Arrest of human oocytes during meiosis I in two sisters of consanguineous parents: first evidence for an autosomal recessive trait in human infertility

Case report

Hardi Schmiady1,3 and Heidemarie Neitzel2

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


    Abstract
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 Abstract
 Introduction
 Case report
 Results
 Discussion
 Acknowledgements
 References
 
Two sisters descended from consanguineous parents underwent unsuccessful IVF treatments. Their oocytes showed neither a first or second polar body, nor pronuclei. After cytogenetic preparation, all oocytes were characterized by condensed maternal metaphase I chromosomes and premature chromosome condensation of the sperm nucleus. Both women exhibited a normal female karyotype (46,XX). The pedigree of the family revealed two other sisters who had delivered children, but also two brothers who had been married for several years without children. There is a strong indication for an autosomal recessive trait responsible for the idiopathic infertility due to the expression of a rare recessive allele inherited from common ancestors. However, neither the mechanism of metaphase I arrest nor the gene(s) involved in this arrest are known in the case of our patients. We discuss molecular mechanism(s) derived from animal models that might be involved in this inherited disorder in human oocytes.

Key words: autosomal recessive infertility factor/consanguinity/meiotic metaphase I arrest/premature chromosome condensation


    Introduction
 Top
 Abstract
 Introduction
 Case report
 Results
 Discussion
 Acknowledgements
 References
 
Meiotic cells, like mitotic cells, have cell cycle checkpoints to ensure that one event is completed properly before another begins. Two such checkpoint controllers have been identified which ensure that meiotic recombination is finished before the meiosis I spindle is set up (Lydall et al., 1996Go) and that anaphase I does not begin until all paired homologues are attached correctly to the spindle (Li and Nicklas, 1995Go). Work on cell signalling pathways in cell cycle control has been essential to our understanding of meiotic arrest in spores of yeast (Murakami and Nurse, 2000Go). In contrast, in human oocytes, few cases of meiosis I arrest have been observed. Hartshorne et al. reported a patient in whom germinal vesicle breakdown occurred in all oocytes, but they failed to enter M-phase (Hartshorne et al., 1999Go). Harrison et al. presented two cases where all oocytes which did not mature in vitro were arrested at the metaphase I stage (Harrison et al., 2000Go). A case of idiopathic infertility with oocyte metaphase I maturation block has also been reported (Bergère et al., 2001Go).

Here, we report our observations on oocytes from two patients who underwent unsuccessful IVF treatments. When the cumulus cells were removed 16–18 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.


    Case report
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 Abstract
 Introduction
 Case report
 Results
 Discussion
 Acknowledgements
 References
 
Patients and methods
Patient 1
A 34-year-old Turkish woman and her 32-year-old husband were admitted to our IVF department. The woman had been unable to conceive during the last 1.5 years with her current husband, and for 9 years before that with her first husband. The diagnostic examination revealed no abnormality. Several homologous and intrauterine inseminations did not lead to a pregnancy. The sperm parameters (World Health Organization, 1992Go) varied between normal and a slight teratozoospermia. The patient requested IVF and in the first treatment ovarian stimulation was performed with the combination of a GnRH agonist (Enantone; Takeda, Aachen, Germany) and recombinant FSH (Gonal-F, 150 IU day 1–10 and 250 IU day 11–15; Serono, Unterschleissheim, Germany). Ovulation was induced with 10 000 IU hCG (Primogonyl; Schering, Berlin, Germany) when the follicles had reached an average diameter of 22 mm.

Follicles were aspirated transvaginally under ultrasound guidance 34–36 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 16–18 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, 1966Go). 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., 1995Go).

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.


    Results
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 Abstract
 Introduction
 Case report
 Results
 Discussion
 Acknowledgements
 References
 
All the oocytes from patient 1 were characterized by condensed maternal metaphase I chromosomes and the prematurely condensed chromosomes (PCC) of the sperm nucleus (Figure 1Go). Individual chromosomal bivalents lay close together or were linked by chiasmata.



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Figure 1. Chromosome spread (A: overall view x200) of anin-vitro inseminated human oocyte (patient 1) exhibiting neither a polar body nor pronuclei (B: x120). Individual bivalent chromosomes of the metaphase I spread and the prematurely condensed chromosomes (PCC) of the sperm nucleus could be seen at higher magnification in insets 1–6 (x1000).

 
In patient 2, 19 inseminated and prepared oocytes, one haploid set of strongly condensed chromosomes and PCC of the sperm nucleus were observed. These compact bivalents were previously interpreted as metaphase II chromosomes accompanied by degeneration of the first polar body (Eichenlaub-Ritter et al., 1995Go). However, the finding of the metaphase I arrest in her sister made this previous interpretation unlikely, as it would implicate two different mechanisms of meiotic arrest in two siblings from consanguineous parents. Therefore, we re-examined the preparations made at that time and, although the chromosomal structure had suffered from ageing, metaphase chromosomes in a less compact state showed bivalents, indicating that these oocytes were blocked in metaphase I (Figure 2Go).



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Figure 2. Part of the chromosome spread of an in-vitro inseminated oocyte of patient 2 after re-examination. The bivalent nature of the chromosomes is still perceptible beside the sperm nucleus with signs of PCC (x1600).

 
Five oocytes from the last IVF trial were processed for anti-tubulin immunofluorescence after being aged in vitro for 24 h (Eichenlaub-Ritter et al., 1995Go). Chromosomes in several oocytes were still aligned in one plate, and a degenerate spindle typical of post-ovulatory aged oocytes was observed.

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 3Go) revealed that the sisters were descended from a consanguineous marriage of their parents, who were first cousins.



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Figure 3. The pedigree of the family indicates that both sisters (•) descend from consanguineous parents who were first cousins. Two brothers ({blacksquare}) may also be infertile, because although they have both been married for several years, they have remained childless.

 
There are two further brothers who have been married for several years without producing any offspring, indicating that these two men may also be infertile. Unfortunately, the brothers’ apparent infertility could not be proven directly, because it was not possible to obtain a semen sample for analysis. Two other sisters delivered two and four children respectively, consistent with an autosomal recessive pattern of inheritance.


    Discussion
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 Abstract
 Introduction
 Case report
 Results
 Discussion
 Acknowledgements
 References
 
Both familial cases reported here seem very similar to that reported by Bergère et al. (Bergère et al., 2001Go) with respect to the metaphase I arrest of the oocytes and their failure to mature in vitro. Since several regimens of hormonal stimulation and hormonal parameters were in the normal range and the oocytes were of normal size, it seems extremely unlikely that the block in oocyte development in both patients is due to inappropriate hormonal treatment. The results of anti-tubulin immunofluorescence in some oocytes from patient 2 (Eichenlaub-Ritter et al., 1995Go) led to the interpretation that the degenerate metaphase spindles in the aged oocytes were probably responsible for the developmental block. However, this interpretation was under the erroneous assumption of a rapid maturation to metaphase II before retrieval and prolonged arrest in this stage before fertilization, accompanied by degeneration of the first polar body. Now, the distinct representation of metaphase I chromosomes in patient 2’s oocytes is evident and could give new insights into the nature of this meiotic block. So far, neither the mechanism of metaphase I arrest nor the gene participating in this arrest is known in the case of our patients. However, the defect must be specific, acting only during meiosis I and not affecting mitosis, as revealed by normal mitotic progression of lymphocytes.

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., 1991Go). 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, 1995Go). 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., 1999Go).

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., 1986Go). 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, 1997Go) and studies have indicated that a failure to regulate protein kinase C clearly participates in the abnormal oocyte behaviour (Viveiros et al., 2001Go).

Therefore, the only way to identify the underlying gene defect in this autosomal recessive trait (Bittles et al., 1991Go) 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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Case report
 Results
 Discussion
 Acknowledgements
 References
 
We gratefully acknowledge the support of the attending physicians and surgical staff at Campus Virchow-Klinikum for their participation in the ovarian aspirations, especially B.Remberg.


    Notes
 
3 To whom correspondence should be addressed. E-mail: hardi.schmiady{at}charite.de Back


    References
 Top
 Abstract
 Introduction
 Case report
 Results
 Discussion
 Acknowledgements
 References
 
Bergère, M., Lombroso, R., Gombault, M., Wainer, R. and Selva, J. (2001) An idiopathic infertility with oocytes metaphase I maturation block. Hum. Reprod., 16, 2136–2138.[Abstract/Free Full Text]

Bittles, A.H., Mason, W.M., Greene, J. and Rao, N.A. (1991) Reproductive behavior and health in consanguineous marriages. Science, 252, 789–794.[ISI][Medline]

Choi, T., Aoki, F., Mori, M., Yamashita, M., Nagahama, Y. and Kohmoto, K. (1991) Activation of p34cdc2 protein kinase activity in meiotic and mitotic cell cycles in mouse oocytes and embryos. Development, 113, 789–795.[Abstract]

Eichenlaub-Ritter, U., Schmiady, H., Kentenich, H. and Soewarto, D. (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]

Hampl, A. and Eppig, J.J. (1995) Analysis of the mechanism(s) of metaphase I arrest in maturing mouse oocytes. Development, 121, 925–933.[Abstract/Free Full Text]

Harrison, K.L., Sherrin, D.A. and Keeping, J.D.(2000) Repeated oocyte maturation block. J. Assist. Reprod. Genet., 17, 231–233.[ISI][Medline]

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

Herbert, M., Morgan, J., Levasseur, M., Murdoch, A.P. and McDougall, A. (1999) A non-degradable form of cyclin can be used to induce arrest at the first meiotic metaphase in human oocytes. Hum. Reprod., 14 (Abstract Book 1), 135–136.

Hirao, Y. and Eppig, J.J. (1997) Analysis of the mechanism(s) of metaphase I arrest in strain LT mouse oocytes: participation of MOS. Development, 124, 5107–5113.[Abstract/Free Full Text]

Li, X. and Nicklas, R.B. (1995) Mitotic forces control a cell-cycle checkpoint. Nature, 373, 630–632.[ISI][Medline]

Lydall, D., Nikolsky, Y., Bishop, D.K. and Weinert, T. (1996) A meiotic recombination checkpoint controlled by mitotic checkpoint genes. Nature, 383, 840–843.[ISI][Medline]

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Schmiady, H., Sperling, K., Kentenich, H. and Stauber, M. (1986) Prematurely condensed human sperm chromosomes after in vitro fertilization (IVF). Hum. Genet., 74, 441–443.[ISI][Medline]

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Viveiros, M.M., Hirao, Y. and Eppig, J.J. (2001) Evidence that protein kinase C (PKC) participates in the meiosis I to meiosis II transition in mouse oocytes. Dev. Biol., 235, 330–342.[ISI][Medline]

World Health Organization (1992). World Health Organization Laboratory Manual for the Examination of Human Semen and Sperm–Cervical Mucus Interaction. Cambridge University Press, Cambridge.

Submitted on March 22, 2002; accepted on May 29, 2002.