The Assisted Reproduction Unit, American Hospital of Istanbul
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Abstract |
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Key words: blastocyst/spermatid/ICSI/IVF
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Introduction |
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Materials and methods |
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Round spermatid identification
Identification, isolation, culture and injection of round spermatids have been described previously in detail (Balaban et al., 2000b). Although in stained preparations round spermatid identification is relatively easy, in wet preparations difficulties arise due to its resemblance to other cells in the extracted testicular tissue. Round spermatids were distinguished from other cells, i.e. spermatogonia, spermatocytes and polymorphonuclear leukocytes, according to their smaller size. The diameter of the round spermatid (6.58 µm) is similar to that of an erythrocyte and also to small lymphocytes (Tesarik et al., 1996
; Angelopoulos et al., 1997
; Vanderzwalmen et al., 1998
; Verheyen et al., 1998
).
Round spermatids may be observed at the Golgi, cap and acrosome phases (movement of the nucleus to a peripheral position). Golgi phase is the first phase after secondary spermatocyte division. The size of a Golgi phase spermatid (78 µm) is similar to that of a red blood cell and small lymphocyte and it contains a centrally located, rounded and thickened nucleus. Golgi phase round spermatids are very difficult to identify accurately, as they may be easily confused with haematopoetic cells. The round spermatids used in this study were in the acrosome phase (Sa1 spermatid). Sa spermatids, as recently described by Sousa et al., are round cells with a smooth outline, having a small rim of cytoplasm around the central nucleus and a bright spot corresponding to the round acrosomal vesicle that lies at the apical nuclear surface (Sousa et al., 1999). There are no visible flagella.
ROSI
Once a round spermatid was chosen as a potential candidate for injection, an attempt was made to aspirate it into the microinjection needle. The behaviour of the cell during this step was decisive for its final acceptance or rejection. Round spermatids were slightly larger than the internal diameter of the microinjection needle and were thus deformed as they entered the needle. The cells that were deformed but did not disintegrate upon aspiration were assumed to be viable spermatids and were deemed suitable for injection. These spermatids were transferred to a polyvinylpyrrolidone solution, and if they resumed their initial round forms during this step then they were thought to be alive. The spermatid injection technique has been previously and extensively described (Tesarik and Mendosa 1996; Vanderzwalmen et al., 1996
, 1997
). Following the aspiration of round spermatids into an injection pipette, the pipette was pushed through the zona pellucida and the oolemma at the equatorial level. Vigorous aspiration of the ooplasma was performed prior to injection. Oocyte activation was achieved mechanically by aspiration of the ooplasma during the ICSI procedure.
In-vitro culture of embryos to the blastocyst stage
In-vitro culture of spermatid-derived embryos was undertaken in a similar method to that of sperm-derived embryos (Gardner et al., 1998; Balaban et al., 1999
). A sequential media system (S1 and S2 media; Scandinavian Science AB Products, Gotenborg, Sweden) designed for further embryonic development was used for blastocyst culturing. Embryos were cultured in groups in S1 medium until day 3 and in S2 medium until the blastocyst stage. Culture medium was refreshed every day. Cleavage stage embryos were graded as follows: grade 1 embryo, no fragmentation with equal sized homogeneous blastomeres; grade 2 embryo, <20% fragmentation with equal sized homogeneous blastomeres; grade 3 embryo, 2050% fragmentation with unequal sized blastomeres; grade 4 embryo, >50% fragmentation with unequal sized blastomeres. Blastocyst grading was according to Dokras et al., (Dokras et al., 1993
). Grade 1 blastocysts were characterized by early cavitation, resulting in the formation of an eccentric and then expanded cavity lined by a distinct inner cell mass region and trophectoderm layer. Grade 2 blastocysts exhibited a transitional phase where single or multiple vacuoles were seen, which over subsequent days developed into the typical blastocyst appearance of the grade 1 blastocysts. Grade 3 blastocysts were defined as blastocysts with several degenerative foci in the inner cell mass, with cells appearing dark and necrotic.
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Results |
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Discussion |
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Sousa et al., studied the outcome of spermatid injection in relation to a spermatid developmental stage classification adapted to this purpose (Sousa et al., 1999). A complete block was detected at the earliest stage (Sa1). No pregnancies were achieved in ROSI cycles.
Conceptions after ROSI have been reported, albeit in only very few patients and by select groups (Tesarik et al., 1996; Antinori et al., 1997
). Failure of ROSI may be due to several factors, including failure of oocyte activation, the use of apoptotic cells and development of genetically abnormal embryos (Tesarik et al., 1999a
). However, pregnancies have been reported after in-vitro maturation of round spermatids (Tesarik et al., 1999b
). Cells with a higher degree of maturity were observed at the end of the in-vitro culture period.
We recently reported a lower and slower rate of blastocyst formation after ROSI (Balaban et al., 2000b). As the azoospermic men who formed the basis of this report had incomplete spermatogenic failure, some of the oocytes were injected with elongated or mature sperm, and round spermatids were used when no mature sperm cells were available for the remaining oocytes. Embryos derived from round spermatids were not transferred and instead were observed in culture up to the blastocyst stage. All blastocysts resulting from ROSI were of poor quality (grade 3) and none showed spontaneous hatching. The pregnancy potential of these blastocysts was not assessed as none were transferred.
We have previously shown a clear association between blastocyst quality and implantation rates (Balaban et al., 2000a). Transfer of poor quality (grade 3) blastocysts was associated with very low implantation rates (7.1%); however, occasional pregnancies occurred yielding a clinical pregnancy rate per transfer of 15.3%. These results prompted us to reconsider transferring blastocysts derived from testicular sperm. We assumed that the few embryos reaching the blastocyst stage might have the potential to implant. Low chances of success were explained to the patient and, in patients who gave informed consent, round spermatid-derived blastocysts were transferred on day 5 or 6. Only 12 out of 58 couples (20.1%) progressed to the embryo transfer stage. Of the 16 blastocysts transferred in these patients, none implanted.
Several concerns may be voiced regarding our study. Accurate identification of the round spermatid is a difficult task. It may be argued that some of the cells injected into the oocytes were not round spermatids. This is unlikely in the case of 2PN embryos. For 1PN embryos, parthenogenetic activation cannot be ruled out. However, not all 1PN embryos can be discarded in that sense, as observation of two pronuclei may have been missed due to earlier occurrence of syngamy in ROSI oocytes. (Ogura and Yanagimachi, 1993; Sofikitis et al., 1994
).
We can conclude that blastocyst transfer is not feasible for patients having round spermatid-derived embryos. Accumulated data in the literature and our own experiences suggest that ROSI should not be offered to azoospermic subjects, as there appears to be no realistic prospect for success.
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Notes |
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Submitted on February 16, 2001
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References |
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Submitted on February 16, 2001; accepted on October 29, 2001.