Reproductive Medicine, BioScience Centre, International Centre for Life, Newcastle upon Tyne NE1 4EP, UK
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Abstract |
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Key words: blastocyst formation/ICSI/IVF/oocyte/spermatozoa
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Introduction |
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It is not known whether the reduced development to the blastocyst stage reported by Shoukir et al. 1998 was unique to the system of co-culture used in that study or whether it might be a consequence of the ICSI procedure itself, or of a patient effect. The possibility of a paternal influence was favoured by Shoukir et al. (1998), who suggested that the reduced blastocyst formation might be a consequence of centrosomal or genetic defects in the spermatozoa of infertile men. While it has been reported that the incidence of sperm aneuploidy is higher in men with abnormal semen parameters compared with normal controls (Preffer et al., 1999; Colombero et al., 1999
), the influence of this on embryonic development is unclear. It was reported (Colombero et al., 1999
) that the increased incidence of aneuploidy in men with oligoteratozoospermia did not influence the incidence of miscarriage or neonatal malformation. Furthermore, Munné et al. (1998) showed that ICSI embryos do not have a higher incidence of numerical chromosomal abnormalities than embryos from conventional IVF. Thus, the significance of sperm aneuploidy as a causative factor in impaired embryonic development following ICSI remains to be established.
The ICSI procedure itself may be detrimental to embryonic development. During normal (unassisted) fertilization, the fertilizing spermatozoon loses its acrosome while penetrating the zona pellucida. The sperm membrane then fuses with the oolemma, and triggers a series of transient increases in cytoplasmic Ca2+ that initiate the cortical reaction and the completion of meiosis (reviewed by Schultz and Kopf, 1995). By contrast, when spermatozoa are injected directly into oocytes, the sperm acrosome and membrane may be intact, fusion of oocyte and sperm membranes does not occur, and the timing and pattern of Ca2+ transients may be altered (Tesarik et al., 1994; Tesarik and Sousa, 1994
). While the injection of intact spermatozoa does not appear to reduce fertilization rates in humans (Liu et al., 1994
) or embryonic development in mice (Ahmadi and Ng, 1997
), the possible effects of altered dynamics of the sperm-induced Ca2+ transients or other ICSI-specific phenomena on developmental competence of ICSI embryos remain to be comprehensively investigated. Furthermore, procedures such as cumulus cell removal, penetration of the oolemma, injection of medium and deposition of spermatozoa in the cytoplasm may have a negative effect on the developmental capacity of human oocytes.
The aims of this study were (i) to compare blastocyst formation of embryos following IVF for tubal infertility and ICSI for male infertility and (ii) to investigate the effect of the ICSI procedure in isolation by comparing blastocyst formation in an auto-controlled series of sibling oocytes subjected to IVF or ICSI with homologous spermatozoa. This latter series effectively controlled for all extrinsic factors.
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Materials and methods |
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Patient treatment
Ovarian stimulation was carried out using a long course gonadotrophin releasing hormone agonist (GnRHa) protocol, using intranasal spray (Suprefact®; Hoechst Marion Rousell Ltd, Denham, Middx, UK) in combination with gonadotrophins (Metrodin HP® or Gonal F®; Serono Laboratories, Welwyn Garden City, Herts, UK or Menogon®; Ferring Pharmaceuticals, Langley, Berks, UK) and HCG (Profasi®; Serono or Choragon®; Ferring). Intravaginally administered progesterone (Cyclogest®; Shire Pharmaceuticals Ltd, East Anton, Andover, Hants, UK) was used for luteal phase support. Oocytes were retrieved transvaginally under ultrasound guidance 3739 h post-HCG, and were placed in Earle's balanced salt solution (EBSS) supplemented with 10% human serum albumin (HSA) and pyruvate, in a 5% CO2 incubator at 37°C.
Sperm preparation
Semen samples were produced on the day of oocyte retrieval and motile spermatozoa obtained using discontinuous density gradient centrifugation. The sperm pellet was re-suspended in HEPES-buffered EBSS for ICSI or in bicarbonate-buffered EBSS for IVF, supplemented with 10% HSA and pyruvate.
ICSI and IVF procedures
For the ICSI procedure cumulus and corona radiata cells were removed 23 h after oocyte retrieval, by aspiration of the oocytes through glass pipettes (150200 µm inner diameter) in HEPES-buffered EBSS containing 80 IU hyaluronidase/ml (Type I-S; Sigma-Aldrich Company Ltd, Poole, Dorset, UK). Only those oocytes found to be at metaphase II were injected. Injections were performed at 4244 h post-HCG in HEPES buffered EBSS with pyruvate and 10% HSA, on a heated microscope stage (37°C). The injection and holding pipettes were made from 1 mm borosilicate glass capillaries (Clarke Electromedical Ltd, Reading, UK) using a pipette puller, microforge and beveller from Research Instruments Ltd (Penryn, Cornwall, UK). Sperm were immobilized in HEPES-buffered EBSS with 10% HSA, by breaking the tail with the shaft of the injection pipette. Oocytes were held in position by gentle suction on a holding pipette with the polar body in the 6 o'clock or 12 o'clock position. The immobilized spermatozoon was advanced to the tip of the injection pipette, which was then pushed through the zona at the 3 o'clock position. Oolemma puncture was confirmed by a sudden shift of ooplasm into the injection pipette, before depositing the spermatozoon. The IVF oocytes were inseminated with 300 000500 000 spermatozoa/ml at 41.544 h post-HCG. Oocytes from both IVF and ICSI were cultured individually in 100 µl droplets of EBSS containing 10% HSA and pyruvate for 1820 h when they were checked for fertilization. Normal fertilization was determined by the presence of two pronuclei (2PN) and two polar bodies. Normally fertilized zygotes were then transferred to EBSS containing 15% HSA and pyruvate.
Embryo transfer
Embryo transfer was performed on day 2, or day 3 of development (day of insemination = day 0). All embryos were scored for cell number and morphology. The criteria used for the morphology score were cytoplasmic fragmentation, clarity of the cytoplasm in the blastomeres and evenness of blastomere size. The morphology score was expressed as a percentage, with a perfect embryo scoring 100%. The highest scoring embryos were selected for replacement. In the case of the eight `split' cycles (series II), embryos for replacement were selected either from the IVF or ICSI cohorts, in compliance with the Human Fertilisation and Embryo Authority (HFEA) guidelines [HFEA directive: Annex A. Ref: CE (97)(4)] at the time of this study. On this basis four patients had IVF embryos replaced and four patients had ICSI embryos replaced. All the remaining embryos were then transferred to Dulbecco's modified Eagle's medium (DMEM): F12 supplemented with 2% Ultrosera G® (Life Technologies, Paisley, UK), and were examined on day 6 and 7 for blastocyst formation.
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Results |
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Discussion |
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Our results together with those of Shoukir et al. (1998) highlight an apparent paradox of in-vivo and in-vitro observations, since, despite the lower proportion of ICSI embryos developing to the blastocyst stage in vitro, the implantation rate was equivalent to that observed in the IVF group. One possible explanation for this is that the female partners of couples undergoing ICSI may be inherently more fertile, demonstrating higher receptivity than those undergoing IVF treatment. Thus, once fertilization is achieved with ICSI, the probability of implantation occurring may be higher than in IVF, which may in turn compensate for the reduced viability of ICSI embryos. Alternatively, there may be a true difference between in-vitro and in-vivo viability of ICSI embryos. It is possible that embryos are able to recover from the trauma of the ICSI procedure more readily in vivo than in vitro. It may be that stress induced by the ICSI procedure causes the secretion of factors into the culture medium that reduce developmental potential. However, if this were the case, one would have expected to see increased fragmentation and/or reduced cleavage rates in the early cleavage stages, and our analysis of embryo quality on day 2 showed no difference in the quality of IVF and ICSI embryos.
While the results reported here indicate a negative effect of the ICSI procedure on embryonic development, it is not clear exactly what aspect of the procedure is detrimental. A study of the effect of the mechanical aspects of ICSI on in-vitro matured bovine oocytes showed that insertion of a pipette and injection of culture medium alone had no effect on blastocyst formation (Motoishi et al., 1996). However, the significance of these findings for clinical ICSI, which typically involves the use of in-vivo matured oocytes, is unclear, since the membranes of in-vivo matured human oocytes are generally more elastic than those matured in vitro (personal observation). Observations of ICSI practice with in-vivo matured human oocytes indicate that the ease of oolemma penetration varies between oocytes because of differences in membrane elasticity (Nagy et al., 1995; Palermo et al., 1996
). Penetration of highly elastic membranes involves repeated stabbing with the pipette, strong cytoplasmic suction and severe distortion of the oocyte (Nagy et al., 1995; Palermo et al., 1996
). The extent of oocyte deformation and cytoplasmic suction required to rupture the membrane appears to be negatively correlated with morphology of cleavage stage embryos (Nagy et al., 1995). These effects could be overcome by the use of a piezo micropipette driving unit, which permits penetration of the oolemma without cytoplasmic suction, and with minimal distortion of oocytes (Kimura and Yanagimachi, 1995
). This approach has been reported to result in improved fertilization and pregnancy rates after ICSI (Yanagida et al., 1998
).
The site of pipette insertion is another variable that may affect the outcome of ICSI. The established practice is to orientate the oocyte on the holding pipette with the polar body in the 6 o'clock or 12 o'clock position and to insert the injection pipette at the 3 o'clock position, so as to minimize the chance of disrupting the meiotic spindle. Recent studies in hamster oocytes cast doubt on the logic of this, since it was found that the polar body position does not necessarily predict the location of the spindle (Silva et al., 1999). Observations on human oocytes also indicate displacement between the first polar body and the meiotic spindle (Payne et al., 1997
; Hewitson et al., 1999
) and it was suggested that this was due to movement of the polar body during the cumulus denuding process (Payne et al., 1997
; Hewitson et al., 1999
). Studies on the effect of site of spermatozoa deposition in relation to the polar body position indicate an influence on fertilization rates (Van der Westerlaken et al., 1999
) and embryo quality (Nagy et al., 1995; Blake et al., 2000
). However, these studies did not provide consensus on the optimal orientation. Investigation of the possible use of polarized optics (Silva et al., 1999
) to enable visualization of the spindle during ICSI is warranted.
Biological differences between the process of fertilization during ICSI and conventional IVF may contribute to the reduced blastocyst formation of ICSI embryos. By performing ICSI we override the early events of fertilization, such as acrosome loss and spermatozoa-oocyte membrane fusion. The injection of intact spermatozoa was found not to be detrimental to fertilization of human oocytes (Liu et al., 1994) or to embryonic development of mouse zygotes (Lacham-Kaplan and Trounson, 1995
; Ahmadi and Ng, 1997
). Curiously, the injection of acrosome-intact spermatozoa was associated with reduced fertilization of mouse oocytes (Lacham-Kaplan and Trounson, 1995
) but this was not substantiated by other work (Ahmadi and Ng, 1997
). While the evidence suggests no negative effect on embryonic development due to the presence of intact sperm membranes, differences in the membrane structure of zygotes arising from IVF and ICSI may have physiological significance. Zygotes obtained by conventional fertilization have a mosaic membrane structure resulting from the incorporation of the fusing sperm membrane and the cortical granules with the oolemma (Shapiro et al., 1981
; Wolf and Ziomek, 1983
). The incorporation of the sperm membrane into the oolemma was found to have a role in the inhibition of polyspermy that was independent of cortical granule release (Sengoku et al., 1999
). The physiological significance of oocyte membrane restructuring at the time of spermatozoaoocyte fusion may not be confined to the early post-fertilization stages of embryonic development. It may also have a role in the later stages of preimplantation development, for example in the formation of intercellular junctions at the time of compaction (Collins and Fleming, 1995
). These possibilities remain to be investigated.
Differences in the pattern of sperm-induced Ca2+ oscillations could also account for the reduced developmental competence of ICSI embryos. Ozil (1990) demonstrated an association between the pattern of Ca2+ signalling at the time of rabbit oocyte activation and the ability to undergo subsequent blastocyst formation (Ozil, 1990), and there is some evidence that the pattern of Ca2+ signalling after ICSI differs from that seen after normal spermatozoaegg fusion (Tesarik and Sousa, 1994
; Tesarik et al., 1994
). Tesarik and Sousa (1994) measured Ca2+ responses in human oocytes subjected to ICSI or subzonal insemination (SUZI). Oocytes that fertilized after SUZI showed a uniform Ca2+ response consisting of an initial rapid series of 36 oscillations (frequency <1 min) followed by oscillations of lower frequency. By contrast, oocytes that fertilized after ICSI showed a variety of Ca2+ responses (Tesarik and Sousa, 1994
; Tesarik et al., 1994
). Half of them did not have oscillations but underwent instead a prolonged Ca2+ increase. The remainder had Ca2+ oscillations but without the initial series of rapid transients seen in SUZI oocytes. Given the evidence for a frequency/amplitude encoded influence of Ca2+ signalling on blastocyst formation (Ozil, 1990
), it is possible that the different dynamics of the Ca2+ responses observed after SUZI and ICSI may contribute to the lower blastocyst formation of ICSI embryos. However, a similar study of mouse oocytes did not detect any marked differences, apart from a delay in the onset of Ca2+ oscillations, between the pattern of oscillations seen after ICSI and conventional IVF (Nakano et al., 1997
).
In addition to its impairment of embryonic viability, ICSI resulted in fewer embryos per oocyte than IVF. The main clinical implication of these findings is that ICSI treatment should, where possible, be confined to cases where failure of fertilization is anticipated, on the grounds that embryonic viability is compromised and fewer embryos are available for cryopreservation. Taken together with the increased incidence of miscarriage after replacement of frozen/thawed ICSI embryos (Aytoz et al., 1999), the chances of benefit from an embryo cryopreservation programme are likely to be substantially lower with ICSI than with IVF.
In conclusion, this study shows that the ICSI procedure is detrimental to embryonic development in vitro. It is not clear whether this is due to damage incurred during the injection process, or to a negative effect of overriding or altering the physiological events of normal spermatozoaoocyte fusion.
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Acknowledgments |
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Notes |
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References |
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Submitted on December 30, 1999; accepted on April 10, 2000.