Birth after treatment of a male with seminoma and azoospermia with cryopreserved–thawed testicular tissue: Case report

Ursula Res1,4, Primoz Res1, Darja Kastelic1, Marjeta Stanovnik2, Andrej Kmetec3 and Aleksander Merlo1

1 Centre for Infertility Treatment Postojna, 6320 Postojna and 2 Institute of Oncology, 1000 Ljubljana and 3 Department of Urology, University Medical Centre, 1000 Ljubljana, Slovenia


    Abstract
 Top
 Abstract
 Introduction
 Case report
 Discussion
 Conclusion
 References
 
The case of an infertile couple in which a testicular seminoma and azoospermia were discovered in the husband during infertility treatment is described. A small piece of testicular tissue, obtained by biopsy from the healthy testis [testicular sperm extraction (TESE)], was deep-frozen before oncology therapy was initiated. The patient's lymphocyte karyotype was normal and no Y microdeletions were found. After conclusion of oncology treatment, the tissue was thawed and successfully used in the intracytoplasmic sperm injection (ICSI) procedure. A healthy girl was born. Testicular tumours are known to impair fertility in the majority of patients, and fertility deteriorates further after cytotoxic and surgical oncology treatment. Until recently in Slovenia, for young oncology patients cryopreservation was applied only to high quality ejaculate fulfilling the criteria for intrauterine insemination or in-vitro fertilization after thawing. Failing that, the only remaining options were fertilization by donor spermatozoa or child adoption. New assisted reproductive technologies, of which the ICSI procedure is the most successful, are suitable for the treatment of only the most severe cases of male infertility. It is reasonable to cryopreserve even poor quality ejaculate prior to the oncology therapy, as well as testicular tissue in cases of azoospermia.

Key words: azoospermia/cryopreservation/ICSI/seminoma/TESE


    Introduction
 Top
 Abstract
 Introduction
 Case report
 Discussion
 Conclusion
 References
 
Testicular cancer is the most frequent malignant tumour in men aged 15–35 years (Kovac, 1994Go). In Slovenia its incidence in 1994 was 6.6/100 000 males (64 newly discovered cases per year) and remains on the increase (Incidenca raka v Sloveniji, 1997Go). Testicular cancer patients have a very good prognosis for survival; patients with local/regional disease are cured in nearly 100% of cases, and those with an advanced disease are cured in 70–80 percent of cases (Kovac, 1994Go). As young patients dominate, it is important to consider the consequences of treatment that may affect quality of life, e.g. fertility, before cancer treatment is initiated. Chemotherapy and radiotherapy damage germ cells, while surgical treatment impairs the capability to ejaculate; 80% of cases develop retrograde ejaculation (Res et al., 1994Go). The undesirable side-effects of oncology treatment may be avoided in part by selective lymphadenectomy with preservation of thoracolumbar ganglia and by shielding the contralateral testis from radiation. It is of extreme importance, however, that each oncology patient should be informed and counselled regarding the option of cryopreserving ejaculate (Res et al., 1994Go) or testicular tissue before treatment.


    Case report
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 Abstract
 Introduction
 Case report
 Discussion
 Conclusion
 References
 
A 35 year old man was referred to the Infertility Treatment Clinic in mid-March 1997 because his wife had failed to conceive during the previous 4 years. The results of initial tests performed on his wife were within normal limits. The patient's medical history revealed that during the preceding month he had been taking antibiotics for presumed epididymitis, that 6 years ago he had suffered a blunt injury to both testicles, and that he was a heavy smoker. Clinical examination revealed an enlarged, knotty, firm and tender tumour which had completely overgrown the right testicle and which had a volume of 25 ml. The left testicle was of normal consistency to palpation, with a volume of 20 ml. Both epididymides and both spermatic ducts were normal. The scrotum was normal, as was the result of the rectal examination. After 2 days of sexual abstinence 3 ml of ejaculate were obtained, but no sperm cells were found in the sediment. A repeat ejaculate confirmed the absence of sperm cells.

Fructose concentration in the ejaculate was normal. An ultrasound examination of the right testicle revealed an extensive tumour growth with the normal testicular structure virtually replaced. The left testicle appeared normal by ultrasound examination. Surgical treatment was immediately arranged with a urologist. In view of the azoospermia and at the request of the couple, a tissue biopsy (3x3x3 mm) was obtained from the healthy left testicle under local xylocaine anaesthesia on 22 March, 1997, prior to semi-castration. A biopsy of the healthy testicle was undertaken following the normal ultrasound examination, and the need to exclude the possibility of carcinoma in situ. The couple were informed and counselled regarding the procedures and possible consequences of the treatment.

A native unstained portion of the biopsy sample was examined and individual sperm cells identified. A small piece was processed for histology, and the remainder of the sample was prepared for cryopreservation. The testicular tissue was minced and suspended in an equal volume of a freezing medium (SpermFreeze; FertiPRO N.V., Aalter, Belgium). The samples were distributed into three 0.2 ml aliquots in plastic straws, and then stored in a refrigerator at 4°C for 1 h before transfer into liquid nitrogen vapour for 20 min and plunged into liquid nitrogen (Res, 1981Go). Histological examination of the biopsy taken from the left testicle revealed degeneration of all sperm cell precursors. A few spermatogonia, spermatocytes, spermatids and sperm cells were preserved. Degeneration of Leydig cells was also observed. The spermatogenesis scored 8 on the Johnson scale (Johnson, 1970Go). An excellent relationship was demonstrated between spermatid count per tubule cross-section and fluid sperm count (Silber et al., 1981). In that study, spermatid/tubule counts of 40, 20, and 6–10 corresponded to sperm counts of 45, 10 and 3x106/ml respectively. This relationship existed for the patient with no obstruction. As possible obstruction aetiology in our patient was excluded, i.e. the vas deferens was palpable, and volume of ejaculate and fructose concentration was normal, we can estimate that the number of spermatids in the testicular tissue corresponded to a concentration of 6x106 sperm cells per ml of ejaculate.

The patient underwent radical right-sided orchidectomy at the Department of Urology of the Ljubljana Medical Centre, with ligation of the spermatic cord. The removed testicle was examined microscopically in the operating theatre, and no healthy tissue suitable for freezing was found. The testicle, together with the spermatic cord, weighed 80 g and contained a lobulated tumour, 5.5 cm in diameter, which was semi-solid and whitish on the cut surface. Pathohistology confirmed the diagnosis of testicular seminoma. After the orchidectomy the patient was referred to the Department of Oncology for further diagnostic tests and treatment. Staging tests [ultrasound scan of the abdomen, lung X-rays, a computed tomography (CT) scan of the retroperitoneal space] were performed. The CT scan of the abdomen showed several pathologically enlarged lymph nodes, up to 3 cm in diameter, between the aorta and the inferior vena cava. No metastases were observed in the parenchymal organs. FSH assays and concentrations of the tumour markers {alpha}-fetoprotein (AFP) and ß-human chorionic gonadotrophin (ßHCG) preceding the orchidectomy were within normal limits. The above tests established a clinical radiological stage of IIA. In accordance with the treatment regime, combined chemotherapy (etoposide, cisplatin and bleomycin) was administered to the patient on three occasions, commencing on 14 April, 1997.

Almost complete remission was achieved after three chemotherapy treatment cycles. A control CT scan of the retroperitoneal space showed only remains of metastases up to 0.5 cm in diameter. In view of this finding we decided not to continue the treatment. No significant toxic side-effects were observed during the course of treatment or during the period following it, and the treatment was terminated on 29 May, 1997. The patient remains under regular specialist supervision.

Five months after treatment at the Department of Oncology the couple decided to continue infertility treatment. Azoospermia was again ascertained in the ejaculate. The growth of an increased number of follicles in the wife was achieved with 24 ampoules of human menopausal gonadotrophin (HMG) i.m. The growth of the follicles was monitored by ultrasound, and 10 000 IU HCG was administered on day 13 of the menstrual cycle. Needle aspiration of the follicles was performed under local anaesthesia 36 h after HCG administration. Eight oocytes were retrieved and cultured in Medicult culture medium for 4 h (37°C, 5% CO2). After a 30 s incubation in hyaluronidase, cumulus cells were removed from the oocyte by pipetting. After repeated washing, the degree of maturity of the oocytes was assessed by the presence of the polar body under microscope. A 1 ml aliquot of sperm preparation medium (SPM; MediCult, Jyllinge, Denmark) was added to one 0.2 ml aliquot of thawed testicular tissue. After centrifugation, a few immotile sperm cells were found in the sediment, of which only a small proportion was morphologically normal. These were matured in vitro for 4 h, in a culture medium under paraffin oil using identical conditions to those used for the oocytes. After incubation their viability was confirmed by weak movement of the tail. Morphologically normal spermatozoa were selected and used for fertilization by direct injection into the oocyte (intracytoplasmic sperm injection; ICSI). Six oocytes were found to be in metaphase II and suitable for fertilization. Successful fertilization of four oocytes was confirmed by the presence of two pronuclei after 18 h. Forty-eight hours after follicle aspiration four 4-cell embryos were transferred into the uterus with a Frydman catheter (CCD, Paris, France). A raised HCG concentration indicated a positive pregnancy test, and the pregnancy was confirmed by ultrasound scan on 28 December, 1997. A single gestation sac with a fetal heartbeat was observed in the uterus. The couple had had prior genetic counselling. The husband's karyotype was normal and no Y microdeletion was found (AZFa, AZFb, AZFc). The couple opted to refuse amniocentesis. The course of the pregnancy was uneventful, and a healthy baby girl was born at term on 20 August, 1998. The couple wished to return for assisted reproductive treatment within a few months, as the husband still had two aliquots of frozen testicular tissue in storage.


    Discussion
 Top
 Abstract
 Introduction
 Case report
 Discussion
 Conclusion
 References
 
This case demonstrates that young male cancer patients have a greatly improved chance of fatherhood after completing their oncology treatment, thanks to new methods in assisted reproduction methods. Modern oncology treatment -by surgery, chemotherapy and radiotherapy makes long-term survival a reality for the majority of patients. As patients with testicular tumours are often young, and treatment is becoming more and more successful, it is important that they should be able to live as normal and high quality lives after treatment as possible. An integral part of such a life is the option to plan parenthood. In Slovenia until recently, the only ejaculate that was frozen had to be of high quality and deemed capable of fertilization after thawing. Patients with poor ejaculate or azoospermia were given the alternative of fertilization by donor or child adoption. The proportion of motile sperm cells after thawing is known to decrease in different cases to various degrees, but generally speaking up to 50% survival is expected (Res et al., 1994Go). Thawed ejaculate may be, and indeed have been, used for intrauterine insemination, and for classical in-vitro fertilization (IVF). The probability of fertilization may be increased by centrifugation, by swim-up of sperm cells or by the use of density gradients. These methods require a large number of motile sperm cells. However, the use of ICSI requires only a few motile, i.e. living, sperm cells. This method of directly injecting a single sperm cell into a mature oocyte is professionally and technically very demanding, but markedly improves the chances of fatherhood for men who previously had none. It is therefore expedient to freeze all ejaculates, regardless of the inferior quality of the sample.

Virant (Virant, 1997Go) reported no pregnancies in five cases in which thawed sperm cells obtained from testicular tissue were used. A review (Devroey,1998Go) described the results obtained with frozen testicular tissue. Up to that time, 12 pregnancies after fertilization by the ICSI method with thawed sperm cells obtained from testicular tissue had been reported worldwide. The birth of the first child after this treatment was described by Khalifeh (Khalifeh et al., 1997Go), who had frozen the testicular tissue by using the ultrarapid freezing method. The first case published (Yavetz et al., 1997Go) worldwide in which a biopsy of healthy testicular tissue from a patient with azoospermia and testicular seminoma was frozen. After completion of oncology treatment, the sample was successfully used for fertilization by ICSI, and a healthy child was born. Al-Hasani (Al-Hasani et al., 1999Go) described pregnancies achieved using frozen–thawed pronuclear oocytes obtained by ICSI with spermatozoa extracted from frozen–thawed testicular tissue.

Four births and two ongoing pregnancies using sperm cells from thawed testicular tissue for fertilization by ICSI have been recorded at our Postojna Centre for Infertility Treatment. The results of ICSI with frozen–thawed testicular spermatozoa are similar to those obtained using fresh testicular spermatozoa (Gil-Salmon et al., 1996Go; Fidler et al., 1997Go). It is very important to incubate the thawing testicular tissue in sperm medium, because most vital spermatozoa acquire motility within 1–2 h (Ben-Yosef et al., 1999Go). When immotile spermatozoa are injected, fertilization rate decreases (Ben-Yosef et al., 1999Go; Shulman et al., 1999Go). Normal spermatogenesis in testicular biopsy predicts a greater probability, and maturation arrest a lower probability of recovering motile testicular spermatozoa (Nagy et al., 1998Go).

The stage of spermatogenesis is also very important, as the fertilization rate with spermatozoa is 64%, and 40% with round spermatids (Gianaroli et al., 1999Go). Novero (Novero et al., 1996Go) described the first successful pregnancy using ICSI of fresh sperm cells obtained from cryopreserved–thawed testicular tissue (TESE) from a patient with azoospermia with a seminoma as an incidental finding; he emphasized the importance of histological diagnosis in order to exclude malignancy on the other side. The incidence of malignant tumours in infertile men is 0.4–1.1%. A testicular tumour is more likely in men with undescended testes, in those with an already diagnosed tumour in the contralateral testis or in men with sexual differentiation disturbances. The incidence of seminoma in patients with undescended testes is 2% and 50–70% of testicular cancer patients are known to have inferior quality ejaculate prior to the onset of oncology treatment (Botchan, 1997), although the mechanism of this phenomenon has not been fully elucidated.

Ejaculate in non-seminoma testicular tumours is statistically significantly inferior to the ejaculate in seminoma patients (Botchan et al., 1997Go). In 14 patients with bilateral testicular tumours or a unilateral testicular tumor and carcinoma in situ in the contralateral testis, azoospermia was described (Kliesch et al., 1997Go) in four patients and oligozoospermia in the remaining patients before the onset of oncology treatment; fertility subsequently deteriorated further. Two cases of spontaneous pregnancy in the partners of men with bilateral testicular tumours have been described (Heidenreich et al., 1997Go). Biopsy of the healthy testis was used in order to eliminate as far as possible the presence of a carcinoma in situ, which arises in 5% (Kliesch et al., 1997Go) of testicular cancer patients on the contralateral side. The technique of choice for carcinoma in situ detection is biopsy, which reveals pathological changes in 85% of cases (Giwereman et al., 1990Go).

Seminoma is a tumour of the undeveloped germ cell. The spermatozoa used for ICSI was a matured cell, so it is not a malignant cell. However, spermatozoa can carry chromosomal or gene defects and all patients with testicular tumours should remain under long-term medical supervision. Devroey (Devroey, 1998Go) cites studies by different authors who describe an increased incidence of Klinefelter syndrome (3–19%) and other sex chromosome disorders (0.6–3%), and autosomal anomalies (0.6–3%) in patients with azoospermia. He recommends peripheral blood karyotyping in all patients with azoospermia and this was performed in our patient. There is a possibility of transfer of the AZF gene (azoospermia factor), located on the Y chromosome, in non-obstructive azoospermia. Male offspring might thus inherit their father's defect. Our patient was free of Y deletions. A distinct increase in spontaneous chromosomal aberrations was reported (Gundy et al., 1992Go) in blood lymphocytes over the level of healthy controls (0.48% cells) in patients with testicular cancer (1.96% cell). The chromosomal instability may be a factor in the development of malignancy for testicular tumours. The structural chromosome aberrations were categorized as chromosomal breaks, eccentric fragments, dicentric and ring chromosomes. Alvarez (Alvarez, 1999) reported that there were no significant differences in the frequencies of chromosomal aberrations between control and testicular cancer patients (9.7 and 10.3% respectively). In our case it was not possible to carry out more sophisticated tests on humal spermatozoa, such as karyotypes or multicolour fluorescence in-situ hybridization (FISH) analysis. Sperm karyotypes were studied (Martin, 1998Go) using the human sperm–hamster oocyte system in four patients with testicular cancer: the results in cancer patients were not significantly different from control donors. In another study (Martin et al., 1991Go), it was demonstrated that cryopreservation of human spermatozoa does not affect the freqency of numerical or structural chromosome anomalies or the `sex ratio' of spermatozoa.


    Conclusion
 Top
 Abstract
 Introduction
 Case report
 Discussion
 Conclusion
 References
 
This report describes the birth of the first child in Slovenia conceived by means of fertilization with thawed sperm cells from testicular tissue of a patient with seminoma and azoospermia. The case demonstrates successful co-operation between the Infertility Treatment Centre, Department of Urology, The Institute of Oncology, Postojna Hospital of Gynaecology and Obstetrics, a pathohistologist and a genetics specialist. Of great importance is the co-ordinated and synchronized action of all specialists participating in the treatment procedures of young oncology patients. It is advisable that patients with testicular tumours who might later wish to father a child be offered options prior to the initiation of oncology therapy, including deep-freezing of ejaculate, even in cases of very poor ejaculate quality, and freezing a biopsy of healthy testicular tissue in cases of azoospermia. The new assisted reproduction techniques offer greatly increased options in the treatment of infertility. Thus, it is now possible for oncology patients under current treatment to become biological fathers.


    Notes
 
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    References
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 Abstract
 Introduction
 Case report
 Discussion
 Conclusion
 References
 
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Submitted on May 10, 1999; accepted on January 12, 2000.