American Hospital of Istanbul, Istanbul, Turkey
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Abstract |
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Key words: azoospermia/in-vitro culture/motility/testicular sperm extraction
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Introduction |
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Testicular sperm extraction (TESE) procedures may be performed 2448 h prior to human chorionic gonadotrophin (HCG), thus 34 days before oocyte retrieval, and the extracted spermatozoa kept in culture until ICSI. The success of this technique shows the feasibility and safety of culturing testicular spermatozoa. The practice of early sperm retrieval is associated with better patient scheduling in the busy IVF clinic and is appreciated by the operating room personnel and the embryologists (Urman et al., 1998). Testicular spermatozoa may also be obtained at a remote time before a possible oocyte retrieval and frozen for later use. The feasibility of this technique, however, is not well defined, especially in men with very limited numbers of spermatozoa (Nagy et al., 1995b
).
Incubation of testicular spermatozoa is normally performed in commercially available defined culture media. Motility of the extracted spermatozoa appears to be increased after in-vitro culture, although it is not known whether implantation and pregnancy rates are affected from such a practice (Nijs et al., 1997). The improvement in motility, however, facilitates the selection of viable spermatozoa for ICSI. To further improve the favourable results obtained with in-vitro culture of testicular spermatozoa, supplementation of culture media with motility-inducing agents such as pentoxifylline has been investigated, but as yet without corroboration of results (Tasdemir et al., 1998
).
In this study we examined the effect of supplementing the culture medium with recombinant follicle stimulating hormone (recFSH) on motility of testicular spermatozoa from patients with non-obstructive azoospermia, and compared the results of ICSI with spermatozoa cultured in recFSH medium and in simple medium. FSH is believed to participate in the complex process of spermatogenesis and spermiogenesis (Tesarik et al., 1998). Deficiency of FSH in primates has been associated with oligozoospermia and teratozoospermia. However, whether FSH has any role at all in the process of tail formation and the acquisition of motility is currently unknown.
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Materials and methods |
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Following randomization, minced tissue samples were placed in a Falcon tube with approximately 2 ml of EBSS medium (Earle's balanced salt solution, Sigma, Aldrich Co. Ltd, Irvine, Paisley, UK) which was used in the extraction process. This medium was supplemented with 0.5% HSA (human serum albumin, Irvine Scientific, Santa Ana, CA, USA). In-vitro culture at 30°C was undertaken with or without the addition of 25 mIU/ml recFSH (Puregon; Organon, Os, The Netherlands). Motility of the spermatozoa was assessed the next day and graded as: immotile, twitching, and progressive linear movement. The samples were then prepared with Percoll separation method to be used for ICSI. The media surrounding the testicular tissue was placed on top of a discontinuous Percoll gradient (2 ml of 90% and 1 ml of 50% gradients were used for all samples). The first step consisted of centrifugation at 800g for 20 min. EBSS medium (9 ml) supplemented with penicillin-pyruvate and 0.5 HSA was added to the 90% fraction pellet and centrifuged for another 10 min at 1000 g. The pellet was prepared as a swim-out droplet covered with mineral oil (Sigma) to be used for the ICSI procedure. ICSI was performed as previously described (Balaban et al., 1998). The main outcome measures were (i) the effect of recFSH on motility of testicular spermatozoa, (ii) fertilization rates, embryo quality, and implantation rates following ICSI of testicular spermatozoa cultured in recFSH or in simple medium.
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Results |
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Discussion |
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In this study we compared fertilization rates, embryo quality and pregnancy rates resulting from ICSI of testicular spermatozoa incubated in a simple culture medium versus medium supplemented with recFSH. After extraction of the connective tissue, testicular spermatozoa were cultured with the surrounding cells before Percoll separation. Testicular spermatozoa cultured with recFSH for a period of 24h appeared to gain twitching and linear motility significantly more often than testicular spermatozoa cultured in simple medium. This resulted in a greater percentage of motile spermatozoa available for ICSI and appears to have been advantageous in terms of fertilization rates, with a resulting increase in the proportion of better quality embryos available for transfer. In this study, the mean number of transferred embryos was similar in the two groups; however, better selection was possible in the recFSH group in terms of embryo morphology related to the blastomere number.
The effect of recFSH on sperm motility may be mediated through Sertoli cells, as spermatozoa were cultured prior to extraction from the testicular tissue. The distribution of FSH and of the messenger RNA for its receptor in Sertoli cells has been clarified (Heckert and Griswold, 1991, 1993
). It may be argued that testicular spermatozoa lose their contact with Sertoli cells as they become detached during the procedures employed to search for their presence. Therefore, in the subsequent culture period a non-contact action of the Sertoli cells on spermatozoa is most likely. This presumably non-contact action of Sertoli cells on motility of testicular spermatozoa may be similar to the known FSH action on in-vitro viability and differentiation of spermatogenetic cells (Trez and Kierszenbaum, 1983
). Furthermore, a direct action of FSH on testicular spermatozoa, which induces or potentiates motility, may also be possible.
The injection of motile testicular spermatozoa with unquestionable viability should yield better fertilization rates compared with injection of immotile testicular spermatozoa. This was the case in our study as significantly more oocytes were fertilized in the recFSH group. The incidence of slow cleaving embryos was higher in the simple medium group. This may be due to parthenogenetic activation of some of the oocytes injected with immotile and therefore non-viable testicular spermatozoa. Increase in implantation and clinical pregnancy rates, therefore, may be due to transfer of embryos showing appropriate cleavage rates. The conclusions reached in this study, if corroborated by others, may increase the success of ICSI in men with non-obstructive azoospermia. However, lack of biological plausibility necessitates caution before widespread acceptance of this technique. Other techniques of in-vitro sperm culture in defined media with higher concentrations of albumin or media supplemented with motility enhancers such as pentoxifylline should be further investigated in randomized trials.
We conclude that culturing testicular spermatozoa for approximately 24 h in recFSH supplemented medium yields an increased number of motile spermatozoa available for ICSI. This appears to increase fertilization rates and also embryo quality, yielding higher implantation and clinical pregnancy rates.
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Notes |
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References |
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Submitted on April 16, 1999; accepted on July 30, 1999.