Womens General Hospital, IVF-Unit, Linz, Upper Austria, Austria
1 To whom correspondence should be addressed at: Womens General Hospital, IVF Unit, Lederergasse 47, A-4010 Linz, Upper Austria, Austria. e-mail: Thomas.ebner{at}gespag.at
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Abstract |
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Key words: blastocyst formation/cytoplasm viscosity/injection funnel/oocyte morphology/zygote morphology
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Introduction |
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Cytoplasmic maturation involves numerous metabolic and structural modifications in preparation for subsequent fertilization (Eppig et al., 1994). Since enucleated oocytes show similar maturation-associated changes in protein synthesis as their nucleated counterparts (Sun and Moor, 1991
) it is likely that cytoplasmic maturation is, at least in part, under cytoplasmic control (Eppig, 1996
).
Deficient cytoplasmic maturity may be reflected by certain cytoplasmic abnormalities (e.g. cytoplasmic inclusions, vacuoles, smooth endoplasmic reticulum clustering) visible at light microscope level. In contrast with conventional IVF (Bedford and Kim, 1993) cytoplasmic dysmorphism is unlikely to have any impact on fertilization rate and embryo quality in ICSI patients (De Sutter et al., 1996
; Balaban et al., 1998
; Ebner et al., 2000
) though ICSI of dysmorphic gametes can result in embryos with reduced developmental capacity (Alikani et al., 1995
; Serhal et al., 1997
).
In addition to cytoplasmic inclusions, apparent differences in cytoplasmic texture and density have been described (Kahraman et al., 2000; Meriano et al., 2001
). Extensive cytoplasmic granularity may either be homogeneous, affecting the whole gamete, or centrally located. The latter was found to be negatively correlated with ongoing pregnancy rate (Kahraman et al., 2000
). In contrast, slight or moderate granularity has been accepted as a normal feature of oocytes. It is conceivable that an increased viscosity of the cytoplasm may constrain cell organelles and/or pronuclei in their movement preventing the zygote from achieving alignment of both pronuclei or alignment of pronuclei with respect to the polar bodies, thereby severely impairing polarity and further preimplantation development (Edwards and Beard, 1997
; Garello et al., 1999
).
Though it has been reported that granular areas are more viscous than the surrounding cytoplasm (Payne et al., 1997) there is a lack of markers of increased cytoplasmic viscosity. However, everyday laboratory work reveals that not all oocytes are able to restore their spherical shape immediately after injection of a spermatozoon. This phenomenon is possibly due to a decreased fluidity of the cytoplasm, which may result in a reduced intracellular pressure. Therefore, this prospective study was set up to investigate the developmental fate of oocytes with impaired fluidity of the cytoplasm as assessed by the persistence of the injection funnel after withdrawal of the ICSI pipette.
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Materials and methods |
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In preparation for oocyte collection COH was conducted using either a long protocol (n = 39) or an antagonist protocol (n = 98). In the long protocol down-regulation of the pituitary was achieved with the GnRH agonist buserelin (Suprecur®; Aventis Pharma, Vienna, Austria). Stimulation was initiated with hMG (Menogon®; Ferring, Kiel, Germany).
In the GnRH antagonist protocol recombinant FSH (Puregon®; Organon, Vienna, Austria) was started on day 2 of the cycle. In addition, a GnRH antagonist (Orgalutran®; Organon) was administered after 56 days of stimulation, depending on the presence of a 1213 mm follicle in the ultrasound scan.
In all patients ovulation was induced with 500010 000 IU hCG (Pregnyl®; Organon) provided that the lead follicle reached a diameter of 19 mm and serum estradiol appeared adequate. Oocyte retrieval was carried out transvaginally under ultrasound guidance 36 h after hCG administration.
After incubation for 24 h (BM1 medium; NMS Bio-Medical, Switzerland) brief exposure to 80 IU/ml hyaluronidase (MediCult, Copenhagen, Denmark) facilitated mechanical removal of the cumulus cells. Following this procedure all ICSI procedures were carried out by the same embryologist according to our previously published guidelines (Ebner et al., 2001) with mechanical immobilization of the spermatozoa (x400 magnification) and the polar body being held at the 6 oclock position. Documentation was carried out using imaging and archival software (Octax Eyeware®; MTG, Altdorf, Germany), thus minimizing inter-individual differences in scoring.
Immediately prior to injection oocytes showing vacuoles or dark central granulation of the cytoplasm were separated from the others in order to minimize the possible influence of severe forms of cytoplasmic anomalies on ICSI outcome.
The remaining metaphase II (MII) oocytes showed no to moderate granulation with or without minor inclusions. Due to our selection criteria only MII oocytes showing normal response to the injection pipette with a resultant characteristic funnel prior to oolemma breakage were included in our study (Palermo et al., 1996; Ebner et al., 2002
). Within 23 min after injection and withdrawal of the glass pipette mature oocytes were assessed for the presence of an injection funnel (Figure 1). To confirm persistence of the funnel the oocytes were rotated by means of the holding pipette to detect an invagination on the surface of the gametes. According to this behaviour oocytes were cultured in two different groups (BM1 medium).
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Early embryo development (number of blastomeres and percentage of fragmentation) was evaluated 4244 h after injection. From day 2 onwards, the first sequential medium was replaced by Blastassist System Medium 2 (MediCult) which was changed daily until day of transfer (day 3 or 5). Blastocyst quality on day 5 was assessed according to the degree of expansion and morphological criteria. In detail, good quality blastocysts were characterized by a tightly packed inner cell mass (ICM) and the formation of a cohesive trophectoderm (TE).
Transfer was either done on day 3 (n = 94) or on day 5 at the blastocyst stage (n = 42). In one patient, no blastocysts formed in culture and so transfer was cancelled. No priority was given to transferring embryos or blastocysts that derived from oocytes with less viscous cytoplasm. Selection in terms of transfer was exclusively based on morphological assessment. Consequently, approval of our Institutional Review Board was not sought.
All data of this prospective investigation were compared using 2-test, t-test, and MannWhitney U-test. Significance was defined as P < 0.05.
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Results |
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Approximately one-fifth (26/137) of all patients had no oocytes with a persistent funnel after withdrawal of the injection pipette, whereas nine (6.6%) exclusively showed such gametes. In a total of 355 oocytes the injection funnel did not vanish within 23 min (35.2% of all MII oocytes). There was no relation to stimulation protocol (P > 0.05) since frequency of a persistent funnel in the long protocol (36.9%) did not differ from that in the antagonist protocol (34.5%). Female age showed no correlation with funnel persistence.
Table I compares the outcome of the ICSI procedure on day 1 in funnel positive oocytes (group 1) with that in funnel negative ones (group 2). No differences in fertilization rate could be observed between oocytes with persistent injection funnel and oocytes without this feature. The latter group showed a significant increase in degeneration rate after ICSI when compared to their counterparts (P < 0.05).
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Discussion |
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Intracellular pressure and ooplasm fluidity can be estimated by the extent to which the ooplasm rises within the injection pipette immediately following penetration of the oolemma prior to aspiration of cytoplasm. In addition, ooplasm of higher viscosity is more likely to adhere to the spike of the injection pipette which could be seen in a limited number (0.5%) of injections after withdrawal of the glass tool (Ebner et al., 2001
). However, these observations are strictly empirical and cannot serve as prognostic markers. Persistence of the injection funnel after ICSI reflects a deficiency in cytoplasmic texture. Flux characteristics of cytoplasm are altered in the more viscous type which does not allow the oocyte to restore its original spherical shape as fast as seen in gametes with a more aqueous ooplasm.
The injection funnel is likely to be responsible for sealing the breach during injection (Kimura and Yanagimachi, 1995; Palermo et al., 1996
). The absence of such a protective mechanism in oocytes with sudden breakage (without any injection funnel at all) causes an increase in oocyte degeneration (Palermo et al., 1996
; Ebner et al., 2001
). Since all oocytes involved in this prospective study showed an injection funnel during ICSI, the observed difference in degeneration rate (Table I) may be explained by a difference in cytoplasm fluidity. In other words, increased viscosity of the cytoplasm (persistent funnel) may keep it from leakage, whereas oocytes with more aqueous cytoplasm may tend to leak more frequently after ICSI.
Our data suggest that overall fertilization rate is not influenced by cytoplasmic texture in ICSI. However, since differences in ooplasm viscosity have been observed (Payne et al., 1997) it is possible that alterations in viscosity may delay abuttal of pronuclei by severely impairing microtubule organization. In contrast, a more fluid cytoplasm would promote optimal conditions in preparation for further preimplantation development.
Since there is some evidence that pronuclear alignment and alignment of nucleoli at the site of pronuclear contact are time-dependent (Tesarik and Kopecny, 1989; Payne et al., 1997
), this may have been manifested by a higher frequency of pronuclear patterns of good prognosis (Tesarik and Greco, 1999
; Montag and Van der Ven, 2001
) in group 2 (Table II). This finding is consistent with data from literature showing higher proportions of optimal pronuclear patterns in ICSI zygotes compared with IVF zygotes (Montag and Van der Ven, 2001
) since direct placement of one single spermatozoon will shorten the fertilization process (Nagy et al., 1998
). Our results with respect to pronuclear patterns suggest further subdivision of pattern 0 (Tesarik and Greco, 1999
) as recommended by Montag and Van der Ven (2001
).
Additional evidence supporting the hypothesis that increased viscosity hinders optimized cellular processes may be gained from the observation that significantly more zygotes showed a cytoplasmic flare in group 2 (Table II). This phenomenon is called cytoplasmic halo and is the manifestation of a microtubule-mediated withdrawal of mitochondria and other cytoplasmic components to the perinuclear region (Scott and Smith, 1998). Oocytes with affected cytoplasm did not show this positive prognostic feature in terms of developmental competence as frequently as did oocytes with unaffected cytoplasm. It may be speculated that suboptimal cytoplasmic texture compromizes microtubule-organized concentration of mitochondria to perinuclear regions and thus impairs cell cycle regulation severely.
According to the literature, our data suggest that optimal pronuclear patterns (0A, 0B) on day 1 are closely related to embryo morphology on day 2 (Balaban et al., 2001; Montag and Van der Ven, 2001
; Salumets et al., 2001
). Probably due to a dramatic increase in the rate of poor quality embryos (P < 0.01) the mean percentage of fragmentation was significantly higher in embryos from funnel positive oocytes (P < 0.05, Table III). One possible explanation for an increase in fragmentation might be that frequency and periodicity of cytoplasmic waves, which are known to be correlated to embryo quality (Payne et al., 1997
), may have been affected by a higher cytoplasmic viscosity resulting in embryos of lower quality.
However, the similar blastocyst formation rates seen in both groups indicate that embryo morphology on day 2 or 3 may be an inappropriate prognostic factor in some patients (Rijnders and Jansen, 1998). Since approximately half of the embryos reached blastocyst stage in both cohorts it appears that embryos derived from oocytes with persistent funnel after ICSI may overcome the developmental delay found in earlier stages.
However, when considering exclusively those blastocysts which extended beyond the early blastocyst stage and showed a distinct ICM consisting of many cells and a cohesive trophectoderm, a significant difference between both groups could be observed (P < 0.05). In detail, two thirds were top quality blastocysts in the group 2 whereas <50% of the affected embryos formed optimal blastocysts in vitro. This benefit is reflected by an significant increase in clinical pregnancy rate in patients who had homogeneous transfers of embryos or blastocysts that developed from oocytes without a persistent funnel compared with those who had the opposite concepti transferred.
To conclude, our data indicate that MII oocytes with an injection funnel persisting for some time after ICSI are likely to represent a cohort of gametes with an intrinsic ooplasmic defect having a negative influence on developmental competence. In our prospective study, we found numerous indications of an involvement of cytoplasmic viscosity in preimplantation development throughout the time of in-vitro culture. However, this developmental disadvantage could be compensated for during subsequent cleavages as cleavage rate and blastocyst formation were not affected. Nevertheless, in the present study blastocyst quality as well as clinical pregnancy rates indicate that information regarding developmental competence is gained during the oocyte or zygote stage rather than at the cleavage stages.
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References |
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Submitted on November 15, 2002; accepted on February 7, 2003.