1 Fertility Center and 2 Department of Urology, Malmö University Hospital, Lund University, Malmö and 3 Department of Oncology, Lund University Hospital, Lund University, Lund, Sweden
4 To whom correspondence should be addressed: at Department of Oncology, Lund University Hospital, SE 221 85 Lund, Sweden. e-mail; jakob.eberhard{at}kir.mas.lu.se
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Abstract |
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Key words: androgen receptor/chemotherapy/radiotherapy/semen quality/testicular cancer
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Introduction |
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Although available data show that testicular cancer is associated with impaired sperm production as well as decreased fertility potential (Møller and Skakkebæk, 1999; Petersen et al., 1999
), many studies do not discriminate between the effects of different treatment modes including orchidectomy, radiotherapy (RT), CT or retroperitoneal lymph node dissection (RPLND) (Hendry et al., 1983
; Fosså et al., 1985
; Hansen et al., 1990
; Stephenson et al., 1995
; Joos et al., 1997
; Lampe et al., 1997
; Pont and Albrecht, 1997
) and do not take the intensity of the treatment into account (Hansen et al., 1990
; Stephenson et al., 1995
; Bokemeyer et al., 1996
). Information regarding the time course and the degree of the recovery of sperm production as well as the proportion of males becoming permanently azoospermic is also limited.
Some studies have indicated that pre-treatment FSH levels, sperm concentration and the sperm chromatin structure predict post-treatment recovery of spermatogenesis (Fosså et al., 1985, 1990, 1997; Aass et al., 1991
; Lampe et al., 1997
). However, no attention has yet been paid to the impact of genetic factors.
Androgens are crucial for spermatogenesis, the concentration of testosterone being 100 times higher in the testis than in serum (Maddocks et al., 1993). Recent animal studies (Meistrich, 1999
; Shetty et al., 2001
) have shown that the intra-testicular steroid balance probably plays a crucial role for post-treatment recovery of spermatogenesis. Androgen action is the sum effect of available testosterone and 5
-dihydrotestosterone, and the responsiveness of the androgen receptor (AR) in target cells. One critical function of the AR gene product is to activate the expression of other genes. Two polymorphic sequences of CAG and GGN repeats have been shown to be important for the transactivation of other genes and thus play an important role for the fine-tuning of AR function (Tut et al., 1997
; Hsing et al., 2000
; von Eckardstein et al., 2001
).
The aim of this prospective, longitudinal study was to provide data on the effect of CT and RT on semen quality of TGCC patients, with special focus on the doseresponse effect and the time course of recovery. Furthermore, we have investigated the impact of the variation in the lengths of the CAG and GGN repeats of the AR gene on pre-treatment sperm characteristics and as a predictor of sperm regeneration post-treatment.
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Materials and methods |
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Up to April 1, 2003, 112 of 144 eligible patients were included in the study. Nineteen men declined to participate and 13 were excluded for the following reasons: five due to bilateral TGCC; three for psychiatric/psychological reasons; two due to linguistic difficulties; two because of hepatitis C; and one because of physical handicap. Eleven patients developed retrograde ejaculation due to retroperitoneal lymph node dissection. One died in progressive disease and one developed bilateral disease after inclusion (Figure 1). After primary inclusion, none of the men has left the study due to unwillingness to deliver a semen sample.
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The characteristics of the study population are given in Table I.
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Standard chemotherapy of NSGCT was the BEP regimen (bleomycin 30 000 IU days 1, 5 and 15; etoposide 100 mg/m2 days 15; cisplatinum 20 mg/m2 days 15, given every third week). In six patients given ACT diagnosed before 1998, the etoposide was replaced with vinblastine 0.15 mg/kg, maximum 11 mg/day, days 1 and 2 (CVB regimen).
ACT (BEP/CVB) was given to 32 patients. Among these, 27 patients had one and five had two CT cycles.
Forty-two patients with metastatic disease received three or more CT cycles (HCT). Among patients with NSGCT, 14 received three cycles of BEP, 15 four cycles of BEP and one died in progressive disease during treatment. Four cycles of EP (BEP minus bleomycin) were given to seven patients with advanced SGCT. Five patients (three NSGCT and two SGCT) received more intensive CT.
RT was administered to para-aortic and ipsilateral iliacal lymph nodes. A target dose of 25.2 Gy was given in 14 fractions. In order to obtain an estimate of the magnitude of the scattered irradiation of the gonad, the total dose to the remaining testicle was estimated retrospectively in seven randomly selected men to be 0.040.43 Gy.
Biological samples
The patients delivered a blood sample for DNA analysis at the first control after inclusion in the study and semen samples at the fixed time points between T0 and T60. An overview of sample delivery in relation to treatment and follow-up time is given in Figure 2.
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The laboratory participates in the external quality control programme of the Nordic Association for Andrology and the European Society of Human Reproduction and Embryology (Cooper et al., 2002). The data have been expanded with results of analysis of samples delivered for cryopreservation, after orchidectomy but prior to inclusion in this study.
All samples collected after inclusion were analysed in one laboratory (Fertility Centre, Malmö University Hospital). However, the cryopreservation samples were analysed by two different laboratories (Fertility Centre, Malmö University Hospital and Fertility Laboratory, Lund University Hospital). Both laboratories were performing semen analysis according to the most recent WHO recommendations (World Health Organization, 1992, 1999).
DNA analysis
Blood samples for DNA analysis were available from the first 83 men included in the study. These subjects did not differ from the remaining 29 with regard to age, sperm concentration at T0, disease, stage, histological type or treatment given (data not shown). Genomic DNA was prepared from peripheral leukocytes, and the CAG and GGN repeats (in 81 subjects) were amplified by PCR and subsequently analysed externally on a Beckman Coulter CEQ 2000XL (Beckman Coulter, Bromma, Sweden) sequencing gear as previously described (Lundin et al., 2003).
Statistical analysis
Statistical analysis was performed using the SPSS 11.0 software (SPSS Inc., Chicago, IL). Longitudinal analysis of data was performed (Figure 2). Additionally, comparison of semen parameters between groups defined according to the treatment and follow-up time (cross-sectional analysis) was done. In order to obtain sufficient numbers of individuals, the results of samples collected at T24, T36 and T60 were combined into one category (T2460). If a patient delivered more than one sample during this time interval, the one with the highest sperm concentration was included in the analysis. For longitudinal comparisons of more than two samples, Friedmans test was used. For intra-individual comparison of values at two time points only, the Wilcoxon test for paired data was applied. In the cross-sectional analyses, KruskalWallis test and MannWhitney test for unpaired data were used.
Spearmans rho was calculated in order to find the correlation between the CAG or GGN repeat length and the sperm concentration at any of the following time points: T0, T6, T1224 and T3660. These calculations were performed for the randomly selected group of 83 TGCC patients from whom the DNA data were available and separately for the therapy groups ACT, HCT and RT.
Subsequently, in these 83 men, in order to calculate the predictive value on the sperm concentration at T0, multivariate linear regression analysis was used with the type of tumour as the discrete variable, and age and CAG repeat length as continuous variables. A similar type of analysis was done for sperm concentration at T6, T1224 and T3660, with type of therapy as the discrete variable, and CAG repeat lengths and age as well as sperm concentration at T0 as continuous independent variables. Sperm concentrations were log transformed (after adding 0.1 to all sperm concentrations in order to be able to transform 0 values) prior to the analysis.The numbers of patients providing semen analysis data were 56, 23, 42 and 31 for T0, T6, T1224 and T3660, respectively. All statistical tests were two-sided, and P < 0.05 was considered statistically significant.
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Results |
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Sperm concentration
The data are summarized in Table II and Figure 3.
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In the HCT group, the longitudinal comparison showed that the median sperm concentrations at T6 and T12 were significantly lower than at T0, 0.05 x 106/ml at T6 compared with the baseline value of 3.4 x 106/ml (P = 0.043), and 2.4 x 106/ml at T12 compared with 18 x 106/ml at T0 (P = 0.046) (Table II). At T2460, the sperm concentration had recovered to 19 x 106/ml, not different from the T0 value of 12 x 106/ml (P = 0.8)
In the SGCT group treated with RT, the sperm concentration was lower at T6 compared with T0, 0.1 versus 48 x 106/ml (P = 0.04), while the T12 value was significantly higher compared with T6 (6.8 versus 0.9 x 106/ml, P = 0.03). The difference between T12 and T0 levels (median: 6.3 versus 36 x 106/ml, respectively) was close to the level of statistical significance (P = 0.08). Sperm concentration at T2460 in four males, also investigated at baseline, was not different from the T0 value, 47 versus 32 x 106/ml (P = 0.27) (Table II).
There was no significant difference in median sperm concentration at T0 between the NSGCT and SGCT patients (median: 19.6 versus 10.0 x 106/ml, respectively) (P = 0.24). Comparing the different treatment groups at T0, no statistical difference was seen. At T6 and T12, there was a significant difference between the groups (P = 0.0001), the ACT group having significantly higher sperm concentration than both the HCT (P = 0.0001) and the RT group (P = 0.001). T2460 concentrations did not differ between the therapy groups.
The number of observations in the surveillance group was too low to allow statistical analysis.
Data on the period of ejaculation abstinence were available for 91 of the 177 samples. No significant difference in abstinence time was found when T0 samples were compared with samples collected at the other time points (P = 0.11).
Length of CAG repeat, correlation with sperm concentration recovery
There was a significant negative correlation between CAG repeat and sperm concentration after 1224 months ( = 0.72; P = 0.03) (Figure 4) in men treated with three or four cycles of CT. The repeat number did not correlate with sperm concentration for the other therapy forms or at other time points, including at T0. No significant correlation was found for GGN length.
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Sperm motility
The only significant finding was a lower percentage of motile sperm at T12 compared with T0, found in the RT group, 38 versus 68% (P = 0.03). However, for several comparisons, the number of patients included was not sufficient to perform statistical analysis (see Table III).
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Discussion |
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Novel for this study, compared with those already published (Giwercman and Petersen, 2000), is the combination of discrimination between treatment modes and post-treatment sample collection at well-defined time points, in principle allowing for more precise mapping of the process of spermatogenesis recovery (Kuczyk et al., 2000
). The participation rate was close to 80%, reducing the risk of selection bias. From 65% of these men, pre-treatment sperm parameters were available, and 80% of them delivered further samples, allowing a longitudinal follow-up.
Four of the 73 men from whom a pre-treatment semen sample was available had azoospermia already after orchidectomy. None of the patients treated with ACT developed azoospermia. However, the number of patients, especially those given two chemotherapy cycles, is still limited, and until more data are available, we recommend cryopreservation to men receiving ACT. The risk of developing azoospermia was significantly higher for those receiving RT or HCT. Unfortunately, we did not have enough longitudinal observations to allow detailed analysis of the recovery from azoospermia. Previous studies (Hansen et al., 1989) have indicated that 5 years after treatment, the risk of permanent azoospermia is negligible even in men treated with more than two cycles of BEP chemotherapy.
In agreement with earlier reports, we found the negative impact of CT on spermatogenesis to be dose dependent (Petersen et al., 1994). ACT did not have any significant influence on sperm production (Cullen et al., 1996
). The time course of the recovery of sperm production was similar in men treated with RT and HCT. Apart from the already mentioned risk of azoospermia, the pre-treatment levels of sperm concentration were seen after 2460 months. Since the reproductive period of a couple is limited, a prolonged period of azoospermia or very low sperm concentration may seriously reduce the possibility of having a child.
Potentially, the results of our study could be influenced by variation in the length of the abstinence period. Such data were only available for 91 of the 177 samples. However, there was no statistically significant difference between the lengths of the abstinence period at the different time points, and these parameters were therefore not taken into consideration in the analysis of data. Concerning inter-laboratory variation in evaluation of sperm parameters, 41 samples analysed at the time of cryopreservation at another laboratory were included in order to increase the amount of data available for analysis. Both laboratories followed the WHO guidelines for performing semen analysis, and previous studies have shown that assessments of sperm concentration and total percentage of motile sperms are rather robust to inter-laboratory variation, when the same protocols (WHO) are applied (Jørgensen et al., 1997; Giwercman et al., 1999
).
The major conclusions of the study regarding the recovery of sperm concentration are based on longitudinal data and, for some of the analyses, the number of patients was fairly low. Inclusion of additional patients and prolonged follow-up of those who have already entered the study will give more precise information about the post-treatment recovery of spermatogenesis.
We found a negative correlation between the length of CAG repeat in the AR gene and sperm concentration after 1224 months in men who were treated with 34 cycles of BEP; the shorter the CAG stretch, the higher the sperm concentration. Furthermore, in a multivariate analysis, together with sperm concentration at T0, the CAG length was shown to be a significant, independent, predictor of sperm concentration at T1224. Previous in vitro and in vivo studies have shown that the length of the CAG repeat is inversely correlated with the transcriptional activity of the AR and with the sensitivity to the male sex hormones (Tut et al., 1997). Some studies have demonstrated longer CAG repeats in infertile men (Dowsing et al., 1999
) and an inverse correlation between sperm concentration and CAG length (von Eckardstein et al., 2001
). We did not find any correlation between CAG lengths and pre-treatment sperm concentration. Furthermore, the results of the multivariate analysis indicated that the effect of this AR polymorphism is not exerted through regulation of the pre-treatment state of spermatogenesis but rather is implicated in the process of recovery. We did not find any effect of the other repetitive sequence of the AR, i.e. the GGN repeat.
The finding of an association between the CAG segment and recovery of spermatogenesis is intriguing, not only from a clinical, but also from a biological point of view. The impact of androgen action on the rapidity of recovery of spermatogenesis indicates that after HCT, late stages of spermatogenesis (Zhang et al., 2003) play an important role for reaching pre-treatment levels of sperm concentration. Our findings should also be seen in view of the recent (Meistrich et al., 1999
) finding showing that in rats, recovery of spermatogenesis after procarbazine or irradiation treatment could be stimulated by use of GnRH agonists or antagonists. Further, it was shown (Shetty et al., 2000
) that this effect of hormonal treatment was due to lowering intratesticular testosterone levels. Our study indicates that decreased androgen action after BEP treatment rather delays the recovery of sperm production. This apparent discrepancy might be due to one or a combination of interspecies difference, use of another treatment mode or the fact that men with TGCC might also have Leydig cell dysfunction (Willemse et al., 1983
), leading to a relative hypoandrogenic intratesticular milieu of such patients. However, inclusion of larger groups of men is necessary to draw any firm conclusions regarding this issue.
In conclusion, we found a non-significant decrease in sperm concentration with full recovery after 12 months in TGCC patients treated with 12 cycles of cisplatin-based chemotherapy. In those who received RT or more than two cycles of CT, the dip in sperm concentration was more prolonged, not reaching pre-treatment levels until 25 years after completion of therapy. We also found that the AR CAG polymorphism is a significant predictor of the rapidity of recovery after 34 cycles of CT. Future studies including more patients and additional genetic markers may provide us with new powerful tools for an individualized prediction of the gonadotoxic effects of therapy in young males undergoing cancer treatment.
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Acknowledgements |
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Submitted on December 18, 2003; accepted on February 10, 2004.
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