Crha I.1, Pacík D.2, Beharka R.2, Ventruba P.1, Žáková J.1, Huser M.1, Kubešová B.3, Pohanka M.1

1Department of Gynaecology and Obstetrics, Faculty of Medicine, Masaryk University and Faculty Hospital Brno
2Department of Urology, Faculty of Medicine, Masaryk University and Faculty Hospital Brno
3Tissue Bank, Faculty Hospital Brno

Received after revision April 2009



Testicular cancer
Male infertility



Crha I.
Department of Gynaecology and Obstetrics
Faculty of Medicine
Masaryk University and Faculty Hospital Brno
Jihlavská 20
625 00 Brno
Czech Republic


This study was designed to evaluate the use of sperm frozen before testicular cancer treatment and to analyse results of infertility treatment. A total of 166 male patients were referred for sperm freezing before testicular cancer treatment from 1995 to 2007. Cancer treatment, semen analysis, assisted reproduction techniques, and statistical evaluation were basic interventions. Sperm pathology analysis before and after cancer treatment, survival of patients, and pregnancy after infertility treatment were analysed. Testicular cancer was diagnosed and treated in 226 men from 1995 to 2007. A total of 166 men (73.5 %) decided to freeze their semen before cancer treatment. Seminoma was diagnosed in 87 men (52.4 %), nonseminomatous germ cell tumours (NSGCT) in 79 men (47.6 %). A total of 121 men (72.9 %) were of stage I. Azoospermia was diagnosed in 9 men (5.4 %), semen was cryopreserved in 157 patients. Median sperm concentration was 8.5 mil/ml in the seminoma group and 9.4 mil/ml in the NSGCT group - a non-significant difference. Until now, 5 patients (2.9 %) have died and 27 patients (17.2 %) have attended for infertility treatment. Median fresh sperm concentration was 4.4 mil/ml, median progressive sperm motility 3.7 %, azoospermia was found in 4 men (14.8 %). The confounding female infertility factor was found in 19 (70.4 %) female partners. Cryopreserved semen was used in 25 couples (92.6 %), intracytoplasmic sperm injection (ICSI) was the most effective procedure - 36 cycles resulted in 12 pregnancies and 9 deliveries. Testicular cancer survivors have a good chance of fathering a child by using cryopreserved sperm and ICSI procedure.


Damage to reproductive function is a very frequent and well-documented side effect associated with the treatment of malignant testicular tumours. The first work describing chemotherapy-induced azoospermia was published in 1948 [1]. Variation in sperm quality in relation to the type of malignant tumour was also investigated [2]. The increasing success of testicular cancer treatment and determined efforts to improve the quality of life after successful treatment have turned attention to the preservation of reproductive function in young males [3]. The development of assisted reproduction techniques has brought about effective qualitative changes in this field [4, 5]. The collection, freezing, and long-term storage of sperm are currently considered to be the most effective method. The Assisted Reproduction Centre of the Department of Gynaecology and Obstetrics in collaboration with the Department of Urology launched their programme of sperm freezing and long-term storage before testicular cancer treatment in 1995.


The objective of this study is to analyse the sperm counts of cancer patients and a possible correlation between sperm pathology and cancer diagnosis, and to make an overview of using frozen sperm during the 12 years of sperm banking.

Material and Methods

Between October 1995 and the end of December 2007 young men with testicular cancer were referred from the Department of Urology to the Assisted Reproduction Centre for sperm cryopreservation prior to treatment for testicular cancer. An obligatory examination of HIV 1, HIV 2, HBsAg, anti- HCV, and anti-HBC before storage of cryopreserved semen was introduced in 2003. Sperm counts were evaluated according to the respective WHO (World Health Organisation) laboratory manual [6] using the Neubauer counting chamber. Commercial media, Medi-Cult (Jyllinge, Denmark) or Vitrolife (Kungsbacka, Sweden) were used. The semen was mixed with a cryopreservation medium and placed in 2 ml Nunclon CryoTubes (Roskilde, Denmark) and frozen. Cryopreservation technology and the procedures used in the storage of frozen sperm samples were aimed at minimising the potential risks, including mistaken identity and transmission of infection. Sperm samples were frozen in a programmable Planer Kryo F10 (Sunbury-on-Thames, United Kingdom) instrument using a standard cooling curve. Samples from 1-3 collections before starting cancer treatment were frozen. The cryotubes were stored in liquid nitrogen at a temperature of -196 °C in an LS 4800 container Tailor-Wharton HARSCO (Husum, Germany), with an indicator of the surface level and an alarm.
The assisted reproduction methods used complied with the respective standards of the centre [7]. The diagnoses and deceases were verified in the database of the National Oncological Register of the catchment area, in compliance with personal data protection. The Faculty Hospital Brno Ethics Committee's approval was obtained.
The study group was described using the basic descriptive statistics, where categorical variables were characterised using the percentage representations of individual categories, while continuous variables (age, sperm concentration and motility) were described using the mean, the median, the range of values (minimum and maximum), and standard deviations.
Statistical testing was used to confirm the hypothesis of whether or not the results of sperm counts correlated with the patient's diagnosis. The differences among groups of patients were tested using the Kruskal-Wallis test. When the influence of the diagnosis on the sperm count was significant, partial hypotheses were tested to see which particular diagnoses differed by their values (i.e. multiple comparisons of mean ranks). The critical limit for the level of significance was set to p = 0.05.


Testicular cancer was diagnosed and treated in 226 men (age 27.6±6.4) from 1995 to 2007. A total of 166 men (73.5 %) decided to freeze their semen before cancer treatment. The number of men asking for semen cryopreservation increased gradually from 9.1 % in 1995 to 89.5 % in 2007 (Figure 1). Seminoma was diagnosed in 87 men (52.4 %), nonseminomatous germ cell tumours (NSGCT) in 79 men (47.6 %). Patient characteristics including the stage of the tumour are given in Table 1. Azoospermia was diagnosed in 9 men (5.4 %), thus semen was cryopreserved in 157 patients. Sperm concentration was 17.2±21.4 (median 8.5) mil/ml in the seminoma group and 19.7±26.3 (median 9.4) mil/ml in the NSGCT group - a nonsignificant difference (Table 2). Statistical analysis did not find any significant difference of sperm count in relation to the stage of the cancer.
Until now, 5 patients (2.9 %) died and 27 testicular cancer survivors (17.2 %) with semen cryopreserved prior to cancer treatment have undergone infertility treatment. The interval between cryopreservation and infertility treatment ranged from 7 to 70 months (mean 22.2±14.7, median 18 months). In these men the mean fresh sperm concentration was 8.4±16.8 (median 4.4) mil/ml, progressive sperm motility 4.2±17.7 (median 3.7) %, azoospermia was found in 4 men (14.8 %). As these examinations were done after different time intervals following cancer treatment and after different chemotherapy protocols, the data were not sufficient for reliable statistical analysis.
A confounding female infertility factor was found in 19 (70.4 %) female partners. Cryopreserved samples were used in 25 couples (5 cycles of intrauterine insemination, 36 ICSI cycles). Clinical data and results of infertility treatment are given in Table 3. Fresh semen was used in 2 cases for 6 ICSI cycles and one clinical pregnancy resulted in spontaneous abortion. After failure of two ICSI cycles 3 couples (11.1 % of men coming for infertility treatment after sperm cryopreservation) decided to use intrauterine insemination with donor sperm, 5 cycles resulted in 2 pregnancies and deliveries.


The increasing frequency of testicular cancer in our study corresponds to the increasing incidence of this tumour in the Czech Republic. In 1977 the incidence of testicular cancer was 3.2/100 000 men, in 1995 it increased to 6.5, and in 2005 to 8.8/100 000 men. Approximately 68 % of them were in the range from 20 to 39 years. Advances in the diagnostics and management of this cancer and progress in its treatment contributed to a significant improvement in the survival rate of this very common malignancy of young men [8]. Stage I tumours were only 5 % in 1977 and improved gradually to 50 % in 1995 and 55 % in 2005. Similar findings are also reported by the Bourn Hall Clinic [9] and other clinics [10, 11].
Azoospermia was found in 5.4 % of the males referred for sperm cryopreservation, as compared to Lass et al. [9], who reported 17.3 %, and Kelleher et al. [12], who detected only 3.3 %. Severe abnormalities in sperm concentration and progressive motility were detected in both seminoma and NSGCT groups, similar to other studies [13, 14].
The aetiology of impaired spermatogenesis in testicular cancer patients is not fully understood. Damage to the DNA of the sperm due to malignancy was confirmed [15]. However, the extent of damage to spermatogenesis does not correlate with the severity of the cancer [16]. The correlation between sperm pathology and testicular tumours is also known [17]. The impaired quality of sperm production is probably associated with disturbed differentiation of the testicle during the embryonic development of the gonad - the Testicular Dysgenesis Syndrome hypothesis [18]. This syndrome is manifested by the increased incidence of developmental defects of the genital (cryptorchism, hypospadias), spermatogenesis disorders, and testicular carcinomas. Testicular dysgenesis is caused by alteration in the development of the testicle by factors affecting endocrine regulation ("endocrine disruptors"). As spermatogenesis disorders correlate well with testicular carcinoma, close urological examination of men with severe sperm abnormalities is of importance [17]. When analysing impaired spermatogenesis in relation to the type of malignancy, we did not find any significant difference like Agarwal et al. [5].
After the completion of gonadotoxic therapy, the quality of sperm was significantly impaired. The resulting function of the gonad is affected by a number of factors, such as early diagnosis of the malignant disease, the chemotherapy regimens used, and the sperm count as determined prior to the start of therapy [19]. The recovery of spermatogenesis is usually very slow - for example, the mean period of time to achieve the best level of the sperm count is 51 months in patients who completed chemotherapy for leukaemia [20]. In the case of azoospermia, the methods of assisted reproduction based on the surgical collection of sperm provide worse results [21, 22]. Sperm cryopreservation performed prior to cancer therapy is therefore a prerequisite for the successful treatment of subsequent infertility. The main requirement of this programme is to establish a special cryobank to allow safe long-term storage of sperm samples. The operation of such a cryobank requires an exact database of patients and its own records. A proper examination complying with the law of the Czech Republic (Table 4) is a prerequisite of cryopreservation and storage of frozen semen. Another important aspect is that the patients should be given clear and relevant information on the possibilities and conditions of sperm cryopreservation and its use in future.
The fulfilment of this task requires close co-operation between the respective department of urology, the assisted reproduction centre capable of providing this kind of treatment, and the tissue bank. In the Brno Faculty Hospital, this interdisciplinary co-operation is facilitated [23, 24]. Thanks to a strong awareness among urology specialists of the possibilities and easy availability of sperm cryopreservation in our centre, the number of patients referred for this procedure has increased.
In our study 17.2 % of the males have come in for infertility treatment so far. This finding is higher than the data from the literature, for example 7.7 % published by Kelleher [25]. The reasons are not only in the area of patient health, but also in the social area, i.e. patients usually plan to start a family long after they have successfully completed therapy [26]. Another important aspect is that patients are afraid of the increased risk of congenital defects and malignant tumours in their offspring. Many detailed studies which have investigated this risk [3, 16, 27] have failed to prove its increase [28, 29]. Most males from our group who came in for infertility treatment had undergone successful treatment for testicular cancer and usually came 18 months after sperm cryopreservation. Intrauterine insemination was performed in our clinic much less frequently (17.0 %) as compared to Schmidt et al. [29]; ICSI was used in 83.0 % of the treatment cycles.
The mortality rate of our group of patients was also analysed. According to the data obtained from the Oncological Register, only 2.9 % of males referred for sperm cryopreservation died, which corresponds to the total survival rate in patients with the early stage of testicular seminoma, which exceeds 95 % (30).


Sperm cryopreservation prior to gonadotoxic therapy is the basic method used to preserve reproductive potential for the survivors of cancer treatment. Cancer patient sperm banking programmes require close co-operation between the respective oncology department, the assisted reproduction centre, and the tissue bank. Sperm cryopreservation should be offered to every patient before therapy that could damage spermatogenesis.

Acknowle dgements

This work was supported by the Internal Grant Agency (IGA) of the Ministry of Health of the Czech Republic - No. NS 9661-4.


  1. Spitz S. The histological effects of nitrogen mustard on human tumours and tissues. Cancer 1948; 1: 383-398.
  2. Botchan A, Hauser R, Yogev L, et al. Sperm quality in Hodgkin's disease versus non-Hodgkin's lymphoma. Hum Reprod 1997; 12: 73-76.
  3. Howell SJ, Shalet SM. Spermatogenesis after cancer treatment: damage and recovery. J Natl Cancer Inst Monogr 2005; 34: 12-17.
  4. Palermo G, Joris H, Devroey P, Steirteghem AC. Pregnancies after intracytoplasmic injection of single sperm into an oocyte. Lancet 1992; 340: 1718.
  5. Agarwal A, Shekarriz M, Sidhu RK, Thomas AJ. Value of clinical diagnosis in predicting the quality of cryopreserved sperm from cancer patients. J Urol 1996; 155: 934- 938.
  6. World Health Organization Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction, 3rd ed. Cambridge: Cambridge University Press, 1992: 67 pp.
  7. Crha I, Ventruba P, Žáková J, et al. Kryokonzervace spermatu před gonadotoxickou léčbou - 11 let zkušeností [Sperm banking before gonadotoxic treatment - 11 years of experience]. Čes Gynek 2007; 72: 320-326.
  8. Paduch DA. Testicular cancer and male infertility. Current Opinion in Urology 2006; 16: 419-427.
  9. Lass A, Akagbosu F, Brinsden P. Sperm banking and assisted reproduction treatment for couples following cancer treatment of the male partner. Hum Reprod Update 2001; 7: 370-377.
  10. Agarwal A, Ranganathan P, Kattal N, et al. Fertility after cancer: a prospective review of assisted reproductive outcome with banked specimens. Fertil Steril 2004; 81: 342-348.
  11. Hallak J, Kolettis PN, Sekhon VS. Sperm cryopreservation in patients with testicular cancer. Urology 1999; 54: 894-899.
  12. Kelleher S, Wishart SM, Liu PY, et al. Long-term outcomes of elective human sperm cryostorage. Human Reprod 2001; 16: 2632-2639.
  13. Fossa SD, Theodorsen L, Norman N, Aabyholm T. Recovery of impaired pretreatment spermatogenesis in testicular cancer. Fertil Steril 1990; 54: 493-496.
  14. Bahadur G, Ling KLE, Hart R, et al. Semen quality and cryopreservation in adolescent cancer patients. Hum Reprod 2002; 17: 3157-3161.
  15. Kobayashi H, Larson K, Sharma RK, et al. DNA damage in patients with untreated cancer as measured by the sperm chromatin assay. Fertil Steril 2001; 75: 469-475.
  16. Colpi GM, Contalbi GF, Nerva F, et al. Testicular function following chemo-radiotherapy. Eur J Obstet Gynecol Reprod Biol 2004; S1: 2-6.
  17. Jacobsen R, Bostofte E, Engholm G, et al. Risk of testicular cancer in men with abnormal semen characteristics: cohort study. Br Med J 2000; 321: 781-782.
  18. Skakkebaek NE, Rajpert-De Meyts E, Main KM. Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects: opinion. Hum Reprod 2001; 16: 972-978.
  19. Huser M, Crha I, Ventruba P, et al. Prevention of ovarian function damage by a G nRH analogue during chemotherapy in Hodgkin lymphoma patients. Human Reproduction 2008; 23: 863-868.
  20. Bahadur G, Ozturk O, Muneer A, et al. Semen quality before and after gonadotoxic treatment. Hum Reprod 2005; 20: 774-781.
  21. Chan PT, Palermo GD, Veeck LL, Rosenwaks Z, Schlegel PN. Testicular sperm extraction combined with intracytoplasmic sperm injection in the treatment of men with persistent azoospermia postchemotherapy. Cancer 2001; 15: 1632-1637.
  22. Ragni G, Arnoldi M, Somigliano E, et al. Reproductive prognosis in male patients with azoospermia at the time of cancer diagnosis. Fertil Steril 2005; 83: 1674-1675.
  23. Žáková J, Ventruba P, Crha I, Lousová E. Sperm donation programme at the Centre of Assisted Reproduction in Brno: results from 1995-2005. Scripta Med (Brno) 2005; 78 (6): 323-328.
  24. Hudeček R, Ventruba P, Unzeitig V, Vavrušková R, Račanská E, Šarmanová J. Analysis of ovarian hyperstimulation syndrome development using data mining. Scripta Med (Brno) 2005; 78 (6): 329-340.
  25. Kelleher S, Wishart SM, Liu PY, et al. Long-term outcomes of elective human sperm cryostorage. Human Reprod 2001; 16: 2632-2639.
  26. Neal MS, Nagel K, Duckworth J, et al. Effectiveness of sperm banking in adolescents and young adults with cancer: a regional experience. Cancer 2007; 110: 1125-1129.
  27. Meirow D, Schiff E. Appraisal of chemotherapy effects on reproductive outcome according to animal studies and clinical data. J Natl Cancer Inst Monogr 2005; 34: 21-25.
  28. Sankila R, Olsen JH, Anderson H, et al. Risk of cancer among offspring of childhood-cancer survivors. New Engl J Med 1998; 338: 1339-1344.
  29. Schmidt KLT, Larsen E, Bangsboll S, et al. Assisted reproduction in male cancer survivors: fertility treatment and outcome in 67 couples. Hum Reprod 2004; 19: 2806- 2810.
  30. Sant M, Aareleid T, Artioli ME, et al. Ten-year survival and risk of relapse for testicular cancer: a EUROCARE high resolution study. Eur J Cancer 2007; 43: 585-592.
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