Human Reproduction, Vol.25, No.1 pp. 179–191, 2010
Advanced Access publication on October 14, 2009 doi:10.1093/humrep/dep347
ORIGINAL ARTICLE Reproductive biology
Cytogenetic analyses of human oocytes
provide new data on non-disjunction
mechanisms and the origin
of trisomy 16
R. Garcia-Cruz 1,5, A. Casanovas 1, M. Brieño-Enrı́quez 1, P. Robles1,
I. Roig2, A. Pujol 4, L. Cabero3, M. Durban 4, and M. Garcia Caldés 1
1
Unitat de Biologia Cel.lular i Genètica Mèdica, Facultat de Medicina, Universitat Autònoma de Barcelona, Barcelona, Spain 2Molecular
Biology Program, Memorial Sloan-Kettering Cancer Center, NY, USA 3Servei de Ginecologia i Obstetrı́cia, Hospital de la Vall d’Hebrón,
Barcelona, Spain 4Clı́nica Eugin, Barcelona, Spain
5
Correspondence address. E-mail: [email protected]
background: Nowadays, oocyte donation is an extended practise in IVF programmes. However, to date, little information on aneuploidy frequency in oocytes from donors is available. Aneuploidy is one of the major causes of embryo and fetal wastage as well as of congenital mental and developmental disabilities. It is known that most aneuploidies are due to non-disjunction events occurring in the maternal
germ line. Linkage studies have associated abnormal patterns of meiotic recombination to the origin of the non-disjunction event in many
aneuploid conditions.
methods and results: In the present study, we analyse the frequency of chromosome imbalances in a series of metaphase I (MI;
n ¼ 44) and metaphase II (MII; n ¼ 103) oocytes from 140 young donors (aged from 18 to 35 years, mean age 26.6) after hormone-induced
superovulation. The aneuploidy frequency found in MII oocytes was 12.6%, and both whole-chromosome non-disjunction (1.94%) and premature separation of sister chromatids (PSSC) (12.6%) have been found. The chromosomes involved have been identified by multiplex fluorescent in situ hybridization (FISH). Achiasmate chromosomes have been identified in MI oocytes (9.1%), with most of them corresponding
to chromosome 16 (6.8%). For this reason, the meiotic recombination pattern of chromosome 16 has been analysed in prophase I oocytes
(n ¼ 81) by immunofluorescence staining against MLH1 protein and subsequent FISH with specific probes. Our results show a percentage of
oocytes with non-crossover bivalent 16 (2.5%) and a high percentage of bivalents 16 with a single exchange (19.8%).
conclusions: In the present study, we report the finding of a considerable frequency of aneuploidy in oocytes from young donors,
with the frequency of PSSC being higher than the frequency of whole-chromosome non-disjunction. In addition, we report vulnerable patterns of meiotic recombination in chromosome 16 that may be at risk of leading to a non-disjunction event. This gives new data on the
susceptibility of the control population to conceive a trisomic 16 embryo.
Key words: aneuploidy / chromosome non-disjunction / oocyte donor / meiotic recombination / trisomy 16
Introduction
Aneuploidy is the leading cause of inborn mental and developmental
disabilities. Additionally, it constitutes one of the major causes of
embryo implantation failure, miscarriage and pregnancy loss
(Hassold and Hunt, 2001). As a class, trisomy is the most common
type of aneuploidy. The contribution of each particular chromosome
among trisomic individuals varies depending on the population analysed. Hence, the most common trisomy among spontaneous miscarriages is trisomy 16, followed by trisomy 22 and trisomy 21 (Hassold
et al., 1980; Eiben et al., 1990). On the other hand, trisomy 21 and
sex-chromosome trisomies are the most prevalent in newborns,
due to the inviability of most of the other trisomic conditions.
In recent decades, linkage analysis of affected individuals and trisomic conceptuses has traced the origin of the extra chromosome. Such
studies report that most aneuploidies are due to errors which have
arisen in the oocyte by chromosome segregation errors (generically
known as non-disjunction events) and that in general terms (with
the few exceptions of trisomy 18 and 47,XXY) the errors are
mostly originated in the oocyte during first meiotic division (reviewed
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180
in Lamb et al., 2005; Hassold et al., 2007). Nowadays, it is well known
that advanced maternal age increases the likelihood of a nondisjunction event, although the strength of the maternal-age effect
depends on each particular aneuploidy (Risch et al., 1986; Lamb
et al., 2005; Pellestor et al., 2005b).
Besides maternal age, to date, the only factor that has been unequivocally associated with the genesis of aneuploidies is abnormal patterns of meiotic recombination (reviewed in Lamb et al., 2005;
Garcia-Cruz et al., 2009). Besides generating genetic variability, recombination events have another fundamental role during meiosis: they
tether homologous chromosomes together at the places where a
crossover (CO) has occurred (chiasmata). In general terms, linkage
analyses of particular aneuploidies have reported decreased levels of
recombination in the chromosomes implicated in the non-disjunction
event. In fact, reduced recombination in trisomy-generating chromosomes, with respect to normally disjoining chromosomes, mainly
due to an increased proportion of non-exchange tetrads, has been
reported for nearly all acrocentric chromosome trisomies and some
non-acrocentric chromosome trisomies (trisomy 18 and sex chromosome trisomies) (MacDonald et al., 1994; Sherman et al., 1994; Lamb
et al., 1996; Bugge et al., 1998, 2007; Robinson et al., 1998; Hall et al.,
2007a, b). Reduced recombination is also associated with the genesis
of trisomy 16, although a relatively low percentage of cases are
derived from non-exchange bivalents (Hassold et al., 1995). In contrast, the origin of some aneuploidies is related to increased recombination patterns, usually linked to a shift towards pericentromeric
exchanges, as is the case of trisomy 21 of maternal meiosis II origin
(Lamb et al., 1996, 1997).
Direct evaluation of meiotic recombination has been classically
based on the counting of chiasmata in diakinesis/metaphase I (MI)
cells. This approach, which has largely been used in human spermatocytes, has not proved useful for chiasma counts in human oocytes
mainly because female MI preparations lack the clarity of definition of
chromosome structure needed for chiasma counting (Angell, 1995).
During the last decade, an emergence of direct studies of meiotic
recombination has been possible due to the recognition that
certain proteins, including mismatch repair protein MLH1, localize
to sites of COs at the pachytene stage. Specific antibodies have
been used for the visualization of MLH1 foci along the Synaptonemal
Complexes (SC). Unfortunately, data regarding MLH1 distribution in
the human oocyte are very scarce, mainly due to the difficulties
accessing sample material. In mammalian females, meiotic recombination takes place during fetal life, so the obtainment of fetal
ovaries is required for the analysis of patterns of meiotic exchange.
In spite of this intrinsic technical difficulty, to date, analyses of the
number and distribution of COs in human oocytes have been conducted in chromosomes 13, 18, 21 and X. Although exchange configurations susceptible non-disjunction in the adult ovary have been
found, these seem to occur at a low percentage in the fetal ovary
for these particular chromosomes (Tease et al., 2002; Robles
et al., 2009).
Besides maternal age and meiotic recombination, it has been
recently conjectured that gonadal mosaicism may also be involved in
the genesis of aneuploidy (Hulten et al., 2008).
Two different mechanisms of non-disjunction occurring at the first
meiotic division have been identified. The imbalances found at the
resulting MII oocyte are different:
Garcia-Cruz et al.
(i) whole-chromosome non-disjunction, where both homologues are
segregated to the same pole; as a result, an extra or missing univalent is found at the metaphase II (MII) oocyte, and
(ii) pre-division (PD) (Angell, 1991), also known as precocious separation of sister chromatids (PSSC), where the connection between
sister chromatids is abnormally lost at meiosis I; therefore, missing
or extra chromatids are found in the oocyte. In addition, several
studies have reported the finding of dissociated chromatids from
one univalent chromosome in MII oocytes (balanced PD).
During the last few decades, cytogenetic studies on human MII
oocytes have provided relevant data on the mechanisms of nondisjunction that lead to chromosome imbalances. Despite discrepancies in the frequency of whole-chromosome non-disjunction and
PSSC in the overall aneuploidy frequency, most authors agree
that both mechanisms are involved in the origin of aneuploidy in
human oocytes (reviewed in Pellestor et al., 2005a; Rosenbusch,
2006).
The use of oocyte donation offers the possibility for infertile women
to become pregnant, and it is now commonplace in the countries
where this practise is authorized. However, until now, most of the
studies on oocyte aneuploidy and mechanisms of non-disjunction
have been conducted on oocytes from women undergoing IVF due
to infertility of various aetiologies. Nowadays, little information is available for oocytes and embryos from young donors, although it is commonly expected that the frequency of imbalanced oocytes should be
lower due to the young age of the women eligible to become an
oocyte donor and to the assumption that they are fertile. However,
so far, studies in that area report diverse rates of aneuploidy in
oocytes (3% in Fragouli et al., 2009; 29% in Sandalinas et al., 2002;
65% in Sher et al., 2007) and embryos (40.7% in Reis Soares et al.,
2003; 25% in Munne et al., 2006) from young donors. Apart from
the obvious interest for the clinical community, the analysis of
oocytes from donors has a scientific significance, as it offers the possibility to study the mechanisms of non-disjunction in a less biased
population.
Herein, the results of the cytogenetic analysis of a series of human
oocytes at the MI and MII stages coming from women included in
oocyte donation programmes are presented. Chromosome imbalances were detected in MI and MII oocytes by examination of
chromosome size and centromere position as determined by immunofluorescence (IF). The chromosomes involved in the imbalances
were then identified by multiplex fluorescence in situ hybridization
(M-FISH). Finally, the meiotic recombination pattern of chromosomes
more commonly found as achiasmate in MI oocytes was analysed in
fetal oocytes by IF against MLH1 and subsequent FISH with specific
probes.
Materials and Methods
MI and MII oocyte processing
Oocyte recovery and fixation for the obtainment of chromosome spreads
A total of 263 oocytes were obtained from 140 oocyte donors (18– 35
years old, mean age: 26.6) after hormone-induced superovulation performed at the Clı́nica Eugin (Barcelona, Spain). Written informed
consent was obtained from all women. Oocytes were retrieved by transvaginal follicular puncture and denuded. Most oocytes were at the MII
181
Oocyte non-disjunction and trisomy 16 origin
stage (MII) but a small proportion of the retrieved oocytes were found to
be immature at the germinal vesicle (GV) or at the MI stages. The oocytes
used for this study did not undergo any further procedure for reproductive
purposes and were collected from the clinic a few hours after retrieval.
Mean number of oocytes per donor was 1.8 oocytes (range 1 – 9 oocytes).
MI and MII oocytes were fixed at the University laboratories upon
arrival (lapse between follicular puncture and fixation did not exceed
7 h). From the 191 MII oocytes fixed, 148 came from MI oocytes that
underwent spontaneous maturation to MII within few hours after retrieval.
In vitro matured MII oocytes were not used for reproductive purposes due
to Eugin clinic guidelines and were donated for the present study. Results
from in vivo and in vitro matured MII oocytes have been combined due to
sample size.
GV oocytes were cultured in G-FERT medium (Vitrolife) to promote
GV breakdown and resumption of meiosis for the obtainment of MI
oocytes. Those oocytes that did not show signs of maturation after
15 – 17 h in culture were discarded from the analysis. From the 71
MI oocytes fixed, 20 came from GV oocytes matured in G-FERT
medium. Results from these groups have been combined due to
sample size.
Fixation of oocytes at the MI and MII stages was performed following
the method described by Hodges and Hunt (2002), with some minor
modifications. Briefly, the zona pellucida was removed by using Tyrode’s
acid (Sigma) and the oocytes were rinsed in the medium. Zona-free
oocytes were placed on slides soaked in fresh fixative solution (1% paraformaldehyde, 0.18% Triton X-100, 3 mM dithiothreitol in water, pH 9.2)
under a stereomicroscope. Slides were rapidly placed in a humid chamber
and kept for several hours before rinsing in 1% photoflo (Kodak). DNA
was counterstained by applying an antifade solution containing 0.1 mg/
ml 4’,6’-diamidino-2-phenylindole (DAPI) and the slides were checked
using an epifluorescence microscope. Only those preparations showing
good chromosome morphology and no cytoplasm were further processed
by IF.
Immunofluorescence
A standard protocol was used for the immunostaining of oocyte preparations. Briefly, slides were blocked in 0.2% bovine serum albumin,
0.05% Tween-20 for 30 min. CREST patient serum (a kind gift of
M. Fritzler, University of Calgary), which is enriched with anti-centromeric
protein antibodies, was used for the identification of centromeres. A FITCconjugated secondary antibody (Jackson ImmunoResearch) was used for
detection. DNA was counterstained by applying an antifade solution containing 0.1 mg/ml DAPI.
Both fetuses had a euploid karyotype as confirmed by prenatal cytogenetic diagnosis. Ovaries were removed within 1 h after delivery by the
team of the Foetal Tissue Bank of the hospital and transported to our laboratory in Modified Eagle medium on ice.
The fixation protocol has been previously described by us (Roig et al.,
2005). Briefly, ovaries free of adjacent tissues were pricked with entomologic needles to allow oocyte release. Twenty microlitres of the resulting
cellular suspension were centrifuged with 500 ml of 0.1 M sucrose in a
cytocentrifuge chamber containing a slide. After centrifugation, the slide
was placed in a humid chamber for 2 h. Five-hundred microlitres of
fresh fixative solution (1% paraformaldehyde, 0.15% Triton X-100 in
water, pH 9.8) were added to the slide and allowed to fix for several
hours. After rinsing in 1% photoflo (Kodak), the slides were processed
for IF.
Immunofluorescence
As mentioned for MI and MII oocytes, a standard protocol was used for
immunostaining of prophase oocyte preparations. In prophase I oocyte
preparations, the following primary antibodies were used: rabbit polyclonal
antibody against SC protein SYCP3 (a kind gift of C. Heyting, Wageningen
University), mouse monoclonal antibody against the CO marker protein
MLH1 (BD Pharmingen) and CREST serum (a kind gift of M. Fritzler, University of Calgary). Cy3-, FITC- and Cy5-conjugated secondary antibodies
(all from Jackson ImmunoResearch) were used for detection. DNA
was counterstained by applying an antifade solution containing
0.1 mg/ml DAPI.
FISH on immunostained preparations of fetal oocytes
Probes for chromosome 16 centromere (labelled with SpectrumGreen)
and q arm telomere (labelled with SpectrumOrange) (both from Vysis)
were used for the identification of chromosome 16 p and q arms. A
probe for chromosome 17 centromere (labelled with SpectrumAqua)
(from Vysis) was used for the detection of chromosome 17. Chromosome
17 p and q arms were identified by length. A mix of the three probes was
hybridized on previously immunostained preparations. Briefly, slides were
denatured in 70% formamide in 2 SSC at 738C for 5 min. A treatment
with sodium thiocyanate (NaSCN) was applied for 4 h at 658C before a
second denaturation of the slide was performed. The probe mix was
denatured following the manufacturer’s instructions. Slides and probes
were allowed to hybridize for 48 h at 378C.
Microscopy and image analysis
Multiplex FISH
Briefly, slides were incubated with 0.005% pepsin for 5 min before
denaturation in 70% formamide at 738C for 1 min. SpectraVysion probe
(Vysis) was denatured following the manufacturer’s instructions,
applied to the target area on the slide and allowed to hybridize at 378C
overnight.
Prophase oocytes processing
Sample processing for the obtainment of SC spreads
In mammalian females, the meiotic prophase initiates during fetal life.
Therefore, the obtainment of fetal ovaries is required for the analysis of
events occurring at prophase I, such as meiotic recombination.
In the present study, ovaries were obtained from two fetuses after legal
interruption of pregnancy performed at the Hospital de la Vall d’Hebrón
(Barcelona, Spain) according to the Ethics Rules Committee of the Hospital. Gestational ages were 19 and 21 weeks. Indication for pregnancy
interruption was Arnold Chiari malformation and Glutaric Aciduria type I.
MI and MII oocyte preparations were visualized on an Olympus BX60
epifluorescence microscope equipped with the appropriate filters and
a charge-coupled device (CCD) camera. Images from IF- and
M-FISH-processed preparations were captured, produced and analysed
by PathVysion and SpectraVysion software, respectively (both from Vysis).
SC preparations were visualized on a Nikon Eclipse 90i epifluorescence microscope equipped with the appropriate filters and a CCD
camera. Images were captured and produced by Isis software
(Metasystems).
Analysis of MLH1 distribution
Micromeasure
software
(http://www.biology.colostate.edu/Micro
Measure/) was used for the analysis of the distribution of MLH1 on
bivalent 16 and bivalent 17. Micromeasure is an application that allows
the measurement of different parameters from digitally captured images.
In the present study, the total length of the SC, the position of the centromere and the position of MLH1 foci were determined for SC16 and SC17.
182
Garcia-Cruz et al.
Results
MII oocyte analysis
A total of 191 MII oocytes were fixed. From those, 88 oocytes could
not be further analysed because excessive chromosome superposition, poor chromosome morphology or evident signs of degeneration,
as deduced by the presence of DNA with no chromosome morphology, were observed or because they were covered by cellular
debris. The analysable oocytes (n ¼ 103) were further processed by
IF to identify the position of the centromeres, which, together with
chromosome size, allowed for the classification of the oocytes
(Table I).
For the 103 analysable oocytes, 53 (51.4%) were euploid (Fig. 1a),
27 (26.2%) had alterations in the number of whole chromosomes, 11
(10.7%) had extra and/or missing chromatids (PSSC) and 11 (10.7%)
were diploid. In addition, regardless of whether they were aneuploid
or not, two oocytes (1.94%) showed two separate chromatids replacing a missing chromosome (Balanced PD).
The most common alteration involving whole chromosomes was
hypohaploidy (less than 23 univalents) (n ¼ 26; 25.2%), whereas
hyperhaploidy of a whole chromosome was only found in one
oocyte (0.97%) (Fig. 1b). Of the oocytes showing whole-chromosome
hypohaploidy, 15 oocytes (57.7%) had 22 univalents, 5 oocytes
(19.2%) had 21 univalents and 6 oocytes (23.1%) had 20 or fewer
univalents. Chromosome hypohaploidy may be due to chromosome
artifactual loss during the fixation procedure, thus not all cases can
be considered to be an actual oocyte abnormality. Therefore, according to convention, a conservative estimate of the total frequency of
whole-chromosome non-disjunction was calculated by doubling the
frequency of hyperhaploidy found (in the present study, conservative
estimate of total whole-chromosome non-disjunction is 1.94%).
For the 11 oocytes showing PSSC (Fig. 1c–e), eight oocytes showed
chromatid loss (seven oocytes had the loss of a chromatid from one
Table I Number of chromosome abnormalities in MII
oocytes
n
Percentage
........................................................................................
Oocytes analysed
103
Euploid
53
51.4
Missing chromosome
26
25.2
Extra chromosome
1
0.97
21
1.94
Missing chromatid
10
9.7
Extra chromatid
3
2.9
Total unbalanced PD
13
12.6
Balanced PD
2
Alterations in the number of chromosomes
Total whole-chromosome non-disjunction
(conservative estimate)
Alterations involving single chromatids*
1.94
Total aneuploidy (excluding balanced PD) *
13
12.6
Diploidy
11
10.7
*Oocytes with more than one type of abnormality are counted in each line that applies,
but only once in the total of aneuploidy.
chromosome and one oocyte had the loss of chromatids from two
different chromosomes), two oocytes showed chromatid gain and
one oocyte showed gain and loss of chromatids from different
chromosomes.
Subsequent to IF, M-FISH was performed on the preparations of MII
oocytes showing imbalances to identify the chromosomes involved in
the non-disjunction event (Fig. 1d2, d3, e2 and e3). Individual karyotypes and donor ages are summarized in Table II.
The chromosome most involved in PSSC events was chromosome
22 (3 of 10 events involving missing chromatids and 1 of 3 events involving extra chromatids) (Fig. 1d and e) followed by chromosome 15
(2 of 10 involving missing chromatids and 1 of 3 events involving
extra chromatids). Other chromosomes identified in PSSC events
include chromosomes 4, 8, 12, 14, 16 and 19. The chromosomes
involved in balanced PD events were chromosomes 16 and 22.
In a few cases, the corresponding first polar body (PB) could also be
analysed. In 100% of the cases, the PB karyotype was complementary
to the corresponding MII oocyte (Table II).
When extra chromatids were present, they were normally found as
single extra chromatids dissociated from its homologous chromosomes. However, the singular case of an oocyte was found with an
extra chromatid 4 attached to the univalent 4 at the centromeric
area (Fig. 1c).
Unfortunately, we were unable to identify the extra chromosome in
the oocyte showing hyperploidy due to a technical problem during the
hybridization protocol. However, chromosome size and centromere
position allowed us to identify it as a B-group chromosome (chromosome 4 or 5). Curiously, the extra chromosome was superposed onto
another B chromosome at the pericentromeric area as if they were
actually attached to each other (Fig. 1b).
MI oocyte analysis
A total of 71 MI oocytes were fixed. From those, 24 could not be
further analysed mainly because of the absence of chromosome condensation, poor chromosome morphology or excessive chromosome
superposition. In addition, three oocytes had already started the transition to anaphase I at the moment of fixation as deduced by the
finding of segregating homologues. These oocytes were classified as
anaphase I and were discarded from the present analysis. The analysable oocytes (n ¼ 44) were further processed by IF to identify the
position of the centromeres, which allowed for the classification of
oocytes (Table III). For the 44 analysable oocytes, 30 (68.2%) were
euploid (Fig. 2a), 10 (22.7%) were hypohaploid (having 22 or fewer
bivalents), most likely as a consequence of artifactual chromosome
loss during spreading, and four oocytes (9.1%) showed a pair of univalent chromosomes (achiasmate) (Fig. 2b1).
M-FISH performed on the preparations of MI oocytes with achiasmate chromosomes showed that chromosome 16 was clearly the
most commonly involved (three out of four cases) (Fig. 2b2 and
b3). The other case of achiasmate chromosomes corresponded to
chromosome 15. Individual karyotypes and donor ages are summarized in Table II.
MLH1 analysis
The finding that three out of four achiasmate chromosomes in MI
oocytes corresponded to chromosome 16 led us to analyse the
183
Oocyte non-disjunction and trisomy 16 origin
Figure 1 Oocytes at the MII stage.
(a) MII with an euploid karyotype. (b) MII with an extra chromosome (arrowhead). (c) MII with an extra chromatid attached to its homologue (arrowhead). (d1) MII showing a
single chromatid (arrowhead). The same metaphase after M-FISH hybridization (d2) and karyotype (d3) showed a single chromatid 22. (e1) MII showing a single chromatid
(arrowhead). The same metaphase after M-FISH hybridization (e2) and karyotype (e3) showed a single chromatid 22 plus a chromosome 22 (also arrowhead in e2).
recombination profile of this chromosome. We decided to compare it
with that of chromosome 17, as this is a similar sized chromosome not
involved in the most commonly recognized aneuploidies in spontaneous miscarriages. Analysis was carried out by IF for the identification of MLH1, SCP3 and centromeres (Fig. 3a1). SCP3 is a
component of the lateral element of the SC and consecutive FISH
with specific probes for the identification of the SC16 centromere
and q arm telomere and for the SC17 centromere (Fig. 3a2).
Number and position of MLH1 foci were analysed in SC16 and
SC17 from prophase oocytes obtained from two fetal samples.
MLH1 frequencies in the analysed bivalents did not show statistical
differences between the two samples analysed, so data from both
cases were added up. A total of 81 SC16 and 93 SC17 were analysed.
MLH1 values
The number of MLH1 foci on both SC16 and SC17 ranged from 0 to 4
foci. The mean MLH1 focus values were 2.1 for SC16 and 2.35 for
SC17 (Table IV). In both SC16 and SC17, the presence of two
MLH1 foci was the most commonly observed pattern, followed by
three MLH1 foci and one MLH1 focus (Table V). Nevertheless, frequency of bivalents showing one MLH1 focus was significantly higher
for SC16 compared with SC17 (19.8% and 6.4%, respectively;
Fisher’s test, P-value: 0.0112). In contrast, frequencies of bivalents
showing two, three and four MLH1 foci were lower for SC16 with
respect to SC17 (48.1% versus 53.8%; 24.7% versus 33.3%; 4.9%
versus 5.4%), although no statistical differences were found between
both bivalents in these groups. Bivalents lacking MLH1 were found
in both SC16 and SC17 (2.5% and 1.1% bivalents, respectively).
MLH1 distribution
For the analysis of the distribution of COs on SC16 and SC17, both
chromosome arms were divided into 10 segments of equal length
and the presence of MLH1 foci in each section was scored (Fig. 3b).
When a single MLH1 focus was found on SC16, this was generally
located on the p arm (11 of 16; 68.8%). In contrast, a single MLH1
focus on SC17 is normally found on the q arm (5 of 6; 83.3%).
When two MLH1 foci were present, the common distribution in
both SC16 and SC17 was one focus per each chromosome arm.
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Garcia-Cruz et al.
Table II Abnormal karyotypes found in MII and MI
oocytes and corresponding donor age
Karyotype
Donor age
........................................................................................
MII,24,X,þB
23
MII,23,X,þ15cht
29
MII,22,X,-16þ16cht*,1
31
MII,20,X,-2,-15,þ15cht,-16,þ16cht,þ16cht
30
MII,22,X,-22,þ22cht*,2
31
MII,22,X,þ4cht,-22,þ22cht
21
MII,21,X,-14,þ14cht,-19,þ19cht
35
,3
MII,23,X,þ22cht*
35
MII,22,X,-8,þ8cht*,4
35
MII,22,X,-22,þ22cht
27
MII,22,X,-22,þ22cht,þ22cht
27
MII,22,X,-15,þ15cht
19
MII,22,X,-12,þ12cht
31
MI,24,XX,-II(16),þI(16),þI(16)
29
MI,24,XX,-II(16),þI(16),þI(16)
32
MI,24,XX,-II(16),þI(16),þI(16)
34
MI,24,XX,-II(15),þI(15),þI(15)
31
*The first polar body (PB) of these oocytes was available for analysis.
The corresponding karyotypes were: 123,X,þ16cht; 223,X,þ22cht; 322,X,þ22cht;
4
23,X,þ8cht.
commonly observed in SC17 with four MLH1 foci, although two bivalents (40%) were found with two MLH1 foci on each arm.
In general terms, MLH1 foci were located in interstitial regions along
the chromosome arms in both SC16 and SC17. MLH1 foci close to
the centromere (i.e. within 10% from the centromere at both chromosome arms) were never seen on SC16 and seldom seen on SC17 (4 of
93; 4.3%). However, the finding of one SC17 with a single MLH1 focus
in the whole bivalent, which was located extremely close to the centromere (within only 4.4% of the q arm length), was intriguing. MLH1
foci were seldom seen close to the telomeres in SC16: only one bivalent 16 (1 of 81; 1.2%) had an MLH1 focus within 10% of the q arm
telomere. Meanwhile, nine SC17 (9 of 93; 9.7%) were found with
an MLH1 focus within 10% of the q arm telomere, although only
one of them was extremely close to the telomere (less than 4.5%
of the q arm telomere).
To facilitate comparisons between MLH1 distribution in SC16 and
SC17, cumulative frequency plots were created for the p and q
arms of both chromosomes (Fig. 3c). Despite differences in the distribution of MLH1 per chromosome arm between SC16 and SC17,
these were not statistically different (Kolmogorov –Smirnov test,
P-value: 0.787 and 0.884 for the p and q arms, respectively). Notwithstanding, the absence was noticeable of MLH1 foci from the centromere until almost 20% of the q arm length in SC16, attributable to
the presence of the pericentromeric heterochromatin block in
chromosome 16 (16qh).
Discussion
Table III Cytogenetic classification of the analysable
MI oocytes
n
%
........................................................................................
Oocytes analysed
44
Euploid
30
68.2
Hypohaploid
10
22.7
3
6.8
Containing achiasmate
MI,24,XX,-II(16),þI(16),þI(16)
MI,24,XX,-II(15),þI(15),þI(15)
1
2.3
Total
4
9.1
However, in some cases, both MLH1 foci were located in the same
arm, which was always the q arm. This was quite common in SC17
(13 of 50, 26%), but very rare in SC16 (1 of 39, 2.6%), most probably
due to the fact that chromosome 17 q arm is longer than chromosome 16 q arm.
When three MLH1 foci were present, one was generally positioned
on the p arm and two on the q arm in both SC16 and SC17. Nevertheless, a small proportion of SC16 showed two MLH1 foci on the p
arm and only one MLH1 on the q arm (4 of 20; 20%). In contrast, in
some SC17, the three MLH1 foci were located on the q arm, whereas
no MLH1 focus was found on the p arm (3 of 31; 9.7%).
When four MLH1 foci were present, they normally distributed two
foci per arm in SC16, although one SC16 had one focus on the p arm
and three foci on the q arm. This last distribution pattern was the most
In the present study, a series of MII and MI human oocytes from
women participating in oocyte donation programmes have been analysed. Different mechanisms of chromosome non-disjunction were
clearly identified by chromosome morphology and centromere detection by IF. Moreover, chromosomes involved in these events were
next identified by M-FISH. MII analysis revealed that PSSC is more frequent than whole-chromosome non-disjunction and that the chromosomes most involved are chromosome 22 followed by chromosome
15. Chromosomes 16 and 22 have been found in balanced PD
events. MI analysis revealed the presence of achiasmate chromosomes, generally involving chromosome 16. Subsequent analysis of
meiotic recombination in chromosome 16 was performed by IF
against MLH1 in prophase-stage oocytes. This analysis revealed a percentage of non-exchange bivalents and a high percentage of bivalents
with a single CO.
Aneuploidy and mechanisms of
non-disjunction in MII and MI oocytes from
oocyte donors
During the last few decades numerous studies have reported aneuploidy frequencies in human MII oocytes. In most of them, the
oocytes used for the analysis came from patients undergoing IVF for
infertility due to different aetiologies. In addition, aneuploidy studies
are usually based on the analysis of oocytes discarded because they
failed to fertilize, although aneuploidy frequencies have also been
reported from the analysis of polar bodies of non-discarded oocytes
during PGD procedures (Kuliev et al., 2005; Montag et al., 2005;
Vialard et al., 2007). The frequency of aneuploidy reported for the
Oocyte non-disjunction and trisomy 16 origin
185
Figure 2 Oocytes at the MI stage.
(a) MI with an euploid karyotype. (b1) MI showing two univalents (arrowheads). The same metaphase after M-FISH hybridization (b2) and karyotype (b3) showed two
chromosome 16 univalents.
different studies display substantial variations, but the values go as high
as 33% in conventional karyotyping studies using the gradual fixation
technique (reviewed in Pellestor et al., 2005a, 2006), 47.5% in FISH
studies analysing nine chromosomes (Pujol et al., 2003), 57.1% in comparative genomic hybridization (CGH) studies (Gutierrez-Mateo et al.,
2004a; Gutierrez-Mateo et al., 2004b) and 53.7% in PB analysis by
FISH (Montag et al., 2005). In the current study, oocytes from
young women, with no apparent fertility problems, which have been
included in donation programmes for reproductive purposes, have
been analysed. Thus, this sample constitutes a less biased population
for the evaluation of the aneuploidy frequency in human MII oocytes,
which was of 12.6% in the present study. In addition, the oocytes
donated had not undergone any further procedure for reproductive
purposes after retrieval and were donated within few hours after
the follicular puncture, so influence of prolonged time in culture or
other manipulation could be excluded. However, conclusions should
also be considered with care as the superovulation treatment is also
known to induce aneuploidy (Reis Soares et al., 2003; Munne et al.,
2006). In addition, some of the MII oocytes analysed in the current
study had undergone spontaneous in vitro maturation, which could
also predispose to aneuploidy. In the present study, the chromosomes
most frequently involved in chromosome abnormalities were chromosomes 22 and 15 at MII and chromosome 16 at MI. Chromosomes 15,
16 and 22 have also been frequently involved in aneuploidy in previous
studies of polar bodies, oocytes, embryos and spontaneous
miscarriages.
It has been reported that frequencies of both whole-chromosome
non-disjunction and PSSC events are positively correlated with
maternal age, but that the effect is more pronounced for PSSC
events (Sandalinas et al., 2002; Pellestor et al., 2003; Vialard et al.,
2007). In the present study, we have also identified both mechanisms
leading to non-disjunction in oocytes from young oocyte donors,
although the frequency of PSSC is much higher than the one found
for whole-chromosome non-disjunction (12.6% versus 1.94%). This
is in agreement with other reports which cite PSSC as the main contributor to oocyte aneuploidy (Angell, 1991; Verlinsky et al., 1999,
2001; Sandalinas et al., 2002; Pellestor et al., 2003; Gutierrez-Mateo
et al., 2005). A previous study performed on fresh non-inseminated
oocytes from fertile donors reported a similar frequency of imbalanced PSSC in the oocytes from women aged 20–34 as the present
study (Sandalinas et al., 2002). Nevertheless, their frequency of wholechromosome non-disjunction is higher than the one reported in the
present study, but this may be explained by the fact that they included
hypohaploid oocytes in the overall frequency of whole-chromosome
non-disjunction rather than that calculating the Conservative estimate.
In the present study, some of the hypohaploid oocytes may reflect an
actual loss, instead of an artifactual loss during fixation, so we cannot
exclude the fact that we may be undervaluing the actual frequency of
186
Garcia-Cruz et al.
Figure 3 (a1) Pachytene oocyte with SC shown in red, MLH1 in yellow and centromeres in blue. (a2) Subsequent FISH on the same oocyte allowed
for the identification of chromosomes 16 and 17 centromeres and chromosome 16 q arm telomere (all arrowheads). (b) Histograms showing MLH1
distribution for SC16 and SC17 when the number of MLH1 foci is 1, 2, 3 and 4. (c) Cumulative frequency plots comparing MLH1 distribution between
SC16 and SC17 p and q arms.
187
Oocyte non-disjunction and trisomy 16 origin
Table IV MLH1 foci frequencies in SC16 and SC17
n
pARM
qARM
.............................................
Mean
SD
.............................................
Range
Mean
SD
Range
Whole SC
.............................................
Mean
SD
Range
.............................................................................................................................................................................................
SC16
81
1.00
0.42
0 –2
1.1
0.68
0– 3
2.1
0.86
0 –4
SC17
93
0.81
0.45
0 –2
1.55
0.65
0– 3
2.35
0.73
0 –4
Table V Frequency of SC16 and SC17 with 0, 1, 2, 3 and
4 MLH1 foci per bivalent
Number of
MLH1
0
1
2
3
4
........................................................................................
SC16 [% (n)]
2.5 (2)
19.8 (16)
48.1 (39)
24.7 (20)
4.9 (4)
SC17 [% (n)]
1.1 (1)
6.4 (6)
53.8 (50)
33.3 (31)
5.4 (5)
whole-chromosome non-disjunction by calculating the conservative
estimate.
Balanced PD phenomena have been related to a prolonged time in
culture (Kamiguchi et al., 1993; Munne et al., 1995; Dailey et al., 1996).
In this study, the oocytes showing balanced PD had been in culture
little time, as only few hours transpired between follicular puncture
and oocyte fixation. Therefore, although it has been proved that
extended time in culture predisposes to detachment of sister chromatids, our results suggest that balanced PD also occurs in fresh noninseminated oocytes as a consequence of an abnormal loss of sister
chromatid cohesion. A previous study performed in fresh noninseminated oocytes also reported balanced PD cases, with a
marked increase with maternal age (Sandalinas et al., 2002). These
oocytes, although genetically balanced, may be important contributors
to embryo aneuploidy as they face, in the case of fertilization, the
second meiotic division with a high chance of mis-segregation.
In contrast to MII, studies reporting analysis of MI oocytes are
extremely scarce, although the information that they may provide
regarding the frequency of achiasmate chromosomes in human
oocytes is highly valuable. One of the main reasons for such a lack
of studies in MI oocytes is the difficulty in the obtainment of
samples at this particular stage of maturation. The aim of the superovulation treatments is to obtain mature oocytes at the MII stage, and
only full-grown follicles are aspirated during the follicular puncture procedure. Hence, only occasionally, some of the retrieved oocytes are
found to be immature at the MI stage. Another approach that has
been employed for the obtainment of MI oocytes is maturation of
oocytes at the GV stage, but only few studies have analysed the
chromosome constitution of these MI oocytes (Polani et al., 1982;
Angell, 1995). Angell’s examinations of 22 MI oocytes reported the
finding of oocytes showing pairs of homologous univalents. In our
study, 4 oocytes out of 46 were found showing homologous univalents
at MI. The advantage of our study, with respect to Angell’s, is that we
were able to determine the achiasmate chromosomes by FISH. The
finding that three out of the four achiasmate homologues corresponded to chromosome 16 represent an interesting result as it
may provide new data on the origin of trisomy 16. The presence of
univalents 16 at MI could lead to segregation errors during the first
meiotic division. Achiasmate non-disjunction would result in both univalents segregating to the same pole during the first meiotic division.
Alternatively, it has been recently proposed that alignment of one
or both univalents onto the metaphase plate could lead to the equational segregation of sister chromatids into opposite poles, in mice
(Kouznetsova et al., 2007). This abnormal segregation mechanism
confirms what had been hypothesized by Angell (1991) to explain
the PSSC phenomenon.
In this study, PSSC involving chromosome 16 was found, but the most
prevalent chromosome involved in PSSC events was chromosome 22,
followed by chromosome 15. Findings from indirect studies revealed
that the percentage of maternal meiosis I errors leading to trisomy 22
(94%) and trisomy 15 (76%) are the highest, after trisomy 16, among
all the trisomic conditions (Hassold and Hunt, 2001).
In the present study, two MII oocytes were found showing an extra
chromatid and an extra chromosome, respectively, which appeared to
be attached to its corresponding homologue at the pericentromeric
area. Pericentromeric regions generally show repression for CO
establishment. However, sometimes pericentromeric exchanges do
occur and these misplaced COs may originate non-disjunction
events. In fact, they seem to be the major contributors to maternal
and paternal meiosis II trisomy 21 cases (Lamb et al., 1996; Savage
et al., 1998). The non-disjunction events found in the oocytes mentioned above could have been originated by an exchange so close
to the centromere that it fell within the area of cohesin protection.
This abnormal situation could have been resolved by an abnormal segregation of the bivalent, either leading to whole chromosome nondisjunction or to PSSC. Indeed, both mechanisms have been reported
in animal models: entanglement of homologous chromosomes due to
pericentromeric exchanges, leading to both homologues segregating
together has been proposed to explain non-disjunction events in
Drosophila (Koehler et al., 1996); on the other hand, PSSC due
to centromere-proximal COs seems to be the major contributor to
aneuploidy in yeast (Rockmill et al., 2006). It is intriguing that in
both cases of non-disjunction, hypothetically due to a pericentromeric
CO found in the present study, the chromosomes implicated are from
group B, with at least one of them corresponding to chromosome 4. A
CGH survey found that chromosome 4 is among the chromosomes
most frequently involved in aneuploidy in human oocytes (GutierrezMateo et al., 2004a, b; Sher et al., 2007). Analysis of the recombination pattern of chromosomes 4 and 5 would be of great interest to
establish the frequency of pericentromeric COs in these chromosomes and their possible implication in the origin of non-disjunction
events.
In the present study, a high percentage of metaphases showing
extreme hypohaploidy were found, which is attributable to an
188
artefactual loss during the fixation procedure. Oocyte fixation is the
most important limitation in both conventional karyotyping and FISH
techniques, and chromosome artefactual loss, chromosome superposition and poor morphology are common issues reported in most
studies. Nonetheless, the use of paraformaldehyde-based fixation represents an advantage, with respect to methanol:acetic fixation, as it
allows for subsequent identification of centromeres by IF. This
permits unequivocal identification of chromosome and chromatid
number. Thus, our protocol offers an advantage, with respect to conventional karyotyping techniques, as errors in the classification of
single chromatids and whole chromosomes are avoided. MulticolourFISH techniques such as M-FISH, spectral karyotyping (SKY) and
cenM-FISH have been previously used in the analysis of MII oocytes
(Marquez et al., 1998; Clyde et al., 2001, 2003; Sandalinas et al.,
2002; Gutierrez-Mateo et al., 2005). They offer the advantage, with
regard to FISH techniques with specific probes, in that the whole
chromosome set is analysed. However, chromatid number may be difficult to discern after M-FISH and SKY if chromosome morphology is
not optimal, thus complicating the elucidation of the mechanism of
aneuploidy (Marquez et al., 1998; Sandalinas et al., 2002). The combination of centromere identification by IF followed by M-FISH used in
the present study helps to overcome this difficulty.
Direct analysis of meiotic recombination on chromosome 16
Although unviable, trisomy 16 is the most common human trisomy,
occurring in at least 1% of all clinically recognized pregnancies. In
addition, indirect studies have proved that virtually all of the cases originate at the maternal first meiotic division. Linkage studies support a
primary role of meiotic recombination in the genesis of aneuploidy.
In fact, abnormal patterns of meiotic recombination have been
linked to many trisomies (Lamb et al., 2005). In our study, the frequency of non-recombinant bivalents is 2.5% for chromosome 16
and 1.1% for chromosome 17. Meiotic recombination between homologues is needed for their tethering until the first meiotic division.
Therefore, failure to recombine would result in these bivalents
becoming achiasmate and could explain, at least in part, our finding
of detached univalents 16 at the MI stage. Similar studies in human
oocytes reported frequencies of non-CO bivalents of 1.5% for
chromosome 18 (Tease et al., 2002) and around 3.5% for chromosome 21 (Tease et al., 2002; Robles et al., 2009), although no
non-CO bivalents were found for chromosomes 13 and X (Tease
et al., 2002).
In the current study, the frequency of achiasmate chromosomes 16
found in MI oocytes from young women was of 6.8%. If data presented
in the current MLH1 analysis reflect the prevailing recombination
pattern in female meiosis, it seems unlikely that absence of COs
alone could explain the high frequency of achiasmate chromosomes
16 in MI oocytes. That led us to presume that some achiasmate
chromosomes 16 could come from bivalents 16 which failed to
mature the CO into a chiasma or that lost its chiasma connection
during the long prolapse between CO establishment (during fetal
life) and resumption of meiosis (in adult fertile life). When analysing
the frequencies of MLH1 foci per bivalent, we were amazed by the
fact that the frequency of bivalents with a single CO was more than
3-fold in bivalent 16, when compared with the similar sized bivalent
17 (19.8% versus 6.4%). Chromosome 16 is a metacentric chromosome which optimally should bear a minimum of two COs (one per
Garcia-Cruz et al.
chromosome arm) to result in a stable configuration. Therefore, it is
likely that a single CO may not be the optimal scenario for chromosome 16. We hypothesize that chromosome 16 showing a single
CO event may be at risk of non-disjunction in the first meiotic division.
Indeed, indirect studies conducted on trisomy 16 fetuses concluded
that 100% of the cases originated at maternal meiosis I, but that
only a relatively low percentage of cases (around 21%) were derived
from non-exchange bivalents (Hassold et al., 1995). In contrast, the
authors found that nearly 50% of the cases of trisomy 16 had their
origin in bivalents that had a single CO, confirming the role of
reduced, but not absent, recombination in the genesis of trisomy
16. The study also revealed that recombination in proximal regions
of both arms is reduced in trisomy 16 generating chromosomes
suggesting that the presence of chiasmata in proximal regions is
needed to stabilize bivalent 16. If the position of the markers used
in the Hassold et al. (1995) study are extrapolated to SC16, it is
inferred that 87.5% of the oocytes with a single-exchange in SC16
(i.e. 17% of the fetal oocytes) found in the present study would
have their single chiasmata in a suboptimal position (i.e. too distant
from the centromere) and are, presumably, at risk of non-disjunction.
Hassold et al. (1995) conclude that the mere presence of a chiasma
does not ensure normal segregation. We go further and suggest
that homologues 16 with a single CO, especially if it is located in a suboptimal position, may not be so efficient in maintaining their connection and may be at risk of detachment during the long period of
meiotic arrest. The factors that would lead to the loss of a single
chiasma in chromosome 16 are unknown, but the presence of a
large heterochromatic block may be involved. Heterochromatic
domains show particular characteristics of chromatin compaction,
which could also have a distorting effect in chiasma maintenance. In
fact, those chromosomes that carry large heterochromatic blocks in
their pericentromeric regions (1, 9 and 16) show increased frequency
of disomy in the sperm from normal men, suggesting that this factor
may also have a role in male meiotic non-disjunction (reviewed in
Guttenbach et al., 1997).
Mean MLH1 frequency in chromosome 16 in males (Codina-Pascual
et al., 2006; Sun et al., 2006) is lower than the one reported here for
females. However, when comparing the percentage of bivalents with
no MLH1 and of bivalents with a single focus, these are higher in
females. On top of that, it is expected that the processing of suboptimal CO configurations may be worse in females than in males because
of the existence of the long dyctiate arrest that could last up to 50
years plus to the evidence, from animal models, that meiotic checkpoints are less stringent in females. This sex differences may provide
some clues about the maternal origin of trisomy 16.
Comparison of MLH1 distribution between the p and q arms of
SC16 and SC17 showed that although some variation exists, these
are not statistically different. As expected, the area adjacent to the
centromere in both arms is a cold recombination region for both bivalents. Nevertheless, in SC16, the pericentromeric area free of COs in
the q arm extends to almost 20% of the total length of the corresponding arm, whereas in SC17 COs are observed beginning at
around 5% of the total length of the q arm. This difference is attributable to the presence of the pericentromeric heterochromatin block in
chromosome 16 (16qh). In the present study, COs in SC16 p arm are
also observed from around 20% of the total length of the p arm
onwards. Hence, according to our observations, the area where
Oocyte non-disjunction and trisomy 16 origin
proximal ‘anchoring’ chiasmata establish seems to be limited to a small
region in both arms of chromosome 16.
Overall, we have identified suboptimal levels of recombination on
chromosome 16 in fetal oocytes, in the form of non-CO and
single-CO bivalents, which are believed to increase the risk of
chromosome 16 non-disjunction in the adult ovary. Lamb et al.
(1996) proposed that human non-disjunction could respond to a
‘two-hit’ model, in which the first hit is the establishment of a suboptimal CO configuration in the fetal oocyte and the second hit is the
abnormal processing of such configurations in the adult ovary.
Maternal age is the factor that modulates the second hit, which
includes any factor (known or unknown) of the meiotic machinery
that may be affected by maternal age. To date, cohesion weakening
(Hodges et al., 2005), spindle aberrations and chromosome misalignment (Volarcik et al., 1998), depletion of oocyte pools (Kline
et al., 2000) or impaired spindle checkpoint (LeMaire-Adkins et al.,
1997; Steuerwald et al., 2001; Vogt et al., 2008) are some of the
factors that have been proposed in relation to age-related nondisjunction. Trisomy 16 is extremely frequent at all maternal ages
but a linear relationship between increasing maternal age and its frequency exists, suggesting that trisomy 16 is entirely dependent on
maternal age (Risch et al., 1986; Morton et al., 1988). Hence, the
outcome of the suboptimal CO configurations found in the present
study for chromosome 16 is likely to depend on the time that the
oocyte remains arrested in the ovary and on the ability of the
meiotic machinery to process them with success.
Acknowledgements
The authors wish to thank the teams from the Embryology Laboratory
of the Clı́nica Eugin and of the Foetal Tissue Bank of the Hospital de la
Vall d’Hebrón for their collaboration in the collection of samples. We
also wish to thank Dr Christa Heyting and Dr Marvin Fritzler for providing SYCP3 antibody and CREST serum, respectively.
Funding
RG-C was a recipient of a grant from the Agència de Gestió d’Ajuts
Universitaris i de Recerca de la Generalitat de Catalunya
(2004FI00953). This study has been supported by the Ministerio de
Ciencia y Tecnologı́a (BFU2006-1295).
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Submitted on May 18, 2009; resubmitted on August 26, 2009; accepted on
August 27, 2009