Centre for Reproductive Medicine, University Hospital of the Dutch-speaking Brussels Free University, Laarbeeklaan 101, B-1090 Brussels, Belgium
1 To whom correspondence should be addressed. Email: greta.verheyen{at}az.vub.ac.be
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Abstract |
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Key words: cryopreservation/ICSI/non-obstructive azoospermia/pregnancies/testicular sperm
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Introduction |
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Preliminary diagnostic biopsy retrieval combined with cryopreservation of the tissue may overcome these problems. After its introduction for ICSI (Romero et al., 1996), several groups reported comparable results in terms of fertilization, embryo quality, pregnancy and implantation rates with the use of fresh and frozen testicular sperm for patients with obstructive azoospermia (Prins et al., 1999
; Gil-Salom et al., 2000
; Wood et al., 2002
). The first reports on the use of frozenthawed testicular sperm in non-obstructive patients were case reports (Romero et al., 1996
; Perraguin-Jayot et al., 1997
) or present data on a mixed population of mainly OA and some NOA patients (Gil-Salom et al., 1996
; De Croo et al., 1998
; Palermo et al., 1999
; Huang et al., 2000
; Windt et al., 2002
). An additional problem in these early publications is the definition of testicular failure (NOA), which was not always clearly described and/or not always based on testicular histology. Nevertheless, acceptable results of ICSI with frozenthawed testicular sperm of NOA patients have been described (Friedler et al., 1997
; Oates et al., 1997
; Ben-Yosef et al., 1999
; Habermann et al., 2000
; Kupker et al., 2000
; Sousa et al., 2002
).
In contrast to OA patients, however, where viable sperm can easily be retrieved from the frozen specimens, the impaired quality of the testicular tissue of NOA patients does not allow for cryopreservation and later use for ICSI in all cases. As has been demonstrated for ejaculated sperm, a significant decrease in sperm motility and viability by freezing and thawing also occurred for testicular sperm (Verheyen et al., 1997). This implies that cases with extremely low numbers of sperm retrieved can hardly be considered candidates for cryopreservation. Preliminary diagnostic surgery and freezing can therefore still be considered a controversial approach for them. There is indeed a high chance that sperm may not be found at diagnostic retrieval due to its complete absence or to limited tissue retrieval and/or efforts for sperm searching. In order to overcome the risk that the frozen material is inadequate for injection upon thawing, some IVF centres define limits for testicular sperm quality suitable for freezing, and others only allocate patients for treatment on the basis of sufficient quality (motility) of a preliminary-thawed testicular sperm fraction. In this way, patient populations with NOA may differ greatly from one IVF clinic to another, which may define the results of ICSI.
In our IVF clinic, we offer couples with non-obstructive azoospermia any chance for having their own genetic offspring. While, historically, the use of freshly retrieved sperm has been our first-line approach, diagnostic surgery combined with cryopreservation is also offered. Couples are carefully counselled about the pros and cons of both strategies.
The aim of this retrospective study was to analyse the efficiency of the use of frozenthawed testicular sperm from NOA patients in an IVF programme with low-restrictive criteria for treatment allocation and for cryopreservation of diagnostically retrieved sperm. The first part analysed the overall results of ICSI cycles planned with frozenthawed sperm. The second part studied a subgroup of patients who underwent both cycles with frozen and cycles with fresh testicular sperm.
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Materials and methods |
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For all patients, the diagnosis of testicular failure was based on the clinical findings and on the histological examination of the testicular biopsy. Patients with germ-cell aplasia (Sertoli-cell only syndrome), maturation arrest or tubular sclerosis/atrophy, either with or without focal spermatogenesis on histology were included in the study. Patients with hypospermatogenesis were excluded.
Out of the 69 patients, 37 patients underwent only treatments with frozen testicular sperm while a population of 32 patients (mean male age 36.5, range 2455) underwent ICSI cycles with frozen testicular sperm (42 cycles) as well as ICSI cycles with fresh testicular sperm (44 cycles). Some of them first had a fresh TESE cycle followed by a frozen TESE cycle, or vice versa. For those 32 patients, the results of ICSI with fresh testicular sperm and of ICSI with frozen testicular sperm were compared in a second part of the study.
At the start of the treatment, the mean age of the female patients was 32.0 (range 2144) in the first part of the study. In part II of the study, the mean female age was 32.0 (range 2145) in the fresh TESE cycles and 32.8 (range 2245) in the frozen TESE cycles.
All couples were counselled about the procedure and signed an informed consent for their treatment.
Testicular sperm recovery
In patients with a clinical diagnosis of non-obstructive azoospermia, open excisional testicular biopsy retrieval was carried out under general anesthesia or occasionally under locoregional anesthesia, as previously described (Tournaye et al., 1997b). After excision, testicular biopsy specimens were placed in a Petri dish with HEPES-buffered culture medium. Concomitant with the surgery, testicular biopsies were processed by mechanical shredding (Verheyen et al., 1995
) and the suspension was explored under an inverted microscope at 400xmagnification. Whenever appropriate, additional biopsies from different regions of the testis were retrieved bilaterally. The surgery was discontinued when few, preferably motile, sperm had been observed or when a representative number of small biopsies had been excised randomly from both testes or when testicular volume did not allow for further excision. When no or only immotile spermatozoa were observed after mechanical shredding of all retrieved biopsies, lysis of the red blood cells (Verheyen et al., 1995
; Nagy et al., 1997
) and/or enzymatic digestion of the remaining tissue fractions with collagenase type IV (Crabbé et al., 1997
, 1998
) was carried out. During surgery, a single small biopsy of each testis was sent for histological examination.
Testicular biopsies were retrieved either for diagnostic reasons (n=37 patients) followed by freezing and later ICSI cycles with frozenthawed testicular sperm, or for immediate fresh therapeutic use (ICSI) at the day of oocyte retrieval (n=32 patients) and followed by ICSI cycles with frozenthawed testicular sperm. The testicular cell suspension was frozen for later use, if at least one, preferably motile, sperm was observed after diagnostic retrieval or if, after injection of the mature oocytes at the day of biopsy retrieval, sufficient remaining spermatozoa were supposed to be available for a next ICSI treatment.
Preparation and cryopreservation of testicular suspensions
Testicular biopsies, either treated by mechanical methods or additionally by enzymatic methods, were cryopreserved if appropriate. The quality of the material was assessed for concentration and motility, if possible. The information obtained was mostly based on very few spermatozoa observed. The suspensions were prepared for freezing by dropwise dilution with Test-Yolk buffer (TYB) containing glycerol as cryoprotectant (Irvine Scientific, Santa Anna, CA), in a proportion 1:1 (v/v). The straws (Instruments de Médecine Véterinaire IMV, 61300 L'Aigle, France, or CBS high security straws, Cryo Bio System, L'Aigle, France) were loaded and equilibrated for 10 min in a 37°C water bath. The straws were rapidly frozen in liquid nitrogen vapour as follows: straws were horizontally placed at 20 cm above the liquid nitrogen surface for 8 min, followed by 8 min at 10 cm above the surface. After this two-step procedure, the straws were finally immersed and stored in liquid nitrogen.
Thawing and preparation of testicular suspensions
At the day of oocyte retrieval, testicular suspensions were thawed only when mature metaphase II oocytes were available for injection. Thawing was performed at room temperature for 510 min. According to the quality of the fractions before freezing, only one or immediately more straws were thawed. The thawed suspension was washed by dropwise dilution with 5 ml Hepes-buffered medium. During addition, the tube was shaken in order to continuously obtain an homogeneous suspension. The mixture was centrifuged at 750 g for 5 min and the supernatant was discarded. After a second washing step, the remaining pellet was resuspended. Droplets of 10 µl were smeared at the bottom of a Petri dish and overlayed with 3.5 ml of paraffin oil. Individual spermatozoa were identified and selected on the basis of (twitching) motility and appropriate morphology, whenever possible. If no selection was possible due to an extremely low number of sperm present, also morphologically subnormal sperm and in some cases even immotile sperm were used for injection. Searching for sperm was mostly carried out by two or three technicians/embryologists and the duration of this procedure was registered. Whenever necessary, additional straw(s) were thawed in order to find motile sperm for injection. If no sperm at all were found after thawing and exploring all the frozen material, or occasionally if only immotile sperm were found after extensive search, a fresh testicular biopsy retrieval was carried out after consulting the patient. If the patient refused new surgery or if repeated surgery was not indicated, donor sperm was used as anticipated from a preliminary counselling, or the treatment was cancelled. Considering the maturity of the injected sperm, only morphologically mature-looking spermatozoa or elongated spermatids with a fully developed tail but a cytoplasmic droplet were injected. Round spermatids were never used for ICSI.
Ovarian stimulation, oocyte retrieval and ICSI
Female patients underwent ovarian stimulation using urinary or recombinant FSH in combination with GnRH antagonists or agonists. Oocyte retrieval was carried out 36 h after injection of 10 000 IU human chorionic gonadotropin (hCG) by vaginal ultrasound-guided puncture of the ovarian follicles. Denudation of the cumulus cells was carried out by exposure to 10 IU of hyaluronidase (Van de Velde et al., 1997). Nuclear maturation of the oocytes was inspected under a stereomicroscope. Only mature, metaphase II oocytes were used for ICSI. Searching for sperm and intracytoplasmic sperm injection were started shortly after thawing and preparation of the testicular suspensions. The procedure of intracytoplasmic sperm injection has been described previously (Joris et al., 1998
). Individual culture of the injected oocytes and embryos was performed in 25-µl droplets under paraffin oil during the entire culture period.
Fertilization, embryo evaluation and embryo transfer
Survival and fertilization of the oocytes were observed 1618 h after injection under an inverted microscope at 400xmagnification. Normal fertilization was confirmed by the presence of two distinct pronuclei (PN) with two distinct or fragmented polar bodies. Fertilization rates were expressed as the percentage of 2PN per injected metaphase II oocyte. Embryo development and quality were assessed daily until the moment of intrauterine transfer. Embryo quality was scored according to the number of blastomeres and the rate of fragmentation. Type A embryos had no anucleate fragments, type B embryos showed 20% fragments, type C embryos >20% and
50% fragments, and type D embryos >50% fragments. Only the results of embryo quality at day 2 are presented in this study.
Normal cleaving embryos up to type C for fragmentation were considered eligible for transfer. Intrauterine transfer was carried out at day 2 or 3, and occasionally at day 5 after injection.
A rise in serum hCG on two consecutive occasions from 11 days on after embryo transfer indicated pregnancy. A clinical pregnancy was defined by the presence of a gestational sac with fetal heart beat at ultrasonography after 7 weeks of pregnancy. An ongoing clinical pregnancy was defined as a clinical pregnancy with fetal heartbeat beyond 20 weeks of pregnancy. The implantation rate is considered as the percentage of fetal sacs with heart beat on the number of embryos transferred.
Statistical analysis
Comparison of parameters between the pregnant and non-pregnant patients in the first part of the study was performed using the MannWhitney U test.
In the second part of the study on ICSI cycles with fresh and ICSI cycles with frozen testicular sperm in the same patients, comparison of sperm parameters, fertilization and embryo quality was performed using the MannWhitney U test. Data on embryo transfer, pregnancy and implantation rates were analyzed using the Chi-square test. Statistical tests were performed at the 5% level of significance.
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Results |
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The proportion of cycles with successful sperm recovery after thawing was comparable for the different histological subgroups: in cycles of patients with germ-cell aplasia, frozenthawed sperm could be used for injection in 41 of the 52 cycles (79%). In 16 of the 20 cycles of patients with maturation arrest (80%), and in 10 out of 12 cycles of patients with sclerosis/atrophy (83%), frozenthawed testicular sperm could be injected. Sperm retrieval from the frozenthawed suspensions of patients with Klinefelter syndrome was successful in 10 out of the 13 cycles (77%).
In the 77 cycles with frozenthawed sperm used for ICSI, the average time needed per cycle to find the sperm was 113 min (range 3384), and the average time per sperm was 17 min (range 285). In 63 cycles (82%), only motile sperm were injected, while only immotile sperm were used in six cycles (8%). In the eight remaining cyles (10%), insufficient motile sperm were found to inject all mature oocytes after extensive search, so that part of the oocytes were injected with immotile sperm. Overall, 87% of all mature oocytes were injected with a motile sperm. In all 41 cycles of patients with germ-cell aplasia, and in all 10 cycles of patients with sclerosis/atrophy, motile sperm were found to inject almost all the oocytes. The six cycles with only immotile sperm available for injection were carried out for patients with maturation arrest (4/16 cycles) and for patients with Klinefelter syndrome (2/10 cycles).
Fertilization and embryo development
The number of oocytes retrieved and the results of fertilization are presented in Table I. The mean values presented are the averages of the percentages of fertilization calculated per cycle. In the present study with frozen testicular sperm, the mean fertilization rate was 58.4% in 77 cycles. In six out of the 77 cycles (8%), none of the oocytes showed normal fertilization. The cycle characteristics that probably contributed to complete fertilization failure were: low number (14) of oocytes retrieved (three cycles), injection with only immotile sperm (three cycles), injection with a high proportion of immotile sperm (one cycle), injection of a proportion (5/18) of the M-II oocytes (one cycle) only. For six treatment cycles (8%) in which only immotile sperm were injected, total fertilization failure was observed in three cycles (with low oocyte numbers: 1, 1, 4). In three other cycles, however, fertilization was obtained but oocytes did not cleave in two of the cycles.
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Association between sperm characteristics and outcome parameters
In order to assess any parameter at the level of the IVF laboratory that could be associated with pregnancy, characteristics of the pregnant cycles and non-pregnant cycles were compared. The total duration of searching for sperma parameter that reflects the ability of sperm selection for ICSI and indirectly also the quality of the injected spermwas comparable between the pregnant and non-pregnant group (112 min vs 103 min). Similarly, the mean search time per spermatozoon (15 min vs 14 min) and the proportion of oocytes injected with motile sperm (93% vs 96%) showed no difference. Also fertilization rate (64.3% vs 64.9%), proportion of good-quality embryos (98% vs 91%) and number of embryos transferred (2.5 vs 2.4) did not differ between the pregnant and non-pregnant groups.
For most of the scheduled cycles with frozenthawed testicular sperm, the pre-freeze quality characteristics of the testicular suspensions could be registered. In order to check for possible relationships, a comparison in sperm characteristics was made between cycles in which the frozenthawed suspension could not be used for ICSI, cycles in which the frozenthawed sperm could be used for injection, and out of these the subgroup of cycles that resulted in a clinical pregnancy. It should be taken into account that the characteristics of sperm quality are often based on the observation of a very limited number of sperm (often <10) and should therefore be considered as rough estimates rather than quantitative measurements. Results of concentration and motility are presented in Table IV. The cycles where no frozenthawed sperm was used for injection were characterized by a significantly lower sperm concentration (0.0042 millions/ml) before freezing than in the cycles with frozen sperm injected (0.0332 millions/ml) and in the cycles that resulted in a clinical pregnancy (0.0565 millions/ml). The percentage motility, however, was similar in the three groups.
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The rank of trial was 1.5 for the cycles with fresh and 2.2 for the cycles with frozen sperm (P < 0.001 in the MannWhitney U test). The characteristics of the cycles considering the sperm searching procedure are summarized in Table V. The data show that sperm suitable for injection can be found more rapidly in the fresh than in the frozen specimens. The proportions of cycles and the proportion of oocytes injected with motile sperm, however, were comparable.
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Table VI shows the results of embryo transfer, pregnancy rates and implantation rates. When fresh testicular sperm was used, significantly more cycles (P=0.028 in the Chi-square test) resulted in embryo transfer than with frozen testicular sperm (93.2% vs 76.2%). The reasons for having no embryos available for transfer in cycles with fresh and frozen sperm were: no fertilization (one vs three cycles), no or poor cleavage (two vs six cycles) and only aneuploid embryos (no vs one cycle). The rates of positive hCG and the ongoing clinical pregnancy rates were comparable with the use of fresh and frozen sperm. Similarly, no differences were observed in the implantation rates.
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Discussion |
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One drawback associated with using frozen sperm for NOA patients within our current attitude is the substantial risk (20%) of not finding sperm suitable for injection, despite extensive efforts. It is evident that this observation is linked to our unrestrictive criteria for testicular sperm freezing. Unfortunately, information on the probability of using the frozen testicular suspensions for ICSI is hardly presented in the literature. On the one hand, sufficient numbers of motile sperm may have been ensured either before freezing, or after diagnostic thawing of a fraction before starting treatment in some studies. On the other hand, the cycles in which no cryopreserved sperm was available for injection may have been excluded from the literature report. The information, however, is of paramount importance in establishing the limits to using frozen testicular sperm for NOA patients. In our series of 20 cycles without frozen sperm, a back-up fresh retrieval was successfully carried out in 14 cycles.
A second point of discussion is the use of immotile frozenthawed sperm for injection. In the ICSI programme, motility is used as an indicator for viability. It has been shown previously that immotile fresh testicular sperm can successfully be used for ICSI, notwithstanding the fact that the fertilization rate was lower (46% in 14 cycles) than with motile spermatozoa (65% in 159 cycles, Nagy et al., 1998). The difference was more pronounced when only non-obstructive cases were considered (31.2% vs 60.8%). In general, the use of immotile sperm for ICSI results in reduced fertilization rates, and complete immotility is one of the major causes of fertilization failure in the ICSI programme, as demonstrated by Liu et al. (1995)
in 29 out of the 76 cycles without fertilization. Despite the extremely low numbers and poor motility of testicular NOA sperm in our programme, motile sperm could be found for injection in an unexpectedly high proportion of cycles (92%). The proportion of cycles with only motile sperm injected (82%) is comparable to the observation by Ben-Yosef et al. (1999
; 72%). Besides its effect on fertilization, the use of immotile sperm also affects pregnancy and implantation rates. Only in one cycle (of a Klinefelter patient), an ongoing clinical pregnancy occurred but resulted in a stillborn delivery. A reduced pregnancy rate with immotile frozenthawed sperm was also described by Fischer et al. (1998)
. Although in vitro culture of frozenthawed testicular sperm may improve motility in obstructive cases, it was ineffective and unpredictable when only immotile sperm of non-obstructive cases were cultured (Liu et al., 1997
). These observations stress the importance of motility for ICSI with cryopreserved testicular sperm of NOA patients. Surprisingly, Gianaroli et al. (1999)
needed to inject almost exclusively immotile sperm after thawing and obtained a fertilization rate of 64%. It may be questioned whether real immotility was distinguished from the hardly visible twitching motility in their study. Based on our results and those achieved by most other authors, it seems preferable to consider a fresh biopsy retrieval if the embryologist is faced with the presence of only real immotile sperm after thawing. For each individual case, this option should be discussed beforehand between the clinician and the patient.
A third drawback of using cryopreserved testicular sperm in a low restrictive programme is the high proportion of cycles without embryo transfer. The transfer rate of 83% in the present study was similar to the transfer rate (86%) in the study with fresh testicular sperm by Vernaeve et al. (2003). Within the same patient population (Part II), however, the transfer rate was significantly lower (P=0.028) in the frozen (76%) than in the fresh cycles (93.2%). This observation was due to the higher prevalence of fertilization failure but also to absent or impaired embryo cleavage in the frozen cycles.
This is the first study to report on the duration of sperm searching in patients with testicular failure. It is well known and confirmed by this report that the search procedure is cumbersome and time-consuming for embryologists and technicians, depending on the severity of the cases that are allocated for treatment. In suspensions that showed no sperm after mechanical disruption, enzymatic digestion was mostly carried out and proved effective in 10 out of 18 patients. The long searching time indicates the severity of testicular failure and the low-restrictive attitude for sperm freezing in our patient population.
Based on the pre-freezing quality of the testicular suspensions, it seems difficult to define cut-offs for what to freeze and what not to freeze (Table IV). The lowest sperm numbers before freezing were indeed observed in samples that could not be used for ICSI upon thawing, but individual data indicate that no clear prediction is possible. Extremely low sperm numbers were also observed in samples that were used for ICSI and even in samples that resulted in ongoing pregnancies. Also the parameter motility had no predictive value. Even in one third of the samples that had led to ongoing pregnancies, no motility was observed before cryopreservation, while predominantly motile sperm could be injected from the thawed specimens after extensive search. This means that testicular suspensions can hardly be excluded for freezing on the basis of extremely low numbers and/or absence of motility.
For the different histological patterns in the testis, the probability of sperm availability after thawing was similar. Most of the pregnancies were, however, obtained for patients with germ-cell aplasia or tubular sclerosis/atrophy. Only two children were born from patients with maturation arrest (1/16 cycles) or patients with Klinefelter syndrome (1/10 cycles). For Klinefelter patients, this reduced chance is not related to the abnormal karyotype, as PGD was performed on the transferred embryos. It might, however, be correlated with the higher probability of only finding immotile frozenthawed sperm for injection as observed in these populations. Recently, Staessen et al. (2003) reported an implantation rate of 12.8% obtained after 26 transfers of PGD-tested embryos for 20 couples of which the man had a 47,XXY karyotype.
In the present study, the overall outcome of cycles carried out with frozenthawed testicular sperm is not inferior to that with fresh testicular sperm in the same patient group, despite the greater difficulty of finding sperm suitable for injection, and despite the higher risk of having no embryos of sufficient quality for transfer. The results seemed not to be biased by a difference in female age between the two arms of the study, being 32.0 years for the fresh TESE and 32.8 years for the frozen TESE cycles. Although there was a difference in rank of trial, showing that more patients first had a fresh TESE cycle followed by a frozen TESE cycle, the opposite also occurred. These observations indicate that, besides its damaging effects (Verheyen et al., 1997; Nogueira et al., 1999
), freezing testicular sperm eliminates the most deficient sperm. This selection may, on the one hand, also explain why fertilization (48.5%), clinical pregnancy (15.4%) and implantation rates (8.6%) in the study by Vernaeve et al. (2003)
with fresh testicular sperm were somewhat lower than those in the present study with frozen testicular sperm. On the other hand, it may be postulated that the population by Vernaeve et al. (2003)
includes proportionally more difficult cases (without cryopreservation) than the population in the present study.
Although diagnostic retrieval and cryopreservation of testicular sperm is the first-line approach in several IVF centres, the limits of this approach should not be overlooked. Notwithstanding our minimal restriction criteria for sperm freezing, the results of ICSI with frozenthawed testicular sperm were acceptable, at least once sperm were found for injection. Cryopreservation has several advantages as well as a number of drawbacks. Careful counselling with information on the pros and cons of cryopreservation is therefore of paramount importance.
It may be concluded from the present study that, even in a programme with low-restrictive criteria for patient allocation and cryopreservation of testicular sperm, diagnostic testicular sperm retrieval followed by cryopreservation can be the procedure of choice. In order to counteract the reasonable risk of not finding sperm or only immotile sperm, scheduling fresh surgery as back-up or counselling the couple for donor sperm as back-up is recommended. The use of totally immotile sperm after thawing should be discouraged on the basis of the present data.
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Acknowledgements |
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Submitted on March 5, 2004; resubmitted on June 24, 2004; accepted on August 2, 2004.