Human Reproduction, Vol. 14, No. 11, 2808-2811,
November 1999
© 1999 European Society of Human Reproduction and Embryology
In-vitro culture of spermatozoa induces motility and increases implantation and pregnancy rates after testicular sperm extraction and intracytoplasmic sperm injection
American Hospital of Istanbul, Istanbul, Turkey
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Abstract |
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The aim of this study was to determine the effect of 24-h in-vitro culture of testicular spermatozoa in recombinant follicle stimulating hormone (recFSH) supplemented medium versus simple medium on sperm motility, and to analyse the outcome of intracytoplasmic sperm injection (ICSI) of such spermatozoa. A total of 143 positive testicular sperm extraction procedures in men with non-obstructive azoospermia was evaluated prospectively. Extracted testicular tissue samples were randomized to be cultured in vitro for 24 h in simple medium or recFSH supplemented media. ICSI was performed with spermatozoa cultured in recFSH (n = 73) or in simple medium (n = 70). Sperm motility following in-vitro culture, embryo quality after ICSI, and implantation and pregnancy rates were assessed. Of the 898 MII oocytes available in the recFSH group, 646 (71.9%) were injected with spermatozoa showing either twitching or progressive motility. However, only 29.1% of the oocytes in the simple medium group (245/841) were injected with motile spermatozoa (P < 0.05). Fertilization rate (68.8 versus 42.1%), implantation rate per embryo (20.1 versus 13.2%), and clinical pregnancy rate (47.9 versus 30%) were significantly increased in the recFSH group compared with the simple medium group respectively (P < 0.05). In conclusion, in-vitro culture with recFSH appears to increase the motility of testicular spermatozoa, thus increasing the success of ICSI.
Key words: azoospermia/in-vitro culture/motility/testicular sperm extraction
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The use of testicular spermatozoa for intracytoplasmic sperm injection (ICSI) has become an established treatment for the indication of obstructive or non-obstructive azoospermia (Silber et al., 1995
). Testicular spermatozoa are often immotile or only show twitching movements upon extraction (Zhu et al., 1996; Liu et al., 1996). They are presumed to be viable although lower fertilization rates (17–46%) obtained with testicular spermatozoa imply the possible utilization of some non-viable spermatozoa for ICSI (Schoysman et al., 1993; Nagy et al., 1995a; Silber et al., 1995). Injection of motile and thus viable testicular spermatozoa may be theoretically more beneficial regarding fertilization rates. To this end, testicular spermatozoa have been incubated in defined simple culture media which induced progressive motility (Craft et al., 1995; Zhu et al., 1997). Higher fertilization rates obtained with the injection of motile spermatozoa will produce more embryos and afford better embryo selection for transfer. It is currently unknown whether embryo quality will also be favourably affected from such a practice.
Testicular sperm extraction (TESE) procedures may be performed 24–48 h prior to human chorionic gonadotrophin (HCG), thus 3–4 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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All couples with infertility due to non-obstructive azoospermia were eligible for the study. The study, conducted in 1997, included data from 143 sperm-positive TESE cycles. Ovarian stimulation, oocyte retrieval, embryo transfer, and luteal phase support were performed as described previously (Balaban et al., 1998
). Embryo transfer was performed ~48 h after oocyte retrieval using the Wallace catheter (The Edwards–Wallace Embryo Replacement Catheter, Sims, Portex Ltd, Kent, UK) for easy transfers, and the Frydman (Set de Frydman Pour Transfert Difficile, Laboratoire C.C.D., Paris, France) catheter for difficult transfers. TESE was performed ~24 h prior to oocyte retrieval. A previously described technique was used (Urman et al., 1998). After extraction of the connective tissue surrounding the seminiferous tubules with glass slides, an inverted microscope was used to detect the presence of spermatozoa at x200 and x400 magnifications. Sperm positive tissue samples were randomized at this point to either of the treatment groups: culture for 24 h in recFSH supplemented medium versus culture in simple medium. Simple randomization using computer generated random numbers was used. Informed consent was obtained from all patients prior to the procedure, and institutional review board approval was obtained prior to the study.
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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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Motility of testicular spermatozoa following 24 h culture in recFSH supplemented and simple media is shown in Table I
. Spermatozoa showing twitching and linear motility were significantly increased in the recFSH group.
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Of the 898 MII oocytes available in the recFSH group, 646 (71.9%) were injected with spermatozoa showing either twitching or progressive motility. Conversely, only 29.1% of the oocytes in the simple media group (245/841) were injected with motile spermatozoa. Patient characteristics and the results of ICSI with differently cultured spermatozoa are shown in Table II
. Fertilization rate was significantly increased in the recFSH group. However, the cleavage rate and the incidence of G1 + G2 (high quality) embryos were similar. The mean number of transferred embryos in the two groups was similar. However, implantation and clinical pregnancy rates were significantly increased in the recFSH group (P < 0.05).
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Embryos derived from ICSI with the two types of spermatozoa are shown in Table III
. Of the total 337 embryos derived from ICSI with spermatozoa cultured in recFSH medium, 45.7% were in the >4-cell stage on the day of embryo transfer. This was a significantly greater proportion than for the simple medium group (25.3%). Conversely, the number of 2-cell embryos in the simple medium group was significantly increased compared with the recFSH group. Of the transferred embryos in the recFSH group, 50.1% (169/337) were either G1 or G2 (Table II
). In the simple medium group, 59.6% (195/327) of the embryos were either G1 or G2 (not significantly different). However, when transferred embryos were analysed according to grade and blastomere number on day 2, significantly more G1 embryos with four or more blastomeres were transferred in the recFSH group (Table IV). Of the 169 G1 and G2 embryos transferred in the recFSH group, only 7.1% (12/169) were at the 2-cell stage on the day of transfer compared with 26.6% (52/195) in the simple medium group (P < 0.05). The same pattern of results was also observed for G3 and G4 embryos.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The utilization of testicular spermatozoa for ICSI has been associated with lower fertilization rates (Tournaye et al., 1996
). This may be attributed to the injection of immotile and therefore non-viable spermatozoa into the oocyte, especially in cases of germ cell aplasia and maturation arrest. In most instances only immotile spermatozoa are observed after TESE. Incubation of such immotile testicular spermatozoa in simple culture media has been associated with increased motility (Craft et al., 1995; Zhu et al., 1996, 1997). Theoretically, utilization of motile and therefore viable spermatozoa for ICSI should yield higher fertilization rates. This notwithstanding, comparable fertilization rates have been shown when motile, initially immotile, or totally immotile epididymal or testicular spermatozoa were used for ICSI (Nijs et al., 1996). However, embryos of lower quality tended to be produced when totally immotile spermatozoa were used. Furthermore, in 26 patients who underwent ICSI with totally immotile testicular spermatozoa, there were eight pregnancies (31%) of which five (19%) were ongoing. The numbers were too small to reach definite conclusions. 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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1 To whom correspondence should be addressed at: Reproduction Unit, VKV American Hospital of Istanbul, Guzelbahce sok No: 20, Nisantasi 80200, Istanbul, Turkey
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References |
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Submitted on April 16, 1999; accepted on July 30, 1999.
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