1 Women's General Hospital, IVF-Unit, Lederergasse 47, A-4010 Linz, Austria
2 To whom correspondence should be addressed. Email: thomas.ebner{at}gespag.at
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Abstract |
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Key words: cytoplasmic maturation/fertilization failure/mitochondria/mitochondrial membrane potential/oocyte activation
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Introduction |
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Two steps are considered essential for successful fertilization, namely immobilization of the spermatozoon and rupture of the oolemma (Svalander et al., 1995; Van den Bergh et al., 1995
; Vanderzwalmen et al., 1996
). The mechanism of human oocyte activation during ICSI is not exactly known (Edwards and Van Steirteghem, 1993
). However, a sperm-associated factor is likely to be responsible for initiation of egg activation in ICSI (Dozortsev et al., 1995
, 1997
; Parrington et al., 1996
) apart from a parthenogenetic effect caused by the injection procedure per se (Winston et al., 1991
; De Sutter et al., 1992
). In addition, a certain oocytesperm interaction, e.g. ooplasmic factors triggering sperm chromatin decondensation (Tesarik and Kopecny, 1989
; Van Blerkom et al., 1994
; Dozortsev et al., 1997
), is required.
The complexity of the fertilization process may explain its susceptibility to disturbances which may cause complete fertilization failure in spite of the presence of a presumably normal spermatozoon (Sousa and Tesarik, 1994; Dozortsev et al., 1995
; Ludwig et al., 1999
). The frequency of total fertilization failure cycles was reported to be
13% (Liu et al., 1995
; Moomjy et al., 1998
; Ludwig et al., 1999
), most of them being the result of impaired semen characteristics or very low oocyte numbers. In such cases, repeated ICSI treatment proved useful (Liu et al., 1995
; Moomjy et al., 1998
); however, some patients will have to face repeated fertilization failure in spite of normal sperm parameters and good ovarian response (Moomjy et al., 1998
; Tesarik et al., 2002
).
In order to rescue such cycles, Tesarik et al. (2002) reported a modified ICSI technique mainly based on a repeated dislocation of the central ooplasm to the periphery, thus increasing the intracellular concentration of free calcium either by creating an influx of calcium ions (Tesarik and Sousa, 1995
) or by the release of calcium stored in cell organelles.
Taking into account a possible negative effect of this rather vigorous injection technique on further preimplantation development (Dumoulin et al., 2001; Eichenlaub-Ritter et al., 2002
), we developed a modified ICSI (mICSI), which is based on the hypothetical accumulation of highly polarized mitochondria, e.g. showing a high inner mitochondrial membrane potential (Van Blerkom et al., 2002
), from pericortical regions (9 o'clock) to the centre of the oocyte, thus, theoretically, supplying more energy (ATP) directly to the place where the spermatozoon was injected. In this regard, it appeared that aggregation patterns of mitochondria correspond well to the light microscopic appearance of the oocyte (Wilding et al., 2001
).
Encouraged by promising preliminary results, we prospectively applied this mICSI approach to patients with previous fertilization failure after ICSI. In parallel, a possible application as a routine method has been evaluated comparing conventional ICSI with the modified technique in sibling oocytes. Once the equivalence of both methods in terms of fertilization and preimplantation development had been established, a randomized prospective study of patients was set up to evaluate the actual influence of mICSI on pregnancy and implantation behaviour.
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Material and methods |
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In the first phase of the study, 14 patients (32.2±4.4 years) having had no fertilization with conventional ICSI in 17 previous cycles were treated with a modified injection technique. Total fertilization failure was not due to low response (>3 oocytes) or severe male factor infertility (>5 x 106/ml).
In the second phase, ICSI was applied on sibling oocytes of 24 consecutive patients (33.0±4.6 years) who showed a good response to controlled ovarian stimulation. Approximately half of the metaphase II (MII) oocytes of each patient were injected using conventional ICSI and half of the gametes using the new technique.
In the last phase, 92 ICSI cycles were allocated alternatively to the conventional ICSI group or the mICSI group. All ICSI patients met our inclusion criteria, which were female age <40 years, additional tubal or unexplained infertility and fewer than four previous stimulated cycles.
All 127 patients presented at our clinic in 2003 and were stimulated with either a long protocol (n=55) using buserelin (Suprecur®; Aventis Pharma, Vienna, Austria) and HMG (Menogon®; Ferring, Kiel, Germany) or an antagonist protocol (n=72) with administration of recombinant FSH (Puregon®; Organon, Vienna, Austria) and GnRH antagonist (Orgalutran®; Organon, Vienna, Austria). In all patients, ovulation was induced with 500010 000 IU of HCG (Pregnyl®; Organon, Vienna, Austria) provided that lead follicles and serum estradiol appeared adequate. Oocyte retrieval was carried out transvaginally under ultrasound guidance 36 h after ovulation induction.
After careful denudation of the oocytes and identification of the mature oocytes, conventional ICSI was performed as previously described (Ebner et al., 2001). In detail, the first polar body was kept at the 6 o'clock position while the injection needle passed the zona pellucida and the oolemma at the 3 o'clock position. Once the centre of the oocyte was reached, aspiration of a small amount of ooplasm was done followed by central deposition of the mechanically immobilized spermatozoon.
Since the mICSI aimed to accumulate peripheral mitochondria of high inner mitochondrial membrane potential to the region of pronuclear formation, the needle tip was pushed close to the membrane opposite the puncture site (9 o'clock position) where careful aspiration of cytoplasm was performed (Figure 1). The peripheral region bearing mitochondria with a high inner mitochondrial membrane potential could be located easily since aggregation patterns of mitochondria corresponded well to the light microscopic appearance of the oocyte (Wilding et al., 2001). The degree of aspiration was comparable with conventional ICSI. If a certain response of the opposite membrane was noted during aspiration, e.g. a slight invagination (Figure 2), the aspiration process was stopped immediately and the sperm was released in the centre of the female gamete. Special care was taken to avoid lateral deviations from the original injection channel.
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On day 2, embryos were checked for the number and size of blastomeres as well as for the percentage of fragmentation. Culture (10 µl per embryo) was prolonged in Blastassist System Medium 2 (MediCult) until day 3 or 5. If day 5 transfer was considered, the extension and quality of blastocysts were checked according to our routine method (Ebner et al., 2003). It has to be kept in mind that the number of blastocysts checked for quality differs from the total number of blastocysts since some early blastocysts did not show a clearly distinguishable inner cell mass and trophectoderm.
Summarizing all three phases of the present study, transfer was done on either one day 3 (n=73) or day 5 (n=54), using a small volume of Blastassist System Medium 2 in an EdwardsWallace Catheter (Smiths Industries, Lancing, UK). To support the luteal phase, 3000 IU of HCG (Pregnyl®; Organon, Vienna, Austria) were injected on day 2, day 5 and day 8. The clinical pregnancy rate and implantation rate were calculated using pooled day 3 and day 5 results. The ratio of day 3/day 5 transfers did not vary in both cohorts. Nineteen days after oocyte collection, the blood concentration of HCG was measured to exclude biochemical pregnancies (<20 mU/ml). Clinical pregnancy was defined by transvaginal ultrasound visualization of a gestational sac with positive heart activity 4 weeks after embryo transfer. Subclinical pregnancy revealed no fetal heartbeat. The implantation rate was determined by ultrasound visualization of a gestational sac per transferred embryo (subclinical pregnancies were included).
Comparison between the two ICSI cohorts was performed using 2 and MannWhitney U hypothesis tests. Statistical significance was defined as P<0.05.
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Results |
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During the second phase of our study which dealt with sibling oocytes (n=277), an overall fertilization rate of 74% (205 out of 277) was seen, 67 (32.7%) of the zygotes showing optimal pronuclear patterns 0. After first cleavages had occurred, 25.4% (52 out of 205) of all embryos were found to have no signs of fragmentation. Overall, 179 cleaved embryos were considered for blastocyst culture, but only 99 (55.3%) reached this stage on day 5. Approximately half (31 out of 70) of the blastocysts showed an inner cell mass of optimal morphology. Table I indicates that both methods, i.e. conventional ICSI and mICSI, trying to concentrate peripheral mitochondria in the centre of the oocyte, are comparable in outcome (P>0.05).
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Discussion |
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In order to evaluate a possible correlation between the respiratory activity of mitochondria in oocytes and the corresponding preimplantation development, two main approaches have been considered: (i) assessment of intracellular ATP content (Van Blerkom et al., 1995, 2000
); and (ii) indirect assessment of mitochondrial activity measuring the inner mitochondrial membrane potential (Wilding et al., 2001
, 2002
; Van Blerkom et al., 2002
, 2003
).
Wilding et al. (2001) described two different mitochondrial aggregation patterns in MII oocytes, one granular aggregation pattern restricted to the periphery (type A), and a smoother pattern found towards the centre of the gamete (type B). Interestingly, both patterns were characterized by similar inner mitochondrial membrane potentials.
On the other hand, Van Blerkom et al. (2002) found mitochondria in oocytes to be heterogeneous with respect to the inner mitochondrial membrane potential and described those organelles considered to be highly polarized, and thus being of high metabolic ATP activity, to be located along the pericortical region. These mitochondria may maintain adequate ATP levels in the subplasmalemmal zone while other mitochondria move to the centre of the ooplasm during the fertilization process (Van Blerkom et al., 2003
) and/or play a key role in Ca2 + regulation at oocyte activation (Ozil and Huneau, 2001
).
The reported clustering of highly polarized mitochondria in subplasmalemmal regions led us to try an accumulation of such peripheral organelles close to the spermatozoon during injection in cases of complete fertilization failure after ICSI. MII oocytes from such patients are thought to have a significantly lower mitochondrial DNA copy number compared with gametes with a normal rate of fertilization (Reynier et al., 2001). This metabolic dilemma is possibly the result of cytoplasmic maturational incompetence (Barritt et al., 2002
). Theoretically, during maturation, transition from the more peripheral mitochondrial aggregation type A (Wilding et al., 2001
), which is found in primary oocytes (Sathananthan and Trounson, 2000
) and fresh (time from ovulation induction) MII oocytes (Wilding et al., 2001
), to the more equal distribution of mitochondria in aggregation type B cannot occur. This would lead to the dilemma that an insufficient number of mitochondria is present in the centre of the gamete.
Another hypothesis would be that minor disturbances in the cytoskeleton at maturation may indirectly affect mitochondrial clustering, which could result in mitochondrial microdomains of impaired function (Eichenlaub-Ritter et al., 2004).
Provided that oocytes from patients with complete fertilization failure after ICSI produce such affected oocytes in subsequent cycles, mICSI would ensure not only the presence of a higher number of mitochondria at the site of pronuclear formation but also the presence of mitochondria of high metabolic ATP activity, both of which are thought to promote fertilization and preimplantation development (Wilding et al., 2001; Barritt et al., 2002
).
However, it has to be clearly stated that this work does not offer evidence that peripheral mitochondria are actually removed to the centre at all. Nevertheless, considering previous data (Van Blerkom et al., 1998), it is quite within the bounds of probability. Specifically, the feasibility of mitochondrial transfer between two oocytes could be demonstrated; furthermore, the persistence of activity in the transferred organelles (e.g. ATP production) was found to be maintained. Maintainance of the biological function of the removed mitochondria is strongly supported by several pregnancies which were achieved after cytoplasmic transfer (Cohen et al., 1998
; Dale et al., 2001
). Consequently, if dislocation of mitochondria works at a distance, it seems highly likely that it will also work within an oocyte. The final proof of the presence of active mitochondria around the spermatozoon would require a separate study, which is, unfortunately, not possible in Austria due to the restictive legislation in place.
In addition to the presumed increase in ATP supply, this new injection technique is likely to enrich intracellular free calcium ions compared with standard ICSI. Similar to the injection technique of Tesarik et al. (2002), the manipulation (e.g. aspiration of the ooplasm, repeated movement of the pipette) during our mICSI will cause an influx of calcium from the surrounding medium (Tesarik and Sousa, 1995
). In addition, calcium stored in the endoplasmic reticulum will be set free due to mechanical damage of this organelle. Enhancement of intracellular calcium ions will positively affect trigger components of calcium-dependent oocyte activation, theoretically alleviating any impairment of the oscillatory component (Tesarik et al., 2002
).
However, it must be emphasized that the positive effect of our mICSI that could be shown in cases of previous fertilization failure after standard ICSI could not be demonstrated in cases without this problem since the fertilization rate and further development were comparable. This implies that a minimum baseline of functionally active mitochondria must have been present in oocytes without impaired fertilizability, as suggested by Barritt et al. (2002). One additional reason would be the patient selection criteria applied for the second and third part of the study with the female range not exceeding an age of 40 years, since it is well documented that mitochondrial activity is negatively correlated to maternal age (Wilding et al., 2001
).
To conclude, mICSI which possibly accumulates mitochondria with a higher inner mitochondrial membrane potential proved to be a reliable and safe alternative to conventional ICSI as could be shown by comparable rates of blastocyst formation, implantation and clinical pregnancy. In particular, the present technology proved useful in terms of previous failure of fertilization in ICSI patients.
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Submitted on January 27, 2004; accepted on April 27, 2004.