1 Andrology Laboratory and Semen Bank, Instituto Valenciano de Infertilidad, 2 Fundación IVI, 3 Department of Paediatrics Obstetrics and Gynaecology and Department of Surgery, Valencia University School of Medicine, 4 Hospital Universitario Dr. Peset, Valencia and 5 Hospital Clínico Universitario, Valencia, Spain
6 To whom correspondence should be addressed at: Instituto Valenciano de Infertilidad, Plaza de la Policía Local, 3, Valencia 46015, Spain. e-mail: marcos.meseguer{at}ivi.es
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Abstract |
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Key words: azoospermia/cancer/chemotherapy/ICSI/TESE
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Introduction |
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Harmful effects of chemotherapy on spermatogenesis are variable, depending on the type of chemotherapeutic agents employed and on their dosage. Moreover, it is not possible to predict with certainty if spermatogenesis will return to normal after therapy (Naysmith et al., 1998). Therefore, sperm banking before starting chemotherapy is highly recommended in young cancer patients (Lass et al., 1998
).
Nevertheless, there remain a group of young cancer survivors that did not freeze sperm samples before gonadotoxic treatments. The reasons for not sperm banking are variable. Some patients may have not been adequately counselled by their oncologists, underestimating future fertility issues when focusing all their efforts in treating the primary disease. In other cases, spermatogenesis may already have been affected by the primary tumour before chemotherapy commenced, as in some testicular cancers. Sperm quality in some of these patients may have not been considered good enough for cryopreservation prior to the ICSI era (Hendry et al., 1983). Finally, sperm storage is not feasible in pre-adolescent cancer patients.
Patients remaining persistently azoospermic after chemotherapy have been considered traditionally as sterile if frozen sperm samples are not available. Recent advances during the last decade combining testicular sperm extraction (TESE) and ICSI in patients suffering from non-obstructive azoospermia (Devroey et al., 1995; Tournaye et al., 1996
) have opened a new alternative for these couples. Non-obstructive azoospermia is caused, however, by a heterogeneous group of diseases, both congenital and acquired. Therefore, it is of interest to evaluate specific groups of patients with uniform aetiologies.
Our aim in this study is to assess the expectations that assisted reproductive techniques (ART) offer to patients with long-term maintained azoospermia caused by chemotherapeutic agents. Hence, we have retrospectively analysed our results in terms of the success of finding sperm using TESE, and the ICSI outcome in a series of young cancer survivors who have previously received chemotherapy.
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Materials and methods |
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For freezing, an equal volume of Sperm Freezing Medium (Medicult) containing glycerol was added to the sperm pellet, and then homogenized and placed at room temperature for 10 min. Sperm samples were frozen in small tablets using a dry ice surface for approximately 1 min and then were transferred to pre-labelled cryotubes that were subsequently plunged into liquid nitrogen tanks until future use (Gil-Salom et al., 1996). For thawing, the pills were removed and transferred into 5 ml Falcon tubes, and placed in an incubator at 37°C and 5% CO2. Then samples were washed again with Sperm Medium (Medicult) and centrifuged at 600 g for 5 min. The supernatant was discarded and samples resuspended in variable amounts of medium. Thereafter, motile spermatozoa were checked again for ICSI.
Ovarian stimulation
Female gynaecological diagnoses were normal except for one case of severe endometriosis. Mean age was 33.5 years (range 2840). Ovarian stimulation protocol was initiated by pituitary desensitization with daily s.c. administration of 1 mg leuprolide acetate (Procrin; Abbott S.A., Madrid, Spain) and began in the luteal phase of the menstrual cycle. Serum estradiol (E2) levels <60 pg/ml (220 pmol/l) and negative vaginal ultrasonographic scans were used to define ovarian quiescence. On days 1 and 2 of ovarian stimulation, 2 ampoules/day hMG (hMG Lepori; Farma-Lepori, Madrid, Spain) were administered together with two ampoules of high purity FSH (Neo-Fertinorm; Serono Laboratories, Madrid, Spain). On days 3, 4 and 5 of ovarian stimulation, 1 ampoule/day hMG plus 1 ampoule/day FSH were administered to each patient. Beginning on day 6, hMG and FSH were administered on an individual basis according to serum E2 and transvaginal ovarian ultrasound scans. The criteria for hCG administration (10 000 IU, Profasi, Serono) were the presence of two or more follicles >1.9 cm in greatest diameter, and serum E2 levels >800 pg/ml (2940 pmol/l). Leuprolide acetate, FSH and hMG injections were discontinued on the day of hCG administration. Oocyte retrieval was scheduled 3638 h after hCG injection. The luteal phase was supported with vaginal administration of 400 mg/day micronized progesterone (Progeffik; Laboratorios Effik, Madrid, Spain).
ICSI
The microinjection was performed as previously described (Palermo et al., 1992). Briefly, morphologically normal spermatozoa were sought in the sperm droplet, and then immobilized and aspirated, tail first, into the tip of the microinjection pipette. A metaphase II oocyte was held on the holding pipette, and the injection pipette was pushed through the zona pellucida, injecting a single spermatozoon.
Injected oocytes were incubated in 20 µl drops and fertilization was assessed after 18 h, and embryo cleavage 24 h thereafter. Embryos were transferred into the uterine cavity 4872 h after ICSI. Supernumerary embryos were frozen for eventual future transfers. Clinical pregnancy was determined by observing a gestational sac with fetal heartbeat at 7 weeks of pregnancy. Prenatal diagnosis included fetal karyotype by amniocentesis at week 1618 of pregnancy.
Statistical analysis
The t-test was used for comparing TESE outcome according to cancer types and treatments. Fishers exact test was used for comparing TESE outcome according to testicular volume and serum FSH. A P-value of <0.05 was considered as statistically significant.
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Results |
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Patients with positive TESE showed significantly higher mean maximum testicular volumes than patients with negative TESE (18.0 versus 14.3 ml, P < 0.01, t-test). Mean serum FSH levels were, however, not significantly different in both groups (30.7 mIU/ml in positive TESE versus 21.3 mIU/ml in negative TESE).
From the histopathological point of view, sperm were recovered in 2/9 (18%) patients with Sertoli cell-only syndrome (SCOS), 2/2 (100%) with severe hypospermatogenesis and 1/1 (100%) with maturation arrest. Mature spermatozoa were observed at histology in four biopsy specimens (two hypospermatogenesis, one with maturation arrest and one SCOS with focal spermatogenesis).
A total of eight ICSI cycles using frozenthawed testicular sperm were performed on the five couples in whom TESE was successful (Table II). There was one fertilization failure. The normal, 2PN fertilization rate in this series was 68%. A total of seven embryo transfers were carried out and one clinical pregnancy was achieved, which resulted in the delivery of a healthy, normal girl.
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Discussion |
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For patients suffering from permanent azoospermia after chemotherapy, the combination of TESE and ICSI has opened new chances of fertility. Motile spermatozoa for cryopreservation and subsequent ICSI were retrieved in 41.5% of patients in the present series. This retrieval rate is in the expected range at our institution (Gil-Salom et al., 1998). It is similar to the 45% successful sperm recovery rate reported by Chan et al. (2001
) after TESE in 20 persistently azoospermic patients receiving chemotherapy for a variety of cancers, and also to the 40% successful retrieval rate reported by Tournaye et al. (2000
) in 10 azoospermic patients after chemotherapy. Recently, Damani et al. (2002
) have reported an excellent 65.2% retrieval rate when analysing 23 chemotherapy-treated patients (including both cancer and non-cancer cases) from a dual-institutional study.
Patients with persistent azoospermia after chemotherapy are a heterogeneous population, comprising different types of cancers treated by distinct chemotherapeutic regimes. So far, only a limited number of such patients have undergone a TESE procedure (Chan et al., 2001; Damani et al., 2002
). Therefore, TESE prognosis in specific tumours after specific treatments is still unknown. Nevertheless, it seems that the TESE outcome in testicular cancer patients with persistent azoospermia after chemotherapy is particularly high, with sperm retrieval rates of 67% in our series and 75% in other studies (Chan et al., 2001
; Damani et al., 2002
). Moreover, 5/6 testicular cancer patients in the present series were persistently azoospermic despite receiving only four cycles of the standard combinations of cisplatin, bleomycin and either vinblastin or etoposide, which have been reported to be unlikely to cause irreversible damage to spermatogenesis (Cullen et al., 1996
; Pont et al., 1996
). It is not possible to know with certainty why these patients remained persistently azoospermic after chemotherapy. Individual susceptibility to gonadotoxic effects of chemotherapy may be implicated in some patients; in others, a pre-existing spermatogenic impairment caused by the testicular tumour itself may have contributed to a more severe degree of spermatogenesis damage than usual.
As with the general population with non-obstructive azoospermia, clinical and histologic parameters were unable to predict with certainty the TESE outcome in an individual patient (Mulhall et al., 1997; Tournaye et al., 1997
; Gil-Salom et al., 1998
). Although maximum testicular volume was significantly higher in those patients with successful sperm retrieval, no patient should be excluded from a TESE procedure solely on the basis of a reduced testicular volume (Gil-Salom et al., 1998
). Similarly, the presence or absence of mature spermatids on histopathology is not absolutely predictive of TESE outcome. Histological findings on a single diagnostic testicular biopsy tend to correlate with TESE outcome using multiple testicular biopsies, because distribution of the minute foci of preserved spermatogenesis in testes from non-obstructive azoospermia patients is diffuse and multifocal (Silber et al., 1997
). Nevertheless, an accurate correlation is lacking (Tournaye et al., 1997
; Gil-Salom et al., 1998
). Therefore, we usually carry out the TESE procedure first and, when successful, retrieved spermatozoa are cryopreserved for performing ICSI later on, so avoiding unnecessary ovarian stimulation treatments in the women should TESE fail (Gil-Salom et al., 1996
; Oates et al., 1997
).
One couple achieved the live birth of a healthy girl in our series. Although the normal, 2PN fertilization rate was high (68%), clinical pregnancy and live birth rates in our patients were low (12.5%). Reported ongoing/delivery rates in other ICSI series in persistently azoospermic patients after chemotherapy are, however, higher33% (Chan et al., 2001) and 30.8% (Damani et al., 2002
). The small number of ICSI cycles included in each study may account for such differences. Although we used frozenthawed spermatozoa for ICSI, in our experience testicular sperm cryopreservation has no impact on ICSI outcome (Gil-Salom et al., 1996
; 2000). More patients should be analysed before concluding that ICSI outcome in this subpopulation is comparable with the success rates obtained in other ICSI candidates.
One important matter of concern is the potential genetic risk associated with the use of ART in cancer survivors with non-obstructive azoospermia. As pointed out by Chan et al. (2001), genetic counselling is particularly relevant in this specific subpopulation regarding both the possibility of an increase in the incidence of tumours in the offspring and the consequences of the known mutagenic effects of chemotherapy on germ cells (Arnon et al., 2001
). Although several studies have not found evidence of a significantly increased risk of non-hereditary cancer among the offspring of childhood-cancer patients (Mulvihill et al., 1987
; Sankila et al., 1998
), early screening for neoplasic conditions during the lifespan of these children should be addressed.
On the other hand, the possibility of genetic damage caused by cytotoxic drugs on germ cells is still controversial. An increased incidence of sperm chromosomal abnormalities has been assessed in cancer patients during and immediately after receiving chemotherapy, but it is considered to be a transient effect of cytotoxic drugs (Robbins et al., 1997; Martin et al., 1999
). Based on experimental studies, the common recommendation has been to not use spermatozoa for 6 months following the completion of chemotherapy (Meistrich, 1993
). However, a recent study has shown that a significant increase in the frequency of sperm aneuploidy may persist until least 18 months after the initiation of chemotherapy (De Mas et al., 2001
). The clinical impact of such a rise in sperm chromosomal abnormalities remains still to be clearly defined, but, theoretically, it could lead to spontaneous abortions, stillbirths or birth defects in chromosomally abnormal children (Hassold et al., 1996
). So far, including our case, at least 14 live births of healthy children have been reported after TESE and ICSI in patients with persistent non-obstructive azoospermia after chemotherapy (Chan et al., 2001
; Damani et al., 2002
). Although these results are encouraging, couples should be counselled regarding the potential risks involved, and prenatal diagnosis is strongly recommended. More patients and children should be screened to assess the safety of the procedure, and to define an eventual role for genetic diagnostic methods such as sperm fluorescence in-situ hybridization analysis or preimplantation genetic diagnosis in these couples.
In conclusion, in some patients with permanent azoospermia after chemotherapy who have not frozen sperm samples before therapy, testicular spermatozoa can be successfully retrieved for ICSI. Since no clinical or histopathological parameters can accurately predict TESE outcome, sperm cryopreservation is advisable before starting ovarian stimulation. This approach can result in pregnancy and the delivery of healthy children, but couples should be aware of the potential genetic risks of the procedure before entering the TESEICSI programme. Therefore, freezing semen before starting gonadotoxic therapy remains the strategy of choice, and patients should be counselled accordingly.
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FOOTNOTES |
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References |
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Submitted on December 31, 2002; accepted on March 4, 2003.