1 The IVF Unit and 2 The Institute for the Study of Fertility, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, affiliated to the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
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Abstract |
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Key words: non-obstructive azoospermia/spermatozoa/testicular sperm cryopreservation/testicular sperm extraction
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Introduction |
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Unfortunately, ~3050% of non-obstructive azoospermic men have no spermatozoa in their testicular tissue (Mulhall et al., 1996; Silber et al., 1997
; Devroey 1998b
). Also regrettable is that there are no reliable pre-operative parameters that can predict whether spermatozoa will be retrievable from the testicular tissue of these men (Mulhall et al., 1997
), and even a prior biopsy does not have a statistically significant predictive value because of the unequal distribution of spermatozoa in all tubules (Devroey 1998a
).
Since the spouses concomitantly undergo ovulation induction, many of them will undergo an unnecessary and potentially dangerous procedure. In order to avoid this and the possible need for repeated testicular sperm sampling in subsequent ICSI cycles, cryopreservation of testicular biopsy-extracted spermatozoa was performed (Oates et al., 1997). However, in contrast to ejaculated and epididymal spermatozoa, the cryopreservation of testicular sperm cells is controversial, mainly because of the low quality of the spermatozoa extracted from the testicular biopsies (Verheyen et al., 1997
). None-the-less, satisfactory fertilization rates were achieved (Romero et al., 1996
). Moreover, pregnancies achieved using frozenthawed testicular spermatozoa obtained from obstructive (Fischer et al., 1996
; Podsiadly et al., 1996
) and non-obstructive azoospermic patients (Hovatta et al., 1996
) were reported, although implantation and live birth rates per transferred embryo were lower compared with fresh specimens (Friedler et al., 1997
; De Croo et al., 1998
).
Cryopreservation of spermatozoa retrieved by testicular sperm extraction (TESE) without involving the spouse would enable ovulation induction to be planned only in those cases where testicular spermatozoa are found and this would be highly advantageous. The present study was conducted to compare the fertilization potency as well as the implantation and pregnancy rates of fresh versus cryopreserved testicular spermatozoa obtained from the same non-obstructive azoospermic patients. Results were also compared with those obtained when only frozenthawed testicular spermatozoa were used at the outset. When only immotile sperm cells were available for oocyte injection, as occurred in some cases, the fertilization and implantation potentials after microinjecting were compared with those of motile sperm cells.
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Materials and methods |
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All 55 testicular sperm retrieval procedures involved a bilateral, scrotal exploration. The scrotum was opened via a medial incision and all layers were cut until full exposure of the testis was accomplished. Testicular tissue was extracted with almost no exception from three locations in each testis. The tissue was placed in sperm medium (Medi-Cult, Copenhagen, Denmark) and transferred immediately to the IVF laboratory for sperm cell isolation. In all cases, testicular tissue was also taken for histological evaluation. No operative or major post-operative complications were recorded in any of the cases. In no instance was there a need for re-hospitalization, no haematoma developed, and no abscess was formed. Mild infections of the scrotum occurred with fever up to 38°C and with moderate tenderness and swelling in three men. All symptoms resolved after a few days of antibiotic treatment on an outpatient basis.
Mature spermatozoa were found in 33 (60%) patients (Figure 1), and were used in 57 ICSI cycles as follows. Fresh testicular spermatozoa were used in 15 ICSI cycles (14 patients; group 1). In each case, excess spermatozoa were cryopreserved and used later in 24 ICSI cycles (for the 10 couples who did not conceive in the `fresh' cycle plus one couple whose pregnancy achieved in the `fresh' cycle ended in a spontaneous first trimester miscarriage; group 2). In order to evaluate the efficacy of performing an elective testicular sperm retrieval without the need to involve the spouse simultaneously, cryopreserved testicular spermatozoa were obtained from 19 additional patients, but the spouses of eight of them had not yet undergone oocyte retrieval, leaving a total of 11 men whose frozen spermatozoa were used in an additional 18 ICSI cycles (group 3).
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For frozen testicular spermatozoa, thawing of testicular tissue was performed at room temperature on the day of ICSI (Edirisinghe et al., 1996). In order to remove the cryopreservation medium, the sample was washed in sperm medium by centrifugation for 5 min at 1160 g. The pellet, which was suspended in 0.20.3 ml of sperm medium, was incubated and examined for the presence of motile spermatozoa in the same manner as was the fresh testicular specimen. Most vital spermatozoa acquire motility within 12 h of culture.
Cryopreservation of spermatozoa
All testicular tissue specimens, including those that were not used for ICSI on the day of ovum retrieval, were evaluated. Only samples in which at least a few motile spermatozoa were observed after preparation were frozen. The samples were cryopreserved in ampoules, each containing a small number of motile spermatozoa to ensure the minimum sperm wastage at the time of thawing for the future ICSI cycle. The cryopreservation medium used was TEST {TES [N-tris(hydroxymethyl)methyl-2-aminoethanesulphonic acid] and Tris} yolk buffer containing glycerol (Irvine Scientific, Irvine, CA, USA), and the freezing procedure was performed as described previously (Yavetz et al., 1991).
Female patients and oocyte preparation
Ovulation induction was performed using a routine protocol of gonadotrophin-releasing hormone analogue {GnRHa; 900 µg/day of buserelin acetate nasal spray, D-Ser[TBU]6-ethylamide-luteinizing hormone-releasing hormone (LH-RH), Suprefact; Hoechst AG, Frankfurt, Germany} and 3 ampoules of human menopausal gonadotrophin (HMG; Pergonal; Teva Pharmaceutical Industries Ltd, Petah Tikva, Israel). Oocyte retrieval was scheduled 3536 h after administration of 10 000 IU human chorionic gonadotrophin (HCG; Chorigon; Teva Pharmaceutical Industries Ltd).
The cumulusoocyte complexes were isolated in IVF medium (Medi-Cult). Cumulus cells were removed with hyaluronidase (Sigma, Rehovot, Israel; H-3757; 80 IU/ml) in flushing medium (Medi-Cult). Denuded oocytes were assessed for their specific meiotic stage according to the presence or absence of germinal vesicle and polar bodies, and metaphase II (MII) oocytes were prepared for injection.
ICSI procedure and embryonic development
The ICSI procedure was carried out according to Van Steirteghem et al. (Van Steirteghem et al., 1993). The spermatozoa were assessed continuously for any indication of movement (i.e. `twitching' of head or tail) in the sperm medium droplet without PVP. In most cases, a sluggish local motility was observed and this served as a sign of vitality. A single motile spermatozoon was aspirated from the separate sperm droplet into the injection pipette and then transferred to the 10% PVP droplet to separate it from attaching cells and debris. When only totally immotile sperm cells were obtained even after exhaustive evaluation, spermatozoa of at least normal morphology were selected for the microinjection procedure. Both motile and immotile spermatozoa were immobilized before their aspiration into the injection pipette. The isolated testicular spermatozoon was then injected immediately into the oocyte retrieved from the spouse. The injection procedure for testicular spermatozoa, although more time-consuming and complicated than ejaculated spermatozoa, did not result in a higher oocyte degeneration rate (98.9% and 95.6% intact oocytes in groups 1 and 2 respectively; Table I
).
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Statistical analysis
Statistical tests were performed at the 5% level of significance. The calculations were carried out using the SPSS statistical package. Test distribution for normality was carried out by KolmogorovSmirnov goodness-of-fit test. Since most parameters presented a non-normal distribution, the non-parametric KruskalWallis one-way analysis of variance (ANOVA) was used to compare female age, number of retrieved oocytes, intactness of the injected oocytes, fertilization, cleavage, embryo quality and embryo transfer. The 2 analysis was used to compare pregnancy and implantation rates.
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Results |
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Fresh versus cryopreserved testicular spermatozoa (groups 1 and 2)
The first part of the study was conducted to compare the potential of a couple to conceive following the first ICSI cycle performed with their `fresh' testicular-extracted-spermatozoa (group 1), with a subsequent 14 trials using their cryopreserved testicular spermatozoa (group 2). Table I displays fertilization and embryo quality after ICSI with fresh or cryopreserved testicular spermatozoa and Table II
shows pregnancy rates.
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The fertilization rate (two pronuclei, 2PN) was also not significantly different when microinjection was carried out with fresh (group 1) as opposed to cryopreserved testicular spermatozoa (Table I). However, it should be emphasized that when analysing only the cycles in which motile testicular spermatozoa were injected, fertilization rates appeared higher than when all oocytes were injected indiscriminately. The percentage of oocytes with 1PN (i.e. activated oocytes lacking the male PN) as well as those with 3PN (i.e. non-extrusion of the second polar body) was very low, and there was no significant difference between the two groups (Table I
). Cleavage rates were high in both groups.
The morphological quality of the 2PN-cleaved embryos was similar in the two groups: ~70% of the embryos were of good quality (grades I and II; Table I).
Normal cleaved embryos were available for transfer in all fresh cycles (group 1) and in 23 of the 24 cryopreserved cycles (group 2) (Table II). The mean number of embryos transferred in couples from both groups was not significantly different (~3 embryos per transfer).
Four patients conceived when the fresh sperm cells were used for ICSI, and five clinical pregnancies were achieved in the subsequent 14 trials in which the cryopreserved spermatozoa obtained from this single testicular operation were used (26.7% and 21.7% clinical pregnancy rate per transfer in groups 1 and 2 respectively). The take home baby rate was not significantly different between the two groups. Of the nine pregnancies achieved, one was triplet, one was twin, and seven were singletons. Implantation rates per transferred embryo were, therefore, 12.5% and 8.5% respectively (not significantly different). Of these pregnancies, two were missed abortions, one was ectopic and six resulted in deliveries of healthy babies, giving a 42.8% (6/14) cumulative take home baby rate per testicular operation.
Use of cryopreserved sperm cells as first line approach (group 3)
In 19 of the 33 non-obstructive azoospermic patients who underwent TESE prior to initiating ovarian stimulation, and in whom testicular spermatozoa were found, all the spermatozoa were directly cryopreserved (Figure 1). Since eight of their spouses had not yet undergone oocyte retrieval, the remaining 11 comprised group 3 of this study (Figure 1
). Eight study patients had one ICSI cycle with their frozen spermatozoa, two had three trials and one had four trials. Table III
shows fertilization, cleavage and embryo quality results for the 18 ICSI cycles performed. The spouse's age and the mean number of oocytes retrieved and injected per cycle were not significantly different from that of groups 1 and 2. A normal fertilization rate was achieved in 61.5% ± 7.2 of all injected oocytes and in 67.4% ± 6.5 of the oocytes injected solely with motile spermatozoa. Cleavage rate and embryo quality were also similar to the other groups.
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Discussion |
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Since the tissue can be divided into multiple aliquots prior to cryopreservation, and since each aliquot can potentially serve as a sperm source for a single ICSI attempt, numerous cycles may benefit from only one testicular tissue surgery. Moreover, the multiplicity of aliquots provides a high cumulative clinical pregnancy rate per single TESE procedure (52.8%; 13/25 including all fresh and cryopreserved samples used for all 25 patients who participated in this study, i.e. groups 1, 2 and 3).
It was recently demonstrated that cryopreservation of epididymal spermatozoa enables multiple ICSI treatment cycles with success rates similar to those of ejaculated spermatozoa (Holden et al., 1997). Gil-Salom et al. (1996) were the first to report the feasibility of cryopreservation of testicular spermatozoa. In another study, the first published report of intentional cryopreservation of testis tissue, two pregnancies were reported in a group of 10 non-obstructive azoospermic men (19 frozenthawed cycles; Oates et al., 1997
). Comparison between the outcome of ICSI using cryopreserved and fresh testicular spermatozoa of the same non-obstructive patient in nine couples (nine fresh and 14 frozenthawed cycles) revealed no differences between the two groups in terms of fertilization (47 and 44% respectively), cleavage (88 and 89%, respectively) and clinical pregnancy rate (26 and 27% respectively) (Friedler et al., 1997
). However, when the ongoing pregnancy rate was considered, a higher value was noted when fresh spermatozoa were used (22 compared with 9%). Similarly, a high fertilization rate after ICSI was shown with both fresh and frozenthawed testicular spermatozoa, but that implantation and live birth rates per transferred embryo were significantly reduced with frozen cells (De Croo et al., 1998
).
Our results demonstrate that cryopreserved and fresh testicular spermatozoa retrieved from non-obstructive azoospermic patients are equally potent for fertilization as well as for implantation. Moreover, the results (including clinical pregnancy rate, delivery or ongoing pregnancy rate and implantation rate per transferred embryo) are not significantly different from those obtained in our unit with ejaculated spermatozoa. This indicates that testicular tissue-extracted spermatozoa can be successfully cryopreserved despite their low numbers and poor motility, and their use does not compromise treatment outcomes. It can be speculated that the difference between our results and those of others is due to the difficulty in selecting a vital testicular spermatozoon, with fertilizing ability, after thawing. It is not clear from the above-mentioned studies whether motile spermatozoa were available for the microinjection of all the oocytes in all cases. Verheyen et al. demonstrated that both motility and vitality are equally affected by freezing and thawing, and that the ratio of motile-to-vital sperm cells is maintained after thawing and preparation of testicular spermatozoa for ICSI (Verheyen et al., 1997).
In the present study, it was found that motile spermatozoa, whether fresh or frozenthawed, produce a normal fertilization rate (58.8%). In contrast, only a limited number of embryos were obtained when immotile spermatozoa were microinjected (13%), and none of them implanted. Our results indicate the crucial importance of motility for a successful treatment. Even sluggish motility is a good indication for testicular sperm viability and fertilizability. Obviously, meticulous scrutiny of the biopsy for motility should be performed when the cells are suspended in the PVP-free medium, in order to avoid PVP motility interference. Nijs et al. (1996) demonstrated that 65% of oocytes were fertilized when totally immotile testicular spermatozoa were injected; however, the overall embryo quality was low. They reported an encouraging pregnancy rate as determined by positive HCG (31%), and the achievement of a 19% ongoing pregnancy rate (Nijs et al., 1996). In a more recent study, the fertilization rate with immotile fresh testicular spermatozoa obtained from patients with non-obstructive azoospermia was 31%, but the use of motile spermatozoa was associated with a much higher fertilization rate (61%; Nagy et al., 1998
). The above results and the dramatically lower fertilization rate obtained in our study using immotile cryopreserved spermatozoa from non-obstructive azoospermic patients highlight the important contribution of the histological findings which characterized the present study group: non-obstructive azoospermia implies pathological spermatogenesis expressed as partial germ cell aplasia (Devroey, 1998b
).
Different dissection methods are used to release the spermatozoa from the testicular tissue. It has been postulated that the reason why the number of non-obstructive azoospermic patients from whom spermatozoa were not obtained in their study group was rather high (52%), was because only one biopsy specimen was obtained and because of the use of sperm separation gradients such as Percoll which decrease the yield of sperm cells (Kaharaman et al., 1996). In our study, spermatozoa were not obtained in only 40% of specimens. This was achieved by multiple biopsies from each testis, as well as by using high speed centrifugation in combination with the `swim-out' technique, thus enabling the isolation of enough motile spermatozoa to microinject the majority of the oocytes and to rescue the rest of the tissue by freezing for subsequent ICSI trials. Furthermore, in contrast to other sperm separation techniques, the `swim-out' technique avoids the exposure of human cells to reagents, the long-term effect of which is not always clear.
The results of the present study favour the first line choice of cryopreserved spermatozoa in cases of non-obstructive azoospermia. This approach (first suggested by Oates et al., 1997), eliminates the need for concomitant preparation of the spouse. The outcomes using these TESE-extracted cryopreserved spermatozoa are comparable with those obtained in our unit with fresh TESE-retrieved and ejaculated spermatozoa. Obviously, just as ejaculated specimens vary in their post-thaw survival, so do pre-ejaculated specimens. Consequently, in a limited number of cases in which only very few motile sperm cells are available, we suggest that synchronous treatment of the spouse may yield better results. However, in order for this to be arranged, improved pre-operative parameters that predict whether spermatozoa will or will not be found in the testicular tissue, must be developed. We believe that our findings warrant further investigation of our proposed approach in a larger series of non-obstructive azoospermic patients. In parallel with careful diagnosis and the therapeutic modalities that are being developed, there is a need to screen couples requiring ICSI, especially in cases of non-obstructive azoospermia (Devroey, 1998a
). The protocols of screening have to be delineated, e.g. karyotyping the husband, carrying out tests for the presence of Yq deletions, analysing the karyotype of the resultant fetus and evaluating the health of the newborn child.
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Acknowledgments |
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Notes |
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References |
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Submitted on November 26, 1998; accepted on March 30, 1999.