Infertility Centre, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium
1 To whom correspondence should be addressed. Email: bjorn.heindryckx{at}ugent.be
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Abstract |
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Key words: calcium ionophore/fertilization failure/heterologous ICSI/oocyte activation
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Introduction |
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Oocyte activation is characterized by a two-step pattern of rises in intracellular Ca2+ concentrations. A first Ca2+ rise (trigger) originates from the oocyte cortex after spermoocyte membrane interaction and is followed 30 min later by a series of shorter Ca2+ transients of high amplitude that continue for 34 h (oscillator) (Tesarik et al., 2000). The oscillator function is dependent on the release of a sperm-associated oocyte activation factor that conditions the oocyte to sustain repetitive Ca2+ releases from intracellular stores (Ben-Yosef and Shalgi, 2001
). This sperm factor is sensitive to heat, is not species-specific and can control the frequency of oscillations. The repetitive nature of this signal is essential for complete oocyte activation. Tesarik et al. (2002)
have also shown that not only a sperm factor but also an oocyte factor is involved in the activation oscillator mechanism. To distinguish between deficiency of the oocyte-activating capacity of sperm or the inability of oocytes to respond to penetrated sperm, a heterologous ICSI model can be used as a diagnostic tool (Rybouchkin et al., 1996
).
We applied heterologous ICSI of human sperm into mouse oocytes (mouse oocyte activation test, MOAT) for diagnosing sperm- or oocyte-related activation deficiencies in a group of 17 patients. AOA in a subsequent ICSI cycle was done by Ca2+-ionophore treatment. We were able to normalize fertilization rates after ICSI and to obtain pregnancies in cases of sperm- or oocyte-related activation deficiencies.
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Materials and methods |
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Heterologous ICSI or mouse oocyte activation test
B6D2 F1 hybrid female mice aged 714 weeks were induced to superovulate with 810 IU eCG (Folligon; Intervet, The Netherlands) followed by 810 IU of hCG (Chorulon, Intervet) 48 h later. Collection of metaphase II oocytes was done 1314 h post-hCG. Potassium-simplex optimized medium (KSOM) (Lawitts and Biggers, 1991) containing 0.2 mmol/l glucose and supplemented with 0.4% bovine serum albumin (BSA; MP Biomedicals, Belgium) was used as oocyte and embryo culture medium. Cumulusoocyte complexes were treated with 200 IU hyaluronidase (type VIII)/ml for 5 min to disperse the surrounding cumulus cells. A sharp needle was used to make a tangential slit in the zona pellucida at 12 o'clock (partial zona dissection, PZD). The chromosomespindle complex, visible as a translucent region in the ooplasm, was located at 34 o'clock to avoid the spindle complex being destroyed during injection later on.
Fresh or frozenthawed sperm from patients were washed twice by centrifugation at 1600 rpm for 10 min. Only motile sperm were selected for injection. For each MOAT, four experimental groups were set up: (i) injection with test-case sperm (30 oocytes); (ii) injection with donated sperm from a man with proven fertilization (positive control) (30 oocytes); (iii) sham injection of medium (negative control) (20 oocytes); (iv) non-manipulated oocytes to examine the occurrence of spontaneous parthenogenetic activation (medium control) (20 oocytes). Sperm were resuspended in injection medium consisting of KSOMHEPES and an equal volume of 8% polyvinylpyrrolidone (PVP; ICSI-100; Vitrolife Scandinavia) in a 5 µl central drop. Oocytes were put in microdrops consisting of KSOMHEPES supplemented with 20% fetal bovine serum (Gibco BRL; Invitrogen, Belgium). Injection was performed at 1517°C as described elsewhere (Rybouchkin et al., 1996). Motile sperm were immobilized by touching their tails with a blunt injection needle against the bottom of the dish. After deep invagination of the oocyte membrane with the injection needle (inner diameter 56 µm) through the slit made by PZD, the oolemma was broken by gentle suction. Cytoplasm was aspirated and then returned into the oocyte together with the spermatozoon. In the negative control group, sham injection of injection medium (KSOM-HEPES + PVP) was performed. Oocyte activation was assessed around 44 h post hCG by examining the percentage of 2-cell formation (number of 2-cells versus the number of surviving injected oocytes). As a standard, the positive control must show >90% 2-cell formation while both the negative control and medium control groups must show <10% 2-cell formation before MOAT results can be considered as reliable.
Assisted oocyte activation in clinical ICSI
The ionophore (ionomycin; cat. no. 159611, MP Biomedicals) was dissolved in cell culture-tested dimethylsulphoxide (DMSO; SigmaAldrich Chemie, Belgium) at a concentration of 1 mmol/l stock solution. The final solution containing 10 µmol/l ionophore was prepared just before ICSI by diluting the stock solution with Cook Cleavage medium (Cook Ireland Ltd, Ireland). For AOA, an immobilized sperm was first aspirated in the injection needle and brought to the tip of the needle. In a separate drop, 5 pl of 0.1 mol/l CaCl2 was aspirated and injected along with the spermatozoon in the ooplasm. Thirty minutes after injection, a first Ca2+-ionophore treatment was done during 10 min. Oocytes were then washed with Cook Cleavage medium and put in the incubator at 37°C in a 6% CO2 air atmosphere. After another 30 min, the Ca2+ ionophore treatment was repeated for 10 min. Oocytes were washed and placed in Cook Cleavage medium in the incubator. Oocytes were checked for pronuclei 1620 h after ICSI. Embryo transfer was carried out on day 2 or 3. Pregnancy was detected by measuring serum hCG on two independent occasions 15 days after embryo replacement. Clinical pregnancy was determined by observation of a gestational sac with fetal heartbeat on transvaginal ultrasound at 67 weeks of pregnancy.
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Results |
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Small or absent acrosomes
MOAT was performed in a case of male infertility where the sperm analysis revealed sperm with very small or absent acrosomes. Because the MOAT resulted in normal oocyte activation (95%), no AOA was proposed. In fact, normal fertilization was obtained by a routine ICSI procedure and two consecutive pregnancies were achieved after transfer of two good quality embryos.
Total fertilization failure
In two couples with a severe male factor no fertilization was obtained by routine ICSI. The MOAT showed defective oocyte activation capacity of the sperm (09%). AOA was done during five subsequent ICSI attempts in these two couples. Twenty-five of 32 mature oocytes showed normal fertilization (78%) but no pregnancy was established.
In another couple which had no oocytes fertilized in two ICSI attempts, the MOAT revealed a normal oocyte activation capacity of the patients' sperm (94% activation). With AOA in two consecutive cycles a normal fertilization rate was obtained (14 out of 19 oocytes, 74%) but pregnancy was not established.
In a fourth couple with failed fertilization after ICSI (none of five mature oocytes) in another centre, the MOAT showed a normal activation capacity of the sperm (94% activation). During the following ICSI attempt, 18 mature oocytes were harvested, seven of nine were normally fertilized without AOA, while six of nine oocytes were fertilized using AOA. One good quality embryo originating from ICSI without AOA was transferred but no pregnancy was established.
Near fertilization failure
Four couples presented at our Infertility Centre because of poor fertilization in previous ICSI attempts elsewhere. From a total of 118 mature oocytes, retrieved in 12 cycles, only 11 (9%) were reportedly normally fertilized. The MOAT with the patients' sperm gave mixed results varying from a normal (97%) to a reduced oocyte activation capacity (65, 87 and 74%). In subsequent ICSI cycles, AOA was applied. The overall fertilization rate was 79% (77 out of 97 mature oocytes) and two couples achieved a singleton pregnancy.
Low fertilization rate and embryo arrest
A first patient was referred because of poor fertilization results in two previous ICSI cycles.
In the first attempt, only two oocytes were retrieved and none of them fertilized. In the second attempt, two out of six oocytes showed normal fertilization but were arrested at the pronuclear stage. The MOAT indicated an 88% activation capacity of the patient's sperm compared to 100% in the positive control. We therefore advised to try AOA in a subsequent cycle. Three out of five oocytes showed normal fertilization but they became temporarily arrested at the pronuclear stage and a delayed and poor embryo development was observed 2 days later. No pregnancy was established in this couple after transfer of two embryos.
The second couple also had a history of poor fertilization and embryonic developmental arrest in three previous ICSI attempts before presenting at our clinic. Because the MOAT showed a decreased activation capacity (72%) of the patient's sperm compared to the positive control (91%), after MOAT, the couple was advised to undergo AOA on all oocytes if the number of oocytes was <10 and AOA on half of the oocytes if the number was >10. In the first cycle, only three mature oocytes were obtained of which two oocytes were normally fertilized after AOA. No pregnancy was achieved from the transfer of one excellent quality embryo. In the second attempt, 13 oocytes were retrieved. ICSI combined with AOA was performed on nine oocytes and four oocytes were treated with ICSI alone. After AOA, six out of nine oocytes were normally fertilized and four embryos of excellent quality were obtained of which two were selected for transfer. Four embryos were frozen. No pregnancy was achieved. Of four oocytes fertilized without assisted activation, two embryos of excellent quality were obtained and frozen for further treatment. Embryo transfer of two thawed (origin with or without AOA not known) embryos resulted in a twin pregnancy.
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Discussion |
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A two-step mechanism, called the trigger and oscillation function, is involved in triggering and maintaining oocyte activation (Tesarik et al., 2002). The initial Ca rise, which is released from internal stores, is the trigger and is initiated by a receptor-mediated interaction between the spermatozoon and the oocyte plasma membrane. During ICSI this natural trigger is replaced by a so-called pseudotrigger whereby a massive influx of Ca2+ into the oocyte is provoked by the injection procedure itself. The second function, the oscillator, is characterized by the development of Ca2+ oscillations, resulting from the release of a soluble sperm factor into the oocyte cytoplasm. The oscillator drives the oocyte's internal calcium stores rendering them capable of supporting the ongoing, largely autonomous series of Ca oscillations for several hours (Tesarik and Mendoza, 1999
; Ben-Yosef and Shalgi, 2001
). In ICSI, the first Ca2+ rise starts 2030 min after ICSI and originates from the oocyte cortex rather than from the vicinity of the injected sperm head. This Ca2+ rise alone is insufficient to fully activate the oocyte (Tesarik and Sousa, 1995
). To sustain the oscillation function, sperm demembranization is necessary to facilitate the liberation of the cytosolic sperm factor responsible for the oscillator function (Dozortsev et al., 1997
; Yanagida et al., 2001
).
In the literature there are reports that success rates of ICSI in cases of globozoospermia are rather variable ranging from total fertilization failure to almost normal fertilization rates with the establishment of pregnancies without any AOA during ICSI (Stone et al., 2000). Overall, fertilization rates remain poor using round-headed sperm for ICSI (Nakamura et al., 2002
) and assistance during ICSI is wanted. In patients with globozoospermia, heterologous ICSI provided evidence for a lack of the sperm factor. Battaglia et al. (1997)
reported that apart from low fertilization rates associated with the use of round-headed sperm, cleavage rates were also compromised and they suggested that these sperm may lack normal centrosomes. After application of AOA during clinical ICSI, we were able to restore fertilization and embryo cleavage and developmental rates were brought to a normal level in these globozoospermic patients.
It has been demonstrated that Ca2+ oscillations in fertilized oocytes regulate not only short-term but also long-term developmental events (Ducibella et al., 2002). It is thus possible that the developmental potential of these embryos may be compromised by not having a [Ca2+]i signalling pattern that is optimal. This raises the question whether patients with embryos that show developmental arrest may also not be helped by AOA.
The first study in which an oocyte-borne oocyte activation deficiency was unequivocally recognized and efficiently treated was reported recently (Tesarik et al., 2002). Another study proposed that the ability of an oocyte to respond to sperm-induced calcium oscillations is dependent on the presence of machinery in the oocyte that is functional only once in mammalian oocytes, and is activated fully by sperm components but not after parthenogenetic activation (Cheung et al., 2000
; Alberio et al., 2001
). They demonstrated that the failure of oocytes to generate Ca2+ transients in response to sperm may be one of the contributing factors to the poor rates of development in embryos from 19 day old mouse oocyte donors compared to 24 day old donors. These data indicated that changes occur in growing oocytes that modify their capacity for releasing Ca2+. The ability to generate Ca2+ transients in response to sperm is initiated during oocyte growth and continues through oocyte maturation. We showed that AOA can overcome this apparent lack of maternal modifications necessary to generate sufficient Ca2+ oscillations in response to sperm.
Micromanipulation-driven Ca2+ activation is a new modified micromanipulation technique using vigorous aspiration of oocyte cytoplasm and may offer a useful alternative for Ca ionophores (Tesarik et al., 2002). The clinical use of ionophores in assisted reproduction is limited by insufficient knowledge about their potential toxic effect on oocytes and embryos. However, preimplantation development seemed not to be affected by ionophore treatment in our study.
In conclusion, we verified whether heterologous mouse ICSI can act as a reliable diagnostic tool when investigating fertilization failure after ICSI. The sperm-borne oocyte deficiency was easily shown by the failure of mouse oocyte activation by the patient's sperm. When the MOAT shows oocyte-borne deficiencies the interpretation is more difficult and clinical treatment by Ca ionophore treatment may not be so straightforward. In the case of suspected oocyte-related fertilization failure it is advisable to seek confirmation of actual fertilization failure by applying AOA only on part of the oocytes. Finally, we were able to restore fertilization rates and to obtain pregnancies in both sperm- and oocyte-related fertilization deficiency groups. Based on these results we recommend AOA as a reasonable and efficient treatment option in cases of fertilization failure after ICSI. If larger-scale studies can confirm and fine-tune the present findings, AOA may become a standard treatment policy in cases of both oocyte- or sperm-related fertilization failure.
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Acknowledgements |
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References |
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Submitted on March 17, 2005; accepted on March 21, 2005.
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