Effect of chemo- or radiotherapy on sperm parameters of testicular cancer patients

Abstract

The aims of our study were to investigate the short- and long-term effects of chemo- or radiotherapy on spermatogenesis in patients with testicular cancer and to establish any correlation between pre-therapy sperm parameters, histotype and treatment type/intensity and the progress of spermatogenesis during the post-therapy period.
We evaluated 166 patients affected by testicular cancer, who cryobanked about 1 month after the removal of the cancerous testis and before beginning chemo- (CH group; n = 71) or radiotherapy (RT group; n = 95).
For the CH group, there was a statistically significant decrease in sperm parameters, which was most significant 3 months after the end of chemotherapy. For the RT group, this decrease was most relevant 6 months after the end of radiotherapy. Two years after therapy, 3% of the CH group and 6% of the RT group remained azoospermic. To evaluate whether spermatogenesis recovery is a function of baseline semen quality, we divided each group into two subgroups by pre-therapy total sperm count (A, <40 x 10(6)/ejaculate; B, >or=40 x 10(6)/ejaculate). At t(24), subgroup A of both the CH and RT groups showed improved sperm parameters over the baseline, whereas subgroup B for both CH and RT groups showed a return of sperm parameters to those of baseline values.
In conclusion, the recovery of spermatogenesis after chemo- or radiotherapy in our group of testicular cancer patients was not a function of pre-therapy sperm parameter quality. Cryopreservation of sperm before performing such therapy is therefore imperative.

Full text

Human Reproduction Vol.21, No.11 pp. 2882–2889, 2006 doi:10.1093/humrep/del167
Advance Access publication September 22, 2006.
2882 © The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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Effect of chemo- or radiotherapy on sperm parameters
of testicular cancer patients
Loredana Gandini
1,3
, Paolo Sgrò
1
, Francesco Lombardo
1
, Donatella Paoli
1
, Franco Culasso
2
,
Lucia Toselli
1
, Petros Tsamatropoulos
1
and Andrea Lenzi
1
1
Laboratory of Seminology and Immunology of Human Reproduction, Department of Medical Pathophysiology, Policlinico ‘Umberto I’
and
2
Department of Experimental Medicine, University of Rome ‘La Sapienza’, Rome, Italy
3
To whom correspondence should be addressed at: Laboratory of Seminology and Immunology of Human Reproduction, Policlinico
‘Umberto I’, University of Rome ‘La Sapienza’, 00161 Rome, Italy. E-mail: loredana.gandini@uniroma1.it
BACKGROUND: The aims of our study were to investigate the short- and long-term effects of chemo- or radiotherapy
on spermatogenesis in patients with testicular cancer and to establish any correlation between pre-therapy sperm
parameters, histotype and treatment type/intensity and the progress of spermatogenesis during the post-therapy
period. METHODS: We evaluated 166 patients affected by testicular cancer, who cryobanked about 1 month after
the removal of the cancerous testis and before beginning chemo- (CH group; n = 71) or radiotherapy (RT group; n = 95).
RESULTS: For the CH group, there was a statistically significant decrease in sperm parameters, which was most sig-
nificant 3 months after the end of chemotherapy. For the RT group, this decrease was most relevant 6 months after
the end of radiotherapy. Two years after therapy, 3% of the CH group and 6% of the RT group remained azoosper-
mic. To evaluate whether spermatogenesis recovery is a function of baseline semen quality, we divided each group
into two subgroups by pre-therapy total sperm count (A, <40 ´ 10
6
/ejaculate; B, ³40 ´ 10
6
/ejaculate). At t
24
, subgroup
A of both the CH and RT groups showed improved sperm parameters over the baseline, whereas subgroup B for
both CH and RT groups showed a return of sperm parameters to those of baseline values. CONCLUSIONS: In con-
clusion, the recovery of spermatogenesis after chemo- or radiotherapy in our group of testicular cancer patients was
not a function of pre-therapy sperm parameter quality. Cryopreservation of sperm before performing such therapy
is therefore imperative.
Key words: chemotherapy/radiotherapy/semen quality/testicular cancer
Introduction
Testicular cancer represents about 1% of all cancers in men but
is the most frequent tumour in men aged 15–39 years. Since
the 1990s, the use of chemo- and radiotherapy combined with
surgical techniques has enabled the cure of 90% of the cases
(Fossa et al., 1986), to the extent that testicular cancer therapy
is today one of the outstanding successes of medical science
(Presti et al., 1993; Dearnaley et al., 2001; Laguna et al., 2001).
However, these treatments can cause serious alterations to
spermatogenesis—with possible transient or permanent
azoospermia (Cullen et al., 1996; Pont et al., 1996; Bahadur,
2000)—and sperm chromatin structure (Martin et al., 1999; De
Mas et al., 2001; Morris, 2002). Given the young age of these
patients, their frequent lack of children and their improved long-
term prognosis, it is essential that they are given the opportunity
to cryopreserve their sperm before undergoing a therapy which
may have an irreversible effect on their fertilizing ability.
Numerous papers in the literature have evaluated the effect
of cancer therapy on sperm parameters. However, they are
sometimes limited by the low number of patients evaluated
(Fossa et al., 1985, 1986; Reiter et al., 1998), different semen
evaluation methods, diversity of cancer pathologies taken into
consideration (Bahadur et al., 2005), varying therapy intensity
and the variety of cancer agents used (Petersen et al., 1994;
Lampe et al., 1997; Huyghe et al., 2004).
The aims of our study were to (i) study the short- and long-
term effects of chemo- or radiotherapy on spermatogenesis in a
significant number of patients with testicular cancer and (ii)
establish any correlation between pre-therapy sperm para-
meters, histotype and treatment type/intensity and the progress
of spermatogenesis during the post-therapy observation period,
to identify any factor predictive of patients’ response to the therapy
in terms of semen quality.
Materials and methods
The study was approved by our University Hospital Ethics Commit-
tee. The sperm parameters of 166 testicular cancer patients attending
the Laboratory of Seminology and Immunology of Reproduction,
Department of Medical Pathophysiology, University of Rome ‘La
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Cancer therapy and semen quality
2883
Sapienza’ to undergo semen cryopreservation were examined. All
patients underwent semen collection about 1 month after the removal
of the cancerous testis and before beginning treatment (t
0
); none had
previously undergone a semen analysis. Patients were divided into
two groups on the basis of their therapeutic treatment according to
their histotype. The chemotherapy (CH) group, consisting of 71
patients with embryonal carcinoma or mixed tumours (variable asso-
ciation of seminoma, teratocarcinoma, choriocarcinoma and yolk sac
tumours), underwent chemotherapy under the PEB regimen (cisplatin,
etoposide and bleomycin). Chemotherapy dose and administration
regimen were as follows: days 1, 2, 3, 4 and 5 cisplatin 20 mg/m
2
i.v.
and etoposide 100 mg/m
2
i.v.; days 2, 9 and 16 bleomycin 18 mg/m
2
i.v. every 3 weeks for a maximum of four cycles. A total of 23 patients
were exposed to two chemotherapy cycles, 24 to three cycles and 23
to four cycles.
The radiotherapy (RT) group included 95 seminoma patients, who
underwent irradiation of the lumbar–aortic lymph nodes (with screen-
ing of remaining testicle). The protocol involved a daily dose of 180
cGy for 15–20 days at a mean dose of 2600 rad (range 1460–4200
rad). Sperm parameters were evaluated at 3 (t
3
), 6 (t
6
), 9 (t
9
), 12 (t
12
)
and 24 (t
24
) months after the end of therapy; some patients missed one
or more follow-ups before returning for a later control. Semen sam-
ples were collected by masturbation directly into a sterile plastic con-
tainer after 3–5 days of sexual abstinence. They were allowed to
liquefy for 60 min at 37°C and then examined by light microscope
according to World Health Organization (WHO) criteria (WHO,
1992, 1999). The following variables were taken into consideration:
ejaculate volume (ml), sperm concentration per ml (n × 10
6
/ml), total
sperm count (n × 10
6
), forward motility (%) and morphology (%
abnormal forms). Owing to the urgent need for patients to begin ther-
apy, seminal fluid was collected and analysed once only for each
patient. All seminal fluid examinations were carried out by the same
biologist (L.G.).
All patients signed their informed consent to both cryopreservation
and follow-up. To evaluate whether spermatogenesis recovery is a
function of baseline semen quality, we divided each group into two
subgroups by pre-therapy total sperm count: subgroup A, <40 × 10
6
/
ejaculate (26 patients for CH group and 31 for RT group) and sub-
group B, ≥40 × 10
6
/ejaculate (64 patients for RT and 45 for CH),
according to WHO (1999).
We also evaluated spermatogenesis recovery versus number of
chemotherapy cycles (two, three or four) or radiation intensity (≤2600
or >2600 cGy) based on the mean dose used to treat our patients (2600
rad, range 1460–4200 rad).
Statistical analysis
Quantitative results are expressed as mean and SD for the entire
patient group and the subgroups CH and RT. Before comparing the
various groups considered (by therapy, dosage and pre-therapy sperm
concentration), uniformity tests were performed on the variables
observed at base time (t
0
). To compare the results for the two groups,
we subsequently calculated the relative efficacy for sperm parameter
X as (X
t
– X
0
)/X
0
, where X
0
is the pre-therapy value and X
t
is the value
of the sperm parameter at time t. The Student’s t-test for paired or
unpaired data was used to evaluate the differences between two mean
values. Non-parametric tests (Wilcoxon and Mann–Whitney tests)
were also calculated where necessary. A two-tailed P-value below
0.05 was considered as statistically significant.
To confirm the results obtained by comparing sperm parameters at
different times, we repeated the same statistical analysis, limited to
patients who had undergone all follow-up sperm examinations.
To evaluate the association of semen variables and the different
covariates, we performed analyses of variance (ANOVA) for repeated
measures, using each semen parameter as the dependent variable,
therapy type as the grouping variable and the number of chemother-
apy cycles or radiotherapy intensity, abstinence duration, age and time
as covariates. The significance of covariates included in the model
was tested by Wald test (Armitage and Colton, 1998).
All statistical analyses were performed using BMDP dynamic soft-
ware (Dixon, 1992; Dixon and Merdian, 1992).
Results
The mean age ± SD of CH and RT group patients was 26.7 ± 4.4
(range 14–40) and 29.8 ± 4.9 (range 20–43) years, respec-
tively; this difference is not significant. The mean period of
abstinence for the CH and RT groups was 3.7 ± 1.1 and 4.3 ±
1.4 days, respectively; this difference is not significant.
Patients becoming azoospermic during the follow-up period
were evaluated separately (Table I). For the CH group, 15 of
40 patients (37%) were azoospermic 3 months after the end of
therapy, 11 of 32 (34%) after 6 months, 5 of 42 (12%) after
9 months and only 3 of 46 (6%) after 1 year, and just 1 of 33
patients (3%) was still azoospermic after 2 years.
For the RT group, 2 of 44 patients (4%) were azoospermic
3 months after the end of therapy, 11 of 43 (26%) after 6 months,
9 of 46 (19%) after 9 months and 6 of 69 (9%) after 1 year, and
just 3 of 57 patients (6%) were still azoospermic after 2 years.
Seminal parameters at t
0
, t
3
, t
6
, t
9
, t
12
and t
24
of patients becoming
azoospermic after therapy were not included in any statistical
analysis to avoid underestimation of mean values. Differences
in mean sperm parameters between the CH and RT groups at t
0
were not significant, that is, the groups were sufficiently
homogenous.
CH group
Means, SDs and significance of sperm parameter variations
(volume, concentration per ml and total sperm count, percent-
age forward motility and percentage abnormal forms) between
t
0
and follow-up periods are reported in Table II. There was a
statistically significant decrease in sperm concentration per ml,
total sperm count and forward motility and a statistically signi-
ficant increase in abnormal forms up to t
9
. Differences between
sperm parameters at t
0
and t
12
were not statistically significant,
indicating that sperm quality had returned to pre-chemotherapy
values. Further significant improvements in sperm concentra-
tion per ml, total sperm count and forward motility were found
at t
24
. The difference in abnormal forms between t
0
and t
24
was
not significant. No significant differences in ejaculate volume
were found at any of the follow-ups.
Table I. Azoospermic patients at t
3
, t
6
, t
9
, t
12
and t
24
Months Chemotherapy Radiotherapy
Total
patients
Azoospermic
patients [n (%)]
Total
patients
Azoospermic
patients [n (%)]
3 40 15 (37) 44 2 (4)
6 32 11 (34) 43 11 (26)
9 42 5 (12) 46 9 (19)
12 46 3 (6) 69 6 (9)
24 33 1 (3) 57 3 (6)
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L.Gandini et al.
2884
Semen evaluation at each follow-up showed that alteration
in sperm parameters was most significant 3 months after the
end of chemotherapy.
RT group
Means, SDs and significance of sperm parameter variations
(volume, concentration per ml and total sperm count, percent-
age forward motility and percentage abnormal forms) between
t
0
and follow-up periods are reported in Table III. There was a
statistically significant decrease in ejaculate volume, sperm
concentration per ml, total sperm count and forward motility
and a statistically significant increase in abnormal forms up to
t
12
. Differences at t
0
and t
24
were not statistically significant for
any parameter except volume, indicating that sperm quality
had returned to pre-radiotherapy values. However, even after
24 months, volume was found to be significantly decreased
from t
0
.
In contrast with the CH group, alteration in sperm parame-
ters was most relevant 6 months after the end of radiotherapy.
Figures 1 and 2 illustrate the progress of sperm parameter
means and SDs over the follow-up period, confirming the dete-
rioration seen in intermediate months and subsequent recovery
12–24 months after the end of therapy.
Recovery of spermatogenesis as a function of total
pre-therapy sperm count
CH and RT groups were divided into two subgroups according
to total sperm count (A, <40 × 10
6
; B, ≥40 × 10
6
). Results
obtained in the subgroups are reported in Tables IV and V.
For CH patients in subgroup A, the total sperm count
showed a non-significant increase at t
12
and a highly signific-
ant increase at t
24
(P < 0.001). The same parameter in subgroup
B appeared reduced at t
12
and increased at t
24
, the difference
being non-significant in both cases. There was a non-significant
alteration in volume in both subgroups, whereas a significant
increase at t
24
was found for forward motility in subgroup A.
Finally, the percentage of abnormal forms showed a statisti-
cally significant decrease at t
24
for subgroup A but was
unchanged in subgroup B (Table IV).
For RT patients in subgroup A, there was a non-significant
increase in total sperm count from t
0
to t
12
and a highly signi-
ficant increase from t
0
to t
24
(P < 0.001). The same parameter
in subgroup B showed a significant decrease at t
12
(P < 0.001)
and non-significant decrease at t
24
. There was a progressive
drop of volume in both subgroups, becoming significant only
in subgroup B at t
24
. Forward motility in subgroup A was simi-
lar at t
0
and t
12
but showed a statistically significant increase at
t
24
, whereas in subgroup B it decreased significantly at t
12
,
returning to the baseline value at t
24
. In subgroup A, there was
a non-significant reduction in abnormal forms at t
12
and t
24
,
whereas subgroup B showed a significant increase at t
12
,
returning to the baseline value at t
24
(Table V).
Spermatogenesis recovery as a function of treatment intensity
There was no significant difference in sperm parameters at t
12
and t
24
with respect to the baseline for the three CH subgroups
of two, three and four therapy cycles. A significant difference
Table II. Comparisons of mean sperm parameters between baseline and follow-up (0/3, 0/6, 0/9, 0/12 and 0/24) of chemotherapy patients
n, number of patients; NS, not significant.
*P < 0.05; **P < 0.01; ***P < 0.001.
Sperm parameter Baseline
(n = 71)
3 months (n = 25) 6 months (n = 21) 9 months (n = 37) 12 months (n = 43) 24 months (n = 32)
M ± SD M ± SD Significance M ± SD Significance M ± SD Significance M ± S.D. Significance M ± S.D. Significance
Volume (ml) 3.2 ± 1.6 3.7 ± 1.7 NS 3.6 ± 1.4 NS 3.6 ± 1.5 NS 3.3 ± 1.6 NS 3.3 ± 1.3 NS
Concentration (×10
6
/ml) 27.2 ± 23.4 3.0 ± 5.4 ** 7.5 ± 11.5 *** 12.5 ± 11.8 *** 22.9 ± 20.2 NS 52.2 ± 43.1 **
Total sperm count (x10
6
) 83.6 ± 78.3 10.9 ± 15.2 *** 23.6 ± 33.8 *** 45.1 ± 59.9 *** 69.7 ± 66.0 NS 146.8 ± 101.4 **
Forward motility (%) 31.6 ± 16.9 11.8 ± 12.8 ** 14.5 ± 17.4 * 28.1 ± 14.7 * 32.4 ± 16.8 NS 41.4 ± 14.0 **
Abnormal forms (%) 66.8 ± 13.3 85.2 ± 13.8 *** 84.4 ± 16.4 *** 70.6 ± 11.4 ** 63.7 ± 19.6 NS 62.7 ± 7.7 NS
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Cancer therapy and semen quality
2885
(P < 0.01) was found between the two RT subgroups (≤2600
and >2600 cGy) between t
0
and t
12
for concentration per ml
and total sperm count only, whereas there was no statistically
significant difference in any parameters between t
0
and t
24
.
ANOVA confirmed that for these specific therapeutic regi-
mens, the only covariate with a different effect on sperm
parameter variations between the CH and RT groups was time.
To confirm the results obtained after chemo- and radiotherapy
in terms of spermatogenesis recovery, we repeated the compar-
isons of the mean sperm parameter values for CH and RT
groups, including only those patients who had undergone all
controls. Twenty-one CH and 19 RT patients underwent all
controls up to t
12
, and 14 CH and 16 RT patients underwent all
controls up to t
24
. The results of the analysis of these patients
were similar to those obtained in the CH and RT groups as a
whole (data not shown). It therefore follows that the differ-
ences seen in sperm parameter values at later follow-ups were
not influenced by missed follow-up appointments.
Discussion
The prognosis of patients with testicular cancer has considera-
bly improved in recent years: whereas in 1970 the mean sur-
vival of such patients was only 10%, since 1990 it has risen to
90%. This can be attributed to notable diagnostic and surgical
advances and new radiotherapy and chemotherapy protocols,
to which testicular tumours are especially sensitive.
The most commonly used chemotherapy regimen is PEB,
consisting of etoposide, bleomycin and cisplatin. Etoposide
works by breaking individual DNA strands and blocking the
cell cycle at the S–G
2
phase. Bleomycin’s cytotoxic activity is
related to its ability to interact with iron and oxygen molecules,
inducing free-radical production capable of fragmenting the
DNA molecules and blocking cells in the G
2
phase. Finally, cis-
platin contains a platinum ion which forms complexes able to
react with DNA, creating both intrachain and interchain cross-
links. It interferes with the S phase of the cell cycle, inhibiting
replication and transcription and inducing coding errors.
In contrast, radiotherapy damages the DNA, impeding the
cell from replicating itself and causing its death. This therapy
is especially effective in cancer cells, which replicate more
quickly, but also affects normal cells, especially those with a
high replication rate such as spermatogonia. Radiation induces
material ionization both directly, through excitation of the
atoms making up the DNA molecule, and indirectly, through
its interaction with non-DNA molecules, which induce the ion-
ization of the genetic material by emitting secondary electrons
(Coogle, 1983; Chabner et al., 2001).
The cytotoxic effect of these therapies on spermatogenetic
cells has led to great interest in studying post-therapy sperm
parameter alterations in testicular cancer subjects. Numerous
literature articles report studies of antineoplastic therapy on
sperm quality but can be limited by the low number of patients
examined and methodological errors which reduce their valid-
ity (Fossa et al., 1985; Aubier et al., 1989; Hansen et al., 1990;
Shafford et al., 1993; Petersen et al., 1994; Palmieri et al.,
1996; Lampe et al., 1997; Reiter et al., 1998; Ishikawa et al.,
2004; Bahadur et al., 2005).
Table III. Comparisons of mean sperm parameters between baseline and follow-up (0/3, 0/6, 0/9, 0/12 and 0/24) of radiotherapy patients
n, number of patients; NS, not significant.
*P < 0.05; **P < 0.01; ***P < 0.001.
Sperm parameter Baseline
(n = 95)
3 months (n = 42) 6 months (n = 32) 9 months (n = 37) 12 months (n = 63) 24 months (n = 54)
M ± SD M ± SD Significance M ± SD Significance M ± SD Significance M ± SD Significance M ± SD Significance
Volume (ml) 3.6 ± 1.6 3.2 ± 1.4 * 3.2 ± 1.9 * 3.1 ± 1.4 ** 3.3 ± 1.7 ** 2.8 ± 1.3 *
Concentration (x10
6
/ml) 34.4 ± 34.2 9.5 ± 15.4 *** 7.9 ± 14.0 *** 13.4 ± 13.0 *** 20.4 ± 24.2 *** 42.9 ± 35.0 NS
Total sperm count (x10
6
)115.9 ± 110.1 30.0 ± 51.9 ** 25.1 ± 46.1 *** 43.3 ± 57.1 *** 67.0 ± 87.9 ** 111.2 ± 81.1 NS
Forward motility (%) 33.2 ± 16.0 17.8 ± 16.3 *** 15.9 ± 19.2 ** 24.2 ± 17.4 * 28.5 ± 17.1 * 39.6 ± 13.1 NS
Abnormal forms (%) 64.6 ± 15.8 80.5 ± 15.8 *** 80.1 ± 22.1 *** 73.2 ± 19.9 *** 71.1 ± 15.2 *** 62.4 ± 12.8 NS
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L.Gandini et al.
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Lampe et al. (1997) studied 178 patients with testicular can-
cer, of whom 170 had recovered spermatogenesis 1 year after
the end of chemotherapy. Of the 89 patients with a normal pre-
therapy sperm concentration per ml, 64% had returned to nor-
mozoospermia, 16% presented oligozoospermia and 20%
azoospermia. It should be noted that these authors arbitrarily
designated all patients with a sperm concentration of less than
1 × 10
6
/ml as azoospermic. The paper concludes that the recov-
ery of spermatogenesis is tied not only to the type of treatment
but also to pre-therapy sperm quality, and therefore these two
factors may be predictive of spermatogenesis recovery after
chemotherapy for testicular cancer.
According to other authors, the most important parameter in
evaluating spermatogenesis recovery is the chemotherapeutic
dose used. Petersen et al. (1994) compared a classic and high-
dose PEB regimen over a 3-year follow-up, with 19% of the
classic group and 47% of the high-dose group becoming
azoospermic. Ishikawa et al. (2004) also found the recovery of
spermatogenesis in only five of 10 patients after high doses of
chemotherapy. The authors found no difference in response to
cumulative therapy dose between two groups of azoospermic
and non-azoospermic patients. They therefore concluded that it
was not possible to establish a priori which patients would
become azoospermic and which would recover spermatogenesis.
Reiter et al. (1998) reported the recovery of a normal sperm
condition in 68% of 22 patients with stage I seminoma 4 years
after chemotherapy. This is in contrast with the results of our
study, which found the recovery of 97% of the patients within
2 years after the end of chemotherapy. It should be stressed
that in our caseload, seminoma patients were subjected to radi-
otherapy, whereas in Reiter’s study the treatment effected was
high-dose carboplatin. In fact, although radiotherapy is more
damaging than chemotherapy for spermatogenesis, it can be
postulated that the massive doses of carboplatin used induced
far more damage.
Fossa et al. (1986) evaluated the recovery of spermatogene-
sis in seminoma patients treated with radiotherapy after shield-
ing of the remaining testicle, under a regimen similar to that
reported in our work but with a more restricted caseload of 29
patients. The authors concluded that spermatogenesis was
much more severely affected in patients with a pre-therapy
sperm concentration of <3 × 10
6
/ml and therefore that the alter-
ations in spermatogenesis observed 2–3 years after the end of
radiotherapy were the result of differing pre-treatment sperm
production rather than the level of irradiation of the remaining
testicle.
The deterioration in semen quality after gonadotoxic therapy
is also confirmed by the retrospective study conducted by
Bahadur et al. (2005). Although this analysis of post-treatment
semen data involved 314 patients, the caseload was somewhat
mixed—including malignant neoplasms with unspecified loca-
tion, benign pathological conditions, lymphomas and leukae-
mia as well as testicular tumours. Furthermore, the paper refers
to a generic gonadotoxic therapy without specifying funda-
mental parameters such as the type and duration of the therapy
undergone by the patients.
Our study was conducted on a uniform caseload of 166
patients. For the chemotherapy group, the number of azoosper-
mic patients was highest 3 months post-treatment, slightly
lower at t
6
and more significantly reduced from t
9
onwards.
This leads us to suppose that the cytostatic effect of the drugs
used is more selectively targeted at cells in the premeiotic
phase—spermatogonia and primary spermatocytes—which suf-
fer serious nuclear damage with a subsequent block of replica-
tion and thus of the spermatogenesis cascade. This halt explains
the absence of sperm in the first 3 months after therapy.
Figure 1. Variation of seminal parameters after chemotherapy over time (in months).
0 3 6 9 12
1
2
3
4
5
6
24
Months
volume (ml)
0 3 6 9 12
0
10
20
30
40
50
60
70
80
90
100
24
Months
Concentration (×10
6
/ml)
0 3 6 9 12
0
20
40
60
80
100
120
140
160
180
200
220
240
260
280
24
Months
Total sperm count (×10
6
)
0 3 6 9 12
0
10
20
30
40
50
60
24
Months
Forward motility (%)
0 3 6 9 12
0
10
20
30
40
50
60
70
80
90
100
24
Months
Abnormal forms (%)
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Cancer therapy and semen quality
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The contrasting progress over time between chemotherapy
and radiotherapy should be stressed. In fact, the peak in
azoospermic patients was delayed in the RT group (t
6
) com-
pared with the CH group (t
3
).
This leads us to hypothesize that not only spermatogonia,
the most radiosensitive cells due to their intense mitotic activ-
ity, but also spermatids are affected by ionizing radiation. The
latter are unprotected, because of the loss of their DNA dam-
age repair mechanisms caused by post-meiotic differentiation
and chromatin condensation. The persistence of an adequate
population of spermatocytes surviving the radioactive impact
and able to mature and produce spermatozoa could explain the
greater time needed to reveal the damage.
Spermatogenesis in chemotherapy patients considerably
worsened in the 3 months after treatment, characterized by a
severe oligoasthenoteratozoospermia exactly coinciding
with the peak in numbers of azoospermic patients. This
again can be explained by the antineoplastic effect of these
substances on spermatogenesis, especially on the spermato-
gonia and primary spermatocytes, cells with a higher mitotic
activity. Spermatogenesis then shows a steady recovery from
9 to 12 months, and at t
24
, these patients presented a sperm
concentration twice that of the analysis made on the day of
cryopreservation.
Spermatogenesis in radiotherapy patients considerably
worsened 6 months after treatment, in line with the number of
subjects becoming azoospermic. This too is explained by the
hypothesis of radiation-induced damage to the spermatogonia
and spermatids as well as the persistence of an adequate popu-
lation of spermatocytes in the maturation phase. In this group
too, most patients had recovered spermatogenesis similar to the
baseline at t
24
: it is self-evident that protection of the remaining
testicle from radiation absorption, as demonstrated by Hansen
et al. (1990) and carried out on all our patients, is essential to
preserve spermatogenesis.
One more important finding of our study is the lack of corre-
lation between pre-therapy sperm parameters and post-therapy
spermatogenesis recovery. In fact, subgroup A (pre-therapy
total sperm count <40 × 10
6
) of both the CH and RT groups
showed improved sperm parameters over the baseline at t
24
. To
explain this, it should be remembered that some patients may
show a poorer sperm quality at the time of cryopreservation
due to the surgical stress of the orchiectomy, the subsequent
antibiotic therapy and the psychological stress related to diag-
nosis of cancer and the need for major therapeutic procedures.
Subgroup B (pre-therapy total sperm count ≥40 × 10
6
) of
both the CH and RT groups recovered their baseline sperm
quality at t
24
. Variations with respect to the baseline were not
statistically or biologically significant. Our data demonstrate
that spermatogenesis recovery is not a function of baseline
spermatogenesis, and thus it is not possible to identify an indi-
cator able to predict either possible azoospermia or the quality
of post-therapy spermatogenesis recovery.
We found that the number of PEB chemotherapy cycles
(two, three or four) and thus the total dose did not affect sper-
matogenesis recovery. There was no statistically significant
difference in recovery between t
0
and t
12
and t
24
(data not
shown) among the three groups. This agrees with literature
data, which describe worsened spermatogenesis with more
than four therapy cycles, if the cumulative cisplatin and etopo-
side dose reaches around twice that taken by the patients
enrolled in our study.
A different result was seen for radiotherapy, considering the
mean dose (2600 rad) as the discriminating value. At t
12
, the
reduction in total sperm count is statistically significant in sub-
jects having undergone a total dose >2600 cGy in comparison
Figure 2. Variation of seminal parameters after radiotherapy over time (in months).
0 3 6 9 12
1
2
3
4
5
6
24
Months
volume (ml)
0 3 6 9 12
0
10
20
30
40
50
60
70
80
90
100
24
Months
Concentration (×10
6
/ml)
0 3 6 9 12
0
20
40
60
80
100
120
140
160
180
200
220
240
260
280
24
Months
Total sperm count (×10
6
)
0 3 6 9 12
0
10
20
30
40
50
60
24
Months
Forward motility (%)
0 3 6 9 12
0
10
20
30
40
50
60
70
80
90
100
24
Months
Abnormal forms (%)
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L.Gandini et al.
2888
Table IV. Comparison of mean sperm parameters between baseline and 12 and 24 months after chemotherapy for subgroups A (total sperm count <40 × 10
6
) and B (total sperm count ≥40 × 10
6
)
n, number of patients; NS, not significant.
*P < 0.05; **P < 0.01; ***P < 0.001.
Sperm parameter Subgroup A Subgroup B
Baseline
(n = 26)
12 months (n = 18) 24 months (n = 11) Baseline
(n = 45)
12 months (n = 25) 24 months (n = 19)
M ± SD M ± SD Significance M ± SD Significance M ± SD M ± SD Significance M ± SD Significance
Volume (ml) 2.5 ± 1.7 3.0 ± 1.6 NS 2.9 ± 1.3 NS 3.7 ± 1.4 3.5 ± 1.6 NS 3.5 ± 1.5 NS
Total sperm count (×10
6
) 17.3 ± 12.8 29.2 ± 30.0 NS 107.8 ± 109.1 *** 119.6 ± 75.4 96.2 ± 70.0 NS 158.5 ± 92.4 NS
Forward motility (%) 18.8 ± 15.0 23.2 ± 17.8 NS 37.3 ± 12.1 ** 38.6 ± 13.5 38.5 ± 13.3 NS 43.6 ± 14.2 NS
Abnormal forms (%) 76.1 ± 13.0 71.0 ± 21.3 NS 64.5 ± 5.4 ** 61.7 ± 10.5 62.4 ± 14.8 NS 62.1 ± 8.6 NS
Table V. Comparison of mean sperm parameters between baseline and 12 and 24 months after radiotherapy for subgroups A (total sperm count <40 × 10
6
) and B (total sperm count ≥40 × 10
6
)
n, number of patients; NS, not significant.
*P < 0.05; **P < 0.01; ***P < 0.001.
Sperm parameter Subgroup A Subgroup B
Baseline
(n = 31)
12 months (n = 21) 24 months (n = 17) Baseline
(n = 64)
12 months (n = 48) 24 months (n = 40)
M ± SD M ± SD Significance M ± SD Significance M ± SD M ± SD Significance M ± SD Significance
Volume (ml) 3.1 ± 1.4 2.8 ± 1.5 NS 2.3 ± 1.1 NS 3.9 ± 1.7 3.4 ± 1.7 NS 2.9 ± 1.3 **
Total sperm count (x10
6
) 17.1 ± 12.5 19.7 ± 22.4 NS 48.3 ± 34.0 *** 163.7 ± 104.3 84.0 ± 96.8 *** 127.9 ± 82.9 NS
Forward motility (%) 19.4 ± 15.2 20.6 ± 14.7 NS 30.3 ± 16.5 * 39.8 ± 11.4 30.9 ± 17.1 ** 42.7 ± 10.6 NS
Abnormal forms (%) 78.2 ± 14.5 78.7 ± 12.6 NS 72.1 ± 14.3 NS 58.6 ± 9.2 68.6 ± 15.2 *** 59.7 ± 10.9 NS
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Cancer therapy and semen quality
2889
with those subjected to a dose ≤2600 cGy. This difference is
not statistically significant at t
24
. This confirms that the total
dose of radiation administered is a discriminating and predic-
tive factor of the time necessary to recover spermatogenesis.
Furthermore, our data demonstrate that these chemo- and
radiotherapy protocols have the most detrimental effect on
spermatogenesis within 3–6 months of the treatment. Sperma-
togenesis recovery is a function of the time since the end of the
therapy, with 94% of patients treated with chemotherapy show-
ing good recovery after 12 months and 97% after 24 months
(93 and 94% for radiotherapy).
No statistically significant differences were seen between
baseline and control ejaculate volume values for the CH group.
The therapy probably acts only on the sperm progenitor cells,
not on the accessory glands. In contrast, a reduction in ejacu-
late volume was seen in the RT group, remaining constant up
to t
24
. This demonstrates the effect of radiotherapy on seminal
vesicles, inducing their partial hypofunction.
In conclusion, it is currently impossible to predict a priori
which patients will recover spermatogenesis and which will
remain azoospermic, and there is no sperm index which might
help predict which patients will remain permanently sterile. In
fact, recovery of spermatogenesis after chemo- or radiotherapy
in our group was not a function of pre-therapy sperm parameter
quality. The detrimental effect on the chromatin condensation
and DNA integrity of spermatozoa of men pre- and post-cancer
therapy (Morris, 2002; Stahl et al., 2004; O’Donovan, 2005)
should also be borne in mind.
Despite all of this, cryopreservation before chemo- or radio-
therapy is an indispensable health tool, giving the patient the
chance of fertility using cryopreserved spermatozoa even in
years immediately after therapy.
Acknowledgements
The authors thank Marie-Hélène Hayles for her assistance in the
English translation of the manuscript. This work was supported by a
grant from the Italian Ministry of Education and Research (MIUR-
COFIN) and the University of Rome ‘La Sapienza’, Faculty of Medicine.
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Submitted on December 27, 2005; resubmitted on February 14, 2006, April 11,
2006; accepted on April 21, 2006
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