Human Reproduction Vol.17, No.10 pp. 2556–2559, 2002
CASE REPORT
Arrest of human oocytes during meiosis I in two sisters of
consanguineous parents: first evidence for an autosomal
recessive trait in human infertility
Hardi Schmiady1,3 and Heidemarie Neitzel2
Charité, Medizinische Fakultät der Humboldt-Universität zu Berlin, Campus Virchow-Klinikum, 1Klinik für Frauenheilkunde und
Geburtshilfe, Reproduktionsmedizin and 2Institut für Humangenetik, Genetische Beratung, Augustenburger Platz 1, 13353 Berlin,
Germany
3To
whom correspondence should be addressed. E-mail: [email protected]
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
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., 1996) and that
anaphase I does not begin until all paired homologues are
attached correctly to the spindle (Li and Nicklas, 1995). 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, 2000). 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., 1999). 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., 2000).
A case of idiopathic infertility with oocyte metaphase I
maturation block has also been reported (Bergère et al., 2001).
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
2556
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
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, 1992) 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.
© European Society of Human Reproduction and Embryology
Autosomal recessive oocyte meiosis I arrest
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, 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.
Results
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 1).
Individual chromosomal bivalents lay close together or were
linked by chiasmata.
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., 1995). 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 2).
Five oocytes from the last IVF trial were processed for antitubulin immunofluorescence after being aged in vitro for 24 h
(Eichenlaub-Ritter et al., 1995). 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 3)
revealed that the sisters were descended from a consanguineous
marriage of their parents, who were first cousins.
There are two further brothers who have been married for
Figure 1. Chromosome spread (A: overall view ⫻200) of an
in-vitro inseminated human oocyte (patient 1) exhibiting neither a
polar body nor pronuclei (B: ⫻120). 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 (⫻1000).
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
Both familial cases reported here seem very similar to that
reported by Bergère et al. (Bergère et al., 2001) with respect
to the metaphase I arrest of the oocytes and their failure to
2557
H.Schmiady and H.Neitzel
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 (⫻1600).
Figure 3. The pedigree of the family indicates that both sisters (d)
descend from consanguineous parents who were first cousins. Two
brothers (j) may also be infertile, because although they have both
been married for several years, they have remained childless.
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., 1995) 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-cyclin2558
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 cyclinB1-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.
Autosomal recessive oocyte meiosis I arrest
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
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.
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.
Bittles, A.H., Mason, W.M., Greene, J. and Rao, N.A. (1991) Reproductive
behavior and health in consanguineous marriages. Science, 252, 789–794.
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.
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.
Hampl, A. and Eppig, J.J. (1995) Analysis of the mechanism(s) of metaphase
I arrest in maturing mouse oocytes. Development, 121, 925–933.
Harrison, K.L., Sherrin, D.A. and Keeping, J.D.(2000) Repeated oocyte
maturation block. J. Assist. Reprod. Genet., 17, 231–233.
Hartshorne, G., Montgomery, S. and Klentzeris, L. (1999) A case of failed
oocyte maturation in vivo and in vitro. Fertil. Steril., 71, 567–570.
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.
Li, X. and Nicklas, R.B. (1995) Mitotic forces control a cell-cycle checkpoint.
Nature, 373, 630–632.
Lydall, D., Nikolsky, Y., Bishop, D.K. and Weinert, T. (1996) A meiotic
recombination checkpoint controlled by mitotic checkpoint genes. Nature,
383, 840–843.
Murakami, H. and Nurse, P. (2000) DNA replication and damage checkpoints
and meiotic cell cycle controls in the fission and budding yeasts. Biochem.
J., 349, 1–12.
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.
Tarkowski, A.K. (1966) An air-drying method for chromosome preparations
from mouse eggs. Cytogenetics, 5, 394–400.
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.
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
2559