Semen quality before and after gonadotoxic treatment

G. Bahadur1,4, O. Ozturk2, A. Muneer3, R. Wafa1, A. Ashraf1, N. Jaman1, S. Patel1, A.W. Oyede1 and D.J. Ralph3

1 Fertility and Reproductive Medicine Laboratories, Department of Obstetrics and Gynaecology, Royal Free and University College Medical School, University College Hospitals Trust, Rosenheim Building, 25 Grafton Way, London WC1E 6DB, 2 Department of Obstetrics and Gynaecology, Royal Free and University College Medical School, 88–96 Chenies Mews, London WC1E 6DB and 3 Middlesex Hospital, The Institute of Urology and Nephrology (St Peters Hospital), London W1N 8AA, UK

4 To whom correspondence should be addressed. Email: g.bahadur{at}ucl.ac.uk


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
BACKGROUND: The aim of this study was to analyse the semen quality of patients before and after gonadotoxic therapy. PATIENTS AND METHODS: We evaluated semen quality in 314 patients over a 26 year period. The diagnostic categories were leukaemia (n=13); lymphoma (n=128); testicular cancer (n=102); benign conditions (n=13); and other malignant neoplasms (n=58). The degree of azoospermia or oligozoospermia for each disease category was recorded. We then analysed the recovery in semen quality over time for each disease category. RESULTS: The mean patient age was 27.9 years (range 13–65 years). A total of 1115 post-treatment semen samples were analysed from 314 patients. There was a significant reduction in the post-treatment sperm concentration, sperm motility and semen volume compared with pre-treatment levels (P<0.05) in the entire cohort. However, the sperm movement and motility grade remained unaffected. Patients with testicular carcinoma had the lowest pre-treatment sperm concentrations but also the lowest incidence of azoospermia after cancer treatment. Patients with lymphoma and leukaemia had the highest incidence of post-treatment azoospermia and oligospermia. Patients having the largest reductions in their sperm concentration after treatment required the longest recovery period for spermatogenesis. The diagnostic category was the only significant predictor of post-treatment azoospermia. CONCLUSION: Gonadotoxic treatment results in a significant reduction in sperm quality. The type of cancer or disease, and the pre-treatment sperm concentrations were found to be the most significant factors governing post-treatment semen quality and recovery of spermatogenesis. All categories of patients displayed varying degrees of azoospermia and oligozoospermia, and recovery of gonadal function from these states was not significant. This highlights the importance of ensuring sperm banking before treatment, including for patients with benign conditions. Several factors and associations are discussed further in order to give an insight into the pre- and post-gonadotoxic treatment effects.

Key words: azoospermia/cancer/gonadotoxicity/oligozoospermia/quality


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Testicular dysfunction is an unwanted and well documented side effect of cancer therapy. Although first reported in 1948, when azoospermia following treatment with nitrogen mustard was described (Spitz, 1948Go), several reports have since assessed the semen quality in various cancer patients (Whitehead et al., 1982Go; Viviani et al., 1985Go; Aubier et al., 1989Go; Palmieri et al., 1996Go; Lampe et al., 1997Go; Tal et al., 2000Go; Ishikawa et al., 2004Go).

Improved patient survival rates coupled with advances in reproductive technologies such as IVF and ICSI mean that adult cancer patients can now be offered sperm cryopreservation (Hallak et al., 1999Go; Bahadur, 2000Go). However, factors such as the post-treatment semen status, recovery of sperm and safety issues have attracted enormous clinical and medico-legal attention (Byrne et al., 1987Go; Meistrich, 1993Go; Robbins et al., 1997Go; Martin et al., 1999Go; Chatterjee et al., 2000Go; Arnon et al., 2001Go; De Mas et al., 2001Go; Bahadur et al., 2002Go; Frias et al., 2003Go; Codrington et al., 2004Go; Deane et al., 2004Go; Seli et al., 2004Go).

This study assesses the semen quality of patients before and after gonadotoxic treatment. The data were also analysed in order to identify predictors or associations of sperm loss and recovery.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
A retrospective study was conducted of 314 patients who underwent gonadotoxic therapy and were referred for sperm cryopreservation between 1976 and 2002 to the combined Fertility Unit Laboratory at the University College and Middlesex Hospitals. Patients were referred from various clinics and seen within 3 days of their referral. Following counselling and informed consent, semen analyses were performed, with all samples obtained by masturbation. The semen analyses were carried out in accordance with WHO guidelines (World Health Organization, 1987Go, 1992Go) using a Neubauer counting chamber or Makler counting chamber (Menkveld et al., 1980Go; Imade et al., 1993Go; Shiran et al., 1995Go). Motility grading was 0–4, with 0 as non-progressive and 4 as very good progression. Semen volume measurements were carried out using a sterile graduated syringe. Morphology data were excluded mainly due to changes in the WHO criteria for sperm normality over the study period.

The patient characteristics such as age and disease were recorded. The patients were placed into one of five groups according to their underlying disease: leukaemia, lymphoma, testicular cancer, benign conditions which required chemotherapy (Crohns disease, renal lupus and musculoaponeurotic fibromatosus) and other malignant neoplasms (soft tissue sarcomas, osteosarcoma, prostate and skin cancers).

Two pre-treatment semen samples were collected from each patient. All patient follow-up remained the same in so far as patients chose a convenient time rather than being given a pre-determined fixed appointment. Therefore, results should be viewed in the context of this pragmatic limitation of study design. Each patient was followed-up after undergoing gonadotoxic treatment with an average of three post-treatment visits (range 1–9 post-treatment visits). Mean values of the two pre-treatment semen analyses were compared with the best post-treatment semen analysis. The follow-up period is variable and depended on the rate of recovery of the sperm concentration. Furthermore, for the lymphoma and testicular cancer groups, the first and last post-treatment semen samples were also compared.

Data analysis
Statistical analysis was performed using SPSS® for Windows® (SPSS Inc, Chicago, IL). Data were analysed using Spearman's rank correlation test, Mann–Whitney U-test, Wilcoxon signed rank test and the Pearson {chi}2 test. A multiple logistic regression analysis was performed using a forward selection model to adjust the existing association between prognostic factors.


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The study cohort consisted of 314 patients (leukaemia n=13; lymphoma n=128; testicular cancer n=102; benign conditions n=13; other malignant neoplasms n=58). The mean age of the patients was 27.9 years (range 13–65 years). A total of 1115 post-treatment samples were available for analysis over a mean follow-up period of 162 weeks (range 13–789 weeks). The results are summarized in Tables IIV and in Figure 1ac.


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Table Ia. Semen analysis values before and after gonadotoxic treatment

 

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Table IIb. Semen analysis values in diagnostic subgroups before gonadotoxic treatment (mean±SD)

 


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Figure 1. (a) Level of decrease in sperm concentrations and recovery period. R2=0.007; F=1.35; P>0.05. (b) Level of decrease in sperm concentrations and recovery period. R2= 0.007; F=0.6; P>0.05. (c) Level of decrease in sperm concentrations and recovery period. R2=0.113; F=5.397; P<0.05.

 
Combining all the available pre- and post-treatment samples showed that the overall sperm concentration, sperm motility and semen volume were significantly reduced following cytotoxic treatment in the whole cohort (n=314, P<0.05) (Table Ia). However, the motility grade remained unaffected after gonadotoxic treatment (P>0.05).

Table Ib illustrates the subgroup analyses of semen quality. In the testicular cancer group, only the concentration and volume were significantly reduced. In the leukaemia group, only the concentration was significantly reduced. The lymphoma group revealed a significant reduction in the concentration, motility and motility grade but not the volume, while in the ‘other malignant neoplasm’ group, both the concentration and motility were significantly reduced. In the ‘benign condition’ group, only the sperm concentration showed a significant reduction.


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Table Ib. Semen analysis values before and after treatment for individual diagnostic groups (P-values)

 
Table II (a–c) illustrates the variation in the concentration, motility grade, motility and semen volume expressed according to the disease category. This shows that the testicular cancer group has the lowest pre-treatment sperm concentration whilst the leukaemia and benign groups have the highest pre-treatment sperm concentrations.


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Table IIa. Semen analysis values in diagnostic subgroups before and after gonadotoxic treatment (mean±SD)

 
The lowest incidence of azoospermia occurred in the testicular cancer and benign groups (Table III). Table IV demonstrates the time taken in order to achieve the best post-treatment sperm concentration for the lymphoma and testicular cancer groups, which represent the groups showing the highest and lowest rates of azoospermia following treatment.


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Table III. Distribution of post-treatment azoo-oligozoo-normospermia in diagnostic subgroups

 

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Table IVa. Recovery period for lymphoma and testicular cancer patients to best post-treatment sperm concentration with different levels of spermatogenetic impairment

 
In order to assess the temporal recovery of sperm concentration, Figure 1ac illustrates that patients who have small changes from their pre-treatment sperm concentrations reached their best post-treatment concentrations at some point within the first 66 months (2000 days, 5.5 years) of their follow-up period. However, those patients who experienced a significant deterioration in sperm concentration had to wait longer than 66 months to reach their peak recovery.


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Although several reports exist in relation to sperm quality before gonadotoxic treatment, very few studies investigate the deterioration in semen quality following such treatment (Aubier et al., 1989Go; Shafford, 1993Go; Hieken et al., 1996Go; Tal et al., 2000Go; Ishikawa et al., 2004Go). In our study, the size of the study cohort, duration of the follow-up period and the inclusion of the benign neoplasm group are unique features allowing useful information and inferences to be made for patients undergoing gonadotoxic treatment.

In our analyses, the sperm quality in terms of density, motility and volume was significantly reduced (P<0.05) following gonadotoxic treatment for all groups (Table I). However, only the magnitude of change in concentration appears to be of clinical relevance. This suggests that the damaged or destroyed germinal stem cells may recover from an initial cytotoxic insult and maintain a reasonable sperm quality in terms of motility and motility grading, although sperm concentration remained variable. The lymphoma and leukaemia groups showed the highest incidence of azoospermia following gonadotoxic treatment, at 46 and 59% of their respective groups (Table III). On the other hand, the benign neoplasms and testicular cancer groups showed the lowest incidence of azoospermia at 16 and 12%, respectively, which may be indicative of less severe gonadotoxic treatment regimens in these conditions compared with, for instance, lymphoma.

The testicular cancer group was found to have the lowest sperm concentrations of all the disease categories before treatment (Table II). However, following cytotoxic treatment, the testicular cancer group showed the lowest level of azoospermia (12%), but the highest level of oligozoospermia (38%). The normospermia levels after treatment for the testicular cancer group were also high (50%), second only to the the benign group (61%) (Table III). The high level of oligozoospermia in these patients may be due to impaired spermatogenesis pre-treatment, and treatment with less gonadotoxic modalities might have spared them from developing azoospermia. This observation was supported by the findings of recent studies (Bahadur et al., 2002Go; Gandini et al., 2003Go). The testicular cancer group had a significant decline in only the concentration and volume. (Table Ib).

Our finding of reduced sperm quality in testicular cancer patients confirms other studies. Patients with testicular cancer have an impaired semen quality at diagnosis which deteriorates further after orchidectomy. Previous studies have shown that 50–70% of patients with testicular cancer after orchidectomy, but before cytotoxic treatments, have impaired spermatogenesis (Hendry et al., 1983Go; Palmieri et al., 1996Go). A link between male factor subfertility and testicular cancer is also recognized (Jacobsen et al., 2000Go). Reduction in sperm concentrations may be linked to disruption of embryonal programming and gonadal development (Skakkebæk et al., 2001Go), elevated scrotal temperature secondary to neovascularization and HCG production by the neoplastic testicle (Berthelsen and Skakkebaeck, 1983Go). Recovery of sperm function after treatment has, so far, been unpredictable (Byrne et al., 1987Go; Siimes and Rautonen, 1990Go), While all patients initially developed azoospermia following bleomycin, cisplatin and etoposide (BEP) chemotherapy, 48% have recovered motile ejaculated sperm after 2 years, and after 5 years this figure rises to 80% (Lampe et al., 1997Go). Although the total sperm count may still be reduced, the potential for fatherhood still exists.

It was reported that impaired pre-treatment semen quality occurs in lymphoma and leukaemia patients (Botchan et al., 1997Go; Hallak et al., 1999Go). Our previous study (Bahadur et al., 2002Go) illustrated that sperm counts before gonadotoxic treatment were significantly lower in Hodgkin's and non-Hodgkin's lymphoma, and in leukaemia patients than in a healthy sperm donor population, reflecting the adverse contribution of the disease. This point receives support elsewhere (Kobayashi et al., 2001Go; Rueffer et al., 2001Go). However, normal semen parameters were also reported in cancer patients (Rofeim and Gilbert, 2004Go).

In this study, the leukaemia and lymphoma groups fared worst following gonadotoxic treatment. There is a body of information to suggest that the nature of treatment regimens could explain some effects on the level of gonadotoxicity, and where sperm prevail, some effect on the sperm function (Reiter et al., 2002Go; Chapman et al., 1979Go; Whitehead et al., 1982Go; Anselmo et al., 1990Go; Longo et al., 1997Go; Viviani et al., 1985Go; Van den berg et al., 2004Go; Kulkarni et al., 1997Go; Pryzant et al., 1993Go). In our analyses, Table IIa reveals the recovery time was longest for the leukaemia and lymphoma groups.

Although leukaemia patients had the highest pre-treatment sperm concentrations (Table II), 46% of patients developed post-treatment azoospermia (Table III). As there is a high proportion of viable stem cells which are subjected to gonadotoxic agents, this results in a larger overall fall in sperm concentration. Germ cell dysfunction occurs over a short interval, as opposed to the situation in testicular cancer. This may account for the longer recovery time in this group of patients. It remains difficult to differentiate the contribution of disease and treatment on semen quality without prospective randomized controlled studies. Despite its large cohort size, retrospective analysis of the clinical information has limited our ability to answer this question completely.


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Table IIc. Semen analysis values in diagnostic subgroups after gonadotoxic treatment (mean±SD)

 
A total of 37% of the cases who had post-treatment azoospermia recovered to a mean sperm concentration of 17.8 x 106/ml (0.01–60) during the post-treatment follow-up period with a mean duration of 48.6 (14–143) months. Recovery among different diagnostic groups did not differ significantly (P=0.25). Information on when sperm recovery first occurs, whilst desirable, would be both unethical and impractical to obtain as this would involve serial weekly or monthly semen analyses studies on each patient with azoospermia following a major treatment.

Although future options for patients may become available with regards to germ cell technology of repopulating the testes with testicular stem cells, or by generating mature sperm cells from embryonic stem cells, the safety and ethical use of these technologies should not be underestimated (Bahadur, 2004Go). Cryopreservation of semen prior to gonadotoxic treatment is the current preferred strategy, but testicular sperm extraction and ICSI after chemotherapy has also been employed successfully (Meseguer et al., 2003Go). Caution needs to be exercised, however, in utilizing sperm obtained after cancer treatment to protect the welfare of the child (Meistrich et al., 1985Go; Meistrich, 1993Go; Robbins et al., 1997Go; Martin et al., 1999Go; Chatterjee et al., 2000Go; Arnon et al., 2001Go; De Mas et al., 2001Go; Kobayashi et al., 2001Go; Reiter et al., 2002Go; Frias et al., 2003Go; Codrington et al., 2004Go; Deane et al., 2004Go; Seli et al., 2004Go). Even if sperm prevail after treatment, it is prudent practice to avoid use of these in vivo sperm for 1.5–2 years to enable sufficient turnover of cells to expel the mutagenic effect. These estimates may be high, and perhaps 6–12 months grace may be more pragmatic and realistic.

It would be most desirable in our field to have predictive tools and recognizable associations (Fossa et al., 1997Go; Tal et al., 2000Go). In our study, we aimed to see if there were any predictors of the semen quality decline and their possible recovery after gonadotoxic treatment. It became clear that the disease category was the single most important predictor of the sperm parameters. This reflects the pathology of the disease and the nature of the gonadotoxic treatment.

As a result of our analyses on our cohort of patients undergoing gonadotoxic treatment, a number of facts and associations emerge, listed below.

  1. There is a positive correlation between the pre-treatment sperm concentration and both post-treatment sperm concentration (Spearman's {rho}=0.181; P=0.01) and sperm motility (Spearman's {rho}=0.258; P=0.000). However, there is a negative correlation between the pre-treatment semen volume and ‘the interval between toxic therapy and the best post-treatment sperm analyses’ (Spearman's {rho}=–0.136, P=0.017), such that men with low pre-treatment semen volume tend to require a longer period to achieve the best sperm analyses after their treatment.
  2. Patients' age showed a positive correlation with the post-treatment sperm concentrations (Spearman's {rho}=1.24, P=0.036) and a negative correlation with the recovery time to achieve the best post-treatment sperm concentration (Spearman's {rho}=–0.264, P=0.000); hence older patients require a shorter interval to obtain a high post-treatment sperm concentration.
  3. The time to achieve the best post-treatment sperm concentration showed a positive correlation with the magnitude of change in sperm concentrations following treatment (pre-treatment concentration–post-treatment concentration: Spearman's {rho}=0.140, P<0.05). Such a correlation was not observed with the final post-treatment sperm concentrations (Spearman's {rho}=–0.067, P=0.241), such that men who suffered from large drops in sperm concentrations during their treatment regardless of the final sperm concentration tend to have longer post-treatment recovery periods in their spermatogenesis. This correlation was still valid even after correcting for age (F=6.26, P=0.002).
    We further evaluated the association between the recovery time to the best post-treatment sperm concentration, and the magnitude of change in sperm concentrations. When we subcategorize the cohort into three groups where group A=recovery period up to 33 months, group B=33–66 months and group C≥66 months, it becomes apparent that the association between the recovery time to the best post-treatment sperm concentrations and the percentage change in sperm concentrations were not significant in groups A and B (Figure 1ac). In essence, men who have small changes from their pre-treatment sperm concentrations reached their best post-treatment concentrations at some point within the first 66 months of their follow-up period. However, those who experienced significant deterioration in sperm concentration had to wait longer than 66 months to reach their peak recovery (R2=0.113, F=5.397, P=0.025). The mean change in sperm concentration was 26x106/ml (0.01–50) in group A, 28x106/ml (1.5–50) in group B and 48x106/ml (7.7–91) in group C.
    The negative numbers in Figure 1 indicate an improvement in sperm concentrations after treatment above the pre-treatment levels, reflecting the positive influence of the treatment which eliminates the effect of the underlying disease on spermatogenesis by overpowering its own gonadotoxic potential. Changes in the post-treatment sperm concentrations which revealed significant differences after 66 months of observation stress that there is not only a possible recovery of stem cell spermatogonia, but there is also a non-recoverable effect on the basic reservoir of these cells after gonadotoxic treatment in a subgroup of patients who may be characterized by their underlying disease process and the type of treatment they received.
  4. A logistic regression analysis to predict the post-treatment azoospermia by using age, diagnostic group and the recovery time revealed that only the diagnostic category is a significant predictor of post-treatment azoospermia. The distribution of patients with oligozoospermia and normospermia also showed significant differences among different diagnostic groups (Table III). Whilst patients with lymphoma had the highest incidence of post-treatment azoospermia, patients with testicular cancer were less likely to be azoospermic after cancer treatment. However, the probability of developing oligospermia in testicular cancer is much higher compared with the other groups. Patients with lymphoma and leukaemia have a higher probability to maintain sperm concentrations >20 x 106/ml if they escaped from developing azoospermia. Patients with benign conditions are most likely to be normo-spermic after their gonadotoxic treatment (Table III).
    Similarly, patients with different diagnoses also exhibited different time intervals to achieve their best post-treatment sperm parameters. This is a function of the percentage of azoospermia in each group (Table IVa). Patients with lymphoma with the highest level of post-treatment azoospermia have the longest recovery period, in contrast to those with testicular carcinoma who have the lowest percentage of post-treatment azoospermia and shortest recovery period. Although some recovery was evident in sperm concentrations with time, this did not reach statistical significance (Table IVb).


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Table IVb. The first and the last semen analyses after treatment in lymphoma and testicular cancer patients

 
On a broader issue, while our study draws together a number of observations, it also uniquely highlights certain features which may be of clinical use. There were significant differences in semen quality amongst the different disease categories, with the lymphoma and leukaemia group showing the largest change. The benign condition group was least affected, thereby serving as a useful internal control. The testicular cancer group overall remained with the lowest sperm numbers before treatment and appeared reasonably unaffected following treatment, thereby reflecting the testicular pathology of the disease. The question as to how long a patient should wait following treatment before the optimal recovery in sperm quality is achieved remained a challenging question for us to answer, and this is also a frequently asked question during clinical consultations. Apart from those patients who experienced the worst deterioration in sperm concentration and had to wait longer than 66 months to reach their peak recovery, there appears to be no significant benefit in prolonging the post-treatment follow-up with the expectation of significant recovery. This adds a new clinical feature to post-treatment counselling and management of patients.

Approximately 63% of patients may develop irreversible azoospermia. The fact that all categories of patients in our cohort suffered variable levels of azoospermia, together with the unpredictable recovery of azoospermia, reinforces the notion that ejaculated sperm should be banked prior to treatment. The significant decline in overall semen parameters after gonadotoxic treatment should lead clinicians to be more proactive in recommending sperm cryopreservation before treatment. This will help obviate patient distress and medico-legal problems, and above all give each patient a sense of reassurance with the knowledge of heightened safe potential use of sperm not previously subjected to toxic therapy. On a cautionary note, our study does not provide either reassurance or caution in the clinical use of post-treatment sperm, and careful counselling especially on genetic issues must follow.

Conclusion
Patient diagnosis remains a significant factor in predicting post-treatment azoospermia. All patient categories displayed varying levels of azoospermia and oligozoospermia. Given that there was an insignificant recovery in spermatogenesis following treatment, or that azoospermia remained, it is essential to offer sperm banking before any gonadotoxic treatment. This should be allied to dedicated counselling and support to cancer patients.

Several additional observations and associations in relation to semen quality are listed further, thereby giving an insight into pre- and post-gonadotoxic treatment effects and relationships. For example, apart from those patients who experienced the worst deterioration in sperm concentration and had to wait longer than 66 months to reach their peak recovery, there appears to be no significant benefit in prolonging the post-treatment follow-up with the expectations of significant recovery, thereby adding a new clinical feature to post-treatment counselling and management of patients. Patients with lymphoma and leukaemia have a higher probability of maintaining sperm concentrations >20 x 106/ml if they escaped from developing azoospermia.. Patients with different diagnoses also exhibited different time intervals to achieve their best post-treatment sperm parameters. The recovery times between treatment and the best post-treatment semen analyses is reflected in the diagnostic category. Finally, the unproven genetic effects of cancer treatment on sperm needs to be kept in perspective.


    References
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
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Submitted on October 5, 2004; resubmitted on November 4, 2004; accepted on November 19, 2004.





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