1 Clínica e Centro de Pesquisa em Reproducião Humana Roger Abdelmassih, São Paulo, Brasil and 2 Mar&Gen, Granada, Spain
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Abstract |
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Key words: epididymal sperm/implantation rate/pregnancy rate/time from vasectomy
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Introduction |
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Epididymal and testicular sperm aspiration are routinely used to obtain spermatozoa in cases of obstructive and non-obstructive azoospermia. The fertilization rates are similar in these cases to those obtained with sperm from the ejaculate (Nagy et al., 1995b; Van Steirteghem et al., 1998
). ICSI, therefore, has become an effective alternative to restore fertility in post vasectomy cases in which a reanastomosis has failed or the time span from vasectomy has resulted in secondary sites of epididymal obstruction. Moreover, ICSI may eliminate the negative effects on fertility of anti-sperm antibodies frequently present in these men (Heidenreich et al., 1994
). In most obstructive cases, including post-vasectomized men, epididymal sperm aspiration is the method of choice to obtain sperm (Craft et al., 1995
). Fine needle aspiration, as a technique, is a simpler and less invasive procedure than microsurgical intervention on the epididymides or on the testes (Tournaye et al., 1998
).
Recent reports suggest that male fertility declines with age (Auroux, 1993). However, it has been previously published that in normal healthy men, semen quality per se does not decline significantly with time (Fisch et al., 1996
). Furthermore, ICSI outcome appears not to be related to paternal age (Spandorfer et al., 1998
).
On the other hand, time-related negative effects on semen quality and fertility have been described in post-vasectomized males (Urry et al., 1990). A higher incidence of anti-sperm antibodies and sperm clumping together with a decrease in the proportion of motile sperm have been reported (Weiske, 2001
).
Because of the above benefits of ICSI over reanastomosis, a significant proportion of obstructive azoospermic patients seeking treatment by ICSI are vasectomized men. Because it is not known at present whether post-vasectomy period has any influence on the outcome of ICSIIVF, we analysed the relationship between the post-vasectomy time period and sperm reproductive capacity after ICSI.
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Materials and methods |
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Ovarian stimulation was performed using a combination of a GnRH agonist (leuprolide acetate, Lupron®; Abbott Laboratórios do Brasil Ltda, São Paulo, Brazil), long protocol and recombinant FSH (Gonal-F®; Serono Laboratorios, São Paulo, Brazil). Follicular development was monitored with periodic measurements of serum estradiol and vaginal ultrasound. HCG 10 000 IU (Profasi®; Serono Laboratorios) was administered when at least two follicles reached a mean diameter of 18 mm and oocyte retrieval was performed by ultrasound guided transvaginal aspiration 34 h later.
Epididymal sperm aspiration
Sperm were retrieved by PESA. A 27.5 gauge needle was introduced into the proximal part of the epididymis (usually located in the upper pole of the testicle) and a delicate suction was performed with a 1 ml syringe (B-D; Becton Dickinson Ind. Cirúrgicas Ltda, Curitiba, Brazil). The aspirate was placed into a Petri dish and was flushed with IVF-50 medium (IVF Science Scandinavian, Gothenburg, Sweden). The sample was assessed for the presence of motile sperm. If in the sample there was no motile sperm present, the epididymal aspiration was repeated.
Embryology laboratory procedures
Oocytes collected in pure follicular fluid were immediately sent to the adjacent embryology laboratory, where they were identified using a dissecting microscope at x50 magnification. After being identified, oocytes were placed in a single well culture dish (3260; Costar, Cambridge, MA, USA) with 1.0 ml of 80 IU/ml of hyaluronidase (Hyase-1; IVF Science Scandinavian) for 30 s and subsequently were washed several times in IVF-50 medium. Each oocyte was placed separately inside one droplet of 30 µl of IVF-50 medium and covered with light weight mineral oil (Ovoil 150; IVF Science Scandinavian) and incubated for 24 h at 37°C in an atmosphere with 5% CO2. At the end of the incubation period, cumulus and corona radiata cells were removed with a 135 µm diameter plastic pipette, connected to a cell stripper (Mid Atlantic Diagnostics Inc., Medford, NJ, USA).
Nuclear maturity and ooplasmic quality were evaluated using an inverted light microscope (Nikon Diaphot Microscope®; Nikon Corporation, Tokyo, Japan) at x200 or x400 magnification. Metaphase-II (MII) oocytes were microinjected within a period of 24 h after collection. Metaphase I (MI) oocytes were observed for the extrusion of the first polar body at 4 h intervals up to 8 h after retrieval and the injection was performed accordingly. The methods for sperm injection have been previously reported (Abdelmassih et al., 1996).
After sperm injection, oocytes were incubated in 1 ml of IVF-50 medium, covered with mineral oil, for 1618 h. Oocytes were observed 1618 h after ICSI for the presence or absence of pronuclei and polar bodies (Nagy et al., 1998a). Fertilization was considered normal when two clearly distinct pronuclei were present. If a single pronucleus was observed, a second evaluation was carried out approximately 4 h later. When the presence of three pronuclei was detected, the oocyte was discarded.
Oocytes containing two pronuclei were cultured separately in droplets of 30 µl of IVF-50 medium covered with mineral oil in a Petri dish (3260; Costar) and were kept until transfer. The embryos were observed at 42 h (day 2) and at 68 h (day 3) after injection and classified according to the criteria proposed by Staessen (Staessen et al., 1989). According to the number of blastomeres and to the relative proportion of anucleate fragments present in the zona pellucida, embryos were classified to one of the four categories: 1, excellent (type A), when no anucleate fragment was present (6- to 8-cell stage on day 3); 2, good (type B), when <20% of the embryo was fragmented (6- to 8-cell stage on day 3 or <6 cells with no fragmentation); 3, fair (type C), when the relative amount of fragments was between 20 and 50%; and 4, poor (type D), when >50% of the embryo was fragmented. Embryos with >50% fragmentation were not transferred.
At the time of transfer, embryos were loaded into 15 µl of IVF-50 medium using an Edwards Wallace Catheter of 23 cm (Simcare Manufacturing Ltd, Hythe, Kent, UK).
The luteal phase was supported by daily administration of 800 mg of vaginal progesterone (Utrogestan®; Laboratoires Besins-Isvovesco, Paris, France) and transdermic 100 µg estradiol patches changed daily (Estraderm TTS100®; Biogalênica Laboratório, São Paulo, Brazil). Serum ß-HCG levels were measured 12 days after the embryo transfer.
Transvaginal ultrasound was performed in all patients with ascending ß-HCG titres; presence and number of intrauterine gestational sacs were assessed, as well as the presence of a fetal pole and cardiac activity. Pregnancy was monitored by the patient's obstetrician and the final outcome was reported to the clinic both by the obstetrician and the patient.
Statistical analysis
The one way analysis of variance (ANOVA) and KruskalWallis tests were used to compare means of female and male age, fertilization rate, embryo quality, number of embryos transferred and time period after vasectomy. Contingency table analysis and Fisher's exact tests were used to compare pregnancy, implantation and abortion rates.
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Results |
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The clinical pregnancy rate and the ongoing pregnancy rate correlated significantly with the time period of vasectomy (P < 0.05; 2 test for trend), and the longer period of vasectomy was associated with fewer pregnancies (Table I
). The implantation rate also decreased from group 1 to 3, and the difference between all groups was statistically significant (P < 0.05, contingency table analyses and Fisher's exact test). The abortion rate showed a tendency to increase from group 1 to 3 but it did not reach statistical significance among the groups.
In order to exclude the effect of female age on the results, we analysed the data of 84 of the 151 cycles in which only those women who were 35 years old were included. The results, organized in the same manner in three groups, are presented in Table II
. The difference in the mean male age of the three groups remained significant (P < 0.0001) but female age did not differ significantly among the three groups. The number of injected and fertilized oocytes were also similar in the three groups as well the number and quality of embryos transferred to the patients. Ongoing pregnancy and implantation rates were significantly higher in groups 1 and 2 when compared with group 3 (Table II
).
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Discussion |
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Importantly, the results of our study suggest, for the first time, a negative correlation between the pregnancy outcome of assisted fertilization and the time span from vasectomy to treatment. The difference in pregnancy and implantation rates persisted after the results were corrected for the age of the women. The only evident clinical differences remaining were the mean age of the males between the groups and the time period of vasectomy. The influence of male age on the results of assisted reproduction is not well determined. Some studies suggest a decline in fertility potential with advancing male age (Auroux, 1993), however most studies did not establish a correlation between the ageing and the reproductive potential of men (Krause and Habermann, 2000
). Also individual follow-up for up to 25 years did not reveal any important changes (Fisch et al., 1996
). More importantly in regard to our study, ICSI results for different diagnostic categories are not affected by the age of the male partner (Spandorfer et al., 1998
) in contrast to the important role of the age of the female partner (Abdelmassih et al., 1996
; Devroey et al., 1996
).
The success rate of vasectomy reversal in restoring fertility is an inverse relationship with the time span from vasectomy (Belker et al., 1991). Until now, the time-related negative effects of vasectomy in relation to fertility were assigned to mechanical factors (the increasing incidence of secondary sites of epididymal obstruction) which made anatomical restoration more difficult, and to the appearance of significant titres of anti-sperm antibodies (Heidenreich et al., 1996). ICSI obviously overcomes the mechanical blockage and the results obtained with the technique are independent of immunological factors (Nagy et al., 1995c
).
All cases included in our study underwent epididymal sperm aspiration. Silber et al. have suggested that in cases of chronically obstructed epididymis, sperm suffer changes related to senescence with a significant decrease in motility towards the distal portion (Silber et al., 1995). On the other hand, Moore has reported that in these cases a disproportionate number of dead or dying sperm is also found in the proximal portion (Moore, 1998
). The author suggests that in normal conditions, epididymal fluid protects viable sperm from acrosomal enzymes liberated by sperm in the process of degeneration. The stagnation secondary to chronic obstruction would interfere with this protective mechanism. Moreover, it has been demonstrated that in men with obstructive azoospermia, testicular sperm DNA is significantly less damaged than proximal epididymal sperm DNA (Steele et al., 1999
). In the same study the authors found that DNA quality in sperm obtained from the testicle is the same as that found in normal men, suggesting that the damage found in epididymal sperm is acquired with time after being produced. It is tempting to speculate that epididymal sperm DNA damage increases with time of obstruction and, in the cases presented here, time from vasectomy. In the same line of thinking, the use of immotile (due to necrozoospermia) ejaculated or epididymal sperm for ICSI was associated with very poor fertilization and embryonic development; however, testicular sperm (from the same patient), even if immotile, provided a much improved outcome of the treatment cycle (Nagy et al., 1995a
; Tournaye et al., 1996
; Nagy et al., 1998b
).
In this study, no significant differences were observed in the fertilization rate and in quality of embryos transferred at 72 h post-fertilization among the three groups. The effects of using molecularly damaged sperm on fertilization could be masked with the use of ICSI, and early stages of embryo development depend mainly on maternal information as the embryonic genome is activated after the 8-cell stage (Braude et al., 1988). However, increased DNA damage of epididymal sperm (used exclusively in the cases reported) could constitute an explanation for the lower embryo implantation rates and for higher rates of abortion found in the cases with a longer period from vasectomy.
Our results differ from the data reported by others (Sukcharoen et al., 2000) but may not be in direct contradiction. The number of cases included in our study was significantly larger and the previously reported data included some cases in which ICSI was performed with sperm obtained from the testes.
The findings reported here may have important clinical implications. Percutaneous epididymal sperm aspiration is a simple, fast and popular procedure to obtain sperm from men with obstructive azoospermia; however, in the light of the present study, fine needle aspiration from the testes may represent a better approach, especially in those cases with prolonged time of obstruction. However, further collection and analyses of data obtained from prospective studies should be performed in order to establish the best intervention in these cases.
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Notes |
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Submitted on September 21, 2001
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References |
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accepted on November 7, 2001.