Assisted Reproduction Unit, American Hospital of Istanbul, Turkey
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Abstract |
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Key words: azoospermia/blastocyst/spermatid/spermatozoa/testicular sperm extraction
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Introduction |
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An overview of the literature suggests a better ICSI outcome when elongating or elongated spermatids are used for ICSI (Fishel et al., 1997). However, extremely poor results have been reported with ROSI. Vanderzwalmen et al. reported a 4% ongoing pregnancy and a 2% live birth rate after the transfer of ROSI-derived embryos (Vanderswalmen et al., 1998). Similarly, our experience with ROSI has been very disappointing; only one biochemical pregnancy was achieved in 33 cleavage stage embryo transfers (unpublished data). The reason for the poor results obtained with ROSI may be the diminished or absent potential of ROSI derived embryos to progress to advanced stages of development. Goto et al. studied blastocyst formation following ICSI of spermatids in the bovine model (Goto et al., 1996
). There were no significant differences in embryo quality of blastocysts obtained by injection of spermatids derived from spermatocytes, spermatids or spermatozoa. They concluded that various types of spermatogenic cells could be used for ICSI to produce viable embryos. However, the implantation potential of these embryos was not assessed. More recently it was shown that round spermatids obtained from hybrid sterile mice can result in normal embryos which progress to the blastocyst stage (Sasagawa et al., 1998
). To our knowledge, no study in the literature has examined the progression of human ROSI embryos to the blastocyst stage. Therefore we aimed to evaluate the progression of human embryos obtained by ROSI to the blastocyst stage. We compared the rate of progression of ROSI embryos to the blastocyst stage with that of sibling embryos injected with testicular spermatozoa.
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Materials and methods |
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Transfer was performed on day 3 only of embryos derived from ICSI of mature spermatozoa. Remaining embryos (immature spermatozoa, elongated spermatids and round spermatid derived) were observed under in-vitro culture in sequential defined media. Excess blastocysts from mature sperm injections were frozen at the blastocyst stage, taking into account their morphological quality.
All men were karyotypically normal. Microdeletions of the Y chromosome were not routinely investigated. Of the 56 men, 21 had undergone a prior diagnostic testis biopsy that showed hypospermatogenesis in four, Sertoli cell-only syndrome in 15 and maturation arrest in two subjects. Ovarian stimulation, oocyte retrieval, embryo transfer and luteal phase support were undertaken as described previously (Balaban et al., 1998). Briefly, gonadotrophin-releasing hormone analogue (GnRHa) was initiated in the luteal phase and was combined with FSH after down-regulation was achieved. In some cycles a flare GnRHa protocol was used. Transvaginal follicle aspiration and oocyte retrieval was performed following the injection of human chorionic gonadotrophin (HCG) at a leading follicle diameter of ~1920 mm.
TESE and processing of testicular tissue
TESE was performed as previously described at ~24 h prior to ICSI (Urman et al., 1998). Briefly, testicular tissue samples obtained by open biopsy were placed into a Falcon 2001 tube containing 2 ml of IVF-50 medium (Scandinavian Science, AB, Gothenburg, Sweden). The tissue was washed several times with IVF-50 and then placed in a Petri dish (Falcon 3004). The cellular contents were extracted by gentle crushing between two needles and sterile glass slides. The suspension thus obtained was transferred into a Falcon 2001 tube and agitated for 60 s on a vortex to separate the different cell types. The vortexed suspension was then placed into a Falcon 3004 tube for observation under an inverted microscope equipped with Hoffman modulation contrast system. Magnifications of x200 (for the presence of spermatozoa) and x400 (for the identification of round spermatids) were used. The suspension was then centrifuged (Vanderzwalmen et al., 1991
) at 600 g for 20 min through a three-layer (50, 70, 90%) discontinuous Percoll gradient (Percoll, Sigma P-1644, Sigma- Aldrich, Chemie GmbH, Stenheim, Germany). Each layer was washed with IVF-50 medium and centrifuged for 5 min at 1000 g. The spermatids were most often found in the 50 and 70% fractions whereas spermatozoa were collected from the 90% fraction. The pellet of each gradient was kept at 2530°C until the ICSI procedure (Tesarik et al., 1998c
). The pellets were prepared in a Falcon 3001 Petri dish as a swim-out droplet covered with mineral oil (Sigma- M-8410 Sigma-Aldrich Co. Ltd., St Louis, MO, USA) for spermatozoa or spermatid collection.
The injection of testicular spermatozoa was similar to that of conventional ICSI of mature ejaculated spermatozoa (Vanderzwalmen et al., 1996). However, in view of the different size and form of spermatids some modifications had to be adopted to the instrumentation and technique.
Spermatid identification and classification
The major concern about round spermatid injection is the identification of the cell. There are many other cells in the extracted testicular tissue that need to be distinguished from round spermatids (Figure 1). Although in stained preparations round spermatid identification is relatively easy, in wet preparations difficulties arise due to the resemblance to other cells in the extracted testicular tissue. Round spermatids at the Golgi cap phase and acrosome phase were observed and identified under an Olympus IX70 microscope equipped with a Hoffman modulation contrast system. Magnifications of x200 and x400 were used. Round spermatids were distinguished from other cells, e.g. spermatogonia, spermatocytes and polymorphonuclear leukocytes, by their smaller size (Figure 1
). The diameter of the round spermatid (6.58 µm) is similar to that of an erythrocyte and to a small lymphocyte (Tesarik et al., 1996
; Angelopoulos et al., 1997
; Vanderzwalmen et al., 1998
; Verheyen et al., 1998
). Round spermatids may be observed at the Golgi phase, cap phase and the acrosome phase (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) (Figure 2
). These were the Sa spermatids described previously (de Kretser and Kerr, 1969
). Sa spermatids were recently described (Sousa et al., 1999
) as 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. There were no visible flagella.
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In-vitro culture of embryos to the blastocyst stage
In-vitro culture of spermatid-derived embryos was similar to that of sperm-derived embryos (Balaban et al., 1998; Gardner et al., 1998
). A sequential media system (S1 and S2 media from Scandinavian Science AB Products) 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 (G1): no fragmentation with equal sized homogeneous blastomeres; grade 2 (G2): <20% fragmentation with equal sized homogeneous blastomeres; grade 3 (G3): 2050% fragmentation with unequal sized blastomeres; grade 4 (G4): >50% fragmentation with unequal sized blastomeres. Blastocyst grading was according to a previous method (Dokras et al., 1993
): grade 1 (B1) characterized by early cavitation, resulting in the formation of an eccentric and then expanded cavity lined by a distinct inner cell mass region and trophoectoderm layer; grade 2 (B2) 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 (G3) with several degenerative foci in the inner cell mass with cells appearing dark and necrotic.
Embryo transfer
Embryos derived from intracytoplasmic injection of testicular spermatozoa were transferred 3 days after oocyte retrieval and supernumerary embryos were cryopreserved at the blastocyst stage. Embryos derived from ROSI, however, were not transferred and were observed in culture until they reached the blastocyst stage or for 8 days if blastocyst stage was not reached earlier. The decision not to transfer spermatid-derived embryos was based upon our earlier experience with testicular ROSI that yielded no clinical pregnancies in 33 TESE cycles.
Statistics
The rate of progression of spermatid-derived embryos to the blastocyst stage was compared with that of supernumerary embryos derived from intracytoplasmic injection of testicular spermatozoa. Paired Student's t-test, 2 and Fisher's exact test were used when applicable for data analysis. A P-value < 0.05 was considered as significant.
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Results |
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Discussion |
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Intracytoplasmic injection of round spermatids has been associated with a lower fertilization rate when compared with injection of spermatogenic cells at more advanced stages of maturation (Vanderzwalmen et al., 1998). This is especially true when round spermatids are derived from azoospermic men with complete spermatogenic arrest. Our study group comprised men having mature spermatozoa and spermatids after TESE. These subjects most probably represented a more favourable subset regarding fertilization success. Round spermatids isolated from testes with incomplete or defective spermiogenesis may not be identical to those obtained from testes with complete spermatogenic arrest. The absence of elongated spermatids in all tubules, which negatively affects the quality of spermatid maturation, may have a genetic cause (Vanderzwalmen et al., 1998
). It was suggested (Tesarik et al., 1998a
) that spermatids in patients lacking spermatozoa, i.e. patients with complete spermiogenesis failure, are often deficient in the factors responsible for oocyte activation. This deficiency may result in abnormal fertilization patterns and impairment of further preimplantation and postimplantation development. Furthermore, patients with complete spermiogenesis failure have an unusually high frequency of spermatids undergoing apoptosis and consequent DNA degradation (Tesarik et al., 1998b
). In a small series of patients, Vanderzwalmen et al. injected oocytes with round spermatids from men with normal spermatogenesis, partial spermatogenesis failure or complete spermatogenesis failure. Two pronuclei fertilization rates were 42, 27 and 8% respectively (Vanderzwalmen et al., 1998
). Fishel et al. compared fertilization rate in sibling oocytes injected with testicular spermatozoa or testicular round or elongated spermatids (Fishel et al., 1997
). The fertilization rate was 24% for spermatids and 79% for testicular spermatozoa. Similarly, it was reported (Kahraman et al., 1998
) that a low fertilization rate of 25.6% was obtained with round spermatids. In our series, 2PN fertilization rate with ROSI was 50%. This is significantly lower than the 73.9% fertilization rate obtained with the injection of testicular spermatozoa.
It is not clear whether spermatid-derived embryos are endowed with the potential to progress to further stages of embryonic development. The recent introduction of blastocyst culture has enabled us to observe the in-vitro development of embryos to the advanced stages of cleavage. The scarcity of pregnancies reported after ROSI is most probably due to the lack of or inadequate progression of these embryos to the blastocyst stage. Our results prove that this is indeed so. Not only did spermatid-derived embryos progress to the blastocyst stage at a much lower and slower rate, but none showed spontaneous hatching in vitro. In conclusion, embryos derived from ROSI are impaired in their ability to reach advanced stages of embryonic growth. This appears to be responsible for the dismal results reported with the utilization of this technique.
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Notes |
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References |
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Submitted on October 25, 1999; accepted on March 6, 2000.