1 Infertility Centre, Department of Obstetrics and Gynaecology and 2 Department of Urology, University Hospital of Ghent, De Pintelaan 185, 9000 Ghent, Belgium
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Abstract |
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Key words: azoospermia/ICSI/non-obstructive/obstructive/testicular spermatozoa
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Introduction |
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Azoospermia is found in 10% of male infertility cases. In cases of surgically irreparable obstructive azoospermia or in cases of congenital absence of the vas deferens (CAVD), microsurgical epididymal sperm aspiration (MESA) with IVF has been shown to yield fertilization and pregnancy (Temple-Smith et al., 1985; Silber, 1989
; Silber et al., 1990
). However, the results were poor and unpredictable. Since the introduction of ICSI, the use of epididymal (Tournaye et al., 1994
) or testicular (Craft et al., 1993
; Schoysman et al., 1993a
,b
) spermatozoa has allowed treatment of male infertility due to obstructive azoospermia.
Testicular sperm extraction (TESE) in combination with ICSI is also the method of choice for the treatment of male infertility due to non-obstructive azoospermia (Devroey et al., 1996), except for correctable hormonal problems. The first births of children born after ICSI with testicular spermatozoa from patients with non-obstructive azoospermia were reported in the mid-1990s (Tournaye et al., 1995
). ICSI in combination with testicular spermatozoa can therefore be used to treat male fertility, even in cases of deficient spermatogenesis. Whether spermatozoa can be recovered from azoospermic patients depends on the underlying aetiology. When the testicular histology is normal, or in cases of germ-cell hypoplasia, sperm recovery is nearly always successful. In patients with germ-cell aplasia or maturation arrest, the yield of sperm extraction can be very variable (Tournaye et al., 1996
).
It can be anticipated that in non-obstructive azoospermia, both the quantity and quality of spermatozoa retrieved will be lower than in obstructive azoospermia. Comparative data on fertilization, pregnancy and embryo implantation rates are sparse and, moreover, are not unequivocal. Significantly reduced fertilization and pregnancy rates have been found in cases of non-obstructive azoospermia (Fahmy et al., 1997; Mansour et al., 1997
), in contrast with other findings (Kahraman et al., 1996
; Palermo et al., 1999
) which report a lower fertilization rate in cases of non-obstructive azoospermia, but without affecting pregnancy rate. Therefore, a retrospective analysis of all cases of ICSI with fresh testicular spermatozoa was performed to compare the results in both obstructive and non-obstructive azoospermia in terms of fertilization rate, embryo quality, embryo implantation and pregnancy rates. In cases of non-obstructive azoospermia, this analysis was also extended to different causes of testicular failure.
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Materials and methods |
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All female patients had been treated with either a long or short gonadotrophin-releasing hormone analogue (Decapeptyl®; Ipsen, Destelbergen, Belgium) protocol, followed by ovarian stimulation with human menopausal gonadotrophin (HMG) (Humegon®; Organon, Oss, The Netherlands). Oocytes were retrieved by transvaginal ultrasound-guided puncture of the ovarian follicles 36 h after human chorionic gonadotrophin (HCG) (Pregnyl®; Organon) administration. Oocyte retrieval was carried out either in our centre or in a transport setting as described previously (Coetsier et al., 1997).
All male patients had a general clinical andrological examination. Since the beginning of 1998 all couples with non-obstructive azoospermia were offered and recommended to have a genetic screening with molecular analysis for Y-chromosome deletions as well as a standard karyotype analysis. Among the patients volunteering for screening, no Y-deletions were detected.
In couples where the male partner suffered from CAVD, both the man and woman were screened for possible cystic fibrosis (CF) gene mutations. This included screening for the eight most common CF gene mutations (F508; G542X; N1303K, 1717-1G>A; W1282X; G551D; R553X;
I507) and 21 less common mutations. In addition, a test for Tn allele in intron 8 (5T-7T-9T) was also carried out. Five patients with cystic fibrosis and/or CAVD were positive for CF gene mutations. All the female partners were negative.
Retrieval of testicular spermatozoa
In our clinic, the urologists prefer to perform an open testicular biopsy in cases of obstructive azoospermia rather than MESA because: (i) it is technically more simple; (ii) it is less expensive; and (iii) it can be carried out under local anaesthesia, allowing the patient to leave the clinic a short time afterwards. However, it should be noted that fewer spermatozoa are to be expected from testicular biopsies than from epididymal sperm extraction.
Open excisional testicular biopsies were taken under local anaesthesia in cases of obstructive azoospermia, and under general anaesthesia in cases of non-obstructive azoospermia. A small incision was made in the scrotal skin and carried through the peritoneal tunica vaginalis. One incision was made in the tunica albuginea and the extruding testicular tissue was excised and placed in HEPES-buffered Earle's medium with 0.1% (w/v) heparin. Incisions in the tunica albuginea were performed macroscopically in between the testicular subtunical blood vessels to limit testicular damage. In cases of obstructive azoospermia generally only one biopsy was taken. In cases of non-obstructive azoospermia, if spermatozoa were present in the first wet preparation, then no further testicular incision was made. If no spermatozoa were found, repeated sampling (up to 10 biopsies) was needed and biopsies were also taken from the contralateral testis.
In all patients two small pieces of testicular tissue (one in Bouin's medium for the histological diagnosis of infertility, and one in buffered formaldehyde for exclusion of carcinoma in situ) were sent for pathology. Histological findings were based on published classifications (Levin, 1979). It should be noted that a specimen was classified as incomplete Sertoli cell-only (SCO) where the pathologist diagnosed SCO but spermatozoa were found in the wet preparations by the IVF laboratory.
In the laboratory, the testicular tissue was placed in Earle's medium supplemented with 0.4% (w/v) human serum albumin (HSA) (20%, w/v; Albumin, Red Cross, Brussels, Belgium) and teased apart using microscissors. A small aliquot of testicular cell suspension was placed in a drop of Earle's medium under mineral oil to check for testicular spermatozoa under the inverted microscope (Zeiss Axiovert 135, Zeiss, Zaventem, Belgium) at x320 magnification. Slowly progressive or non-progressively motile spermatozoa were picked up individually and brought to the micromanipulation dish. No non-motile spermatozoa were injected. The most normal-looking spermatozoa were selected for micromanipulation, preference being given to normal head morphology. In every case, enough motile spermatozoa were found to inject all mature oocytes.
ICSI procedure and assessment of fertilization and embryonic development
ICSI was carried out on the stage of an inverted microscope (Zeiss Axiovert 135) at x320 magnification using the Hoffman Modulation Contrast System, as described previously (Dozortsev et al., 1996).
At 1618 h after ICSI, oocytes were checked under the inverted microscope at a magnification of x320 for the presence of two pronuclei. At 48 h after ICSI, embryos were scored for quality according to a system that takes into account the percentage of anucleate fragments and the size of blastomeres (Laverge et al., 1997). In brief, embryos with equally sized blastomeres without or with up to 10% anucleated fragments were classified as excellent-quality embryos. Embryos with from 1020% fragmentation, between 2050% fragmentation or >50% fragmentation were classified as good-, moderate- and poor-quality embryos respectively. Embryo transfer was carried out on either day 2 or 3. In general, the morphologically two best embryos were selected for transfer.
Pregnancy
Pregnancy was detected by measuring serum HCG at two independent occasions at least 15 days after embryo replacement. Clinical pregnancy was determined by observation of a gestational sac with fetal heart beat on transvaginal ultrasound at 67 weeks of pregnancy. The clinical embryo implantation rate was defined as the number of gestational sacs observed at echographic screening at 67 weeks of pregnancy divided by the number of embryos transferred.
Statistical analysis
The 2-test was used to compare rates. For comparing means between two or more groups, Student's t-test or a one-way analysis of variance (ANOVA) followed by a NewmanKeuls post-test were applied, respectively. All statistical tests were carried out two-tailed at the 5% level of significance.
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Results |
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The mean female age was 32.7 ± 4.3 years in group 1, and 30.9 ± 4.1 years in group 2. This difference was statistically significant (P = 0.0078). The fertilization rate in obstructive azoospermia was 74.5%, which was significantly higher than in non-obstructive azoospermia (67.8%; P = 0.0167). Embryo quality was similar for both groups.
Pregnancy and embryo implantation rates after ICSI in obstructive and non-obstructive azoospermia are detailed in Table III. All deliveries gave rise to healthy babies, and no late abortions occurred.
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Discussion |
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Mean male age was significantly higher in obstructive azoospermia than in non-obstructive azoospermia. This was due to the fact that a greater proportion of these patients have acquired an obstruction after vasectomy. However, after a few years and often in a subsequent relationship, a renewed wish for children occurs. This is most likely the reason why these male patients are on average 6 years older. The calendar age itself may be less important than the number of years post-vasectomy in relation to the influence on fertilization rate (De Croo et al., 1999). In this retrospective study, the fertilization rate was higher in obstructive than non-obstructive azoospermia. Mean female age was also significantly higher in obstructive azoospermia than in non-obstructive azoospermia. However, in our general population, female age under 37 years did not influence the results in terms of fertilization and implantation rate.
The fertilization rates observed after ICSI in cases of obstructive azoospermia (74.5%) and in cases of nonobstructive azoospermia (67.8%) were high compared with previously published reports, possibly because extensive experience and excellent skill, as well as a knowledge of the `best-looking' spermatozoa for injection. Earlier, a fertilization rate of 33.9% had been reported in non-obstructive azoospermia and 65.3% in obstructive azoospermia (Kahraman et al., 1996); others (Fahmy et al., 1997
) also reported a significantly lower fertilization rate after ICSI in non-obstructive azoospermia (41.2%) than in obstructive azoospermia (57.9%). Similar findings (54.4% and 39.0% respectively) were detailed in a later study (Mansour et al., 1997
). A possible reason for the poorer fertilization rates after ICSI in non-obstructive azoospermia might be the lower concentration of spermatozoa, so that the probability of choosing a `normal mature' spermatozoon is reduced. A clear correlation has been shown between the number of spermatozoa recovered and the fertilization rate in cases of non-obstructive azoospermia (Lewin et al., 1999
), while others (Devroey et al., 1994
) found that fertilization and cleavage rates were similar in patients with severe spermatogenetic defects and in patients with normal spermatogenesis in cases of obstructive azoospermia. The fertilization rate may be affected differently by different types of spermatogenetic defects in the non-obstructive group. In cases of severe spermatogenetic defects such as SCO syndrome, severe hypospermatogenesis or maturation arrest sometimes non-fully cytoplasmically matured spermatozoa are present for ICSI. This may explain why a significantly lower fertilization rate was found after ICSI with testicular spermatozoa in non-obstructive azoospermia. In addition, in this study fertilization rate using spermatozoa from patients with maturation arrest was significantly lower than in patients with germ cell hypoplasia or SCO syndrome. Lower fertilization rates were also reported in patients with maturation arrest (45.7%) and SCO syndrome (44.0%) compared with germ cell hypoplasia (67.8%) (Tournaye et al., 1996
). Since maturation arrest and germ cell aplasia are assumed to have a genetic origin (Vogt et al., 1992
; Martin-du-Pan and Campana, 1993
), genetic factors might also explain the decrease in fertilization rate. In the present study, a lower fertilization rate in cases of SCO syndrome was not found, but there were only seven transfers in this group.
Another interesting finding is that spermatozoa were found in only 55.5% of the cases in the non-obstructive group. In recent publications (Friedler et al., 1997; Tournaye et al., 1997
; Van Steirteghem et al., 1998
), the sperm recovery rate in non-obstructive azoospermic men varied between 40% and 70%. This raises the question whether a diagnostic testicular biopsy may be useful. In most of the studies, sperm recovery was mainly performed by TESE. On the one hand, performing a testicular biopsy on a day different from the oocyte retrieval makes it possible to avoid stimulation of the women in cases where no spermatozoa are found. On the other hand, when the testicular biopsy and oocyte retrieval are performed simultaneously, more multiple biopsies and a more exhaustive examination of the tissue are likely to be accomplished. Moreover, by performing a diagnostic testicular biopsy, the few spermatozoa obtained could be lost by the freezing and thawing procedure. Unfortunately, no strong predictors for successful testicular sperm recovery are available, except for testicular histopathology (Tournaye et al., 1997
). Especially in patients showing SCO syndrome histopathology, this is an accurate parameter, but this is not true for patients showing maturation arrest. The results of a recent study (Ben-Yosef et al., 1999
) favour the first-line choice of cryopreserved spermatozoa in cases of non-obstructive azoospermia. Thus, especially for non-obstructive azoospermia, a diagnostic testis biopsy can be useful to avoid unnecessary ovarian stimulation.
Pregnancy and embryo implantation rates were similar after ICSI in cases of obstructive and non-obstructive azoospermia. One study (Kahraman et al., 1996) reported a significantly lower pregnancy rate in cases of non-obstructive azoospermia (33.3%) compared with obstructive azoospermia (43.7%), but embryo implantation rates were similar (30.0% and 26.6% respectively). Others (Mansour et al., 1997
) found significantly reduced pregnancy rates in non-obstructive azoospermia (11.3%) compared with obstructive azoospermia (31.9%). A significantly lower implantation rate was also found after ICSI with testicular spermatozoa in non-obstructive azoospermia compared with a matched control group with ejaculated spermatozoa, and with the obstructive group with normal spermatogenesis (Ubaldi et al., 1999
). In a recent study (Ghazzawi et al., 1998
), the live birth rate per embryo transfer was significantly reduced in patients with non-obstructive azoospermia (10.0%) compared with those groups of patients in which ejaculated spermatozoa (21.0%) and epididymal spermatozoa (22.0%) were used.
Recently, a number of reports have been made on the successful use of testicular sperm aspiration (Lewin et al., 1999; Westlander et al., 1999
) or testicular needle biopsy (Tuuri et al., 1999
) in cases of non-obstructive azoospermia. It was reported (Westlander et al., 1999
) that testicular sperm aspiration (TESA) performed under local anaesthesia seems almost as effective as the more invasive procedure (TESE) both in obstructive and non-obstructive azoospermic men, whereas others (Friedler et al., 1997
) have emphasized that TESE is superior, especially in non-obstructive azoospermia.
It may be concluded from this retrospective analysis that after ICSI in combination with TESE, the fertilization rate in non-obstructive azoospermia is significantly lower than in obstructive azoospermia. Once fertilization is achieved, embryonic development and pregnancy rates were comparable across obstructive and non-obstructive azoospermia, as well as across the different types of spermatogenetic defect in non-obstructive azoospermia. The number of observations per type of spermatogenetic defect are, however, still too few to draw valid conclusions. Continued analysis of fertilization, pregnancy and embryo implantation rates of different histopathological subgroups in cases of non-obstructive azoospermia may cast a light on the influence of the type of spermatogenetic defect on the predictability of sperm recovery and the outcome of ICSI.
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Acknowledgments |
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Notes |
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References |
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Submitted on October 29, 1999; accepted on March 3, 2000.