Human Reproduction, Vol. 14, No. 5, 1279-1286,
May 1999
© 1999 European Society of Human Reproduction and Embryology
Clinical efficacy of spermatid conception: analysis using a new spermatid classification scheme
1 Laboratory of Cell Biology, Institute of Biomedical Sciences, 2 Department of Medical Genetics, Faculty of Medicine, University of Porto, 3 Centre for Reproductive Genetics, Porto, Portugal, 4 HART Clinic, Hiroshima, Japan 730 and 5 Laboratoire d`Eylau, Paris, France
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Abstract |
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Fertilization and pregnancy outcomes of 50 round spermatid injection (ROSI) and 20 elongated spermatid injection (ELSI) treatment cycles are related to various characteristics of the cycles, with particular reference to spermatid developmental stage as assessed by using a classification scheme adapted to this purpose. Although this classification includes eight stages, a complete block was mostly detected at the earliest stage (Sa1) or at the latest stages (Sd1 and Sd2). Thus, spermiogenesis was blocked at Sa1 stage in 50 cases (71%), at Sd1 stage in eight cases (11%) and at Sd2 stage in 10 cases (14%). Only in two cases (3%) was spermiogenesis blocked at an intermediate stage (Sb2). Globally, fertilization rates were higher for ELSI than for ROSI. No pregnancy was achieved in the ROSI cycles, whereas nine pregnancies resulted from the ELSI cycles. Two of them (both with Sd2 spermatids) ended in a first trimester spontaneous abortion. Of the seven ongoing pregnancies, five are singleton (two with Sd1 spermatids, two with Sd2 spermatids, and one after a mixed transfer after injection of Sa2 and Sd1 spermatids) and two are twin (one with Sd1 and the other with Sd2 spermatids). No pregnancy was achieved in the two cycles with Sb2 spermatids. One of the two twin pregnancies has already resulted in the birth of two healthy children.
Key words: complete and incomplete spermiogenesis failure/ELSI/non-obstructive azoospermia/ROSI/spermatid conception/spermatid staging
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Introduction |
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Spermatid conception was proposed for the treatment of sterility in humans (Edwards et al., 1994
). The first ongoing pregnancies and births resulting from the use of round (Tesarik et al., 1995, 1996) and elongated (Fishel et al., 1995, 1996) spermatids for human reproduction, and after the first detailed description of the round spermatid injection (ROSI) and elongated spermatid injection (ELSI) techniques (Tesarik and Mendoza, 1996), were followed by a number of reports on ROSI and ELSI treatment cycles (Mansour et al., 1996; Amer et al., 1997; Antinori et al., 1997a,b; Araki et al., 1997; Vanderzwalmen et al., 1997; Yamanaka et al., 1997; Barak et al., 1998; Kahraman et al., 1998; Sofikitis et al., 1998a,b; reviewed in Aslam et al., 1998). In parallel, round spermatid nucleus injection (ROSNI) is used in some centres (Yamanaka et al., 1997; Sofikitis et al., 1998a,b). In fact, the first human spermatid-derived pregnancies (unfortunately all have been terminated by spontaneous abortion) have been achieved by ROSNI (Hannay, 1995). However, only two studies compared the clinical efficacy of spermatid conception in relation to the type of spermatogenic disorder and to the stage of spermatids injected (Amer et al., 1997; Vanderzwalmen et al., 1997). These studies demonstrated a poor prognosis for men in whom progression of spermiogenesis had never been demonstrated in at least a few spermatids during previous examinations. This condition has been termed complete spermiogenesis failure (Amer et al., 1997). In fact, all the four live births after human ROSI that have been reported in the literature (Tesarik et al., 1995, 1996; Vanderzwalmen et al., 1997; Barak et al., 1998) were achieved in patients in whom late elongated spermatids or spermatozoa had previously been detected. Only one study compared the fertilizing ability of round and elongated spermatids obtained either from the ejaculate or by testicular biopsy (Fishel et al., 1997). However, clinical efficacy could not be addressed in that study because it dealt with supernumerary oocytes donated for research, and, consequently, no embryo transfer was performed (Fishel et al., 1997). Moreover, with the exception of one report (Amer et al., 1997), all previous studies on spermatid conception suffered from the lack of vigorous criteria for the identification of the stage of spermatids used for fertilization. In fact, spermatids were mostly classified into two groups, round and elongated, to which some studies added an intermediate category of elongating spermatids. An urgent need for the refinement of the criteria for spermatid staging and their application in the analysis of spermatid conception success and failure has been pointed out (Tesarik, 1997). In this study, we report on a series of 70 spermatid conception treatment cycles in which fertilization and pregnancy outcomes are analysed according to patients' age, type of spermatogenic disorder, source of spermatids (testicular or ejaculated) and spermatid stage. For spermatid staging, we have developed a new spermatid classification scheme, based on that introduced by de Kretser (1969) but also taking into account the specific conditions in which spermatids are observed at the time of ROSI or ELSI.
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Materials and methods |
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There were 70 treatment cycles (64 patients) with spermatid injection for the treatment of non-obstructive azoospermia. In all cases, the gynaecological survey of female patients was normal. Except in three male cases (see below), couples presented normal karyotypes. Female patients were treated with a long gonadotrophin-releasing hormone analogue (GnRHa) suppression protocol combining buserelin acetate (Suprefact; Hoechst, Frankfurt, Germany) with pure follicle stimulating hormone (FSH) (Metrodin HP; Serono, Genève, Switzerland) or recombinant FSH (Gonal F; Serono; Puregon, Organon, Oss, The Netherlands) for controlled ovarian stimulation; ovulation was induced with human chorionic gonadotrophin (HCG) (Pregnyl; Organon, Profasi; Serono). Oocytes were recovered from large ovarian follicles by ultrasonically guided follicular aspiration, 36 h after HCG, using flush medium (Medicult, Copenhagen, Denmark) (Barros et al., 1997
). For intracytoplasmic sperm injection (ICSI), oocytes were prepared and injected as previously reported, with vigorous aspiration of the ooplasm (Tesarik and Sousa, 1995). Briefly, after mechanical removal of the cumulus by using hypodermic needles, oocytes were exposed to 80 IU hyaluronidase in 1 ml Sperm Preparation Medium (SPM) (Medicult, Copenhagen, Denmark) for 30 s, washed several times in SPM, and then the corona layer was removed by gentle in and out aspiration with fine glass pipettes (SweMed, Frolunda, Sweden). Oocytes were injected in SPM, under light mineral oil (embryo tested; Medicult, Copenhagen, Denmark), in an inverted Nikon research microscope equipped with a heated stage (37°C), using Narishige micromanipulators and commercial micropipettes with an internal diameter of 5 and 9 µm for elongated and round spermatids respectively (SweMed, Frolunda, Sweden). Fertilization was assessed 16–18 h after injection by the presence of two pronuclei (PN) and two polar bodies for elongated spermatids and also by the presence of one syngamic PN in the case of round spermatids (Tesarik and Mendoza, 1996). Embryo cleavage and quality were evaluated 42 h after ICSI, according to the blastomere size equality and the relative proportion of anucleate fragments (Staessen et al., 1995). Embryo transfer was performed at about 46–72 h after oocyte retrieval. Pregnancy was confirmed by a rise in serum ß-HCG on 2 consecutive days, 2 weeks after embryo transfer. All patients had luteal supplementation with three times daily intravaginal administration of 200 mg natural-micronized P (Utrogestan; Jaba, Berlin, Germany). A clinical pregnancy was established by ultrasonography at 7 weeks of gestation (Barros et al., 1997). All couples agreed to have a prenatal diagnosis, which would be performed by amniocentesis at 16 weeks of pregnancy. All therapeutic procedures have been approved by the National Ethical Committees for the Life Sciences. Informed consent was obtained from all patients, after careful explanation of the experimental nature of the treatment technique. After liquefaction, ejaculates were washed twice with SPM by centrifuging for 5 min at 1000 r.p.m. An aliquot of the pellet was then diluted in SPM and placed in a plastic culture dish (Falcon). Round spermatids were selected, washed in 10% polyvinylpyrrolidone (PVP) in SPM (Medicult, Copenhagen, Denmark), and then left in culture for 24 h or up to 3 days in a microdrop of SPM under light mineral oil at 37°C with 5% CO2. For isolation of testicle spermatids, tissue samples were collected in SPM, squeezed with surgical blades, and the resultant fluid was then washed in SPM by centrifuging for 5 min at 1000 r.p.m. Testicular round spermatids were not cultured because the biopsy was performed the same day as oocyte retrieval, whenever the search for ejaculated spermatids was negative the days before.
For cryopreservation, samples with spermatids were mixed (1:1) with medium containing egg yolk buffer and glycerol and placed in cryotubes. After 10 min exposure to liquid nitrogen vapours, cryotubes were plunged directly into liquid nitrogen. For thawing, samples in cryotubes were left for 30 min at 37°C and then washed twice with SPM as described (Barros et al., 1998).
Spermatids were staged using a classification based on the criteria suggested by de Kretser (de Kretser, 1969), which were modified to be better suited to the conditions under which native spermatids are observed during ROSI and ELSI, as suggested by us (Tesarik, 1997). Round spermatids could be easily distinguished from Sertoli cell nuclei, based on their smaller size, similar to that of erythrocytes (Figure 1). All larger cells, including giant round spermatids, were excluded from further consideration for eventual use in ROSI. Most round spermatids lacked a flagellum (Figures 1–3), whereas a short (<8 µm) emerging flagellum could be observed in others (Figure 4). These stages are referred to as Sa1 and Sa2 respectively. Some spermatids with a still round cell body showed a longer (at least 8 µm) flagellum (Figures 5–8); those of them that still retain the central position of the nucleus are called Sb1 stage, whereas those with an oval nucleus located in the periphery of the cell are referred to as Sb2 stage. The 8 µm cutoff was chosen because it corresponds to the cell diameter of normal-sized round spermatids and can thus be easily assessed by simple microscopic observation. Sc1 spermatids (Figure 9) were characterized by a slightly elongated cell body and the beginning of nuclear protrusion which, however, was limited to the most apical nuclear region. Interestingly, the cell body of Sc1 spermatids became more elongated and the nuclear protrusion increased following the breakage of the flagellum during the ELSI procedure (Figure 10). As compared to Sc1 spermatids, Sc2 spermatids (Figures 11 and 12) had a more protruding nucleus, such that approximately half of the nucleus was budding out of the cell outline. In Sd1 spermatids (Figure 13), more than half of the nucleus protruded, while part of it was still embedded in the cell body. Even in Sd1 spermatids, the degree of nuclear protrusion increased after the breakage of the flagellum (Figure 14). Finally, the entire cell nucleus projected out of the elongated cell body in Sd2 spermatids (Figures 15 and 16). Spermatids of Sa1 type were further tested by two sequential steps. First, they had to deform when aspirated (size and consistency selection), without sticking to the internal tip of the microneedle. Sticking is characteristic of lymphocytes (Figures 17 and 18) and of deformed ejaculated round spermatids. All cells showing this phenomenon were excluded from this study. Viability of cells was also checked at this point, as described (Tesarik and Mendoza, 1996). Second, they were washed in 10% PVP-SPM, and only those cells that did not shrink, did not lose flagella, or became sticky in this medium were then selected. All spermatids with flagella had their tails crushed before injection, as in the regular ICSI technique for spermatozoa injection. Oocytes were injected with the most advanced (blocking) stage of spermatids identified in the sample, by using the previously described methodology (Tesarik and Mendoza, 1996).
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Results |
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ROSI with ejaculated spermatids in patients with complete spermiogenesis failure
There were 29 cycles (25 couples) treated with injection of ejaculate retrieved Sa1 spermatids after 1 day of culturing (Table I
). In all cases, previous history showed absence of elongated spermatids or spermatozoa in the ejaculate or in diagnostic testicular biopsy, but round spermatids were found. The mean ages were 33 (male) and 30 (female) years, with a mean length of infertility of 4 years. Of the 262 mature oocytes injected, 19% degenerated after injection. Of the surviving injected oocytes, 48% did not fertilize, 37% showed one nucleus (1N), and 15% had two pronuclei (2PN) (52% fertilization rate). The cleavage rate was 93% (90% from 1PN and 100% from 2PN eggs), with 37% of grade A, 54% of grade B, and 9% of grade C embryos. No pregnancy was obtained. No previous diagnostic biopsy has been obtained in 28% of the patients, the remaining presenting Sertoli cell only (SCO) syndrome (72%). One patient had a Klinefelter syndrome and another one a Johnson 2–4 syndrome (Table I).
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ROSI with testicular spermatids in patients with complete spermiogenesis failure
There were 21 cycles (21 couples) treated with injection of testicular Sa1 spermatids (Table II
). In all cases, previous history showed absence of elongated spermatids or spermatozoa in the ejaculate or in diagnostic testicle biopsy. Mean ages were 36 (male) and 32 (female) years, with a mean time of infertility of 7 years. Of 132 mature oocytes injected, 15% degenerated after injection. Of the surviving injected oocytes, 52% did not fertilize, 38% showed 1N, and 11% 2PN (48% fertilization rate). The cleavage rate was 87% (83% from 1N and 100% from 2PN eggs), with 38% of grade A, 54% of grade B, 6% of grade C and 2% of grade D embryos. In four couples, no embryo transfer was performed due to the absence of fertilization. No previous diagnostic biopsy has been obtained in 10% of the patients, the remaining presenting Sertoli cell only (SCO) syndrome (48%) or maturation arrest at the spermatocyte stage (43%). One patient had Klinefelter syndrome (Table II).
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ELSI with testicular spermatids
Eight cycles of ELSI were performed in seven patients in whom previous examinations indicated the absence of elongated spermatids or spermatozoa in the ejaculate or in diagnostic testicle biopsy (nos 1–8 in Table III
). Mean ages were 39 (male) and 35 (female) years, with the mean time of infertility of 5 years. Of 64 mature oocytes injected, 20% degenerated soon after injection. Of the surviving injected oocytes, 45% did not fertilize, 16% showed 1N and 39% 2PN (55% fertilization rate). The cleavage rate was 89% (75% from 1N and 95% from 2PN eggs), with 32% of grade A, 60% of grade B, and 8% of grade C embryos. Two pregnancies were obtained (ongoing). Previous diagnostic biopsies showed SCO syndrome in three patients and maturation arrest at the spermatocyte stage in four patients. As to the type of spermatids injected, these were Sb2 in 2 cycles, Sd1 in one cycle, Sd2 in four cycles and mixed Sc2 + Sd1 in one cycle, although only embryos resulting from Sc2 spermatid injections had a quality compatible with transfer. Two singleton pregnancies were obtained, one with Sd1 spermatids in a case of previously diagnosed SCO syndrome and one with Sd2 spermatids in a case of previously diagnosed maturation arrest (nos 2 and 7 in Table III).
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Another 12 cycles (11 couples) of ELSI (nos 9–20 in Table III
) were performed in patients in whom previous examinations showed the presence of elongated spermatids in the ejaculate (four patients without diagnostic testicle biopsy) or of foci of complete spermatogenesis (<2% of tubules) in diagnostic testicle biopsy (seven patients with maturation arrest at the primary spermatocyte stage). Mean ages were 35 (male) and 32 (female) years, with a mean time of infertility of 6 years. Of the 102 mature oocytes injected, 5% degenerated soon after injection. Of the surviving injected oocytes, 31% did not fertilize, 9% showed 1N and 60% 2PN (69% fertilization rate). The cleavage rate was 90% (67% from 1N and 93% from 2PN eggs), with 20% of grade A, 65% of grade B, and 15% of grade C embryos. As to the type of spermatids injected, these were Sd1 in six cycles (in two together with Sc2 and in one together with Sa2) and Sd2 in six cycles (in one together with Sa1 and Sa2). Seven pregnancies were achieved (four with Sd2 spermatids, two with Sd1 spermatids and one after transfer of one embryo from Sa2 and 2 embryos from Sd1 spermatids). Two of these pregnancies (both with Sd2 spermatids) ended with a first trimester spontaneous abortion. Of the remaining five ongoing pregnancies, two were twin (one with Sd1 spermatids and the other with Sd2 spermatids). The first of these twins, a male (2.3 kg) and a female (1.9 kg), were born in February 1998, at 35 weeks of gestation, by Caesarean section and are well at present. |
Discussion |
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As pointed out recently (Tesarik, 1997
), a more accurate staging of spermatids used in ROSI and ELSI treatment cycles, as compared to the usual `round/elongating/elongated' classification scheme, is urgently needed to assess both the efficacy and the potential risk of spermatid conception. To be applicable in a standard assisted reproductive technology laboratory, these staging criteria also have to be adapted for the assessment with the use of inverted microscopes equipped with Hoffman contrast optics that are currently used in most laboratories performing ROSI and ELSI. The classification described in this study has been tailored to cope with these practical demands, even though other criteria may be more useful with other methodological approaches. Among those, transmission electron microscopy, confocal laser scanning microscopy, and computer-guided morphometric analysis have already been used for spermatid identification (Yamanaka et al., 1997; Sofikitis et al., 1998a,b).
Spermatid classification used in this study is based on the criteria used for spermatid staging in electron microscopical preparations (de Kretser, 1969). This background was chosen because the classification suggested by de Kretser (1969) is currently used in many andrological laboratories for evaluation of histological sections of seminiferous tubules, because it is relatively simple and thus easily applicable to routine use, and because the key features distinguishing individual stages can be assessed during observation of living spermatids with an inverted microscope equipped with Hoffman optics. However, the obvious limitations of the resolving power of this system, as compared to electron microscopy, makes it impossible to use some of the originally used criteria (de Kretser, 1969) and has led to the need for slight modifications. When comparing this modified classification scheme with other classifications, it must also be taken into account that our classification is based on observations on whole, living cells, whereas most other classifications have been developed for the assessment of sectioned, fixed cells.
The results of this study confirm the previous findings (Amer et al., 1997; Vanderzwalmen et al., 1997), showing that the use of spermatids from patients with a complete spermiogenesis failure (Sa1 stage) is associated with a very low pregnancy success (no pregnancy in 50 cycles in this study). Early round spermatids appear to have a better reproductive capacity when recovered from patients with incomplete spermiogenesis failure (Tesarik et al., 1995, 1996; Vanderzwalmen et al., 1997; Barak et al., 1998). However, no such case was involved in this study.
The poor reproductive capacity of spermatids from patients with complete spermiogenesis failure may be at least partly due to apoptosis (Tesarik et al., 1998a), leading to the lack of oocyte activating factors (Tesarik et al., 1998b,c), whereas these factors are present in round spermatids from men with normal spermatogenesis (Sousa et al., 1996) and probably also in many spermatids from patients with incomplete spermiogenesis failure. The impact of the underlying testicular pathology on spermatid reproductive capacity has also been demonstrated by the finding of decreased fertilizing potential of round spermatids from men with primary testicular damage (Sofikitis et al., 1996b). Since previous results suggested that a crucial developmental step may occur at the Sa1 round spermatid stage (Sofikitis et al., 1997), and the progression of cytoplasmic maturation has been achieved in human spermatids during in-vitro culture (Tesarik et al., 1998d), we tried to culture ejaculated Sa1 round spermatids before injection to ascertain if the removal from the seminiferous tubular environment could favour maturation progression. Compared to the culture of testicular spermatids (Tesarik et al., 1998d), FSH was not included in culture medium because the presumptive FSH target, Sertoli cell, was absent in this system.
Although culturing for 1–3 days, without hormonal supplementation, did not influence cell morphology, it elicited a much better fertilization rate (52%) as compared to ROSI performed with freshly recovered spermatids in a similar group of patients (22%) (Amer et al., 1997), even though most of the injected oocytes developed a single large nucleus rather than two pronuclei. According to the previously described criteria (Tesarik and Mendoza, 1996), the characteristics of these nuclei corresponded to the syngamy nucleus, arising through the fusion of previously separated two pronuclei (Tesarik and Mendoza, 1996). Cleaving embryos developing from such zygotes can give rise to a term pregnancy (Barak et al., 1998). However, in the absence of an earlier observation on pronuclei, we cannot exclude the possibility that, in some cases, a single nucleus observed in a spermatid-injected oocyte was actually a female pronucleus, occurring along prematurely condensed spermatid chromosomes. With this reservation, all spermatid-injected oocytes developing either two pronuclei or one nucleus are referred to as fertilized in this study. Because an early appearance of pronuclei (8–10 h after injection) has been reported after ROSI in both rabbits (Sofikitis et al., 1996a, 1997) and humans (Tesarik and Mendoza, 1996; Yamanaka et al., 1997; Sofikitis et al., 1998a,b), repeated observations on injected oocytes, including those early time intervals after ROSI, will hopefully make this point clear in the future.
Unfortunately, the use of in-vitro cultured spermatids did not lead to an improvement of the clinical outcome. One may speculate that results might have been better if the culture had been carried out at a lower temperature, closer to the physiological condition of the human testis. In fact, a rapid progression of human spermatogenesis in vitro has been achieved at 30°C (Tesarik et al., 1998d,e). However, even with testicular spermatids from men with normal spermatogenesis, in-vitro development was dependent on the presence and viability of Sertoli cells in cultured samples (Tesarik et al., 1998e). In terms of clinical efficacy, in-vitro culture of ejaculated spermatids does not appear to bring any improvement. On the other hand, in-vitro culture of testicular biopsy samples (Tesarik et al., 1998d,e) is a promising approach which can lead to post-meiotic differentiation of round spermatids even in cases of complete spermiogenesis failure (Tesarik, 1998). The choice of the best culture medium among those reported by different groups to support spermatid survival and development during in-vitro culture (Aslam and Fishel, 1998; Tesarik et al., 1998d,e; Yamanaka et al., 1998) is another challenge for future research. Finally, the use of commercial microinjection needles (internal diameter of 9 µm) may result in an insufficient destabilization of the spermatid plasma membrane before ROSI compared with the originally described ROSI technique in which smaller microneedles, prepared ad hoc in the laboratory, were used (Tesarik and Mendoza, 1996). The larger microneedle diameter may have also been at the origin of the higher oocyte degeneration rate in the present study. These questions are currently under investigation in our laboratories.
Compared to round spermatids, injection of late elongated (Sd1 and Sd2) testicular spermatids seems to have an excellent prognosis, in terms of oocyte activation, fertilization and pregnancy rates, even in cases in which there is no previous finding of elongated spermatids in the ejaculates or in diagnostic testicle biopsies. This is in agreement with the results reported by others (Araki et al., 1997; Vanderzwalmen et al., 1997; Sofikitis et al., 1998a). A complete block at intermediate stages of spermatid elongation (Sb1–Sc2) appears to be relatively less frequent and was found only in two cases in this study. Even though the injection of Sb2 spermatids did not lead to a pregnancy in these two cases, more treatment cycles are necessary to evaluate the actual reproductive capacity of these intermediate spermatid stages.
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Acknowledgments |
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We would like to acknowledge the work of Drs Vasco Almeida, Jorge Beires, Nuno Montenegro, Luis Ferraz and Paulo Viana. We would also like to thank Mr João Carvalheiro for the photographic work. This work was partially funded by grants from JNICT-PRAXIS XXI (Project PCNA/C//BIA/100/96) and Eng. António de Almeida Foundation.
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Notes |
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6 To whom correspondence should be addressed
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References |
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Amer, M., Soliman, E., El-Sadek, M. et al. (1997) Is complete spermiogenesis failure a good indication for spermatid conception? Lancet, 350, 116.
Antinori, S., Versaci, C., Dani, G. et al. (1997a) Successful fertilization and pregnancy after injection of frozen-thawed round spermatids into human oocytes. Hum. Reprod., 12, 554–556.
Antinori, S., Versaci, C., Dani, G. et al. (1997b) Fertilization with human testicular spermatids: four successful pregnancies. Hum. Reprod., 12, 286–291.
Araki,Y., Motoyama, M., Yoshida, A. et al. (1997) Intracytoplasmic injection with late spermatids: a successful procedure in achieving childbirth for couples in which the male partner suffers from azoospermia due to deficient spermatogenesis. Fertil. Steril., 67, 559–561.[Web of Science][Medline]
Aslam, I. and Fishel, S. (1998) Short-term in-vitro culture and cryopreservation of spermatogenic cells used for human in-vitro conception. Hum. Reprod., 13, 634–638.
Aslam, I., Fishel, S., Green, S. et al. (1998) Can we justify spermatid microinjection for severe male factor infertility? Hum. Reprod. Update., 4, 213–222.
Barak, Y., Kogosowski, A., Goldman, S. et al. (1998) Pregnancy and birth after transfer of embryos that developed from single-nucleated zygotes obtained by injection of round spermatids into oocytes. Fertil. Steril., 70, 67–70.[Web of Science][Medline]
Barros, A., Sousa, M., Oliveira, C. et al. (1997) Pregnancy and birth after intracytoplasmic sperm injection with totally immotile sperm recovered from the ejaculate. Fertil. Steril., 67, 1091–1094.[Web of Science][Medline]
Barros, A., Sousa, M., Andrade, M.J. et al. (1998) Birth after electroejaculation coupled to intracytoplasmic sperm injection in a gun-shot spinal cord-injured man. Arch. Androl., 41, 5–9.[Web of Science][Medline]
de Kretser, D.M. (1969) Ultrastructural features of human spermiogenesis. Z. Zellforsch., 98, 477–505.[Web of Science][Medline]
Edwards, R.G., Tarin, J.J., Dean, N. et al. (1994) Are spermatid injections into human oocytes now mandatory? Hum. Reprod., 9, 2217–2219.
Fishel, S., Green, S., Bishop, M. et al. (1995) Pregnancy after intracytoplasmic injection of spermatid. Lancet, 345, 1641–1642.
Fishel, S., Aslam, I. and Tesarik, J. (1996) Spermatid conception: a stage too early, or a time too soon? Hum. Reprod., 11, 1371–1375.
Fishel, S., Green, S., Hunter, A. et al. (1997) Human fertilization with round and elongated spermatids. Hum. Reprod., 12, 336–340.
Hannay, T. (1995) New Japanese IVF method finally made available in Japan. Nature Med., 1, 289–290.
Kahraman, S., Polat, G., Samli, M. et al. (1998) Multiple pregnancies obtained by testicular spermatid injection in combination with intracytoplasmic sperm injection. Hum. Reprod., 13, 104–110.
Mansour, R.T., Aboulghar, M.A., Serour G.I., et al. (1996) Pregnancy and delivery after intracytoplasmic injection of spermatids into human oocytes. Middle East Fertil. Soc. J., 1, 223–225.
Sofikitis, N.V., Toda, T., Miyagawa, I. et al. (1996a) Beneficial effects of electrical stimulation before round spermatid nuclei injections into rabbit oocytes on fertilization and subsequent embryonic development. Fertil. Steril., 65, 176–185.[Web of Science][Medline]
Sofikitis, N.V., Miyagawa, I., Andrighetti, S. et al. (1996b) Detrimental effect of left varicocele on the reproductive capacity of the early haploid male gamete. J. Urol., 156, 267–270.[Web of Science][Medline]
Sofikitis, N., Yamamoto, Y. and Miyagawa, I. (1997) The early haploid male gamete develops a capacity for fertilization after the coalescence of the proacrosomal granules. Hum. Reprod., 12, 2713–2719.
Sofikitis, N.V., Yamamoto, Y., Miyagawa, I. et al. (1998a) Ooplasmic injection of elongating spermatids for the treatment of non-obstructive azoospermia. Hum. Reprod., 13, 709–714.
Sofikitis, N., Miyagawa, I., Yamamoto, Y. et al. (1998b) Micro- and macro-consequences of ooplasmic injections of early haploid male gametes. Hum. Reprod., Update, 4, 197–212.
Sousa, M., Mendoza, C., Barros, A. and Tesarik, J. (1996) Calcium responses of human oocytes after intracytoplasmic injection of leukocytes, spermatocytes and round spermatids. Mol. Hum. Reprod., 2, 853–857.
Staessen, C., Nagy, Z.P., Liu, J. et al. (1995) One year's experience with elective transfer of two good quality embryos in the human in-vitro fertilization and intracytoplasmic sperm injection programmes. Hum. Reprod., 10, 3305–3312.
Tesarik, J. (1997) Sperm or spermatid conception? Fertil. Steril., 68, 214–216.[Web of Science][Medline]
Tesarik, J. (1998) Spermatid conception. Hum. Reprod., 13, 2977–2979.
Tesarik, J. and Mendoza, C. (1996) Spermatid injection into human oocytes. I. Laboratory techniques and special features of zygote development. Hum. Reprod., 11, 772–779.
Tesarik, J. and Sousa, M. (1995) Key elements of a highly efficient intracytoplasmic sperm injection technique: Ca2+ fluxes and oocyte cytoplasmic dislocation. Fertil. Steril., 64, 770–776.[Web of Science][Medline]
Tesarik, J., Mendoza, C. and Testart, J. (1995) Viable embryos from injection of round spermatids into oocytes. N. Engl. J. Med., 333, 525.
Tesarik, J., Rolet, F., Brami, C. et al. (1996) Spermatid injection into human oocytes. II. Clinical application in the treatment of infertility due to non-obstructive azoospermia. Hum. Reprod., 11, 780–783.
Tesarik, J., Greco, E., Cohen-Bacrie, P. and Mendoza, C. (1998a) Germ cell apoptosis in men with complete and incomplete spermiogenesis failure. Mol. Hum. Reprod., 4, 757–762.
Tesarik, J., Sousa, M., Greco, E. and Mendoza, C. (1998b) Spermatids as gametes: indications and limitations. Hum. Reprod., 13 (Suppl. 3), 89–107.
Tesarik, J., Greco, E. and Mendoza, C. (1998c) ROSI, instructions for use: 1997 update. Hum. Reprod., 13, 519–523.[Web of Science][Medline]
Tesarik, J., Greco, E., Rienzi, L. et al. (1998d) Differentiation of spermatogenic cells during in-vitro culture of testicular biopsy samples from patients with obstructive azoospermia: effect of recombinant follicle stimulating hormone. Hum. Reprod., 13, 2772–2781.
Tesarik, J., Guido, M., Mendoza, C. et al. (1998e) Human spermatogenesis in vitro: respective effects of follicle-stimulating hormone and testosterone on meiosis, spermiogenesis, and Sertoli cell apoptosis. J. Clin. Endocrinol. Metab., 83, 4467–4473.
Vanderzwalmen, P., Zech, H., Birkenfeld, A. et al. (1997) Intracytoplasmic injection of spermatids retrieved from testicular tissue: influence of testicular pathology, type of selected spermatids and oocyte activation. Hum. Reprod., 12, 1203–1213.
Yamanaka, K., Sofikitis, N.V., Miyagawa, I. et al. (1997) Ooplasmic round spermatid nuclear injection procedures as an experimental treatment for nonobstructive azoospermia. J. Assist. Reprod. Genet., 14, 55–62.[Web of Science][Medline]
Submitted on September 9, 1998; accepted on January 8, 1999.
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