1 Department of Medical Genetics, Faculty of Medicine, University of Porto, 2 Centre for Reproductive Genetics, Porto and Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar, University of Porto, Portugal
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Abstract |
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Key words: non-obstructive azoospermia/spermatids/spermatogenesis/testicular sperm/testicular histopathology
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Introduction |
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Most of the reported clinical series related to non-obstructive azoospermia describe only the clinical outcome associated with sperm microinjection, either fresh or frozenthawed. Those studies evidenced relatively low fertilization rates (3867%) but rather high pregnancy rates (4060%) after ICSI in cases of SCOS, MA and hypoplasia (Silber et al., 1996, 1997
; Tournaye et al., 1996
; Mansour, 1998
; Al-Hasani et al., 1999a
; De Croo et al., 2000
). On the contrary, analysis of the few studies which have used spermatids for treatment reveals that whereas late spermatid injections (n = 127) seem associated with low fertilization (48.2%) and acceptable clinical pregnancy (29.9%) rates, round spermatid injections (n = 216) appear not to be clinically useful (22.5% of fertilization and 3.2% of clinical pregnancy rates) (Fishel et al., 1995
, 1996
; Hannay et al., 1995
; Tesarik et al., 1995
, 1996
, 1999
; Chen et al., 1996
; Mansour et al., 1996
, 1997
; Tanaka et al., 1996
; Amer et al., 1997
; Antinori et al., 1997a
,b
; Araki et al., 1997
; Sofikitis et al., 1997
, 1998b
,c
; Vanderzwalmen et al., 1997
; Yamanaka et al., 1997
; Barak et al., 1998
; Bernabeu et al., 1998
; Kahraman et al., 1998
; Al-Hasani et al., 1999b
; Sousa et al., 1999
).
In the present study we present the Portuguese clinical and laboratory data from the consecutive treatment of 148 non-obstructive azoospermic patients, with normal karyotypes. It is shown that histopathology allows further subdivisions of patients with incomplete SCOS and MA, and that these subgroups evidence distinct prognostic values. The outcome with both fresh and frozenthawed testicular-retrieved sperm and different subtypes of spermatids is also presented.
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Materials and methods |
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Ovarian stimulation
Female patients were treated with a long GnRH analogue suppression protocol combining buserelin acetate (Suprefact; Hoechst, Frankfurt, Germany) with pure FSH (pFSH) (Metrodin HP; Serono, Geneva, Switzerland) or recombinant FSH (rFSH) (Gonal F; Serono, Puregon; Organon, Oss, The Netherlands). Ovulation was induced with HCG (Pregnyl; Organon, Profasi; Serono). Oocytes were recovered from large ovarian follicles by ultrasonically-guided follicular aspiration, 35 h after HCG, using flush medium (Medicult, Copenhagen, Denmark).
Treatment testicular biopsy
The spermatic cord block was performed according to the three-finger technique (Li et al., 1992; Gorgy et al., 1998
; Nudell et al., 1998
). Local anaesthesia was achieved with 56 ml of a 1:1 mixture of 1% lidocaine hydrochloride solution (Xylocaine 2% without epinephrine; Astra Pharmaceuticals International, Sweden) and 0.5% bupivacaine (Marcaine 0.5% without epinephrine; Astra). After a few minutes, a skin weal was raised in the scrotum adjacent to the middle region of the testis, a 1 cm transverse incision was made and the tunica vaginalis space entered. An incision of 0.5 cm then enabled excision of a small piece of the seminiferous tubules. A preliminary sample microscopic check at the end of each biopsy avoided unnecessary tissue sampling. In general, almost all cases with hypoplasia had sperm or spermatids in the first three samples collected at one testis, and these were enough for treatment and frozen storage. In MA, SCOS and, occasionally, in hypoplasia cases, 510 biopsies of the same testis at different locations were needed to find sperm or spermatids. When such cells were not found, the contralateral testis was also analysed whenever possible. After careful cleaning and haemostasis, the tunica albuginea, the vaginal, the scrotum layers and the skin were closed. The procedure took about 2030 min and was performed entirely on an outpatient basis, enabling a rapid recovery with minimal complaints and total absence of surgical complications. Tramadol and nimesulide per os were given to relieve any discomfort in the first 24 h. Where needed, a new biopsy was scheduled only after a period of 6 months (Schlegel and Su, 1997
).
Preparation of testicular samples
Each sample was expressed in Sperm Preparation Medium (SPM, Medicult) with surgical blades, and 10 µl were observed to confirm the presence or absence of sperm or elongated spermatids. The resultant fluid was washed with SPM, 2x5 min, by centrifuging at 500600 g, and the pellet resuspended for 5 min in 2 ml of erythrocyte-lysing buffer (Verheyen et al., 1995) using endotoxin-free, embryo and cell culture tested chemicals (Sigma, Barcelona, Spain). After washing, samples were digested (Crabbé et al., 1997
) for 1 h at 37°C, in a solution of SPM containing 25 µg/ml of crude DNase and 1000 IU/ml of collagenase-IV (Sigma). After a new wash, the pellet was resuspended in 50100 µl of IVF medium (Medicult) and then incubated at 3032°C, 5% CO2 in air until use. For freezing, the sample was diluted with Sperm Freezing Medium (Medicult), exposed for 1015 min to liquid nitrogen (LN2) vapours, and finally immersed and stored in LN2.
Selection of cells for microinjection
The distinction between round spermatids and Sertoli cell nuclei, cytoplasmic remnants and lymphocytes, as also the distinction between elongating spermatids, elongated spermatids and testicular sperm have been the subject of much debate, but clear criteria have been established (Tesarik, 1997, 1998
; Aslam et al., 1998
; Lewis and McClure, 1998
; Mansour et al., 1998
; Silber and Johnson, 1998
; Silber et al., 1998
; Sofikitis et al., 1998b
; Sousa et al., 1998
, 1999
; Tesarik et al., 1998
; Vanderzwalmen et al., 1998
). Briefly, isolated nuclei of Sertoli cells have an elevated border, a large nucleolus, no other visible internal structures, and shrink in 10% PVP-SPM (Medicult); round-shaped cytoplasmic remnants, blebbed-out from degenerating cells, have no internal visible organelles and shrink in PVP; lymphocytes have internal nuclear irregularities due to condensed patches of chromatin, they stick to the tip of a 6 µm inner micropipette and stretch with aspiration, and if left in culture they tend to attach and develop cytoplasmic extensions (pseudopodes) within 24 h. On the contrary, round spermatids have a smooth outline and inner aspect, the nuclear limit is clearly visible, the acrosomal vesicle is distinguishable as 12 large round vesicles (Golgi phase) or as a fine dark elongating region at one nuclear pole (cap phase), they deform and adapt their shape to the aspirating 6 µm inner micropipette, and do not shrink in PVP. Round spermatid injection (ROSI) was used when patients did not accept donor sperm. We have used intact round spermatids for injection because a slightly larger injection pipette is not associated with a higher rate of oocyte degeneration (Sousa et al., 1999
), at this stage the proximal centriole may still not be attached to the nuclear envelope (Holstein and Roosen-Runge, 1981
), and because the cytoplasmic membrane and the nuclear envelope of the round spermatid is ruptured soon after contact with the ooplasm, enabling diffusion of the spermatid oocyte-activating substance and proper pronucleus formation (Sousa et al., 1996
, 1999
). Because ionophores are not allowed for clinical use, oocytes were not activated through an induced intracellular calcium rise after ROSI (Tesarik and Sousa, 1995b
).
On the contrary, elongating and elongated spermatids are very easy to distinguish. In the present series, whenever we found normal elongating spermatids, we also found elongated spermatids, albeit after several hours of searching. When elongated spermatids could not be found but elongating spermatids were present, the latter had extensive nuclear and tail malformations and were not used for treatments. This means that we have only used elongated spermatids for injection (ELSI). However, we did not use elongated spermatids without a full length flagellum or with abnormal head morphology.
The elongating spermatid is differentiated from the elongated spermatid mainly based on nuclear morphology (Holstein and Roosen-Runge, 1981). The former has a nucleus that is not fully elongated nor condensed and protrudes only slightly beyond the apical cell region. The elongated spermatid is differentiated from the testicular spermatozoon based on the upper limit of the basal cytoplasm. In a spermatozoon, the basal cytoplasm is confined to the midpiece region, which does not exhibit any continuity with the equatorial region of the spermatozoon head. This is made possible through the coalescence between the upper limit of the midpiece cytoplasmic membrane and the basal nuclear envelope. This point of fusion is named the posterior ring (Holstein and Roosen-Runge, 1981
). So, the elongated spermatid is differentiated from a testicular spermatozoon when the upper limit of the basal cytoplasm is still above the nuclear base (Holstein and Roosen-Runge, 1981
; Sousa et al., 1999
). It is also at the end of the elongated spermatid stage that the distal centriole is depolymerized. Because the distal centriole is committed to synthesize the axoneme, to which it remains tightly linked from the round spermatid stage (Holstein and Roosen-Runge, 1981
), it has no intrinsic centrosome capability and thus spermatid microinjection does not introduce into the ooplasm an additional microtubule polymerizing centre (Sousa and Tesarik, 1994
; El Shafie et al., 2000
). That is why, and as clinical series have already demonstrated, the distal centriole of microinjected spermatids does not preclude normal fertilization and embryo cleavage.
Microinjection and embryo culture
Cells were selected using an inverted Nikon microscope with Narishige micromanipulators (Nikon, Tokyo, Japan) and commercial micropipettes (45 µm for late spermatids/sperm, 68 µm for round spermatids; SweMed; Frolunda, Sweden) according to a published method (Sousa et al., 1998, 1999
). Oocytes were injected using the strong dislocation of the ooplasmTesarik et al., 1994
; Tesarik and Sousa, 1995a
). Injected oocytes were cultured in IVF medium at 37°C with 5% CO2 in air. After 2 days, they were transferred to M3 medium (Medicult). Normal fertilization was assessed 1418 h after injection, and embryo quality was evaluated (Staessen et al., 1995
). Supernumerary embryos were frozen with embryo freezing medium or with the blastocyst freezing pack (Medicult), in an automatic freezing apparatus (Kryo 10 Series III; Planer, UK). All patients had luteal supplementation with three times daily intravaginal administration of 200 mg natural-micronized progesterone (Utrogestan; Jaba, Berlin, Germany). Pregnancy was confirmed by a rise in serum ßHCG on 2 consecutive days, 2 weeks after embryo transfer. A clinical pregnancy was established by ultrasonography at 7 weeks gestation. All couples agreed to have a prenatal diagnosis, which was performed by amniocentesis at 16 weeks of pregnancy.
Statistics
When appropriate, correlation analysis were performed, and the significance of difference between the percentages of two groups were evaluated with the 2-test. Significance was accepted where P < 0.05.
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Results |
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Hypoplasia
In all patients, the diagnostic biopsy showed spermatogenesis up to the late spermatid or spermatozoa stages, which were recovered for treatment in 43 cases (97.7%). In one case, a total cell aplasia was found at treatment despite an extensive bilateral search of the testicular bioptic specimens. This may be explained by an intratesticular injury that has progressively destroyed the germinal epithelium during the 5 years between the diagnostic and the treatment testis biopsies or, alternatively, by an unfortunous diagnostic testis biopsy that was taken over a unique focus of spermatogenesis. In total, there were 53 cycles with ICSI, 11 cycles with ELSI, and three cycles using IVF or intra-uterine insemination (IUI) with donor sperm (one case with no remaining spermatids and one case with total cell aplasia).
The mean female age was 33 years (range 2240), the mean male age was 36.1 years (range 2755), and the mean duration of infertility was 6.7 years (range 118). Only 11.4% of the women showed associated pathology (three cases with hyperprolactinaemia, one case of endometriosis, and one case with tubal obstruction). About 50% of the male patients had increased FSH levels, 38.6% showed decreased testicular volume, and 13.6% had associated testicular pathology (three varicocele, two orchitis, one cryptorchidia). Most of the patients with decreased testicular volume exhibited increased FSH levels and had no associated local pathology.
In ICSI cycles using fresh sperm, there have been nine normal evolving pregnancies (five clinical, four ongoing), and four deliveries of healthy children. Of these 13 pregnancies, three are twin and two triplet. In ICSI with frozenthawed sperm there were two biochemical pregnancies, one is presently clinical and three have delivered healthy babies. Of these four successful pregnancies, one is twin and one triplet. In fresh ELSI cycles, there is one ongoing pregnancy and three deliveries of healthy children. Of these four pregnancies, one is twin (Table I).
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The mean female age was 31.9 years (range 2441), the mean male age was 34.9 years (range 2453), and the mean duration of infertility was 5.7 years (range 118). About 27.7% of the women showed associated pathology [six polycystic ovarian syndrome (PCOS), four hyperprolactinaemia, one endometriosis, one tubal obstruction, and one ovarian insufficiency]. About 38.3% of the male patients had increased FSH levels, 42.6% showed decreased testicular volume, and 25.5% had associated testicular pathology (three varicocele, nine cryptorchidia). In patients where no late spermatids/sperm could be recovered, there was a predominance of normal values of FSH (72.2 versus 58.6%), of normal testicular volume (77.8 versus 57.4%), and fewer cases of local pathology (5.6 versus 37.9%). Correspondingly, normal testicular volume appeared to be associated with normal FSH values (75%), decreased testicular volume was associated with higher FSH values (71.4%), and 45% of the cases with decreased testicular volume were associated with local pathology (chryptorchidia).
In ICSI cycles using fresh sperm, to date there is one ongoing pregnancy and three deliveries of healthy babies. Of these four pregnancies, one is twin. In ICSI with frozenthawed sperm there is one ongoing pregnancy and two deliveries (healthy babies). In fresh ELSI cycles there were two biochemical and one clinical pregnancies, and two healthy deliveries. Of these three successful pregnancies, one is twin (Table II). Of the cycles with donor sperm, there were four IVF term pregnancies and one IUI term pregnancy with delivery of healthy children.
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The mean female age was 30.8 years (range 1942), the mean male age was 34.2 years (range 2348), and the mean duration of infertility was 6 years (range 119). About 22.8% of the women showed associated pathology (eight PCOS, three hyperprolactinaemia, one endometriosis, one miomatosis). About 75.4% of the male patients had increased FSH levels, 71.9% showed decreased testicular volume, and 36.8% had associated testicular pathology (eight varicocele, six cryptorchidia, four adult parotiditis, one chemotherapy, two hypogonadism). Increased FSH levels were present in 58.8% of the patients with sperm/late spermatids, in 37.5% of the patients where only round spermatids were recovered, and in 93.8% of the patients with complete SCOS. Similarly, in these three groups there was a predominance of decreased testicular volume (82.4, 75 and 65.6% respectively). Because the large majority of the patients in the study had decreased testicular volume and increased FSH, no association was found between these two parameters. There was also no association found between local pathology and any of the other parameters or subgroups of patients.
In ICSI cycles with fresh sperm there was one biochemical pregnancy, and in cycles with frozenthawed sperm there were two deliveries of healthy babies (one twin). In fresh ELSI cycles there is one ongoing pregnancy and two deliveries of healthy children (Table III). Of the cycles with donor sperm, there are currently one ongoing and 15 normal term pregnancies in IVF cycles, and three normal term pregnancies after IUI.
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Discussion |
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In non-obstructive azoospermia, several studies have suggested that the testicular open-biopsy method appears superior for those cases that are expected to be difficult. Nevertheless excellent sperm retrieval success has been achieved with percutaneous testis aspiration, particularly in patients with a better prognosis as given by the presence of elongated spermatids or spermatozoa in the diagnostic testicular biopsy (Lewin et al., 1996, 1999
; Rosenlund et al., 1998
; Meng et al., 2000
). However, other studies suggest that even in cases of hypoplasia, percutaneous aspiration does not enable the recovery of sufficient sperm for injection and freezing (Ezeh et al., 1998b
; Mercan et al., 2000
).
Each biopsy should be immediately checked in order to avoid excessive multiple and bilateral sampling, but in most of the cases where the probability of finding sperm is worst, multiple biopsies seem to be needed (Hauser et al., 1998; Amer et al., 1999
; Sousa et al., 1999
), although the recommended maximum number of biopsies ranges from one (Silber et al., 1995
, 1997
; Verheyen et al., 1995
) to three (Hauser et al., 1998
; Amer et al., 1999
). Our present series confirm these findings, but establishes different approaches regarding the different subtypes of histopathologies, which show distinct inherent difficulties of finding a focus of spermiogenesis. In general, the number of cases with multiple and bilateral biopsies increased according to the severity of the diagnosis and to the type of spermatid retrieved. In hypoplasia, most of the patients had unilateral surgery (97% with sperm, 70% with late spermatids), with 13 fragments giving enough gametes for treatment and storage (72% with sperm, 57% with late spermatids). In MA, most of the patients also had unilateral surgery (83% with sperm, 71% with late spermatids), but these did not have enough gametes in 13 fragments (60% with sperm, 42% with late spermatids). In SCOS, the majority of the patients also had unilateral surgery (50% with sperm, 71% with late spermatids), but of these many fewer had enough gametes in 13 fragments removed (40% with sperm, 25% with late spermatids). In all other cases needing more biopsies, mature sperm or late spermatids were always found in the later specimens, whereas in bilateral biopsies they were retrieved in at least three samples from the contralateral testis. In cases with round spermatids or absence of gamete retrieval, only 31% had an unilateral sampling, and most had more than three specimens removed. This approach has thus enabled us to rescue mature gametes for treatment where the diagnostic testicular biopsy did not show any focus of late spermatids or spermatozoa, including 47% of the cases with MA (15/32) and 29% of the cases with SCOS (16/55). To achieve this difficult goal of careful search for a rare focus of spermiogenesis, we carried out small size (3 mm) testicular biopsies, enabling the exploration of different regions of the testis while avoiding excessive sampling, although this was also limited by the size and quality of the testis.
Another helpful tip that could guide successful sperm retrieval was given by the observation that seminiferous tubules are most probable to contain gametes if they appear larger and opaque (Schlegel, 1999; Amer et al., 2000
). Although we did not use microsurgery, our present series of 148 patients showed that very often dilated and opaque seminiferous tubules contained detached early germ cells but not spermatids or sperm. Nevertheless, because there is no individual certainty of success, it has also been suggested that the treatment testis biopsy should be performed before female ovarian stimulation, with the subsequent use of frozenthawed gametes, which give normal fertilization and pregnancy rates (Gil-Salom et al., 1996
; Romero et al., 1996
; Friedler et al., 1997
; Al-Hasani et al., 1999a
; Ben-Yosef et al., 1999
; Sousa et al., 1999
, 2000
). Another possibility is to perform the testis biopsy at least 12 days before oocyte retrieval, with the tissue being left in culture to enable a better gamete maturation and an increase in sperm motility (Zhu et al., 1996
; Liu et al., 1997
; Balaban et al., 1999
; Sousa et al., 1999
, 2000
). The latter observation appears rather important, since immotile testicular sperm have been shown to elicit lower fertilization rates than motile testicular sperm (31 versus 61%), although motile sperm were found in the majority of reported cases, including SCOS (n = 34, 79%), MA (n = 26, 54%), or hypoplasia (n = 10, 70%) (Nagy et al., 1998a
). Regarding the present cases with microinjection, in hypoplasia most of the biopsies followed oocyte retrieval (40; 93%), and only three were performed 1 day (one case) or 2 days (two cases) before oocyte retrieval. The same applied to MA cases (41 cases at the same day: 93%; three cases 3 days before) and SCOS patients (18 cases in the same day; five cases 1 day and three cases 2 days before). Although this data does not allow statistical comparisons and we did not perform a quantitative study on the percentage of gametes showing motility during the period when motile sperm or late spermatids were found, motile gametes could also be recovered for treatment in all cases after 13 days of culture without any evidence of decreased quality. Furthermore, when gametes were initially immotile, after the culture period we were able to recover for injection at least in-situ motile cells, and when they initially exhibited only in-situ movements, in the majority of the cases we could find slow progressively motile gametes for injection. Similarly, we found no significant differences in the fertilization rate after injection of fresh (n = 50, 76%) or frozenthawed (n = 37, 64.5%) sperm, and also after injection of fresh (n = 33, 55.4%) or frozenthawed (n = 6, 44.7%) elongated spermatids, although frozenthawed gametes showed a tendency for worse results. These findings were confirmed by pathology, except in the case of patients with hypoplasia, where the fertilization rate with frozenthawed sperm was significantly lower.
Some clinical series showed that the relative frequency of finding sperm at treatment varied according to the diagnostic testicular biopsy, being ~25% for SCOS (n = 6111), 48% for MA (n = 176), and 74% for hypoplasia (n = 486) (Jow et al., 1993; Devroey et al., 1995
; Lewin et al., 1996
, 1999
; Friedler et al., 1997
; Amer et al., 1999
; Meng et al., 2000
). Some other studies presented a more detailed histopathological description, and showed that SCOS and MA cases could be subdivided into complete and incomplete syndromes. With this subdivision, the prognosis of finding sperm at treatment changed, with some studies showing drastic results, being 0% in complete SCOS (n = 311) or MA (n = 28), and 100% in incomplete SCOS (n = 27) or MA (n = 111) (Gil-Salom et al., 1995
; Yemini et al., 1995
; Kahraman et al., 1996
; Mulhall et al., 1997
; Ubaldi et al., 1999
; Westlander et al., 1999
). However, those findings could be due to the low number of patients studied, and thus contrasted with other larger clinical series, where the probability of finding sperm at treatment showed mean values of 95% in hypoplasia (n = 1016), 52% in complete MA (n = 260), 69% in incomplete MA (n = 316), 22% in complete SCOS (n = 662) and 90% in incomplete SCOS (n = 250) (Tournaye et al., 1996
, 1997
; Silber et al., 1997
; De Croo et al., 2000
). Our present results from 148 consecutive patients revealed very similar figures for hypoplasia (97.7%, n = 44), complete MA (53.3%, n = 15) and incomplete MA (65.6%, n = 32), but showed divergent results regarding SCOS cases (2.9% for complete SCOS, n = 35; 72.7% for incomplete SCOS, n = 22). However, in successful hypoplasia cycles, 82.8% (n = 53) had sperm for injection whereas 17.2% (n = 11) needed late spermatids; in MA cases the success rate was worst (61.7%, n = 29), with about half of the cycles using sperm (n = 22) and the other half using late spermatids (n = 21); and in SCOS cases the picture was again poor, with only 29.8% of the patients enabling retrieval of gametes for treatment (63.2% with sperm, n = 12; 36.8% with elongated spermatids, n = 7). These differences may be due to the differing origins of the populations. In fact, some cases of incomplete MA showed one focus of early spermiogenesis, and this subgroup had a similar rate of success for sperm retrieval as cases with complete MA, whereas patients with one focus of late spermiogenesis had a much higher chance of success (93.3%). Similarly, incomplete SCOS cases showed two further subgroups, one with a focus of premeiotic cells and another with one focus of early spermiogenesis, but these subgroups all had a very high chance of sucess for sperm retrieval.
In relation to the clinical outcome, most of the series only reported ICSI cycles, without specifying results per pathology or making reference to spermatid injection cycles. In those series, the fertilization rate after ICSI was shown to vary between 3969% and the cleavage rate between 6697% (n = 13179), the high quality embryo rate was 5677% (n = 19) and the pregnancy rate per cycle with embryo transfer showed a mean of 33% (Devroey et al., 1995, 1996
; Silber et al., 1995
; Kahraman et al., 1996
; Fahmy et al., 1997
; Friedler et al., 1997
; Mansour et al., 1997
; Ghazzawi et al., 1998
; Houritz et al., 1998; Madgar et al., 1998
; Lewin et al., 1999
; De Croo et al., 2000
; Mercan et al., 2000
). In our present series of 87 ICSI cycles, the fertilization (71.4%), cleavage (91.7%), high quality embryo (85.9%) and clinical pregnancy (31.7%) rates were similar.
On the contrary, only a few series presented detailed clinical outcomes after sperm injection per type of pathology. In SCOS cases, the fertilization rate after ICSI was 3844% (n = 40), the cleavage rate was 79% (n = 22), the high quality embryo rate was 5761% (n = 40), and the mean pregnancy rate per cycle with embryo transfer was 40% (n = 59). In cases of MA, the fertilization rate was similar (4246%, n = 20), the cleavage rate was 61% (n = 13), the high quality embryo rate was higher (8086%, n = 20), and the mean pregnancy rate per cycle with embryo transfer was 57% (n = 40). Finally, in hypoplasia, the fertilization rate was higher (67%, n = 11), the cleavage rate was 83% (n = 11), the high quality embryo rate was 71% (n = 11), and the pregnancy rate per cycle with embryo transfer was 5460% (n = 46) (Tournaye et al., 1996; Silber et al., 1996
, 1997
; Al-Hasani et al., 1999a
; De Croo et al., 2000
). These rates support previous findings (Nagy et al., 1998b
), which showed no significant differences between ejaculated and testicular sperm regarding timing of oocyte activation, pronucleus formation and embryo cleavage. In our 87 ICSI cycles, the rates of fertilization, cleavage and high embryo quality were relatively higher in all three syndromes (63.5, 85, 88.2% respectively in SCOS, n = 12; 66.3, 84.1, 86.2% respectively in MA, n = 22; and 74.8, 95.5, 85.4% respectively in hypoplasia, n = 53), although the clinical pregnancy rate was lower (20% in SCOS, 35% in MA, 32.1% in hypoplasia), showing a similar rate to the largest series of cycles using either ejaculated or testicular sperm injection (Mansour, 1998
; Bonduelle et al., 1999
).
Including the largest clinical series presented by our group (n = 59), the analysis of all reported cases using elongated spermatid injections (n = 166) demonstrates that, in comparison with ICSI, late spermatids appear to be associated with a significantly lower fertilization rate (48.4%), but relatively similar cleavage (90%) and pregnancy (28.9%) rates (Table IV) (Fishel et al., 1995
, 1996
; Tesarik et al., 1995
, 1996
, 1999
; Chen et al., 1996
; Mansour et al., 1996
; Amer et al., 1997
; Antinori et al., 1997a
; Araki et al., 1997
; Vanderzwalmen et al., 1997
; Barak et al., 1998
; Bernabeu et al., 1998
; Kahraman et al., 1998
; Sofikitis et al., 1998c
; Al-Hasani et al., 1999b
; Sousa et al., 1999
). However, the present analysis by type of pathology did not show significant differences between ICSI and ELSI cycles, with either fresh or frozenthawed gametes, except in the fertilization rate in hypoplasia cases. Although two cases of congenital malformations and chromosome aneuploidy were recently described (Zech et al., 2000
), the overall results do not signal an increase of risk in comparison with ejaculated or testicular sperm (Tarlatzis and Grimbizis, 1999).
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Acknowledgements |
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Notes |
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Note added in proof All ongoing pregnancies from HP, MA and SCOs have resulted in the birth of healthy babies.
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References |
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Submitted on May 1, 2001; resubmitted on November 8, 2001; accepted on February 19, 2002.