IVF and Infertility Unit, Assaf Harofeh Medical Center, Tel Aviv University, Israel
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Abstract |
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Key words: azoospermia/intracytoplasmic sperm injection/Klinefelter's syndrome/TESE/47XXY
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Introduction |
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Materials and methods |
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Following informed consent from the patients regarding the genetic risks of their potential offspring, extensive sperm preparation (ESP) (Ron-El et al., 1997) from ejaculated spermatozoa was offered as first line treatment.
Sperm retrieval and preparation
On the day of oocyte retrieval, the male partners produced fresh ejaculates, and ESP revealed mature, non-motile spermatozoa in two out of the 12 patients. In 10 patients ESP confirmed that no spermatozoa were found in the specimen and TESE was performed immediately. In the first patient with spermatozoa in the ejaculate, ICSI was performed using non-motile ejaculated spermatozoa; however, no fertilization was achieved. Therefore, TESE was offered in the following cycle. In the second patient with ejaculated immotile spermatozoa, TESE was offered immediately. Finally, all 12 patients underwent TESE.
Methodology of TESE procedures
The technique of surgical sperm retrieval by TESE, sperm preparation and ICSI has been described in detail elsewhere (Friedler et al., 1997). In all patients, tissue taken from their testicular biopsy was sent for histological evaluation.
When testicular spermatozoa were found enabling ICSI, excess tissue was cryopreserved using a simple freezing protocol (Friedler et al., 1997). This enabled performance of ICSI using testicular spermatozoa extracted from the thawed specimen, in consecutive cycles, of the same patients.
Ovulation induction and oocyte retrieval were performed using a routine long protocol of mid-luteal pituitary suppression with gonadotrophin-releasing hormone (GnRH) agonist, followed by human menopausal gonadotrophins for ovarian stimulation. Oocytes were retrieved by vaginal ultrasound guided follicular puncture. After assessment of fertilization ~24 h later, embryo transfer was performed on day 2 or 3, after recording embryonic cleavage and morphological quality. No more than three embryos were transferred, in the first trial and up to four, if available, in the repeated attempt. The reason for transferring one more embryo (if available) was to increase the chances of the couple to achieve pregnancy, according to their wish, fearing that the performance of cycles using cryopreservedthawed testicular spermatozoa might be somewhat inferior compared with that with fresh spermatozoa. Routine progestative luteal support was given, as described previously (Friedler et al., 1997). Only clinical pregnancies, including sonographic demonstration of a gestational sac, were counted. As only a few spermatozoa were needed in all ICSI cycles (up to 16 in fresh and up to 11 in cryopreservedthawed cycles), not all the testicular biopsy specimen was extracted and survival rate was not quantified. However, after thawing in four out of five cycles, enough motile spermatozoa were found to enable ICSI of the available oocytes, giving the impression that cryopreservation was quite efficient.
Outcome of ICSI using fresh or crypreservedthawed testicular spermatozoa in these patients was compared.
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Statistical analysis |
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Results |
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Discussion |
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Testicular spermatozoa in KS patients may have a different genetic composition, with an increased rate of hyperhaploidy (Foresta et al., 1999) than those suffering from azoospermia from a different cause and their freezability might differ. It appears that the outcome of ICSI using fresh or cryopreservedthawed testicular spermatozoa in patients with KS has not yet been compared.
The results in the current study show that testicular tissue may be successfully cryopreserved in patients with non-mosaic KS without compromising significantly fertilization and implantation rates. Two pregnancies and a single delivery were previously reported using cryopreservedthawed testicular spermatozoa among patients with non-mosaic KS (Ron-El et al., 2000a).
The ability to use successfully cryopreservedthawed testicular spermatozoa for ICSI in patients with non-obstructive azoospermia (NOA) has been evaluated in several reports. Following the use of fresh or cryopreservedthawed testicular spermatozoa, in patients with NOA, a similar fertilization rate of 47 versus 44%, embryo cleavage rate of 94 versus 89% and implantation rates of 9 versus 11% were observed (Friedler et al., 1997). Interestingly, ICSI using both fresh or cryopreserved testicular spermatozoa from patients with non-mosaic KS patients resulted in somewhat better fertilization rates and implantation rates compared with those from patients with NOA; however to reach significant conclusions the size of the groups should be enlarged.
The risk of transmission of gonosomal aneuploidy by using spermatozoa from non-mosaic KS patient is probably not high. This finding should in fact not be surprising, as it has been demonstrated recently that 47,XXY spermatogonia are probably capable of undergoing meiosis, completing the spermatogenic process culminating in formation of cytogenetically normal spermatozoa (Foresta et al., 1999; Bielanska et al., 2000
). However, in one of the conceptions in our series, prenatal diagnosis of a 47,XXY fetus was performed, demonstrating the risk of injecting an abnormal spermatozoon during ICSI in these patients. This is in concordance with the higher prevalence of sex chromosome disomy found in the spermatozoa of non-mosaic KS patients (Guttenbach et al., 1977; Estop et al., 1998
; Foresta et al., 1998; Okada et al., 1999
). Of course, non-dysjunction in the oocyte injected may not be ruled out completely. Nevertheless, there is no doubt that genetic counselling is warranted in these cases. Preimplantation genetic diagnosis (PGD) by embryo biopsy may serve as a potent tool for embryo selection. As PGD cannot be considered yet as an absolutely reliable technique (Munné et al., 1995
) nor is it available in each unit, prenatal genetic diagnosis should be recommended in all pregnancies achieved. So far, all 15 newborns after ICSI of testicular spermatozoa in non-mosaic KS patients are healthy with normal karyotype (Tournaye et al., 1997
, two healthy newborns) (Palermo et al., 1998
, 1999
, five healthy newborns) (Reubinoff et al., 1998
, one healthy newborn) (Ron-El et al., 1999
, 2000a
,b
, five healthy newborns) (Nodar et al., 1999
, two healthy newborns). One conception was prenatally diagnosed as 47,XXY, and terminated (Ron-El et al., 2000b
).
In conclusion, patients with KS, even the non-mosaic type, may be offered treatment by ICSI. Preferably motile spermatozoa should be identified by ESP in their ejaculates. If only non-motile spermatozoa are found, there is still a possibility of finding motile testicular spermatozoa following TESE that may lead to pregnancy and delivery. Outcome of ICSI using cryopreservedthawed testicular spermatozoa seems comparable with that following use of fresh spermatozoa. The genetic implications for the future offspring should be explained to the patients, warranting pretransfer or prenatal genetic evaluation of their potential offspring.
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Notes |
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References |
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Submitted on December 29, 2000; accepted on September 8, 2001.