Impact of chemotherapy and radiotherapy for testicular

ORIGINAL ARTICLE: ANDROLOGY
Impact of chemotherapy and
radiotherapy for testicular germ cell
tumors on spermatogenesis and
sperm DNA: a multicenter
prospective study from the
CECOS network
Louis Bujan, M.D., Ph.D.,a,b Marie Walschaerts, Ph.D.,b Nathalie Moinard, D.Pharm.,b
Sylvianne Hennebicq, M.D., Ph.D.,a,c Jacqueline Saias, M.D.,a,d Florence Brugnon, M.D., Ph.D.,a,e
Jacques Auger, M.D., Ph.D.,a,f Isabelle Berthaut, Ph.D.,a,g Ethel Szerman, Ph.D.,a,h Myriam Daudin, M.D.,a,b
and Nathalie Rives, M.D., Ph.D.a,i
a
de
ration Française des Centre d'Etude
de
Fe
et de Conservation des Œufs et du Sperme Humains (CECOS), Paris; b Universite
Humaine (EA 3694, Human Fertility Research Group), CECOS, Toulouse;
Toulouse; UPS; Groupe de Recherche en Fertilite
c
la Procre
ation-CECOS, Laboratoire AGIM, CNRS-FRE3405, Equipe
ne
tique-Infertilite
Laboratoire d'Aide a
Ge
rapeutique, Faculte
de Me
decine de Grenoble, Grenoble; d Laboratoire de Biologie de la Reproduction-Cecos,
The
dicale a
la Procre
ation, CECOS, CHU Estaing, and Laboratoire
^ pital La Conception, Marseille; e Assistance Me
Ho
ne
tique Reproduction et De
veloppement, GReD, UMR CNRS 6293, INSERM U1103, Universite
d'Auvergne, Faculte
de
Ge
partement d'Histologie-Embryologie, Biologie de la Reproduction/CECOS, Site Portdecine, Clermont-Ferrand; f De
Me
^ pital Tenon,
Royal, Paris Centre University Hospitals, Paris; g Service d'Histologie, Biologie de la Reproduction-CECOS, Ho
de BDR-CECOS, Pole de Biologie, CHU Co
^ te de Nacre, Caen; and i CECOS Biologie de la Reproduction, CHU
Paris; h Unite
togene
se et Qualite
du Game
te, Universite
de Rouen, Rouen, France
Rouen, and EA 4308 Game
Objective: To determine the consequences of adjuvant testicular germ cell tumor treatment (TGCT) on sperm characteristics and sperm
DNA, and to evaluate the predictors of sperm recovery.
Design: Multicenter prospective longitudinal study of patients analyzed before treatment and after 3, 6, 12, and 24 months.
Setting: University hospitals.
Patient(s): One hundred twenty-nine volunteer TGCT patients and a control group of 257 fertile men.
Intervention(s): Routine semen analyses, sperm DNA, and chromatin assessments.
Main Outcome Measure(s): Comparisons of mean sperm characteristics before and after treatment, with sperm recovery analyzed by
the Kaplan-Meier method.
Result(s): The quantitative and qualitative sperm characteristics decreased after treatment, with lowest values at 3 and 6 months
and with variations according to treatment type. The mean total sperm count recovered to pretreatment values at 12 months
after treatment after two or fewer bleomycin, etoposide, and cisplatin (BEP) cycles, but not after radiotherapy or more than two
BEP cycles. Only the treatment modalities and pretreatment sperm production were related to recovery of the World Health Organization reference sperm values. An increased proportion of patients had elevated high sperm DNA stainability at 6 months after
radiotherapy.
Received March 4, 2013; revised and accepted May 3, 2013.
L.B. has nothing to disclose. M.W. has nothing to disclose. N.M. has nothing to disclose. S.H. has nothing to disclose. J.S. has nothing to disclose. F.B. has
nothing to disclose. J.A. has nothing to disclose. I.B. has nothing to disclose. E.S. has nothing to disclose. M.D. has nothing to disclose. N.R. has nothing
to disclose.
Supported by a grant from the French Ministry of Health, PHRC No. 20030222: GAMATOX project. Regulatory and ethical submissions were performed by
the University Hospital of Toulouse. All samples were registered with the GERMETHEQUE biobank (France).
^ pital Paule de Viguier, 330 avenue de Grande Bretagne,
Reprint requests: Louis Bujan, M.D., Ph.D., EA 3694 Human Fertility Research Group, CECOS, Ho
31059 Toulouse Cedex 09, France (E-mail: [email protected]).
Fertility and Sterility® Vol. -, No. -, - 2013 0015-0282/$36.00
Copyright ©2013 American Society for Reproductive Medicine, Published by Elsevier Inc.
http://dx.doi.org/10.1016/j.fertnstert.2013.05.018
VOL. - NO. - / - 2013
1
ORIGINAL ARTICLE: ANDROLOGY
Conclusion(s): Adjuvant treatments for testicular germ cell tumor have drastic effects on spermatogenesis and sperm chromatin
quality. These new data on both the recovery period according to treatment modalities and the post-treatment chromatin status
of sperm are useful tools for counseling patients wishing to conceive. (Fertil SterilÒ 2013;-:-–-. Ó2013 by American Society
for Reproductive Medicine.)
Key Words: Adverse effects, BEP chemotherapy, radiotherapy, spermatogenesis, sperm chromatin
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T
esticular germ cell tumor (TGCT) is the most common
cancer in young men, and TGCT incidence has
increased in several countries over the past 50 years
(1). Diagnosis and treatment have drastically improved the
long-term survival rate (2). Usually, orchidectomy is followed
by radiotherapy or chemotherapy according to tumor type
and disease stage, except in stage I disease when surveillance
alone may be an alternative. Chemotherapy or radiotherapy
have deleterious consequences on spermatogenesis (3, 4),
and sperm banking is recommended before treatment (5).
Sperm quality may already be altered before any treatment,
due to the effect of the tumor itself or because TGCT is more
frequent in men with other testicular disease (2, 6–8).
Several studies have reported the effects of TGCT treatment on spermatogenesis. However, their value is often
limited because of the small series included (9–14) or
because of their retrospective nature, with various follow-up
designs. Spermatogenesis recovery after cancer treatment
was linked to diagnosis (15), sperm parameters before treatment (16), type of treatment (9, 12, 17), to both of the latter
(18), or was unrelated to any of these factors (14, 19).
The impact of TGCT treatment on sperm DNA and chromatin during recovery is of paramount concern as damage
to the paternal genome may have detrimental effects on the
progeny. Posttreatment alterations of sperm DNA remain
controversial: increased DNA damage was reported in a prospective study 6 to 24 months after treatment (20), but
another study reported no such increase (21). Our prospective
study of 129 TGCT patients [1] evaluated sperm characteristics and DNA damage before and after treatment, [2] evaluated the impact of TGCT treatment on spermatogenesis at
the various time points during follow-up observation (3 to
24 months), and [3] identiﬁed predictive factors of posttreatment spermatogenesis recovery.
MATERIALS AND METHODS
Patients
This prospective study enrolled 129 patients who had been
directed to the Centres d'Etudes
et de Conservation des Oeufs
et du Sperme humain (CECOS) for sperm banking before TGCT
treatment. Eight CECOS sites participated: Caen, ClermontFerrand, Grenoble, Marseille, Paris Cochin, Paris Tenon,
Rouen, and Toulouse. The sperm analyses were subject to
external quality control in all centers. This study was supported by a national research grant (PHRC no. 20030222, GAMATOX project) and was approved by the institutional ethics
review board. All patients gave written informed consent.
2
Usually, two to three semen samples were collected for
sperm banking. Patients taking part in the study provided
an additional semen sample before treatment (T0). In this prospective study, patients were asked to provide additional
semen samples at 3, 6, 12, and 24 months after the end of
treatment (Supplemental Fig. 1, available online). Age, andrologic and reproductive histories, tobacco exposure, and febrile
episodes were recorded, and at each visit the participants
completed a standard questionnaire about any unusual events
since the last visit to the laboratory (disease, febrile episodes,
and any change in lifestyle habits). To evaluate sperm alterations before treatment, the pretreatment sperm characteristics were compared with those of a control group of 257
fertile men. Fifty-one of these fertile men also underwent
sperm DNA evaluation (DNA control group).
Semen Analyses
Semen samples were collected by masturbation after a recommended 3 to 5 days of sexual abstinence. The semen analysis
was performed according to the World Health Organization
(WHO) guidelines (22) with similar methodology in the eight
laboratories. The characteristics considered were sexual abstinence (days), ejaculate volume (mL) and pH, sperm concentration (SC, 106 spermatozoa/mL), round cell concentration
(RC, 106/mL), spermatozoa vitality (V, %) and forward
motility (M, a þ b: %), total sperm count (product of volume
by SC ¼ TSC, 106 spermatozoa/ejaculate), and total motile
sperm count (TMSC ¼ TSC M, 106/ejaculate). The remaining semen sample was mixed with a cryoprotectant, frozen
in straws, and stored in liquid nitrogen until used for further
analyses, which were performed in the Toulouse CECOS by a
single technician.
Sperm Chromatin Structure Assay
The sperm chromatin structure assay (SCSA) to evaluate
sperm chromatin integrity (23) was performed as previously
described elsewhere (24) in the routine manner of our laboratory (25, 26). The extent of DNA denaturation was expressed
as detected ﬂuorescence intensity (DFI), which is the ratio of
red to total (red plus green) ﬂuorescence intensity. We also
calculated the fraction of sperm with high DNA stainability
(HDS), which represents sperm with immature chromatin.
One aliquot of the quality control sperm was analyzed in
pooled samples (results not shown). The analytic coefﬁcient
of variation was <5%, as calculated from the values
obtained from aliquots of a semen sample.
VOL. - NO. - / - 2013
Fertility and Sterility®
TABLE 1
Sperm characteristics in the control group (fertile men) and the testicular germ cell tumor (TGCT) group.
Seminoma group
Sperm
characteristic
Control group
(n [ 257)
TGCT group
(n [ 129)
Without
cryptorchidism
(n [ 67)
Volume (mL)
3.95 1.90
3.60 1.65*
3.41 1.57*,**
,
8.01 0.28*
pH
7.94 0.28
8.08 0.33* **
98.67 90.91
31.26 31.80*,**
36.05 36.94*,**
Sperm count
6
(10 /mL)
2.10 2.62
1.16 1.61*,**
1.11 1.42*,**
Round cells
6
(10 /mL)
Vitality (%)
69.87 13.44
67.38 13.83**
67.14 15.38**
Motility (%)
43.05 13.53
42.08 13.84**
42.94 14.72
,
Total sperm count 361.95 343.25 115.16 142.48* ** 124.86 153.93*,**
(106/ejaculate)
150.49 126.37 53.93 69.08*,**
Total motile
59.45 72.85*,**
sperm count
(106/ejaculate)
Nonseminoma group
With
cryptorchidism
(n [ 3)
Without
cryptorchidism
(n [ 49)
With
cryptorchidism
(n [ 10)
5.73 3.22
8.17 0.31
7.83 4.25*
3.80 1.69
8.17 0.39*,**
29.08 25.15*,**
3.26 1.07
8.03 0.23*
16.84 19.67*,**
0.57 0.51
1.29 1.89*,**
1.04 1.59*
63.33 15.28
67.96 11.40
67.30 15.43
30.00 17.32
43.67 12.19
32.10 9.89*,**
,
47.45 47.31* 118.87 139.99* ** 52.29 56.78*,**
16.71 21.00*
55.60 70.10*,**
19.95 25.57*,**
Note: Values are mean standard deviation.
* P< .05, difference between control group and TGCT group, seminoma group without cryptorchidism, seminoma group with cryptorchidism, nonseminoma group without cryptorchidism, and
nonseminoma group with cryptorchidism.
** P< .05, adjusted for abstinence duration and age, difference between control group and TGCT group, seminoma group without cryptorchidism, seminoma group with cryptorchidism, nonseminoma group without cryptorchidism, and nonseminoma group with cryptorchidism.
Bujan. TGCT cancer treatment effect on sperm. Fertil Steril 2013.
DNA Fragmentation
Sperm DNA strand breaks were detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL
assay) as previously described elsewhere (25). Sperm DNA
fragmentation and propidium iodide labeling were measured
on a FACScan ﬂow cytometer. Each analysis included a minimum of 10,000 stained spermatozoa. The FL1 signals (green
ﬂuorescence from ﬂuorescein isothiocyanate conjugate)
were detected through a 525 25 nm band pass ﬁlter and
FL2 signals (red ﬂuorescence from propidium iodide) through
a 575 25 nm ﬁlter. The TUNEL analysis consisted of subtracting control (no TdT enzyme) green ﬂuorescence histograms from TdT-positive green ﬂuorescence histograms,
yielding the percentage of cells showing DNA strand breaks.
Data Collection and Statistical Analyses
All data were reported on centralized case report forms by Web
access and the data ﬁles were veriﬁed by the coordinating center in Toulouse. Data were compared between the control
group (fertile men) and the TGCT group using the nonparametric Mann-Whitney test. Linear regression was used to
adjust the data on sexual abstinence and patient age because
both parameters may inﬂuence sperm characteristics.
The sperm characteristics of TGCT patients were
compared before and after treatment at T3, T6, T12, and
T24 by the Wilcoxon signed rank-sum test. The treatment
regimens were compared using the Mann-Whitney test. The
Kaplan-Meier method was used to estimate the cumulative
rates of successful semen recovery and the 95% conﬁdence
interval (CI). Censors were deﬁned as patients who did not
succeed in semen recovery—that is, the patients with TSC
<39 106 spermatozoa/ejaculate (27) at their last visit. Statistically signiﬁcant differences between the groups were
VOL. - NO. - / - 2013
calculated by the log-rank test. Univariate and multivariate
Cox models were performed to describe factors associated
with successful or unsuccessful semen recovery using crude
and adjusted hazard ratios (HR) and 95% CIs. The statistical
analysis was performed using SAS software (9.0, SAS Institute), and P< .05 was considered statistically signiﬁcant.
RESULTS
Patients
The study enrolled 129 patients aged 20 to 44 years (mean age
standard deviation [SD]: 30.9 4.9). Seventy patients
(54%) had pure seminoma, and 59 (46%) had mixed or nonseminoma tumors. Thirteen patients (10%) had a history of
cryptorchidism.
Sixty-seven patients with pure seminoma were treated by
radiotherapy, and the other 62 patients underwent chemotherapy with the BEP regimen (bleomycin, etoposide, and
cisplatin): 17 patients received one or two chemotherapy cycles, and 45 patients had more than two cycles (three or four).
Semen Characteristics before Cancer Treatment
The frequency of the risk factors that could inﬂuence sperm
characteristics was not statistically signiﬁcantly different between the controls and the TGCT group (P>.05, results not
shown). Orchidectomy was performed before semen collection in 83 patients (85%), but no statistically signiﬁcant difference was observed between the sperm characteristics
collected before or after surgery (P>.05, data not shown).
Compared with the fertile group, the TGCT patients had a
higher pH and lower ejaculate volume, SC, TSC, and TMSC
(P< .05) (Table 1). Moreover, statistically signiﬁcant decreases
of round cell concentration and percentage of motility and
3
ORIGINAL ARTICLE: ANDROLOGY
TABLE 2
Sperm characteristics before and after treatment and during follow-up observation.
Treatment type and sperm
characteristic
Chemotherapy, %2 cycles
Volume (mL)
pH
Sperm count (106/mL)
Round cells (106/mL)
Vitality (%)
Motility (%)
Total sperm count (106/ejaculate)
Total motile sperm count
(106/ejaculate)
Chemotherapy, >2 cycles
Volume (mL)
pH
Sperm count (106/mL)
Round cells (106/mL)
Vitality (%)
Motility (%)
Total sperm count (106/ejaculate)
Total motile sperm count
(106/ejaculate)
Radiotherapy
Volume (mL)
pH
Sperm count (106/mL)
Round cells (106/mL)
Vitality (%)
Motility (%)
Total sperm count (106/ejaculate)
Total motile sperm count
(106/ejaculate)
After treatment
Before treatment
3 mo
6 mo
12 mo
24 mo
(n ¼ 15)
3.67 1.33
8.19 0.30
32.01 29.51
1.31 1.70
68.07 16.73
43.73 14.75
105.94 90.96
48.78 38.37
(n ¼ 14)
3.79 1.09
7.99 0.40*
4.10 5.95*
2.09 6.72*
61.73 25.18*
31.29 25.51
12.73 15.05*
5.01 7.39*
(n ¼ 13)
4.26 1.50
7.98 0.41*
13.98 23.40*
0.45 0.48*
66.08 12.66
39.23 16.01
63.83 135.95*
37.93 96.62*
(n ¼ 11)
3.74 0.79
7.90 0.37*
30.88 41.77
0.55 0.64
65.91 20.20
43.27 20.32
100.34 106.28
55.39 75.21
(n ¼ 10)
3.76 1.25
8.02 0.29
32.48 25.03
1.58 3.49
66.50 15.94
48.50 24.27
122.29 102.58
73.73 83.86
(n ¼ 45)
3.46 1.61
8.09 0.40
23.46 21.37
1.16 1.87
67.62 11.77
39.33 13.65
88.08 106.20
41.20 60.62
(n ¼ 34)
3.17 1.48*
8.04 0.37
0.24 0.59*
0.48 0.93*
47.78 30.78
12.48 16.51*
0.76 1.52*
0.24 0.46*
(n ¼ 40)
3.53 1.85
7.97 0.43*
4.40 14.97*
0.28 0.43*
45.89 30.80*
25.45 20.15*
16.02 47.98*
5.29 9.67*
(n ¼ 35)
3.32 1.97
8.05 0.48
10.01 15.24*
0.56 0.98*
62.32 21.63
30.76 18.70*
31.46 49.53*
13.62 20.84*
(n ¼ 32)
3.66 1.71
8.03 0.39
30.99 29.08
0.65 0.86
65.53 15.07
38.93 13.76
114.77 127.45
46.99 46.67
(n ¼ 67)
3.68 1.77
8.05 0.29
36.48 37.37
1.12 1.43
67.17 14.79
43.69 13.80
135.69 170.33
63.91 78.99
(n ¼ 58)
3.42 1.59*
8.11 0.41
11.14 15.59*
0.92 1.04
63.10 15.45
35.58 17.25*
41.68 70.37*
18.52 32.53*
(n ¼ 60)
3.53 1.64
8.05 0.34
12.13 27.65*
0.66 1.36*
62.00 21.83
33.41 19.51*
48.24 143.91*
24.50 74.44*
(n ¼ 57)
4.08 1.90
8.03 0.35
22.01 22.45*
0.88 1.44*
67.04 17.43
42.61 16.97
84.82 107.36*
41.60 61.34*
(n ¼ 49)
3.69 1.69
8.00 0.36
37.36 36.90
0.98 0.96
67.73 12.84
40.92 15.40
135.82 156.73
61.42 87.75
Note: Values are mean standard deviation.
* P< .05, difference between values before and after treatment (3, 6, 12, and 24 months).
Bujan. TGCT cancer treatment effect on sperm. Fertil Steril 2013.
Sperm characteristics (SC, TSC, TMSC) decreased after treatment. The lowest values occurred at T3 and were particularly
marked after chemotherapy with more than two cycles and
after radiotherapy (Table 2, Fig. 1). For qualitative characteristics such as motility, the mean values decreased at T3 and T6
in patients treated with more than two cycles of chemotherapy or with radiotherapy. The mean ejaculate volume
differed only at T3 in both treatment groups. The semen characteristics recovered to pretreatment values 12 months after
chemotherapy after fewer than two cycles, but only at 24
months after other treatments. The percentage of men with
azoospermia increased from 0 before treatment to 16% and
17% at T3 and T6, respectively, and remained high at T12
(6%) (P< .05) but decreased to 2% at T24 (P< .05).
production R39 106/ejaculate reached 92% (95% CI, 70–
99) in patients with two or fewer chemotherapy cycles, 63%
(95% CI, 48–79) in patients with more than two cycles, and
86% (95% CI, 76–94) for the radiotherapy group (P< .05). A
statistically signiﬁcant difference in recovery was also
observed according to history of cryptorchidism—that is,
81% (95% CI, 73–89) for patients without a history of cryptorchidism versus 55% (95% CI, 30–84) for those with cryptorchidism—and according to pretreatment TSC value—that is,
89% (95% CI, 80–96) for patients with TSC R39 106 versus
61% (95% CI, 46–76) for TSC <39 106. However, no difference was observed according to TGCT type (Fig. 2).
Adjusted for patient age, history of cryptorchidism,
smoking status, and type of TGCT in a multivariate Cox
model, only the type of treatment (chemotherapy >2 cycles
vs. chemotherapy %2 cycles, HR ¼ 0.44 [95% CI, 0.22–
0.90], and radiotherapy vs. chemotherapy %2 cycles, HR ¼
0.77 [95% CI, 0.29–2.00]) and TSC before treatment (TSC
<39 106 versus TSC R39 106, HR ¼ 1.88 [95% CI,
1.16–3.05]) remained statistically signiﬁcant.
Predictors of Sperm Recovery after Treatment
DNA Damage
Stratiﬁed according to treatments, the Kaplan-Meier estimates showed that the cumulative rates of recovery of sperm
To compare the results of the cancer patients versus the controls, we deﬁned the reference thresholds as the 90th percentiles
vitality were also observed (P< .05). The TSC was lower in nonseminoma patients with a history of cryptorchidism (P¼ .001).
Change in Semen Parameters after Cancer
Treatment
4
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Fertility and Sterility®
FIGURE 1
Mean of total sperm count and standard error of the mean before treatment and during posttreatment follow-up observation according to
treatment type (radiotherapy, 1–2 BEP cycles, >2 BEP cycles). *P<.05, before and after treatment difference. #P<.05, difference between
treatments. Mean standard deviation is given at each time point.
Bujan. TGCT cancer treatment effect on sperm. Fertil Steril 2013.
in controls: DFI ¼ 20%, HDS ¼ 7.5%, DFI þ HDS ¼ 26.1%, TUNEL ¼ 16.7%. After adjusting for age and sexual abstinence,
before chemotherapy or radiotherapy the TGCT patients had
statistically signiﬁcantly higher mean values than the controls
for DFI (17.1 9.8 vs. 11.4 7.6%, P< .05) and DFI þ HDS
(22.7 10.3 vs. 16.9 8.1%, P< .05) but not for DNA fragmentation (TUNEL assay). Before chemotherapy or radiotherapy,
the DFI and TUNEL values were above normal in 30% and
11% of the TGCT patients, respectively. The proportion of patients with abnormal HDS increased at T6 (Supplemental
Table 1).
DISCUSSION
To our knowledge, ours is the ﬁrst prospective study based on
a standardized protocol, involving the largest TGCT population to date who were serially assessed after TGCT treatment.
Before treatment, TGCT patients have altered sperm characteristics compared with fertile men (28). Several explanations
have been proposed for sperm alteration before cancer treatment. Cryptorchidism is a well-documented risk factor for
TGCT (29) and decreased semen quality (30). However, we
found sperm alterations in cancer patients who had no cryptorchidism. Orchidectomy could explain sperm alterations
(28), but like Fraietta et al. (31) we found no difference in
sperm production before or after orchidectomy. Other causes
could be suggested, such as tumor-associated secreted factors
or stress. Moreover, it is postulated that TGCT is part of the
testicular dysgenesis syndrome deﬁned by Skakkebaek et al.
(32), which also leads to defective spermatogenesis.
VOL. - NO. - / - 2013
The TSC values were lowest 3 months after the end of
treatment, particularly when more than two BEP cycles
were performed. Decreased TSC was reported at 3 months in
one study (19), but an analysis according to the number of
BEP cycles was not performed. Radiotherapy induced a TSC
decrease at 3 months which persisted at 6 months, whereas
two BEP cycles had little effect at that time; the TSC values
began to increase after more than two cycles. Gandini et al.
(19) reported that decreased sperm production was of longer
duration after radiotherapy (6 months) than after chemotherapy (3 months). This could reﬂect the different impacts
of these treatments on spermatogenesis and sperm maturation. We found that chemotherapy and radiotherapy had
detrimental effects for at least 1 year after treatment with
more than two BEP cycles or with radiotherapy. Moreover,
at 3 months, decreased semen volume after radiotherapy or
after more than two BEP cycles probably reﬂected transient
dysfunction of genital glands.
It is generally accepted that 74 days are required for one
complete spermatogenic cycle and that around 12 days are
necessary for spermatozoa maturation in the epididymis.
Therefore, 3 months after treatment, one cycle of spermatogenesis and sperm epididymal transit have been completed.
The sperm production changes observed at 3, 6, and 12
months reﬂect treatment-induced germ-cell injury and probably also the impact of treatment on Sertoli-germ cell interactions. These hypothetical effects on the microenvironment of
spermatogenesis could explain the long-lasting damage.
To identify predictors of recovery of sperm production
R39 106/ejaculate, we used a Cox model integrating the
5
ORIGINAL ARTICLE: ANDROLOGY
FIGURE 2
Recovery of total sperm count R39 106/ejaculate after treatment. P value indicates the difference between groups using the log-rank test.
Hazard ratios (HR) and 95% conﬁdence intervals (CIs) were obtained from a univariate Cox model. The cumulative success rates of semen
recovery were estimated by the Kaplan-Meier method, stratiﬁed according to: (A) type of testicular germ cell tumor: 85% (95% CI, 75–93) for
seminoma and 70% (95% CI, 57–83) for nonseminoma; (B) the type of treatment: 63% (95% CI, 48–79) for chemotherapy %2 cycles, 92%
(95% CI, 70–99) for chemotherapy >2 cycles, and 86% (95% CI, 76–94) for radiotherapy; and (C) the total sperm count before treatment:
61% (95% CI, 46–76) for total sperm count (TSC) <39 106 and 89% (95% CI, 80–96) for TSC R39 106; (D) history of cryptorchidism:
81% (95% CI, 73–89) for no cryptorchidism and 55% (95% CI, 30–84) for cryptorchidism.
Bujan. TGCT cancer treatment effect on sperm. Fertil Steril 2013.
different parameters that could modify sperm characteristics.
To the best of our knowledge, this is the ﬁrst time such analysis
has been done. Treatment modalities and sperm production
before cancer treatment were the only factors linked with
sperm recovery in the multiple variable analyses.
It is noteworthy that during the ﬁrst year, while mean total
sperm count was decreased, some men had sperm characteristics compatible with natural fertility. Therefore, gamete quality and potential risk for progeny were particularly relevant
questions. We used SCSA to explore chromatin compaction,
and the TUNEL assay to evaluate DNA sperm fragmentation.
After treatment, we did not ﬁnd a higher proportion of
men with increased sperm DNA fragmentation. However,
more men had chromatin defects 6 months after treatment.
6
Other studies using SCSA (21, 33) or a CMA3 assay and
TUNEL assay (34) did not ﬁnd increased DNA/chromatin
alteration after TGCT treatment. However, using the COMET
assay, O'Flaherty et al. (20) demonstrated increased sperm
DNA damage after chemotherapy, which remained elevated
until 24 months despite sperm production recovery. Our
radiotherapy patients had signiﬁcantly a increased mean
HDS 6 months after treatment, reﬂecting defects in
chromatin condensation. Smit et al. (21) also found higher
DFI levels in radiotherapy than in chemotherapy patients.
Discrepancies in the published studies may arise from differences in treatments, populations studied, end points assessed, or the assessment methods, and particularly from
different follow-up protocols. Nevertheless, radiotherapy or
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Fertility and Sterility®
chemotherapy could induce genome alterations not assessed
by the methods used. We previously reported increased aneuploidy during the 17 months after BEP chemotherapy in cancer patients (35), which was conﬁrmed by some studies in the
ﬁrst 2 years after treatment (36, 37) but not by others (38). It is
interesting that in rats exposed to a BEP regimen similar to
that used in humans, increased preimplantation loss was
noted when these males were mated with healthy females
although normal sperm characteristics were recovered (39).
Other animal studies have demonstrated that BEP affects
spermatozoa quality (40, 41), germ-cell gene expression
(42), and spermatozoa methylation patterns (43). Furthermore, radiation induced transgenerational genome instability
and DNA damage in recent animal studies (44).
It is still debated whether cancer treatments have such effects on humans. Studies of children of fathers treated for cancer have not shown evidence of more frequent abnormalities
in offspring. However, it seems that these studies did not
have sufﬁcient power to detect relative risks of <3–5 (45),
and the interval between the end of cancer treatment and
conception was not precisely analyzed. Moreover, it seems
particularly important to note the paradigm difference (the
‘‘fundamental mystery’’) (46) between the results from animal
and from human studies, underlining the need to conduct
further human studies using new technologies.
CONCLUSION
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
We have demonstrated drastic deleterious effects of chemotherapy or radiotherapy on spermatogenesis of TGCT patients,
with possible recovery 2 years after treatment. Treatment modalities and pretreatment sperm production are predictive of
recovery of sperm production R39 106/ejaculate. Radiotherapy induced slight chromatin changes 6 months after
treatment. In view of differences in published data, the drastic
effects on spermatogenesis during follow-up evaluations and
the results of animal studies, we believe that other prospective
studies are necessary, using new methods of genome and epigenome exploration in humans to evaluate the effects of the
treatment on spermatozoa quality and the possible risk for
progeny (male-mediated developmental toxicity) (47).
Currently, couples are advised to use contraception for 1 to
2 years after TGCT treatment (20, 36).
15.
Acknowledgments: The authors thank Camille LamareEsquerre for her invaluable technical assistance, all the technicians involved in this project in each center, all the urologists
and medical oncologists as well as the radiotherapists who
referred the patients to the CECOS for fertility preservation,
and Nina Crowte for text editing.
21.
23.
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SUPPLEMENTAL FIGURE 1
Design of the prospective study.
Bujan. TGCT cancer treatment effect on sperm. Fertil Steril 2013.
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8.e1
ORIGINAL ARTICLE: ANDROLOGY
SUPPLEMENTAL TABLE 1
Median and proportion of patients with DNA fragmentation and
abnormal chromatin up to the 90th percentile found in normal men.
After treatment
Before
treatment 3 months 6 months 12 months 24 months
DFI
15.3 (30) 15.0 (38) 17.9 (33) 15.4 (33)
HDS
5.3 (9)
5.3 (15) 5.8 (28)
5.3 (8)
DFI þ HDS 20.9 (32) 20.5 (35) 23.1 (33)* 21.6 (33)
TUNEL
9.4 (11) 8.2 (5)
7.9 (14) 10.1 (14)
14.7 (29)
5.3 (15)
19.2 (32)
9.7 (15)
Note: Values are median (% of number of patients >P90 of control group). For detected ﬂuorescence intensity (DFI), P90 ¼ 20%; for high DNA stainability (HDS), P90 ¼ 7.5%; for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), P90 ¼ 16.7%.
* P¼ .05 compared with pretreatment values.
Bujan. TGCT cancer treatment effect on sperm. Fertil Steril 2013.
8.e2
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