1 Reproductive Biology Unit, Department of Obstetrics and Gynaecology, University of Stellenbosch, 2 Diagnostic Electron Microscopy Unit, Department of Anatomical Pathology, University of Stellenbosch, South Africa and 3 Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar, University of Porto, Portugal
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Abstract |
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Key words: ICSI/maturation arrest/metaphase I oocytes/oocyte spindle/ultrastructure
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Introduction |
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The routine performance of intracytoplasmic sperm injection (ICSI) has provided the opportunity to evaluate oocytes more accurately because of denudation, prior to injection. With inverted light microscopy, genetic subgroups (prophase, MI and MII) and to a lesser extent cytoplasmic subgroups (granularity, cytoplasmic inclusions, oocyte fragmentation), can be identified. In the present study we have used transmission electron microscopy (TEM) to examine oocytes from a patient suffering from recurring maturation arrest at the MI stage of meiosis.
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Case report |
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The couple returned to our clinic in 1998 and it was decided to continue treatment with ICSI. Two cycles were performed (April and November) using a long protocol of pituitary desensitization with triptorelin (Synarel, Searle, South Africa), in association with human menopausal gonadotrophin (Pergonal, Serono, South Africa). HCG (Profasi, Serono) was administered when two follicles had reached >17 mm. Nine and 8 oocytes were recovered by ultrasound guided transvaginal aspiration respectively. After cumulus removal, all oocytes were observed to be arrested at the MI stage of meiosis, showing a morphologically normal ooplasm. They were left to spontaneously mature in vitro in IVF medium (Medicult, HarriLabs, South Africa) under light mineral oil at 37°C with 5% CO2 in air for 48 h. After that period, all oocytes remained at MI. Nonetheless, they were microinjected but none were fertilized. In the second attempt, two of the unfertilized oocytes were processed for TEM evaluation. El-Shafie et al. have described a detailed (TEM) method (El-Shafie et al., 2000). Briefly, the oocytes were fixed in Karnovsky's fixative (pH 7.4) at 4°C then washed in cacodylate buffer (pH 7.4) and stored at 4°C. The washed oocytes were then treated with osmium tetraoxide, rinsed with distilled water and placed in uranyl acetate (2% in 70% ethanol). Dehydration steps in 70%, 96% and 100% ethanol followed and the oocytes were finally embedded in Spurr's resin for 24 h at 60°C (in specific plastic moulds). The blocks from the moulds were processed for thin-sectioning with a diamond or glass knife (7090 nm) and positioned on copper grids. Grids were then stained with Reynold's uranyl acetate/lead citrate method and every section scanned on a Hitachi H 600 electron microscope. Sections with applicable features were photographed and evaluated.
We realise that some of the figures are of less than optimal quality (e.g. Figure 3), but these were the only available micrographs showing the underlying abnormality associated with the case.
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Discussion |
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It is relatively rare to find a whole cohort of oocytes suffering from maturity arrest after ovarian hyperstimulation, especially when this is a recurring phenomenon and when all gametes appear resistant to in-vitro maturation. Rudak et al. documented the first cases of patients suffering from idiopathic oocyte maturation disorders (Rudak et al., 1990). In the cases where no polar body was extruded (MI arrest), they speculated that it was an anomaly related to the formation or functioning of the first meiotic spindle. Eichenlaub-Ritter et al. investigated a patient who had undergone four unsuccessful IVF attempts, with all oocytes showing no signs of a polar body nor pronuclei when examined for fertilization (Eichenlaub-Ritter et al., 1995
). Besides considering that the oocytes were arrested at MI, the authors also suggested that a rapid maturation to MII, ageing in vivo and degeneration of the first polar body before aspiration could also cause this failure. The degenerate MII spindle, typical of aged oocytes, was thus probably responsible for the developmental block and induction of premature chromosome condensation (PCC) of the sperm chromatin. The authors concluded that the patient suffered from an unusual asynchrony in follicular, cytoplasmic and chromosomal maturation kinetics. The suggested treatment for this case was earlier oocyte aspiration (Eichenlaub-Ritter et al., 1995
). More recently, Hartshorne et al. investigated a similar case of maturation arrest (Hartshorne et al., 1999
). Two cycles were investigated and in the second cycle the hyperstimulation was changed from a long to a flare-up protocol. The cumulusoocyte complexes were incubated in medium supplemented with recombinant FSH and HCG for up to 2 days, but there was no success in any of the cycles (extrusion of the first polar body). They observed that the arrangement of the female chromatin in all the oocytes was characteristic of arrest at entry to M-phase.
Recurrent immature oocyte retrieval and resistance to in-vitro maturation may be caused by genetic abnormalities. This has been shown by Racowsky and Kaufman who studied meiotically immature but normal appearing oocytes retrieved after oophorectomy and in-vitro cultured for 946 h (Racowsky and Kaufman, 1992). Of the 101 oocyte chromatin configurations analysed, 71.3% were normal, 11.9% were degenerate and 16.8% displayed meiotic aberrations, with the significant majority (88.2%) of the aberrant chromatin configurations being associated with otherwise normal oocyte morphology. Another possible cause could be related to abnormalities attaining the meiotic spindle itself. In a study by Kim et al. the leading role of microtubules and microfilaments in the reconstruction and proper positioning of chromatin after GV breakdown and during meiotic maturation was shown, with absence or abnormalities of microtubules and microfilaments directly influencing the oocyte maturation process (Kim et al., 1998
).
In the case investigated here, the ultrastructural analysis enabled us to conclude that the abnormality was due to a persistent absence of spindle formation that caused arrest of oocytes at the MI stage with no possibility for overcoming this defect by in-vitro maturation. Because no signs of cytoplasmic vacuolization were found (El-Shafie et al., 2000), it can be assumed that a rapid maturation to MII, ageing in vivo and degeneration of the first polar body before aspiration could not be the cause of this failure. This anomaly was also not related to the female's age, which was 34 years. It could also not be due to ooplasmic immaturity of the major oocyte organelles, since they were morphologically normal and correctly positioned as in a normal, mature MII oocyte (El-Shafie et al., 2000
). This case thus represents the first documentation of developmental arrest due to complete absence of spindle formation in association with an otherwise mature ooplasm. In the present study, ooplasm maturity could also be tested by sperm injection, which showed the oocytes to be able to induce a partial cortical reaction, a complete sperm acrosome and flagellar disruption, and a partial sperm chromatin decondensation process, without signs of premature sperm nuclear condensation. These results may suggest that the cortical reaction, oocyte organelle repositioning, and male pronucleus formation all depend on oocyte meiotic resumption and not only on the spermoocyte activating substance. As previously shown (Sousa and Tesarik, 1994
), absence of complete cytoplasmic activation after sperm injection could also be due to a biochemical defect of the ooplasm unrelated to meiosis resumption. This is partially suggested in this case by the observation of other minor abnormalities, which include zona pellucida immaturity and a partial defect of the SER (foam-body), the latter corresponding to a novel structure not previously related to oocyte immaturity (El-Shafie et al., 2000
).
Treatment options for this patient are unfortunately limited since the couple are not comfortable with oocyte donation. The fact that no GV were observed in the cycles from our patient indicates that nuclear and cytoplasmic factors governing germinal vesicle breakdown and chromosome condensation were functioning normally. The affected step seems to be the tubulin and/or biochemical spindle polymerization machinery. Taking into consideration our limited knowledge on the interactions between the nuclear genome and the cytoplasmic factors, the only means of enabling this patient to have genetically related offspring would be by donor cytoplasm transfer or GV transfer into donor oocytes. However, results from these techniques are still preliminary (Flood et al., 1990; Cohen et al., 1998
; Zhang et al., 1999
) and there are concerns regarding the contribution of mitochondrial DNA from donor oocytes (Tsai et al., 2000
).
Using ultrastructural analysis, we were able to find a reason for this couple's unexplained infertility and with the appropriate stimulation protocol (maximize probability of GV) and micromanipulation techniques, in future the patient's GV could be transferred into donated enucleated oocytes.
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Acknowledgements |
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Notes |
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References |
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Submitted on February 16, 2001; accepted on July 13, 2001.