Assisted Reproduction Unit, American Hospital of Istanbul, Turkey
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Abstract |
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Key words: azoospermia/blastocysts/ICSI/testicular spermatozoa
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Introduction |
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The source and quality of spermatozoa utilized for intracytoplasmic injection differ tremendously. It is therefore not surprising that different outcomes in terms of fertilization, cleavage, blastocyst formation, implantation, and pregnancy were reported for male infertility of varying severity. In the light of recent studies, there appears to be a paternal effect on the implantation potential of the human embryo. Lower rates of blastocyst formation on feeder cells when impaired semen was used for IVF have been reported (Janny and Ménézo, 1994). Similarly it has been shown that embryos derived from men with poor semen parameters were of lower quality (Ron-El et al., 1991
).
Further extending the above observations, it has been shown that blastocysts were produced at a higher rate from spermatozoa that showed progressive motility (Shoukir et al., 1998). They also reported a higher blastocyst formation rate in IVF cycles compared with ICSI cycles, suggesting a paternal effect on embryo viability. However, in this study the authors used a single culture medium throughout the entire culture period that may account for the low rate of blastocyst formation in ICSI cycles. The progression of IVF embryos to the blastocyst stage has been compared with ICSI embryos (Dumoulin et al., 2000
). These workers demonstrated a lower rate of blastocyst formation in ICSI embryos regardless of the culture medium used or different culture conditions showed decreased fertilization and pregnancy rates in men with non-obstructive azoospermia when compared with men undergoing epididymal sperm aspiration for obstructive azoospermia (Palermo et al., 1999
). Spermatozoa from testicular biopsies fertilized 57% of the retrieved oocytes compared with 80.5% of the oocytes being fertilized with spermatozoa from non-obstructive cases (Palermo et al., 1999
). These findings collectively suggest that embryo cleavage, quality and implantation is paternally influenced. The severity of the spermatogenic defect appears to be the crucial factor, with more severe spermatogenic defect probably having the greatest impact.
A paternal effect on embryo development is most likely to manifest itself after embryonic genome activation, i.e. after the appearance of first paternal transcriptional products. Besides acting at the genetic level, negative paternal effect on embryo development can also be observed mediated by other components of the spermatozoa. Incomplete formation of the sperm aster due to a centrosome defect can lead to fertilization or cleavage failure (Asch et al., 1995). Unfavourable paternal influence on embryonic development and viability, therefore, should be expected in men with more severe spermatogenic disorders.
Blastocyst formation appears to be an indicator of embryo quality and viability. Unfavourable paternal influence affecting embryo quality may manifest itself clearly during the phase of extended embryo growth up to the blastocyst phase. We speculated that severity of the spermatogenic defect would hinder the potential of the cleavage stage embryo to develop into a blastocyst. Therefore we aimed to compare the rate of blastocyst formation and pregnancy in couples undergoing ICSI with ejaculated spermatozoa, epididymal spermatozoa, and testicular spermatozoa.
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Materials and methods |
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Ovarian stimulation, oocyte retrieval and embryo transfer procedures
In the majority of treatment cycles, ovarian stimulation was undertaken using s.c. buserelin acetate (Suprefact proinjection; Hoechst AG, Frankfurt am Main, Germany) in a long protocol combined with pure FSH (Metrodin, 75; I.F. Serono, Rome, Italy). Buserelin acetate (0.3 mg/day) was commenced on day 20 or 21 of the preceding cycle and continued until the day of human chorionic gonadotrophin (HCG). In cycles where the female was predicted to respond poorly to ovarian stimulation, a flare gonadotrophin-releasing hormone (GnRH) analogue protocol was used. FSH was initiated on the third day of the menstrual cycle with 26 ampoules depending on the patients' previous or anticipated response. The treatment was then individualized in a step-down fashion. When the leading follicle reached 20 mm in mean diameter with a serum oestradiol level of 200300 pg/ml per mature follicle, 10 000 U HCG (Profasi HP 5000; I.F. Serono) was administered. Oocyte retrieval was performed 36 h after the injection of HCG. ICSI was performed only on metaphase II oocytes. All available oocytes were injected with spermatozoa showing at least twitching motility. Embryo transfer was carried out 56 days after oocyte retrieval. A serum pregnancy test was performed 911 days after embryo transfer. Clinical pregnancy was defined as the presence of gestational sac(s) with a viable embryo shown on vaginal ultrasonography performed ~24 days after embryo transfer.
Testicular sperm aspiration and extraction procedures
Procedures were performed under general anaesthesia or local anaesthesia with i.v. sedation. All procedures were performed 2448 h prior to oocyte retrieval. In men with obstructive azoospermia, PESA was attempted first. The epididymis was secured between the thumb and index finger of the left hand while a 21 gauge butterfly needle was gently inserted into the epididymal body and aspiration was affected through the microtubing attached to a 20 ml syringe. If aspiration failed, PTSA was performed. This was undertaken with a 21 gauge butterfly needle that was inserted into the testes and moved up and down to sample a wide area. An artery forceps was secured across the attached microtubing set before the needle was withdrawn. The aspirate located in the tubing was washed into a Falcon tube with a small volume of media. The presence of spermatozoa was sought under x200 magnification. Percutaneous aspiration was attempted from three different areas of the testis and, if spermatozoa were not observed, TESE was performed. In men with non-obstructive azoospermia, PTSA was attempted first and TESE was performed if the former failed. Tissue samples measuring in size from 0.5x0.5x0.5 cm to 1x1x1 cm were removed until spermatozoa were identified or four or five biopsy pieces were extracted from each testis. Testicular tissue samples obtained in this manner were placed into Falcon tubes (Becton Dickinson, Franklin Lakes, NJ, USA) containing 2 ml of preincubated IVF-100 (IVF Science Scandinavia, Gothenburg, Sweden) medium. The samples were then transferred to Petri dishes (Falcon) containing 2 ml of the same preincubated medium. The tissue was crushed with sterile needles and subsequently with glass slides in order to separate the seminiferous tubules. The crushed tissue was then vortexed for a few seconds to facilitate the dispersal of spermatozoa into the medium. The presence of spermatozoa was sought under an inverted microscope at x20 magnification. The medium of the mixture was then treated with Pure Sperm (Nidacon International AB, Gothenburg, Sweden) gradient system. A two step washing procedure was then undertaken. The sample was first washed with Pure Sperm gradients of 90 and 50% at 500 g for 20 min. Subsequently washing was performed with 10 ml IVF-100 at 800-1000 g for another 10 min. The pellet formed was placed in the cover of a Petri dish, prepared as a swim-out droplet and covered with Ovoil 100 (IVF Science). The final solution was kept in the incubator until the ICSI procedure. ICSI was performed according to standard techniques previously described (Palermo et al., 1999). Spermatozoa from non-obstructive azoospermic subjects were co-incubated with recombinant FSH supplemented medium. This has been shown to increase the motility of testicular spermatozoa and induce motility in non-motile cells (Urman et al., 1998
; Balaban et al., 1999
).
In-vitro culture of embryos to the blastocyst stage and embryo grading
In-vitro culture of embryos was undertaken as previously described (Balaban et al., 1998; Schoolcraft et al., 1999
). All cycles, regardless of the number of oocytes collected, were programmed for blastocyst transfer. Sequential media system (G1 and G2 media; Scandinavian Science AB Products, Gothenburg, Sweden) designed for further embryonic development was used. Embryos were group-cultured in 4-well dishes until day 3 in G1 media. After the evaluation of the embryo cell number and morphology they were transferred to G2 medium for further culture up to the blastocyst stage. 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 previously published criteria (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.
Statistics
Numerical variables were compared using analysis of variance with Bonferroni post-hoc test. Categorical variables were compared using 2 or Fisher exact tests. P < 0.05 was accepted as significant.
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Results |
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Discussion |
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The above are in concordance with the results of previous studies. In one study (Parinaud et al. 1993) on the influence of sperm parameters on embryo quality, it was concluded that the spermatozoon was involved in embryo quality starting from the early stages of development; there was also an association between abnormal sperm morphology and poor embryo quality. Another study (Janny and Ménézo, 1994
) compared 334 IVF cycles performed with normal semen, cryopreserved donor semen, and abnormal fresh semen. All embryos obtained were co-cultured with Vero cells up to the blastocyst stage. The cleavage rate and blastocyst formation was highest in the group with normal fresh semen. Furthermore, 87.5% of the patients in the normal fresh semen group had at least one blastocyst available for transfer. Respective rates for patients with cryopreserved donor semen and fresh abnormal semen were 67.4 and 63.2%. The authors performed a regression analysis and found a linear correlation between cleavage rates and blastocyst formation. Finally a higher pregnancy rate was achieved in the group with normal fresh semen, although this did not reach statistical significance. The results of this study suggest a relationship between semen parameters and subsequent markers of embryo competence and viability.
In contrast to these studies, the paternal effect on implantation and pregnancy outcome in IVF/ICSI cycles was investigated (Oehninger et al., 1998). Although a poorer implantation and pregnancy outcome was noted in OAT versus normozoospermic subjects undergoing IVF, this was attributed to the high insemination concentration that was used. This conclusion was based on the findings of an earlier study undertaken by this group that showed higher implantation rates in teratozoospermic subjects treated with ICSI compared to IVF.
In a study of the development of blastocysts from supernumerary embryos after ICSI (Shoukir et al., 1998), the rate of blastocyst formation after ICSI (26.8%) was significantly lower than that after IVF (47.3%). This was partly affected by the higher quality and increased number of day 3 embryos in the IVF group. In this study, sperm concentration and morphology did not appear to influence blastocyst formation. However, in the ICSI group a higher rate of blastocyst formation was observed when spermatozoa from samples with higher progressive motility were used. More recently, the in-vitro development of embryos originating from either conventional in-vitro fertilization or ICSI was compared (Dumoulin et al., 2000
). Culture of surplus embryos derived from ICSI progressed to the blastocyst stage at a significantly lower rate when compared to embryos originating from IVF.
In a study of the factors affecting the success of blastocyst development following IVF (Jones et al., 1998), besides other parameters such as the number of retrieved oocytes, number of cleavage stage embryos, and embryo quality, male factor aetiology in the absence of identifiable female infertility significantly influenced the rate of blastocyst formation up to day 7 after insemination.
Fertilization, pregnancy, and abortion rates after ICSI of ejaculated and surgically retrieved spermatozoa have been compared (Ghazzawi et al., 1998). Fertilization and pregnancy rates were similar when spermatozoa from the ejaculate, epididymis, or testes were used. However, the abortion rate was significantly higher in the latter, suggesting a diminished developmental competence of testicular sperm-derived embryos.
The above studies collectively demonstrate that embryo development and implantation are under paternal influence. In accordance with the above, our results indicate that severity of the spermatogenic disorder is associated with the success of ICSI. Blastocyst formation that is commonly accepted as an indicator of embryo quality and viability is affected by the source of the spermatozoa utilized for ICSI. When spermatozoa are retrieved from the testes as in non-obstructive azoospermia, a lower rate of blastocyst formation and implantation per blastocyst should be anticipated.
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Notes |
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References |
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Submitted on July 21, 2000; accepted on October 11, 2000.