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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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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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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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 13), 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 58); 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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Discussion |
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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 13 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 (810 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 (Sb1Sc2) 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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Notes |
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References |
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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, 554556.[ISI][Medline]
Antinori, S., Versaci, C., Dani, G. et al. (1997b) Fertilization with human testicular spermatids: four successful pregnancies. Hum. Reprod., 12, 286291.[Abstract]
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, 559561.[ISI][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, 634638.[Abstract]
Aslam, I., Fishel, S., Green, S. et al. (1998) Can we justify spermatid microinjection for severe male factor infertility? Hum. Reprod. Update., 4, 213222.
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, 6770.[ISI][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, 10911094.[ISI][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, 59.[ISI][Medline]
de Kretser, D.M. (1969) Ultrastructural features of human spermiogenesis. Z. Zellforsch., 98, 477505.[ISI][Medline]
Edwards, R.G., Tarin, J.J., Dean, N. et al. (1994) Are spermatid injections into human oocytes now mandatory? Hum. Reprod., 9, 22172219.[ISI][Medline]
Fishel, S., Green, S., Bishop, M. et al. (1995) Pregnancy after intracytoplasmic injection of spermatid. Lancet, 345, 16411642.
Fishel, S., Aslam, I. and Tesarik, J. (1996) Spermatid conception: a stage too early, or a time too soon? Hum. Reprod., 11, 13711375.
Fishel, S., Green, S., Hunter, A. et al. (1997) Human fertilization with round and elongated spermatids. Hum. Reprod., 12, 336340.[Abstract]
Hannay, T. (1995) New Japanese IVF method finally made available in Japan. Nature Med., 1, 289290.[ISI]
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, 104110.[Abstract]
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, 223225.
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, 176185.[ISI][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, 267270.[ISI][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, 27132719.[Abstract]
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, 709714.[Abstract]
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, 197212.
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, 853857.[Abstract]
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, 33053312.[Abstract]
Tesarik, J. (1997) Sperm or spermatid conception? Fertil. Steril., 68, 214216.[ISI][Medline]
Tesarik, J. (1998) Spermatid conception. Hum. Reprod., 13, 29772979.
Tesarik, J. and Mendoza, C. (1996) Spermatid injection into human oocytes. I. Laboratory techniques and special features of zygote development. Hum. Reprod., 11, 772779.[Abstract]
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, 770776.[ISI][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, 780783.[Abstract]
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, 757762.[Abstract]
Tesarik, J., Sousa, M., Greco, E. and Mendoza, C. (1998b) Spermatids as gametes: indications and limitations. Hum. Reprod., 13 (Suppl. 3), 89107.[Medline]
Tesarik, J., Greco, E. and Mendoza, C. (1998c) ROSI, instructions for use: 1997 update. Hum. Reprod., 13, 519523.[ISI][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, 27722781.
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, 44674473.
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, 12031213.[ISI][Medline]
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, 5562.[ISI][Medline]
Submitted on September 9, 1998; accepted on January 8, 1999.