1 Departments of Reproductive Biology and Cytogenetics and 2 Gynecology and Obstetrics, CHI Poissy Saint Germain, 78303 Poissy, Paris-Ouest University, France
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Abstract |
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Key words: ICSI/meiosis block/metaphase I/oocytes
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Introduction |
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Case report |
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IVF cycle
IVF was considered after the failure of four cycles of intrauterine insemination. After pituitary down-regulation, the ovaries were stimulated with 150 IU recombinant FSH daily from days 17 and with 225 IU FSH from days 812. Only the right ovary responded to hormonal stimulation, but follicle progression on ultrasound and serum oestradiol concentrations were considered satisfactory. The patient received 10 000 IU of HCG on day 13 (serum oestradiol concentration was 1138 pg/ml). Thirtysix hours later three oocytes were retrieved by ultrasound-guided follicular puncture. Attempts to achieve IVF with 90 000 motile spermatozoa per oocyte were unsuccessful. On the following day none of these oocytes was fertilized and none had a polar body. The couple was informed of the results, and were offered a second cycle with an ICSI procedure to make it possible to check the oocyte maturity and to increase the fertilization rate.
ICSI cycle
After pituitary down-regulation, the ovaries were stimulated with 225 IU FSH daily from days 111. Both ovaries responded and 10 000 IU HCG was administered on day 12 (serum oestradiol concentration was 3090 pg/ml). Ten follicles were aspirated and 10 oocytes of normal mean diameter were retrieved and treated with hyaluronidase (80 IU/ml). None of the oocytes had extruded the first polar body. They were incubated for a further 4 h in P1 culture medium (Irvine Scientific, Paisley, Renfrewshire, UK) and examined again, but their appearance remained unchanged. It was decided jointly by the couple, the embryologist and the gynaecologist not to inject the immature oocytes. Injection was deferred to the following day, with a new sperm sample, provided that the oocytes reached the MII stage in vitro. Twenty-four hours later all the oocytes were still at the MI stage, therefore ICSI was not performed. Two oocytes were incubated at 37°C in 5% CO2 in P1 culture medium for a further 24 h but neither extruded the polar body. The other eight oocytes were fixed by the Tarkowski technique (Tarkowski, 1966) to evaluate their chromosomal constitution and for cytogenetic staging to identify the stage at which meiosis was arrested. We were able to analyse seven of the eight fixed oocytes after staining with 3% Giemsa solution. All had 46 chromosomes which appeared to be in metaphase with either: (i) replicated chromosomes in homologous pairs close together or linked by chiasmata (chromosomes become competent to adopt this configuration at mid-pachytene stage); or (ii) replicated chromosomes already dissociated (suggestive of anaphase I initiation or of the start of chromosome pair degeneration) (Figure 1
).
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Discussion |
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Reports of meiosis 1 arrest are more frequent for the male than the female germ line, in humans (Handel, 1997; Lange et al., 1997
) and in various animal species (Baker and Plug, 1996; Zhu et al., 1997
). Genetic control was suspected before molecular analysis became available, on the basis of familial history and pathological examinations on the testis (Luciani, 1981
). The molecular mechanisms of chromosomal desynapsis impairment in the male germ line are now partially understood (Okabe et al., 1998
; Escalier, 1999
).
In meiotic arrest of spermatocytes, both paired and unpaired homologous metaphase chromosomes are observed, due to asynapsis or the formation of fragmented synaptonemal complexes as described by Lange (Lange et al., 1997), or desynapsis impairment, leading to the arrest of these cells in meiosis I. Similar observations are often made, but not always investigated in testis biopsies in secretory azoospermic men, in the search for mature spermatozoa that could be used for ICSI. These cases are usually diagnosed as `meiotic arrest'.
The scarcity of observations of a similar disorder in women may be due to a recruitment and observation bias, as the number of available meiotic cells from oocytic puncture is generally lower than that available from testis biopsies. Alternatively, the frequency of this type of syndrome may be lower in the female germ line. Several molecular events regulating meiosis are known to differ in males and females; it is not possible to determine the mechanisms involved or incidence, by extrapolation (Baker et al., 1996; Yuan et al., 2000
).
As human oocytes, whether normal or abnormal, are difficult to obtain and are not used primarily for research, the use of targeted mutagenesis in animals (Simon et al., 1997) seems essential to progress in this field. Indeed, the mechanisms controlling the process of meiosis are intricate and diverse, operating both in the oocyte nucleus (microtubule organizing centre, spindle, centromeric structure) and in the cytoplasm (qualitative and quantitative modifications affecting various organelles: Golgi apparatus, mitochondria, ribosomes). These mechanisms also involve the surrounding environment (culture conditions) and the cells in contact with the oocytes (paracrine intercellular interaction factors) (Fulka et al., 1998
).
As stated previously, a block in oocyte meiosis has previously been reported in animal models, including various strains of mice. The fact that only specific recombinant inbred strains are affected is consistent with the polygenic control of meiosis (Eppig and Wigglesworth, 1994; Eppig et al., 1996
).
From a practical point of view, the couple was informed of the results and of the likelihood that the same outcome would occur if another attempt was made. Oocyte donation and adoption were discussed.
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Acknowledgements |
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Notes |
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References |
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Fulka, J., First, N. and Moor, R. (1998) Nuclear and cytoplasmic determinants involved in the regulation of mammalian oocyte maturation. Mol. Hum. Reprod., 4, 4149.[Abstract]
Eppig, J. and Wigglesworth, K. (1994) Atypical maturation of oocytes of strain I/LnJ mice. Hum. Reprod., 9, 11361142.[Abstract]
Eppig, J., Wigglesworth, K., Varnum, D. et al. (1996) Genetic regulation of traits essential for spontaneous ovarian teratocarcinogenesis in strain LT/Svmice: aberrant meiotic cell cycle, oocyte activation and parthenogenetic development. Cancer Res., 56, 50475054.[Abstract]
Escalier, D. (1999) Mammalian spermatogenesis investigated by genetic engineering. Histol. Histopathol., 14, 945958.[ISI][Medline]
Greenhouse, S., Rankin, T. and Dean, J. (1998) Genetic causes of female infertility: targeted mutagenesis in mice. Am. J. Hum. Genet., 62, 12821287.[ISI][Medline]
Handel, M. (1997) Monitoring meiosis in gametogenesis. Theriogenology, 49, 423430.[ISI]
Harrison, K., Sherring, D. and Keeping, J. (2000) Repeated oocyte maturation block. J. Assist. Reprod. Genet., 17, 231233.[ISI][Medline]
Hartshorne, G., Montgomery, S., Klentzeris, L. and MRCOG (1999) A case of failed oocyte maturation in vivo and in vitro. Fertil. Steril., 71, 567570.[ISI][Medline]
Lange, R., Krause, W. and Engel, W. (1997) Analyses of meiotic chromosomes in testicular biopsies of infertile patients. Hum. Reprod., 12, 215421158.[Abstract]
Luciani, J.M. (1981) Le contrôle génétique de la méïose chez l'homme. In Spira, A. and Jouannet, P. (eds) Les colloques de l'INSERM. Facteurs de la fertilité humaine. INSERM, Paris, vol. 103, p. 153.
Okabe, M., Ikawa, M. and Ashkenas, J. (1998) Gametogenesis `98: male infertility and the genetics of spermatogenesis. Am. J. Hum. Genet., 62, 613.[ISI][Medline]
Pellestor, F. and Sèle, B. (1988) Assessment of aneuploidy in the human female by using cytogenetics of IVF failure. Am. J. Hum. Genet., 42, 274283.[ISI][Medline]
Selva, J., Martin-Pont, B., Hugues, J.N. et al. (1991) Cytogenetic study of human oocytes uncleaved after in vitro fertilization. Hum. Reprod., 6, 709713.[Abstract]
Simon, A., Goodenough, D., Li, E. and Paul, D. (1997) Female infertility in mice lacking connexin 37. Nature, 385, 525529.[ISI][Medline]
Tarkowski, A.K. (1966) An air drying method for chromosome preparations of mouse eggs. Cytogenetics, 3, 393400.
Yuan, L., Liu, J., Zhao, J. et al. (2000) The murine SCP3 gene is required for synaptonemal complex assembly, chromosomes synapsis and male fertility. Mol. Cell., 5, 7383.[ISI][Medline]
Zhu, D., Dix, D. and Eddy, E. (1997) HSP70-2 is required for CDC2 kinase activity in meiosis I of mouse spermatocytes. Development, 124, 30073014.
Submitted on February 7, 2001; accepted on June 26, 2001.