1 Charité, Medizinische Fakultät der Humboldt-Universität zu Berlin, Campus Virchow-Klinikum, Klinik für Frauenheilkunde und Geburtshilfe, Reproduktionsmedizin and 2 Institut für Humangenetik, Genetische Beratung, Augustenburger Platz 1, 13353 Berlin, Germany
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Abstract |
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Key words: autosomal recessive infertility factor/consanguinity/meiotic metaphase I arrest/premature chromosome condensation
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Introduction |
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Here, we report our observations on oocytes from two patients who underwent unsuccessful IVF treatments. When the cumulus cells were removed 1618 h after insemination, the oocytes of both patients did not exhibit either a first or second polar body or pronuclei. A cytogenetic study was employed to determine the nature of the disturbance in these patients during oogenesis. When patient 1 was counselled, it was noted that she had a sister also suffering from idiopathic infertility. Moreover, it became evident that this sister (patient 2) had been treated in our department when she was 35 years old.
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Case report |
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Follicles were aspirated transvaginally under ultrasound guidance 3436 h after hCG administration (day 18). Five oocytes of normal mean diameter were obtained.
The oocytes were transferred to medium drops (50 µl) covered with mineral oil, inseminated each with 50 000 motile sperm and controlled for the appearance of polar bodies and pronuclei 1618 h later. However, neither polar bodies nor pronuclei were observed and, after a further 24 h, they did not cleave and were hypotonically treated (1% sodium citrate, 8 min) and fixed (methanol:acetic acid, 3:1) without previous use of colcemid treatment according to a previously described method (Tarkowski, 1966
). After air-drying, the preparations were stained with Giemsa (Merck, Darmstadt, Germany). In addition, chromosome analysis of GTG-banded lymphocyte metaphase plates was performed.
Patient 2
The patient was 35 years old when first admitted to the IVF programme. The data for medical history and hormonal stimulation as well as the observations of the oocytes have been already published in detail (Eichenlaub-Ritter et al., 1995).
In summary, all the oocytes from this woman, who had undergone four unsuccessful IVF attempts, showed neither a polar body nor pronuclei when examined for fertilization. The oocytes whose sizes were in the normal range did not cleave and were prepared as described above. Lymphocyte chromosomes were used for karyotyping.
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Results |
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The chromosome analysis of the lymphocyte metaphase plates revealed a normal female karyotype (46,XX) in both sisters. There was no suggestion of any disturbance in mitotic progression.
During consultation, the pedigree of the family (Figure 3) revealed that the sisters were descended from a consanguineous marriage of their parents, who were first cousins.
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Discussion |
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To date, some animal models exist which could shed light into the involved pathways of mammalian metaphase I arrest. In normal mammalian oocytes, entry into metaphase I is facilitated by the accumulation of activated cyclin-B1-cyclin-dependent kinase (Cdk1), also known as mitosis promoting factor (MPF). The transition to anaphase I is correlated with a decline in the activity of cyclin-B1-Cdk1 due to the degradation of cyclin B1. Its activity is again elevated during metaphase II until fertilization (Choi et al., 1991). In contrast to this normal meiotic progression, oocytes of some mouse strains are aberrant in their meiotic maturation. Hampl and Eppig reported that oocytes of the strain LT/Sv become arrested at metaphase I, even when they are fully grown (Hampl and Eppig, 1995
). The initiation of oocyte maturation was correlated with an elevation of cyclin-B1-Cdk1 activity that continued to rise until late metaphase I while normally the transition from metaphase I into anaphase I is correlated with a decrease of cyclin-B1-Cdk1 activity. This study demonstrated that metaphase I arrest is correlated with a sustained elevation of cyclin-B1-Cdk1 activity. Interestingly, in this respect, is the report of a non-degradable, N-terminal truncated form of cyclin B1 that complexes with Cdk1 resulting in an active cyclin-B1-Cdk1 complex. However, at the metaphase to anaphase transition point this cyclin cannot be degraded. Injection of the non-degradable form of cyclin B1 into human oocytes results in arrest at metaphase I and maintenance of the spindle and chromatin configuration (Herbert et al., 1999
).
Our findings in the affected family with idiopathic infertility are consistent with the idea of high MPF activity in the patients oocytes since PCC is induced in the nucleus of the penetrating sperm cell (Schmiady et al., 1986). However, several other cell cycle controllers might be involved in the complex pathway of meiotic I arrest. The protein MOS (v-mos moloney murine sarcoma viral oncogene homologue) participated in sustaining metaphase I arrest in LT (specific recombinant inbred strain) oocytes (Hirao and Eppig, 1997
) and studies have indicated that a failure to regulate protein kinase C clearly participates in the abnormal oocyte behaviour (Viveiros et al., 2001
).
Therefore, the only way to identify the underlying gene defect in this autosomal recessive trait (Bittles et al., 1991) is a systematic genome scan with tightly linked microsatellites, the so-called homozygosity mapping which is in progress.
Both couples were informed of the results and the unknown genetic basis of this failure, and that it cannot be overcome by changing ovarian stimulation protocols. Because oocyte donation is not permitted in Germany, adoption was discussed as an alternative.
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Acknowledgements |
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Notes |
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References |
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Submitted on March 22, 2002; accepted on May 29, 2002.