined participation in SJLIFE or to submit a semen analysis may also have biased our results relative to proportion with azoospermia or oligospermia. Those who provided a semen specimen were less likely to have previously fathered children compared to those who did not. The lower rate of previous parenthood among participants may have resulted in a higher observed frequency of azoospermia and oligospermia.

In summary, this study demonstrates that CRT, as administered to children with ALL, all of whom received alkylating agents, has no demonstrable independent effect on spermatogenesis in the dose range studied (<26 Gy). These data may be used to counsel patients and their families and to guide the development of future therapeutic protocols for children with ALL.

Supplementary data

Supplementary data are available at Human Reproduction online.

Authors’ roles

M.M.H. was the principal investigator and designed the research program. D.M.G., W.C., C.A.S., L.L.R. and M.M.H. developed the study hypotheses and study design. W.H.K. and R.W.K. performed laboratory investigations and participated in writing the manuscript. D.K.S. directed the data analysis by L.Z. and M.W. and reviewed the manuscript. L.Z. and M.W. wrote the statistical analysis section of the manuscript. C.-H.P., L.E.K. and R.C.R. contributed to patient recruitment. All authors were involved in writing the manuscript and have approved the final version.

Funding

The National Institutes of Health (grant numbers CA 21765, CA 195547, CA00874) and the American Lebanese Syrian Associated Charities (ALSAC).

Conflict of interest

None declared.