Ultrastructural evaluation of recurrent and in-vitro maturation resistant metaphase I arrested oocytes

Case report

M.-L. Windt1,4, K. Coetzee1, T.F. Kruger1, H. Marino1, M.S. Kitshoff2 and M. Sousa3

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


    Abstract
 Top
 Abstract
 Introduction
 Case report
 Discussion
 Acknowledgements
 References
 
An infertile couple whose female partner showed recurrent retrieval of immature metaphase I (MI) oocytes that were resistant to in-vitro maturation, was studied. Four spermiograms revealed teratozoospermia. Consistent non-fertilization and negative pregnancy outcomes were obtained after intrauterine insemination, gamete intra-Fallopian transfer and IVF. Two intracytoplasmic sperm injection (ICSI) cycles were finally performed. All oocytes (n = 17) in both cycles were arrested at MI and failed to mature after 48 h culture. ICSI also resulted in total non-fertilization. In the last cycle, two oocytes were analysed by transmission electron microscopy and showed almost identical results. All organelles showed normal characteristics of an MI oocyte. The main abnormality found was related to the MI spindle, with absence of microtubules and dispersion of the female chromosomes. Minor abnormalities were observed (immature fibrous appearance of the zona pellucida; the presence of small vesicle aggregates which formed a foam-like body). The injected sperm nucleus was arrested in the middle of the chromatin decondensation process, with no visible nuclear envelope reformation. Normal disruption of sperm acrosomal and flagellar components were observed. Only a partial cortical reaction was observed. This represents the first documented case of developmental arrest due to complete absence of spindle formation in association with an otherwise mature ooplasm.

Key words: ICSI/maturation arrest/metaphase I oocytes/oocyte spindle/ultrastructure


    Introduction
 Top
 Abstract
 Introduction
 Case report
 Discussion
 Acknowledgements
 References
 
The objective of exogenous hormone administrations used routinely for ovarian hyperstimulation in IVF programmes is to produce a large cohort of mature follicles, with induction of meiotic maturation being accomplished either in response to an endogenous LH surge or to the administration of human chorionic gonadotrophins (HCG). In the human oocyte, meiosis is a complex process that involves both progressive and stage-specific events at the nuclear and cytoplasmic levels, including germinal vesicle (GV) breakdown, chromosomal condensation, polar body extrusion, metaphase II (MII) arrest and cytoplasmic maturation events (Van Blerkom et al., 1994Go). Oocytes are normally assumed to reach full fertilization competence only after this meiotic and cytoplasmic maturation has been completed. However, the inherent variability of nature often causes non-synchronous oocyte maturation, leading to a heterogeneous population of immature (prophase I, metaphase I [MI]) and mature (MII) oocytes, and is also responsible for numerous anomalies in the nuclear and cytoplasmic maturation process (Sousa and Tesarik, 1994Go; Janssenswillen et al., 1995Go; El-Shafie et al., 2000Go; Nogueira et al., 2000Go). Some of these anomalies can result in unexplained infertility, not detectable by regular microscopy.

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.


    Case report
 Top
 Abstract
 Introduction
 Case report
 Discussion
 Acknowledgements
 References
 
In 1995, a couple with primary infertility of 1 year and 3 months duration was studied. Infertility investigations revealed normal female (laparoscopy) and male (clinical evaluation) genitalia. The wife had regular menstrual periods with a cycle length of 28–32 days. She did, however, regularly present with minimally elevated levels of prolactin (35–45 ng/ml). The computed tomography (CT) scan showed a normal pituitary fossa and she was consequently placed on 2.5 mg/day of bromocriptin (Parlodel, Novartis, South Africa). A chromosomal analysis (idiogramme and karyotype) performed on the patient's cultured lymphocytes indicated a normal female chromosomal complement (46,XX). The karyotype of the husband was not known. The four spermiograms performed on the husband's semen sample revealed normal parameters except for a persistent teratozoospermia (Table IGo).


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Table I. Summarized spermiograms of husband
 
Three unsuccessful intrauterine inseminations were performed in consecutive spontaneous cycles at our clinic during the period of August–November 1995. Three more unsuccessful assisted reproductive cycles were performed in 1996 (May, August and November) at another fertility clinic. At the time the female patient was 30 years old. These included a gamete intra-Fallopian transfer (GIFT) cycle (9 oocytes retrieved, 5 oocytes transferred), an IVF cycle (7 oocytes retrieved, 7 oocytes inseminated with total absence of fertilization), and one ICSI cycle (15 oocytes retrieved). In the case of GIFT the nuclear status was unknown, whereas in the IVF (after denudation at 16 h after insemination) and ICSI cycles all oocytes exhibited MI arrest.

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., 2000Go). 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 (70–90 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 3Go), but these were the only available micrographs showing the underlying abnormality associated with the case.



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Figures 2 and 3. Microtubules are absent from the female spindle region (*) and chromosomes (C) appear dispersed and not aligned. Note the normal mature appearance of the ooplasm, with smooth endoplasmic reticulum (SER) small (arrows) and large (V) vesicles associated with mitochondria (M) in the cortical region, and the predominance of SER small vesicles associated with mitochondria in the inner ooplasm. Original magnification x6000 and x12 000 respectively.

 
The ultrastructural examination revealed an ooplasm with characteristics of a mature oocyte, without any signs of degeneration or ageing vacuolization. The normal features included the presence of dense cortical vesicles correctly positioned under the oolemma and small and large smooth endoplasmic reticulum (SER) vesicles with associated mitochondria (Figure 1Go). Also visible were SER tubular aggregates with associated mitochondria in the cortical region (Figure 1Go) and small SER vesicles with associated mitochondria in the inner ooplasm (Figure 2Go). The main abnormality found was related to the MI spindle. Microtubules were absent from the spindle region and the female chromosomes were dispersed, some appearing outside but on the periphery of the spindle region (Figures 2, 3GoGo). Some additional minor abnormalities were also observed. These included an immature fibrous appearance of the zona pellucida, with a more dense inner region and the presence of small SER vesicle aggregates which formed a foam-like body under the oolemma (Figure 1Go). The injected sperm nucleus was found to be arrested at the middle of the chromatin decondensation process, with no visible nuclear envelope (Figure 4Go). The sperm acrosomal and flagellar components were not observed, which indicated a normal disruption process of those structures. In relation to the cortical reaction induced by sperm injection, only a partial reaction was observed, with cortical vesicle contents being found in the perivitelline space (Figure 1Go).



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Figure 1. The zona pellucida (ZP) is immature, being narrow and fibrous, with a dense inner region. The perivitelline space shows cortical vesicle exudes (arrowheads). Intact cortical vesicles (CV) are present under the oolemma. In the cortex, there are small (arrows) and large (V) vesicles and tubular aggregates (T) of the smooth endoplasmic reticulum (SER), associated with mitochondria (M). Near the surface, there is a foam-body formed by an abnormal accumulation of SER small vesicles (*). Original magnification x7500.

 


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Figure 4. Partially decondensed male chromatin of the injected spermatozoon. The nuclear envelope has already disaggregated. Decondensation appears more pronounced at the centre (B) than at the periphery (A), being blocked at the coarse chromatin stage, with a ladder appearance (*), without progression into fine fibrils. Original magnification x36 000.

 

    Discussion
 Top
 Abstract
 Introduction
 Case report
 Discussion
 Acknowledgements
 References
 
Despite controlled administration of exogenous hormones, used routinely for ovarian hyperstimulation and ovulation in IVF programmes, a high number (17–20%) of the retrieved oocytes exhibit GV or MI arrest. Immaturity may be partially overcome with in-vitro culture, being less successful with GV than with MI oocytes. MI oocytes usually mature spontaneously in vitro at a high rate after about 12 h of incubation (Janssenswillen et al., 1995Go; Goud et al., 1998Go). However, other cytogenetic anomalies may remain undetected, resulting in absence of fertilization and/or abnormal embryogenesis (Nogueira et al., 2000Go).

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., 1990Go). 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., 1995Go). 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., 1995Go). More recently, Hartshorne et al. investigated a similar case of maturation arrest (Hartshorne et al., 1999Go). Two cycles were investigated and in the second cycle the hyperstimulation was changed from a long to a flare-up protocol. The cumulus–oocyte 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 9–46 h (Racowsky and Kaufman, 1992Go). 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., 1998Go).

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., 2000Go), 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., 2000Go). 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 sperm–oocyte activating substance. As previously shown (Sousa and Tesarik, 1994Go), 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., 2000Go).

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., 1990Go; Cohen et al., 1998Go; Zhang et al., 1999Go) and there are concerns regarding the contribution of mitochondrial DNA from donor oocytes (Tsai et al., 2000Go).

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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Case report
 Discussion
 Acknowledgements
 References
 
Sincere thanks to the personnel of the Reproductive Biology Unit and theatre, and to Professor P.A.B.Wranz and his personnel from the Diagnostic Electron Microscopy Unit, Department of Anatomical Pathology, University of Stellenbosch, Tygerberg Hospital. This research was supported by the Harry and Doris Crossley Research Fund, University of Stellenbosch, Tygerberg.


    Notes
 
4 To whom correspondence should be addressed at: Reproductive Biology Unit, Department of Obstetrics and Gynaecology, University of Stellenbosch, Tygerberg Hospital, Tygerberg, 7505, South Africa. E-mail: mlw{at}gerga.sun.ac.za Back


    References
 Top
 Abstract
 Introduction
 Case report
 Discussion
 Acknowledgements
 References
 
Cohen, J., Scott, R., Alikani, M. et al. (1998) Ooplasmic transfer into mature human oocytes. Mol. Hum. Reprod., 4, 269–280.[Abstract]

Eichenlaub-Ritter, U., Schmiady, H., Kentenich, H. et al. (1995) Recurrent failure in polar body formation and premature chromosome condensation in oocytes from a human patient: indicators of asynchrony in nuclear and cytoplasmic maturation. Hum. Reprod., 10, 2343–2349.[Abstract]

El-Shafie, M., Sousa, M., Windt, M.-L. and Kruger, T.F. (2000) An Atlas of the Ultrastructure of Human Oocytes. The Parthenon Publishing Group, New York.

Flood, J.T., Chillik, C.F., van Uem, J.F.H.M. et al. (1990) Ooplasmic transfusion: prophase germinal vesicle oocytes made developmentally competent by microinjection of metaphase II egg cytoplasm. Fertil. Steril., 53, 1049–1054.

Goud, P.T., Goud, A.P., Qian, C. et al. (1998) In vitro maturation of human germinal vesicle stage oocytes: role of cumulus cells and epidermal growth factor in the culture medium. Hum. Reprod., 13, 1638–1644.[Abstract]

Hartshorne, G., Montgomery, S. and Klentzeris, L. (1999) A case of failed maturation in vivo and vitro. Fertil. Steril., 71, 567–570.[ISI][Medline]

Janssenswillen, C., Nagy, Z.P. and Van Steirteghem, A. (1995) Maturation of human cumulus-free germinal vesicle-stage oocytes to metaphase II by coculture with monolayer Vero cells. Hum. Reprod., 10, 375–378.[Abstract]

Kim, N.H., Chung, H.M., Cha, K.-Y. et al. (1998) Microtubule and microfilament organization in maturing human oocytes. Hum. Reprod., 13, 2217–2222.[Abstract]

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Sousa, M. and Tesarik, J. (1994) Ultrastructural analysis of fertilization failure after intracytoplasmic sperm injection. Hum. Reprod., 9, 2374–2380.[Abstract]

Tsai, M.C., Takeuchi, T., Bedford, J.M. et al. (2000) Alternative sources of gametes: reality or science fiction? Hum. Reprod., 15, 988–998.[Abstract/Free Full Text]

Van Blerkom, J., Davis, P.W. and Merriam, J. (1994) The developmental ability of human oocytes penetrated at the germinal vesicle stage after insemination in vitro. Hum. Reprod., 9, 697–708.[Abstract]

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Submitted on February 16, 2001; accepted on July 13, 2001.