Molecular Human Reproduction, Vol. 6, No. 6, 510-516,
June 2000
© 2000 European Society of Human Reproduction and Embryology
Embryo development |
Cytoskeletal organization defects and abortive activation in human oocytes after IVF and ICSI failure
1 Centro de Estudios en Ginecología y Reproducción, CEGyR, 1055-Buenos Aires, Argentina and 2 The Jones Institute for Reproductive Medicine, Dept. of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, VA 23507, USA
Abstract
In this study, we analysed the distribution of ß tubulins to detect spindle and cytoplasmic microtubules, 
acetylated tubulins for sperm microtubules and chromatin configuration in oocytes showing fertilization failure after conventional IVF or intracytoplasmic sperm injection (ICSI). A total of 450 human oocytes that failed to fertilize were studied 20–40 h after IVF or ICSI. In all, 287 oocytes were stained for immunofluorescence and chromosomal spreads were performed by Tarkowski's air-drying method in 163 IVF or ICSI oocytes that did not develop pronuclei after the extrusion of a second polar body. Immunofluorescence analysis showed that the main reason of fertilization failure after IVF was no sperm penetration (55.5%). The remaining oocytes showed different abnormal patterns, e.g. oocyte activation failure (15.1%) and defects in pronuclei apposition (19.2%). On the other hand, fertilization failure after ICSI was mainly associated to incomplete oocyte activation (39.9%), and to a lesser extent with defects in pronuclei apposition (22.6%) and failure of sperm penetration (13.3%). A further 13.3% of the ICSI oocytes arrested their development at the metaphase of the first mitotic division. The chromosomal spreads allowed the analysis of abortive activations, in which no pronuclei formed but a second polar body was extruded. Immunofluorescence and cytogenetic analysis provided a useful tool to improve infertility diagnosis and prognosis in each particular case.
abortive activation/cytoskeletal organization/fertilization failure/microtubules
Introduction
Despite the continuous improvement of IVF techniques, fertilization failure is a recurrent phenomenon in humans. This has been mainly explained in terms of chromosomal alterations. Several human studies analysing different types of fertilization failures have focused on the cytogenetics of oocytes (Zenzes et al., 1985
; Wramsby and Fredga, 1987; Plachot and Crozet, 1992; Zenzes and Casper, 1992). They tried to explain the causes of the incomplete fertilization process in terms of chromosomal alterations. Contrary to the well-documented explanations of chromosomic causes of fertilization failure, the participation of sperm decondensation, meiosis resumption, oocyte activation and pronuclei migration has been less studied.
Fertilization failure in mammals may have different causes (Asch et al., 1995; Simerly et al., 1995; Van Blerkom et al., 1995). Firstly, the injected oocyte may be unable to initiate the biochemical processes necessary for oocyte activation (Tesarik and Testart, 1994). Alternatively, this process may be initiated but may not proceed normally, leading to an incomplete activation. Further, the chromatin of the spermatozoon may remain condensed making difficult the accessibility of oocyte factors required for male pronuclear development (Tesarik and Kopecny, 1989a, b). Finally, the injection procedure may entail subtle structural changes in oocyte structures, like microtubules, not detectable by light microscopy.
In animal species studied so far, the oocyte-activating signal uses calcium as a second messenger (Jaffe, 1990). In mammalian oocytes, the source of calcium participating in these signalling events is predominantly intracellular, although calcium influx from the extracellular compartment is also involved (Ozil and Swann, 1995). In human oocytes, the sperm induced-calcium oscillations can last for up to 5–6 h after sperm–oocyte fusion (Sousa et al., 1996); the frequency between individual calcium spikes ranges from one spike every 2 min to one spike every 35 min, and is highly variable from oocyte to oocyte, but relatively stable in a single oocyte during the whole calcium oscillation period (Tesarik and Sousa, 1994; Sousa et al., 1996). In human oocytes, the early calcium reaction to the spermatozoon is characterized by an initial group of three to six rapidly occurring calcium spikes which is then followed by lower frequency spikes (Tesarik and Sousa, 1994). Any perturbation to the pattern of calcium oscillation can cause incomplete metaphase-promoting factor (MPF) inactivation. This situation can generate an abortive activation in mouse oocytes with the formation of a third metaphase plate and an inability to progress to interphase, unless the oocyte is stimulated by another activating stimulus (Kubiak, 1989; Vitullo and Ozil, 1992).
A coordinated mobilization of intracellular calcium stores and a precise organization of the cytoskeletal network are essential for an appropriate activation of the oocyte and chromosome migration during human fertilization. Metaphase II (MII) arrest in mammalian oocytes is supposed to be maintained by persisting high concentrations of MPF stabilized by a cytostatic factor (CSF) (Murray and Kirschner, 1989). MPF inactivation is one of the principal events in oocyte activation that reactivates the cell cycle, allowing the female's chromosomes to resume the second meiotic division and protecting the male nucleus from entering metaphase prematurely.
Oocyte activation abnormalities are suspected to be the cause of abnormal or incomplete fertilization processes and of different embryonic abnormalities including chromosomal aberrations (Tesarik, 1995).
In order to explore the cellular and molecular aspects of fertilization failure, we studied 450 human oocytes which failed to fertilize after IVF or ICSI, and in which no pronuclei were visualized and/or the extrusion of the second polar body had not occurred after 20–40 h post-insemination or injection. We focussed mainly on the distribution of ß tubulins to detect spindle and cytoplasmic microtubules, acetylated tubulins for sperm microtubules and chromatin configuration.
Materials and methods
Ovarian stimulation
Inseminated and injected human oocytes, discarded as `unfertilized' between 20–40 h post-insemination or sperm injection, were obtained from couples undergoing IVF or ICSI procedures in our Assisted Fertilisation Programme, who gave informed consent in writing. Ovarian stimulation was performed using a combination of FSH (Metrodin; Serono Laboratories, México) and human menopausal gonadotrophin (HMG, Pergonal; Serono Laboratories; Humegon; Organon Laboratories, Buenos Aires, Argentina; and HMG, Massone Laboratories, Buenos Aires, Argentina), under leuprolide acetate (Lupron; Abbot Laboratories, Buenos Aires, Argentina) suppression starting on day 21 of the previous menstrual cycle. Oestradiol plasma concentrations and ovarian follicular size were monitored daily. 10 000 IU of human chorionic gonadotrophin (HCG, Profasi; Serono Laboratories, Mexico) were administrated i.m. when two or more follicles 
16 mm were detected at ultrasound. Oocyte retrieval was performed 35 h after HCG administration by ultrasound-guided follicular puncture.
IVF and ICSI procedures
First, a fresh semen sample was processed within 30 min of ejaculation using Percoll (Sigma Laboratories, St Louis, MO, USA) gradients. The final pellet was resuspended in a volume of human tubal fluid (HTF; Irvine Scientific Laboratories, Santa Ana, CA, USA) supplemented with 15% synthetic serum substitute (Irvine Scientific, Santa Ana, California, USA) for IVF cases. HEPES buffer–human tubal fluid (H-HTF, Irvine Scientific Laboratories) supplemented with 1% bovine serum albumin (BSA; Sigma Laboratories, St Louis, MO, USA) was the medium used for ICSI spermatozoa. In azoospermic patients, testicular biopsies were performed under local anaesthesia. The tissue was dispersed in H-HTF+ 1% BSA with the use of two sterile slides in a small Petri dish and examined under inverted microscope (Nikon Diaphot; Nikon Corporation, Tokyo, Japan) with a heated platform (Nikon Corporation) to search for spermatozoa.
Oocyte–cumulus complexes were placed in 4-well dishes with 0.5 ml of HTF + 15% SSS. At that time, in IVF cases, the maturation state of the oocytes was checked and then they were left to stabilize in HTF + 15% SSS at 37°C in 5% CO2 in a humidified atmosphere until the time of insemination with a final concentration of 200 000 motile spermatozoa/ml. For ICSI cases, the oocytes were treated with 80 IU/ml hyaluronidase (Sigma Laboratories) to remove the granulosa cells and immediately washed in H-HTF + 1% BSA.
After stripping off the granulosa cells, the maturation state of the oocytes was recorded and they were left to stabilize in HTF + 15% SSS at 37°C in 5% CO2 in a humidified atmosphere, until the time of injection. Only MII oocytes were injected and immediately after injection, between four and six oocytes were left in a 40 µl drop of HTF + 15% SSS under mineral oil at 37°C in 5% CO2 in a humidified atmosphere, for 12–18 h, before first checking for pronuclear development. Oocytes were classified as `fertilization failures' when no pronuclei were visualized 20–40 h after insemination or sperm injection.
Cytoskeletal and chromatin labelling
A total of 287 oocytes were stained using a modified protocol already described (Messinger and Albertini, 1991). For this purpose, zona pellucida was removed by a brief incubation with Tyrode's acid, and denuded oocytes were fixed and permeabilized for 20 min at 37°C in a microtubule stabilising buffer (0.1 mol/l PIPES, pH 6.9, 5 mmol/l MgCl2 6 H2O, 2.5 mmol/l ethylene glycol-bis EGTA containing 2.0% formaldehyde, 0.5% Triton X-100, and 1 µmol/l taxol). Fixed oocytes were blocked for at least 1 h at 37°C with 2% BSA, 2% powdered milk, 2% normal goat serum, 0.1mol/l glycine and 0.01% Triton X-100 in PBS. If necessary, they were stored for up to 3 days at 4°C in this solution. To identify the sperm tail, oocytes were incubated overnight at 4°C with 1:100 anti--acetylated tubulin monoclonal antibodies in PBS containing 0.1% BSA and 0.02% sodium azide (PBS+BSA), washed in blocking solution, and further incubated in 1:100 fluorescein-conjugated goat anti-mouse immunoglobulin G (IgG), for 1 h at room temperature. To analyse the meiotic spindle, the material was incubated with 1:500 anti-ß-tubulin-Cy3 monoclonal antibodies in PBS+BSA for 1 h at 37°C. Finally, both anti--acetylated tubulin and anti-ß-tubulin treated oocytes were washed three times in PBS+BSA, counterstained with Hoescht 33258 (1 µg/ml) for 30 min at room temperature, washed in PBS, mounted between a slide and a coverslip in PBS+BSA, examined using an Olympus epifluorescent microscope and photographed with Ektachrome film (1600 ASA). Images were processed using Adobe Photoshop 3.0 software (Adobe System Inc). For control staining, PBS+BSA alone replaced the specific antibody solution. Monoclonal antibodies and reagents used were purchased from Sigma. Statistical analysis was carried out using the z test, and differences were considered to be significant when P < 0.05.
Chromosome analysis
Chromosomal spreads were made 20–40 h after injection or insemination by the air-drying method of Tarkowski (1966) in oocytes that had extruded a second polar body but had failed to develop pronuclei. Giemsa-stained chromosomes were analysed by bright field microscopy (x1000) and photographed with AGFA film (25 ASA).
Results
Microtubules and DNA patterns in oocytes that failed to fertilize after IVF or ICSI
A total of 287 human oocytes that failed to fertilize (no pronuclear formation after 20–40 h post-insemination or sperm injection) were studied by conventional fluorescence microscopy. Out of 129 oocytes, 119 (92.2%) were informative after IVF and 150 out of 158 (94.9%) were informative after ICSI. From the results obtained using this methodology, we were able to determine different stages at which fertilization failed. The relative abundance of oocytes in each stage is summarized in Table I. Stages detected by this analysis were as follows: (i) No sperm penetration after IVF or sperm expulsion after ICSI (55.5% and 13.3% respectively, P < 0.001). This stage (Figure 1A) was defined as the absence of both an incorporated sperm tail and sperm chromatin in the oocyte cytoplasm. This was the main reason for fertilization failure after IVF; (ii) Activation failure without pronuclear formation and no second polar body extrusion. This occurred in 15.1% of the IVF cases and 39.9% of ICSI oocytes, and this difference was highly significant (P < 0.001). This was the main reason for fertilization failure after ICSI. In some cases, oocyte activation failure occurred in combination with premature chromosome condensation (PCC) (Figure 1B). In other cases, sperm nucleus decondensation failed (Figure 1C) and different degrees of condensation were found; (iii) Defects in pronuclear formation/migration. In some cases, oocytes that failed to fertilize in which no pronuclei were seen by light microscopy, were later found by immunofluorescence to have a defective pronuclear constitution. They normally displayed groups of condensed chromosomes and/or disorganized chromatin. This defect was found in a similar proportion of oocytes both after IVF (19.2%) and ICSI (22.6%); and (iv) Mitotic metaphase arrest. After ICSI, 13.3% of arrested zygotes were found to be at the first embryonic metaphase plate. This stage was not detected after IVF (Table I). As well as the abnormalities described above, we observed other alterations at lower frequency. Some of them are not related to cytoskeletal organization, e.g. dispermic oocytes (Figure 1D), tail detachment and others are related to the ICSI technique itself, e.g. injection of the spermatozoa on the metaphase plate of the oocyte, which we call MII injection (Table I).
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Chromosome and chromatin patterns in oocytes that failed to show pronuclei but extruded the second polar body after IVF and ICSI
A total of 163 oocytes (71 from IVF and 92 from ICSI) that did not develop pronuclei and had extruded the second polar body 20–40 h after insemination or injection, were processed by Tarkowski's air drying method. A total of 53 out of 71 (74.6%) were informative after IVF and 70 out of 92 (76.1%) after ICSI. Table II
describes the different levels at which fertilization fails in this particular group of oocytes.
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Sperm DNA
Most of the oocytes analysed were informative for sperm DNA both in IVF (77.3%) and ICSI (58.5%) (Table II
). Sperm DNA was observed as intact or slightly decondensed spermatozoa and in a lower proportion, they were seen as highly compacted chromosomes. In this group, female DNA was not observed. This was probably due to the fact that they entered interphase after activation.
Abortive activation
A moderate percentage (9.5% after IVF, 20% after ICSI; P < 0.01) of female chromosomes displayed different types of abortive activation. We were able to determine the presence of MIII oocytes (in which chromosomes remained at a unichromatid stage) in both groups (Figure 2B) and also other manifestations of abortive activation like the presence of reticular and telophase nuclei (Figure 2A) as described by Kubiak (1989). The reticular nucleus stage is characterized by a small nucleus, rounded and retarded in its enlargement. It has the reticular appearance of the chromatin. A telophase nucleus (Figure 2A) can be identified by dense clumps of chromatin present either in the ooplasm or in the ooplasm and the polar body.
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Mitotic metaphase arrest
As seen in the immunofluorescence analysis, we also detected mitotic metaphase arrest in some of these oocytes. Mitotic metaphase arrest was found in 13.2% after IVF and was significantly increased (P < 0.01) to 21.4% after ICSI (Figure 2C
). Discussion
The results presented here indicate that the main reason for IVF failure is the lack of sperm penetration (55.5%). Defects in the zona pellucida architecture, in acrosomal reaction or in oolema fusion may be responsible. Microtubule and DNA analysis revealed that 44.5% of the oocytes initiated the fertilization process, but arrested at specific stages. According to Simerly et al. (1995), about half of the oocytes classified as `fertilization failures' have either been penetrated or activated by the spermatozoa. Of these, half suffer from sperm incorporation defect and the other half from microtubule or centrosome imperfections. This study also showed that a 13.3% of the oocytes that failed to fertilize after ICSI did not show any spermatozoa in the ooplasm. These results are in agreement with those previously described (Flaherty et al., 1995; Sakkas et al., 1996; Lopes et al., 1998) and are probably the consequence of sperm expulsion through the injection tract or a failure in oolema penetration at injection, ending in no sperm incorporation.
The principal reason of fertilization failure after ICSI is the lack of oocyte activation (39.9%). Fertilization achieved by ICSI bypasses the normal fertilization steps, and the procedure has become the treatment of choice for severe male factor infertility, yielding excellent fertilization and pregnancy rates. The mechanism of oocyte activation following ICSI is still controversial. Swann (1990) and Dozortzev et al. (1995) have shown that the spermatozoa contains a temperature-sensitive cytosolic factor called oscillin, that when injected into the oocyte is able to cause oocyte activation. The absence of this factor or the lack of its activity in abnormal spermatozoa, as those used in ICSI procedures, could explain, at least in part, the activation failure observed after ICSI. When activation is sperm- or experimentally-induced, human oocytes show other cellular anomalies that may contribute to early developmental arrest: (i) the inability of the sperm nucleus to migrate, if deposition was cortical and spermatozoa were completely immotile prior to preparation for ICSI; (ii) the absence of pronuclear rotation or establishment of an appropriate centrosomal position; or (iii) a dispersed rather than focal chromatin distribution (Van Blerkom et al., 1995).
A higher paternal DNA fragmentation rate and an even significantly higher rate of DNA fragmentation in sperm DNA that failed to decondense, compared with the sperm DNA that did decondense has been described (Lopes et al., 1998). The authors suggested that a correlation exists between the rate of DNA fragmentation and a loss of oscillin activity.
Premature chromosome condensation was observed in a total of 9.8% of the oocytes studied after IVF or ICSI. This process was first described by Schmiady et al., and the essential prerequisites are no activation of the oocyte and the presence of condensing factors (e.g. MPF) in the cytoplasm preventing the transformation of sperm nuclei into male pronuclei (Schmiady et al., 1986). The overall rate of PCC in oocytes that failed to fertilize is large and varies between 5 and 23% (Schmiady et al., 1986; Plachot and Crozet, 1992; Gook et al., 1998). When sperm nuclei enter fully mature oocytes they regularly transform into swelling pronuclei, whereas those spermatozoa penetrating cytoplasmic immature oocytes, undergo conspicuous chromosome condensation (Zenzes et al., 1990).
Sperm decondensation failure was observed with a higher incidence after ICSI (Table I). We speculate that this fact could be related to the `sperm quality' used in these cases compared to IVF. A failure to maintain low enough concentrations of MPF as a consequence of an inadequate calcium activation may occur (Collas et al., 1993). Interestingly, we found this phenomenon in all the cases with total fertilization failure when testicular spermatozoa were used.
The occasional observation of two presumptive abortive activations after IVF using immunofluorescence, led us to suspect that this event could reflect a not yet clearly described stage in humans, namely MIII. This stage was previously described in mouse and it is characterized by the formation of a third metaphase plate and the inability to progress to interphase (Kubiak, 1989) after IVF or parthenogenetic activation. MIII arrested oocytes arise from an incomplete activation that hampers pronuclear formation since chromosome reorganize in a new spindle (Kubiak, 1989). The lack of chromosome spreads after immunofluorescence treatment makes the assessment of MIII difficult, which is characterized by unichromatid chromosomes. In order to look for the existence of MIII, oocytes showing two polar bodies and no pronuclei 20–40 h after IVF or ICSI were chosen for the purpose of analysing chromosomes. This enabled us to detect 9.5 and 20% of oocytes showing different types of abortive activation after IVF and ICSI respectively. It is noteworthy, however, that the incidence of abortive activation is moderate in the general scope of fertilization failures. Although its incidence is higher after ICSI this may simply reflect the increased chances of incomplete activation due to the technical procedure. It has been shown that metaphase III formation is clearly dependent on the rhythm of Ca2+-oscillations which drive activation. Perturbation in frequency and/or amplitude of Ca2+ repetitive spikes leads to abortive activations (Vitullo and Ozil, 1992; Ozil, 1998).
Defects in pronuclear formation and/or migration have been found in a similar range after IVF and ICSI (19.2 and 22.6% respectively). Schatten (1994) has shown that this can arise from the inability of microtubules to assemble around the paternal centrosome. Specific binding sites and also energy-consuming proteins like dynein can generate attraction of 
tubulins towards the centrosome. Motility of maternal components to the paternal centrosome leads the aster enlargement in humans, until parental pronuclei contact occurs. During this process, the female pronucleus moves towards the male pronucleus along the sperm astral microtubules until the two pronuclei become apposed in an eccentric position. Then, the two pronuclei move towards the centre of the oocytes together. Maternal karyomeres, for example, are the result of incomplete female pronuclear reconstitution after successful meiotic chromosome separation; following oocyte activation.
Interestingly, 13.3% of the oocytes that failed to fertilize after ICSI showed an arrest in the mitotic metaphase plate. This event has been called mitotic cycle arrest or cell division failure and represents ~7% of the unfertilized oocytes (Asch et al., 1995; Hewistson et al., 1996). The difficulties for ICSI oocytes to go through cell cycle checkpoints, could be the consequence of a higher level of abnormalities in the mandatory Ca2+-oscillation pattern. A larger series of events at the first cellular cycle could be affected after ICSI as indicated by the percentage of oocytes that are arrested at this point. In contrast, we did not observe this in oocytes that failed to fertilize after IVF, where the first surface interactions between oocyte and spermatozoa are present and represent the most critical step for a successful fertilization.
According to Hewitson et al. (1999), in human oocytes that fail to fertilize, the first polar body can be displaced with respect to the spindle 10.8 ± 7.6° (mean ± SD, range: 2.7–21.9°). In contrast, ICSI oocytes showed higher angles of second meiotic spindle-first polar body displacement 56.0 ± 27.5° (mean ± SD, range: 22.9–94.9°). Consequently, a considerable percentage of oocytes undergoing ICSI (6% in our study) may fail to fertilize due to the injection of a spermatozoon in the middle of the second metaphase plate of the oocyte. Similar observations have been reported previously (Silva et al., 1999) using the polarization microscope, a non-destructive tool to visualize the meiotic spindle in hamster oocytes. Since polar body position seems not to predict accurately the location of the meiotic spindle, `MII injection' might be one of the stages at which fertilization fails after ICSI. The results presented here enlarge our knowledge on cytoplasm and nuclear defects during human fertilization arrest. This has implications both for improving infertility diagnoses and understanding the cellular basis of early developmental failure.
Acknowledgments
The authors wish to express their appreciation to Germán Galaverna, for his technical assistance and advice in cytogenetic analysis and to Sabrina De Vincentiis and Felicitas Noblía for their dedication in collecting and selecting the material.
Notes
3 To whom correspondence should be addressed at: Centro de Estudios en ginecología y Reproducción, CEGyR, IOSS-Buenos Aires, Argentina. E-mail: Vanerawe{at}hotmail.com 
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Submitted on October 21, 1999; accepted on March 27, 2000.
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