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
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Abstract |
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Key words: azoospermia/cryopreservation/ICSI/seminoma/TESE
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Introduction |
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Case report |
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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 (Re, 1981
). 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, 1970
). 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 610 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 -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.
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Discussion |
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Virant (Virant, 1997) reported no pregnancies in five cases in which thawed sperm cells obtained from testicular tissue were used. A review (Devroey,1998
) 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., 1997
), who had frozen the testicular tissue by using the ultrarapid freezing method. The first case published (Yavetz et al., 1997
) 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., 1999
) described pregnancies achieved using frozenthawed pronuclear oocytes obtained by ICSI with spermatozoa extracted from frozenthawed 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 frozenthawed testicular spermatozoa are similar to those obtained using fresh testicular spermatozoa (Gil-Salmon et al., 1996; Fidler et al., 1997
). It is very important to incubate the thawing testicular tissue in sperm medium, because most vital spermatozoa acquire motility within 12 h (Ben-Yosef et al., 1999
). When immotile spermatozoa are injected, fertilization rate decreases (Ben-Yosef et al., 1999
; Shulman et al., 1999
). Normal spermatogenesis in testicular biopsy predicts a greater probability, and maturation arrest a lower probability of recovering motile testicular spermatozoa (Nagy et al., 1998
).
The stage of spermatogenesis is also very important, as the fertilization rate with spermatozoa is 64%, and 40% with round spermatids (Gianaroli et al., 1999). Novero (Novero et al., 1996
) described the first successful pregnancy using ICSI of fresh sperm cells obtained from cryopreservedthawed 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 0.41.1%. 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 5070% 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., 1997). 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., 1997
) 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., 1997
). 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., 1997
) 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., 1990
).
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, 1998) cites studies by different authors who describe an increased incidence of Klinefelter syndrome (319%) and other sex chromosome disorders (0.63%), and autosomal anomalies (0.63%) 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., 1992
) 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, 1998
) using the human spermhamster 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., 1991
), 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.
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Conclusion |
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Notes |
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References |
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Submitted on May 10, 1999; accepted on January 12, 2000.