Human Reproduction, Vol.26, No.6 pp. 1575– 1584, 2011
Advanced Access publication on March 25, 2011 doi:10.1093/humrep/der080
ORIGINAL ARTICLE Reproductive genetics
Meiotic segregation of Robertsonian
translocations ascertained in
cleavage-stage embryos—implications
for preimplantation genetic diagnosis
S.M. Bint 1,2, C. Mackie Ogilvie 1,3, F.A. Flinter 1,3, Y. Khalaf 1,4,
and P.N. Scriven 1,2,*
1
Guy’s and St Thomas’ Centre for Preimplantation Genetic Diagnosis, Guy’s and St Thomas’ Hospital NHS Foundation Trust, London, UK
Cytogenetics Department, GSTS-Pathology, Guy’s Hospital, 5th Floor Tower Wing, Great Maze Pond, London SE1 9RT, UK 3Genetics
Centre, Guy’s and St Thomas’ Hospital NHS Foundation Trust, London, UK 4Assisted Conception Unit, Guy’s Hospital, London, UK
2
*Correspondence address. E-mail: [email protected]; [email protected]
Submitted on November 9, 2010; resubmitted on February 17, 2011; accepted on February 28, 2011
background: The aim of this study was to ascertain the prevalence of meiotic segregation products in embryos from carriers of 13/14
and 14/21 Robertsonian translocations and to estimate the predictive value of testing single cells using the fluorescence in situ hybridization
(FISH) technique, to provide more information for decision-making about PGD.
methods: In this prospective cohort study, the copy number of translocation chromosomes in nuclei from lysed blastomeres of cleavagestage embryos was ascertained using locus-specific FISH probes. Logistic regression analysis, controlling for translocation type, female age and
fertility status, was used to calculate the odds ratio (OR) of unbalanced segregation products for female and male heterozygotes. The primary
diagnostic measure was the predictive value of the test result. The primary outcome measure was the live birth rate per couple.
results: Female carriers were four times more likely than male carriers to produce embryos with an unbalanced translocation product
(OR 3.8, 95% confidence interval 2.0–7.2, P , 0.001). The prevalence of abnormality for the chromosomes tested in embryos from female
or male heterozygotes was estimated to be 43 or 28%, respectively, while estimates of the predictive value were 93–100 or 96– 100% for a
normal test result and 79 or 57% for an abnormal test result. The live birth rate per couple was 58% for female carriers and 50% for male
carriers.
conclusions: For female carriers, PGD using FISH could reduce the risk of miscarriage from either translocation or the risk of Down
syndrome from the 14/21 Robertsonian translocation. PGD using FISH for male carriers is unlikely to be indicated given the relatively low
prevalence of chromosome imbalance and low predictive value.
Key words: Robertsonian translocation / meiotic segregation / FISH
Introduction
Robertsonian translocations are found in 1 in 1000 births, and the
most common translocations are between chromosomes 13 and 14
and between chromosomes 14 and 21 (Gardner and Sutherland,
2004). Trivalent formation of non-homologous chromosomes at
meiosis I allows synapsis of the homologous chromosomes, and segregation produces eight different products in gametes: normal
chromosomes or the Robertsonian translocation chromosome (alternate segregation), four products with nullisomy or disomy for one
chromosome (adjacent segregation) and two products with nullisomy
or disomy for both chromosomes (3:0 segregation) (Gardner and
Sutherland, 2004). Alternate segregation is favoured for male and
female carriers; however, female carriers are more likely to produce
unbalanced gametes than male carriers (Munné et al., 2000a). There
is a 10-fold excess of Robertsonian translocations found in males
with oligozoospermia (Chandley, 1988), for whom assisted conception with ICSI is likely to be indicated.
Conceptions with trisomy for chromosome 13 or 21 can result in
offspring with Patau and Down syndrome, respectively. Other unbalanced products are not compatible with life and might fail to
implant, or result in occult pregnancy or first-trimester miscarriage.
The risk of miscarriage is increased for some carriers compared
with the general population background risk of 15%. Trisomy rescue
& The Author 2011. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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1576
(or more rarely monosomy rescue) can result in an apparently
balanced (or normal) karyotype with uniparental disomy (UPD). Prenatal diagnosis (PND) is possible, at which the empirical risk of trisomy
21 or UPD 14 is 15% for a female carrier and 1% for a male carrier
of a 14/21 translocation, while the risk of trisomy 13 or UPD 14 for
female and male carriers of a 13/14 Robertsonian translocation is
1% (Gardner and Sutherland, 2004).
PGD offers another reproductive option for some couples.
However, PGD should only be embarked upon following careful
genetic counselling (taking into account the previous obstetric
history of the couple and other carriers in the family), calculation
and discussion of appropriate risk figures, and investigations for recurrent miscarriage when indicated (Scriven et al., 2001).
Successful treatment for carriers of Robertsonian translocations
using PGD and assisted reproduction technology has been applied
clinically for more than a decade using polar body biopsy (Munné
et al., 1998a, 2000a) and blastomere biopsy (Escudero et al., 2000;
Munné et al., 2000b). Fluorescence in situ hybridization (FISH) has
been the primary technique used to test preimplantation oocytes
and embryos. In the largest multi-centre data collection, the clinical
pregnancy rate for PGD cycles reported to the ESHRE PGD Consortium to the end of 2007 was 23% (268/1146 biopsy cycles; Harper
et al., 2010). The largest series from a single centre published to
date achieved a live birth delivery rate of 33% (24/76 couples) for
couples who had at least one cycle of PGD and it was concluded
that PGD is a good reproductive option, especially when there is
also a fertility problem (Keymolen et al., 2009).
The reproductive risks for carriers of 13/14 and 14/21 translocations at PND are well established but less well studied at conception
for female carriers. Our study presents 10 years of experience of PGD
for carriers of 13/14 and 14/21 Robertsonian translocations at the
Guy’s and St Thomas’ Centre for PGD. We have investigated the
prevalence of meiotic segregation products seen in cleavage-stage
embryos from male and female carriers and evaluated the diagnostic
accuracy of testing using the FISH technique and the success of the
treatment.
Bint et al.
Ovarian stimulation, embryo culture
and biopsy, and confirmation of diagnosis
Procedures were performed as described previously (Khalaf et al., 2000;
Scriven et al., 2000; Pickering et al., 2003). In brief, a standard long stimulation protocol for controlled ovarian stimulation was followed by IVF, or
ICSI when the patient had suboptimal semen characteristics, then biopsy
of one or two cells from cleavage-stage embryos 3 days after fertilization
and embryo transfer on Day 4 or 5. Two testing strategies were used over
the period of the study. The first strategy included biopsying one cell from
embryos with five or more cells on Day 3 and a second cell if the first cell
did not have a clear single nucleus after lysis and the embryo had six or
more cells; in the event of 1-cell being mononucleated and the second
cell being multinucleated, only the mononculeated cell was analysed for
diagnosis. The second strategy included biopsying two cells from
embryos with six or more cells on Day 3; embryos diagnosed to be
normal/balanced had a concordant result from both cells tested.
Embryos unsuitable for freezing were spread for confirmation of diagnosis
(COD) and further analysis with FISH in accordance with Human Fertilization and Embryology Authority research licence R0075. Where embryos
had been transferred, G-band karyotyping at PND or of live born offspring
was carried out when possible.
Fluorescence in situ hybridization
FISH probe selection, blastomere spreading, in situ hybridization and signal
scoring protocols have been described in detail previously (Scriven et al.,
2001; Scriven and Ogilvie, 2007). In general, the risk of misdiagnosis was
minimized either by using two diagnostic probes for each viable imbalance
(26 cycles), or by testing two cells and transferring embryos with concordant normal/balanced results from both cells (4 cycles).
Probe mixes and strategies used for der(13;14) cycles were:
Materials and Methods
(1) 1999– 2001, 1-cell biopsy strategy, Oncor QuintEssential 13q Digoxigenin (13q32– q33)/Vysis TelVysion 14q SpectrumOrange
(D14S308), five cycles for male carriers, three cycles for female
carriers.
(2) 2002– 2003, 1-cell biopsy strategy, Vysis LSI 13 SpectrumGreen
(RB1)/Vysis TelVysion 14q SpectrumOrange (D14S308), and two
cycles for male carriers.
(3) 2003– 2008, 1-cell biopsy strategy, Vysis LSI 13 SpectrumGreen (RB1,
13q14)/Cytocell LPT 13q TexasRed (D13S1825)/Vysis TelVysion 14q
SpectrumOrange (D14S1420), nine cycles for male carriers and nine
cycles for female carriers.
Patients
Probe mixes and strategies used for der(14;21) cycles were:
Our prospective cohort study included 28 consecutive couples undergoing
PGD at the Guy’s and St Thomas’ Centre for PGD between February
1999 and May 2008; none of the couples treated had a successful
pregnancy before PGD. There were seven female carriers of 13/14
Robertsonian translocations [45,XX,der(13;14)(q10;q10)], four with a
history of recurrent miscarriage and three with infertility, and eleven
male 13/14 carriers [45,XY,der(13;14)(q10;q10)], nine with infertility
and two with partners with a history of recurrent miscarriage. There
were also five females carriers of 14/21 Robertsonian translocations
[45,XX,der(14;21) (q10;q10)]—two with a family history of translocation
trisomy 21, one with a pregnancy termination for trisomy 21, one with a
partner with male factor infertility and one with a history of recurrent miscarriage— and five male 14/21 carriers [45,XY,der(14;21)(q10;q10)]—
four with infertility and one with a partner with a history of recurrent
miscarriage.
(1) 1999, 1-cell biopsy strategy, non-commercial 14q Biotin (14q32.3)/
Vysis LSI 21 SpectrumOrange (21q22.13 – q22.2) and two cycles for
female carriers.
(2) 2002– 2005, 2-cell biopsy strategy, Qbiogene 14q Green
(D14S1419)/Vysis LSI 21 SpectrumOrange (21q22.13– q22.2), and
four cycles for male carriers.
(3) 2004– 2008, 1-cell biopsy strategy, Cytocell LPT 14q TexasRed
(D14S1420)/Vysis LSI 21 SpectrumOrange (21q22.13 – q22.2)/Cytocell LPT 21q FITC (D21S1575), five cycles for female carriers, and
three cycles for male carriers.
Blastomeres and whole embryos were spread using the Tween/HCl
method (Coonen et al., 1994). Prepared nuclei were hybridized overnight
and analysed using a fluorescence microscope suitably equipped with the
appropriate filter sets for the probes being used.
Segregation of Robertsonian translocations
COD and assigning the mode of segregation
Normal test results and spread embryos were assigned to be consistent
with alternate segregation at meiosis if the nuclei(us) showed two
signals for each chromosome region tested, for the single nucleus from
a transferred embryo where COD was not possible or at least two
nuclei from spread embryos. For the diagnostic accuracy study, spread
embryos were confirmed to be normal/balanced if at least 50% of
nuclei were consistent with normal copy number. Abnormal tests
results were deviations from a normal test result and spread embryos
were confirmed to be abnormal if .50% of nuclei were abnormal, and
assigned to be consistent with adjacent or 3:0 segregation at meiosis if
at least two nuclei obtained showed the appropriate and consistent deviation from two signals for each chromosome region tested. Haploid and
triploid results were differentiated from 3:0 segregation products by
retesting with a probe specific for a chromosome not involved in the translocation. The likely segregation mode was deemed to be unknown if these
criteria were not met.
Statistical analysis
The odds ratio (OR) of unbalanced translocation segregation products for
female and male heterozygotes was calculated using logistic regression,
controlling for the type of translocation, age of the female partner and fertility status. Test results with an unknown outcome were initially allocated
in proportion to normal and abnormal test results with a known outcome;
in a sensitivity analysis, we varied the allocation of normal test results up to
the upper 95% confidence limit of zero.
Of the two types of potential error, a false-negative result (which could
lead to implantation failure, miscarriage or the birth of a chromosomally
abnormal child) has implications different from a false-positive result,
which could involve a chromosomally balanced embryo not being selected
for replacement. The primary diagnostic accuracy measures calculated
were the positive predictive value (the probability that an abnormal test
result is correct) and the negative predictive value (the probability that a
normal test result is correct). Other statistics calculated were: proportion
of false positives and false negatives (using the test perspective and calculated as the proportion of the total outcomes), overall accuracy (the proportion of all test results that are correct), sensitivity (the probability that a
non-transferable genotype has an abnormal test result) and specificity (the
probability that a transferable genotype has a normal test result). The
primary outcome measure was the live birth rate per couple; the live
birth rate per biopsy cycle was also calculated. For study measures, 95%
confidence intervals (CIs) were calculated and Pearson’s goodness of fit
x 2 was calculated for significance probabilities. Log-likelihood ratios
were used to test for patient heterogeneity within the segregation analysis
patient groups.
Results
Our study included a total of 311 embryos tested from 42 cycles for
18 carriers of 13/14 and 10 carriers of 14/21 Robertsonian
translocations.
For 12 female carriers (Table I), there were 7 (58%) healthy live
born deliveries. A total of 129 embryos were tested: 57 (44%) had
a normal test result, 67 (52%) had an abnormal test result and 5
(4%) failed to be diagnosed. Following COD, 23/23 (100%)
embryos diagnosed to be normal were confirmed as normal and
none was found to be abnormal; 34 embryos for which a result
could not be obtained were therefore assigned as normal. For the
embryos diagnosed to be abnormal, 46/58 (79%) were confirmed
1577
to be abnormal and 12 were found to be normal; results were not
obtained for nine embryos and seven were therefore assigned as
abnormal and two as normal. Table III summarizes the proportion
of abnormal cells found in 58 reanalysed embryos; 21 embryos
were mosaic and 15 (71%) had only abnormal cells. Table IV shows
the diagnostic accuracy measures: the prevalence of abnormal
embryos was estimated to be 43% and it was estimated that the
test had 89% accuracy, 100% sensitivity, 80% specificity, 79% positive
predictive value and 100% negative predictive value. Following the sensitivity analysis to adjust for potential undetected abnormal embryos
(see statistical analysis section above), the prevalence changed to
46% and the sensitivity and negative predictive value to 93 and 93%,
respectively.
For 16 male carriers (Table II), there were eight deliveries (50%). A
total of 182 embryos were tested, of which 91 (50%) had a normal
test result, 90 (49%) had an abnormal test result and 1 (,1%)
failed to be diagnosed. Following COD, 36/36 (100%) embryos diagnosed to be normal were confirmed and none was found to be abnormal; 55 embryos for which a result could not be obtained were
therefore assigned as normal. For the embryos diagnosed to be abnormal, 41/73 (56%) embryos were confirmed to be abnormal and 32
were found to be normal; a result was not obtained for 17 embryos
and 10 were therefore assigned as abnormal and 7 as normal.
Table III summarizes the proportion of abnormal cells found in 73 reanalysed embryos; 36 embryos were mosaic and 23 (64%) had only
abnormal cells. The prevalence was estimated to be 28% (Table IV)
and it was estimated that the test had 78% accuracy, 100% sensitivity,
70% specificity, 57% positive predictive value and 100% negative predictive value. Following the sensitivity analysis, the prevalence changed
to 30% and the sensitivity and negative predictive value to 93 and 96%,
respectively.
Excluding one couple who had a termination of pregnancy at 14
weeks gestation for social reasons, 7/10 (70%, 95% CI 35–93%)
fertile Robertsonian couples who had at least one biopsy cycle had
a healthy live birth pregnancy, 3/10 (30%, 95% CI 7 –65%) had a ‘biochemical’ pregnancy and 1/8 (13%, 95% CI ,1–53%) a failed pregnancy after the detection of a foetal heartbeat.
Our segregation analysis included 257 embryos. Tables I and II
show that 40/58 (69%) embryos from female 13/14 carriers were
consistent with alternate segregation as were 76/91 (84%) embryos
from male carriers (difference 21%, 95% CI 10 –47%, P ¼ 0.037).
For the 14/21 translocation, 31/49 (63%) embryos from female carriers were consistent with alternate segregation as were 56/59 (95%)
embryos from male carriers (difference 50%, 95% CI 20–87%, P ,
0.001).
Controlling for the translocation type, embryos of female Robertsonian translocation carriers were approximately four times more likely
to have an embryo with an unbalanced translocation product than
were male carriers (OR 3.8, 95% CI 2.0 –7.2, P , 0.001). Age and fertility status did not confound the association between sex and segregation outcome and, controlling for sex, age and fertility status, were
not important independent predictors of chromosome imbalance.
Trisomy 13 was found in 4/58 (7%) embryos of female 13/14 carriers
and in 2/91 (2%) embryos of male carriers (difference 214%, P ¼
0.155); trisomy 21 was found in 11/49 (22%) embryos of female
14/21 carriers and 0/59 (0%) embryos of male carriers (difference
infinity %, P , 0.001).
1578
Bint et al.
Table I Diagnostic measures for female Robertsonian translocation carriers.
Measures
Female Robertsonian carriers der(A;B)
.........................................................................................................................................
der(13;14)
% (95% CI)
der(14;21)
% (95% CI)
Total
% (95% CI)
.............................................................................................................................................................................................
Couples
7
5
12
Biopsy cycles
12
7
19
Maternal age (mean + SD years)
35.3 + 2.7
35.6 + 3.4
35.4 + 2.9
Range (years)
32–40
31–39
31– 40
Embryo test results
76
53
129
Normal
34
45
23
43
57
Abnormal
38
50
29
55
67
52
Failed
4
5
1
2
5
4
44
Confirmation of diagnosis
Normal:normala
18
5
23
Normal:abnormal
0
0
0
Normal:none
16
18
34
(Assigned normal:abnormal)
(16:0)
(18:0)
(34:0)
Abnormal:abnormal
27
19
46
Abnormal:normal
4
8
12
Abnormal:none
7
2
9
(Assigned abnormal:normal)
(6:1)
(1:1)
(7:2)
Segregation mode ascertained
58
2:1 Alternate
40
69 (55–80)
31
63 (48– 77)
71
66 (57 –75)
2:1 Adjacent
17
29
18
37
35
33
Nullisomy A
4
7
0
0
4
4
49
107
Nullisomy B
4
7
5
10
9
8
Disomy A
4
7 (2– 17)
2
4
6
6
Disomy B
5
9
11
22 (12– 37)
16
15
3:0
1
2
0
0
1
1
Embryo transfers/biopsy cycle
11
92 (65–99)
7
100 (65–100)
18
95 (75 –99)
Embryos transferred
18
14
32
Pregnancies/biopsy cycle
7
58 (32–81)
5
71 (36– 92)
12
63 (41 –81)
Clinical pregnancies/biopsy cycle
6
50 (25–75)
3
43 (16– 75)
9
47 (24 –71)
Fetal heartbeats/rate
9
50 (26–74)
4
29 (8– 58)
13
41 (24 –59)
Live birth pregnancies/couple
5b
71 (36–92)
2c
40 (12– 77)
7
58 (28 –85)
Infants
7
2
9
a
Includes live born infants.
One single pregnancy terminated at 12 weeks with anencephaly, follow-up declined.
One twin pregnancy social termination at 14 weeks.
b
c
Discussion
The objective of our study was to ascertain the prevalence and OR of
unbalanced meiotic segregation products in cleavage-stage embryos
from female and male carriers of 13/14 and 14/21 Robertsonian
translocations and to estimate the predictive value of testing biopsied
blastomeres using FISH, to provide better information for couples and
professionals involved in decision-making about PGD.
Where the likely mode could be ascertained, the majority of
embryos were consistent with alternate segregation (Tables I and
II). Fewer embryos from female carriers (69%) were consistent with
alternate segregation compared with embryos from male carriers
(84%). Female translocation carriers were approximately four times
more likely than male carriers to have embryos with an unbalanced
product of the translocation (OR 3.8; 95% CI 2.0–7.2, P , 0.001).
More embryos from female carriers had potentially viable translocation trisomy products than did embryos from male carriers: 7 and
2%, respectively, for trisomy 13, and 22 and 0%, respectively, for
trisomy 21.
In contrast to a conventional accuracy study, which compares test
results with a clinical reference standard, measuring the diagnostic
accuracy of PGD in practice is complicated by the nature of the
early human embryo and confounded by incomplete results
1579
Segregation of Robertsonian translocations
Table II Diagnostic measures for male Robertsonian translocation carriers.
Measures
Male Robertsonian carriers der(A;B)
.........................................................................................................................................
der(13;14)
% (95% CI)
der(14;21)
% (95% CI)
Total
% (95% CI)
.............................................................................................................................................................................................
Couples
11
5
16
Biopsy cycles
16
7
23
Maternal age (mean + SD years)
34.5 + 3.4
32.3 + 4.2
33.8 + 3.7
Range (years)
26–40
26–37
26– 40
Embryo test results
118
64
182
Normal
55
47
36
56
91
Abnormal
62
53
28
44
90
49
Failed
1
1
0
0
1
1
50
Confirmation of diagnosis
Normal:normala
11
25
Normal:abnormal
0
0
0
Normal:none
44
11
55
(Assigned normal:abnormal)
(44:0)
(11:0)
(55:0)
Abnormal:abnormal
32
9
41
Abnormal:normal
18
14
32
Abnormal:none
12
5
17
(Assigned abnormal:normal)
(8:4)
(2:3)
(10;7)
36
Segregation mode ascertained
91
2:1 Alternate
76
84 (74–90)
56
59
95 (86 –99)
150
132
88 (82 –93)
2:1 Adjacent
13
14
3
5
16
11
Nullisomy A
3
3
2
3
5
3
Nullisomy B
6
7
1
2
7
5
Disomy A
2
2 (,1 –8)
0
0
2
1
Disomy B
2
2
0
0 (0–6)
2
1
3:0
2
2
0
0
2
1
Embryo transfers/biopsy cycle
13
81 (54–95)
7
100 (65–100)
20
87 (65 –97)
Embryos transferred
21
Pregnancies/biopsy cycle
6
38 (15–65)
4
57 (25 –84)
10
43 (23 –66)
Clinical pregnancies/biopsy cycle
6
38 (15–65)
3
43 (16 –75)
9
39 (20 –61)
12
33
Fetal heartbeats/rate
7
33 (15–57)
5
42 (19 –68)
12
36 (20 –55)
Live birth pregnancies/couple
5b
45 (21–72)
3
60 (23 –88)
8
50 (25 –75)
Infants
6
5
11
a
Includes live born infants.
One single pregnancy terminated at 20 weeks with multiple congenital abnormalities and primary trisomy 18.
b
(many embryos transferred fail to implant and might include some with
false normal test results). Our primary testing strategy was to minimize
the risk of transferring an embryo with viable chromosome imbalance
by using two FISH probes for chromosomes 13 and 21, and therefore
it is not unexpected that all the resulting offspring tested were confirmed to have a normal or balanced translocation chromosome
complement, which inevitably biases the accuracy of our study. In
practice, the most appropriate measure of diagnostic accuracy is
likely to be the positive predictive value because all the abnormal
test results have the possibility of being available for COD studies
(Scriven and Bossuyt, 2010). Our approach in this study was to allocate test results with an unknown outcome in proportion to normal
and abnormal test results with a known outcome and then vary the
allocation of normal test results in a sensitivity analysis. Table IV
shows that the prevalence of chromosome imbalance for the chromosomes tested in embryos from female carriers was estimated to be
43%, the predictive value of an abnormal test result was 79% and
the initial predictive value of a normal test result was 100%; following
the sensitivity analysis, the negative predictive value was 93%. The
prevalence of chromosome imbalance for the chromosomes tested
in embryos from male carriers was estimated to be 28%, the predictive value of an abnormal test result was 57% and the initial predictive
value of a normal test result was 100%; following the sensitivity analysis
the negative predictive value was 96%. In addition to the limitations of
the FISH technique, mosaicism has the potential to produce false
abnormal and false normal test results; however, as observed in our
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Bint et al.
study (Table III) and by others (Colls et al., 2007), the majority of
mosaic embryos have only abnormal cells and this is expected to mitigate the error rate associated with mosaicism (Munné et al., 2010).
For female carriers, the overall accuracy of our PGD testing using
FISH was estimated to be 89%, which roughly equates to 96% per
probe for three probes (0.891/3), and is broadly similar to the 80 –
82% accuracy rates testing (mainly) five chromosome pairs (96%
per probe) previously reported for aneuploidy screening using FISH
(Munné et al., 1998b; Silber et al., 2003). More recently, experienced
groups have reported a much higher accuracy for 1- and 2-cell biopsy
testing for six chromosome pairs (93 and 97% accuracy respectively;
99% per probe) (Michiels et al., 2006) and employing a ‘no result
rescue’ technique testing eight chromosome pairs (95% accuracy,
99% per chromosome pair) (Colls et al., 2007). When testing for
chromosome rearrangements, our priority was to avoid transferring
Table III Proportion of abnormal cells in 131
reanalysed embryos.
Female carrier
Male carrier
........................................................................................
Normal
9
23
,25% abnormal
1
4
25–37% abnormal
0
1
38–50% abnormal
2
4
51–62% abnormal
1
2
63–74% abnormal
0
2
Mosaic
75–99% abnormal
100% abnormal
2
0
15
23
Abnormal
28
14
Total
58
73
an embryo with an unbalanced translocation and our practice,
especially when several embryos were available for transfer, was to
err on the side of caution when interpreting closely adjacent ‘split’
signals and scoring normal copy number. It is therefore consistent,
given the significantly lower prevalence of chromosome aneuploidy
found in embryos from male carriers (28 versus 43%) that more
normal embryos were excluded due to error (30 versus 20%), resulting in a lower overall test accuracy for male carriers (78 versus 89%)
(Table IV). The proportion of embryos tested with a transferable test
result was similar for female and male carriers (44 and 50%, respectively) (Tables I and II).
Of our 16 male carriers, 13 presented with infertility. There is a
10-fold excess of Robertsonian heterozygotes in men with oligozoospermia (Chandley, 1988). Studies in males with severe oligozoospermia have found a high proportion (.60%) of cells with non-random
association between the sex vesicle and the Robertsonian trivalent
for 13/14 and 14/21 translocations (Luciani et al., 1984; Rosenmann
et al., 1985). Associations tended to occur where there was pairing
failure in the short arms of the two non-translocated chromosomes.
Whether such non-random contacts are the cause of germ-cell breakdown or a secondary consequence of a primary pairing failure is not
clear (Chandley, 1988), but it is plausible that incomplete synapse
will result in disproportionate death of cells with chromosome aneuploidy and explain, at least in part, the reduced level of aneuploidy
in mature spermatozoa compared with fertilized oocytes. It is probable that Robertsonian translocations have a negative effect on
sperm count for all male carriers and that oligoospermia or azoospermia is only a matter of degree.
In general, most sporadic chromosome aneuploidy originates during
oogenesis (typically meiosis I) and increases with maternal age
(Hassold and Hunt, 2001). Altered recombination appears to be the
most important known factor associated with the origin of human
trisomy (Lamb et al., 2005); homologous chromosomes that fail to
crossover are expected to produce random segregation at metaphase
Table IV Diagnostic measures for female and male translocation carriers.
Group
Female carrier
Measure
Calculation
..............................................................
%
95% CI
Male carrier
..............................................................
Calculation
%
95% CI
.............................................................................................................................................................................................
False positive
14/124
11
6 –18
39/181
22
False negative
0/124
0
0 –3
0/181
0
16– 28
0– 2
Accuracy
110/124
89
82–94
142/181
78
72– 84
Sensitivity
48/48
100
93–100
51/51
100
93– 100
Specificity
57/76
80
69–89
91/130
70
61– 78
Prevalence
53/124
43
34–52
51/181
28
22– 35
Positive predictive value
53/67
79
67–88
51/90
57
46– 67
Negative predictive value
57/57
100
94–100
91/91
100
96– 100
4/181
Sensitivity analysis
False negative
4/124
3
1 –8
2
1– 6
Accuracy
106/124
85
78–91
138/181
76
69– 82
Sensitivity
53/57
93
83–97
51/55
93
82– 98
Prevalence
57/124
46
37–55
55/181
30
24– 38
Negative predictive value
53/57
93
83–97
87/91
96
89– 99
Mode
.........................
Stage (references)
Total
Alternate
%
Mode
........................
Adjacent
%
Translocation chromosomes
..............................................................................................
1A
%
1B
%
–A
%
–B
%
Mode
........................
3:0/other
Segregation of Robertsonian translocations
Table V Segregation frequencies of 13/14 and 14/21 Robertsonian translocations in gametes, embryos and pregnancies.
%
..........................................................................................................................................................................................................................................................
Female 13/14
Polar body/oocyte (1,2)
Preimplantation embryos (3,4)
Prenatal diagnosis (5,6,7,8)
(113)
81
52
64
27
(+14)
(213)
(214)
33
8
10
6
7
10
12
3
4
2
2
63
42
67
20
32
5
8
5
8
5
8
5
8
1
2
276
273
99
3
1
3
1
0
0
0
0
0
0
0
0
Male 13/14
(113)
(+14)
(213)
(214)
Spermatocytes (9,10,11,12,13)
29 453
25 539
87
3689
13
782
3
742
3
1094
4
1071
4
225
1
Preimplantation embryos (3,4)
124
105
85
17
14
3
2
3
2
3
2
8
6
2
2
Prenatal diagnosis (5,6,7,8)
123
122
99
1
1
1
1
0
0
0
0
0
0
0
0
Female 14/21
Polar body/oocyte (1,2)
Preimplantation embryos (4)
Prenatal diagnosis (5,7,8)
(+14)
93
53
57
38
41
4
(214)
(221)
17
18
3
3
14
15
2
2
49
31
63
18
37
2
4
11
22
0
0
5
10
0
0
208
177
85
31
15
0
0
31
15
0
0
0
0
0
0
Male 14/21
Spermatocytes (11,13,14,15)
4
(121)
(+14)
(121)
(214)
(221)
228 63
20 122
88
2557
11
537
2
565
2
725
3
730
3
184
1
Preimplantation embryos (3,4)
63
60
95
3
5
0
0
0
0
2
3
1
2
0
0
Prenatal diagnosis (5,7,8)
74
73
99
1
1
0
0
1
1
0
0
0
0
0
0
Figures in bold show the prevalence of viable abnormal translocation products found at different stages.
1 Munné et al. (2000a), 2 Durban et al. (2001), 3 Alves et al. (2002), 4 Present study, 5 Boué and Gallano (1984), 6 Engels et al. (2008), 7 Daniel et al. (1989) and corrected for prenatal bias in 8 Daniel (2002), 9 Anton et al. (2004), 10 Escudero
et al. (2000), 11 Frydman et al. (2001), 12 Morel et al. (2001), 13 Honda et al. (2000), 14 Rousseaux et al. (1995), 15 Ogur et al. (2006).
1581
1582
I and a 50% chance of non-disjunction. Other possible factors include
the position of crossovers relative to the centromere and loss of sister
chromatid cohesion or defects in spindle assembly or disassembly
(Hassold et al., 2007). It is therefore consistent that, compared with
males, these mechanisms are likely to be contributing to the additional
chromosome aneuploidy in embryos from female Robertsonian
heterozygotes.
Table V incorporates our study and previous studies of 13/14 and
14/21 Robertsonian translocations that have investigated segregation
products in spermatozoa, polar bodies and oocytes, embryos and
pregnancies. In general, the proportion of products consistent with
alternate segregation is similar in gametes and preimplantation
embryos and is greater for male carriers (85– 95%) than female carriers (57 –67%). Conceptions with whole chromosome imbalance,
particularly monosomy, are more likely to fail than those with a
normal or balanced chromosome complement, and the proportion
of pregnancies with an unbalanced translocation at PND is therefore
expected to be reduced; the remaining risk is associated with translocation trisomy for chromosomes 13 or 21 (1% for male carriers of
both translocations and female 13/14 carriers, and 15% for female
14/21 carriers). Expressing the number of embryos with trisomy 13
or 21 as a proportion of the embryos with trisomy 13 or 21 plus
those with a normal/balanced chromosome complement (the total
products with potential to survive to PND), for female and male carriers, respectively, 11% (95% CI 4–23%) and 3% (95% CI 1 –8%) had
trisomy 13, and 26% (95% CI 14 –42%) and 0% (95% CI 0–6%) had
trisomy 21. The much lower prevalence of these abnormalities at
PND (Table V) demonstrates a substantial degree of embryo loss
between conception and prenatal diagnosis. A significant rate of
foetal death is also expected for trisomy 13 and 21 (Hook, 1978,
1980) and it is logical to conclude that the risk of miscarriage for
Robertsonian heterozygotes might be increased compared with the
general population risk of 15%. The 13/14 Robertsonian study of
Harris et al. (1979) included 86 sibships and found that 16% (95%
CI 11– 22%) of pregnancies ended in miscarriage for female carriers
compared with 9% for male carriers. A criticism of this study is that
exclusion of all miscarriages with an unknown karyotype might have
over-corrected for ascertainment bias (Engels et al., 2008). The
ascertainment-corrected study of Engels et al. (2008) which included
101 pedigrees of carriers of 13/14 Robertsonian translocations,
found that 28% (95% CI 20 –36%) of pregnancies for female carriers
and 20% (95% CI 11 –31%) for male carriers resulted in miscarriage.
Evidence from studies of gametes, embryos and pregnancies indicates that the risk of a translocation trisomy 21 conception and pregnancy is significant for a female 14/21 heterozygote; PGD can
therefore be considered a realistic alternative to PND for fertile as
well as infertile couples, in order to avoid Down syndrome (Fig. 1
summarizes the decision pathway for PGD). The risk of an unbalanced
translocation for a male 13/14 or 14/21 heterozygote is low and PGD
for fertile couples is therefore unlikely to be indicated; our study indicates that even for infertile couples (accepting that the test will have
some utility in detecting triploid and chromosomally chaotic
embryos), the predictive value of an abnormal test result using the
FISH technique is relatively low and embryo transfer without PGD
(and additional cost) should be considered a reasonable choice,
especially where few embryos are available for testing. Male and
female 13/14 heterozygotes have a low risk of translocation trisomy
Bint et al.
Figure 1 Pathways to PGD for carriers of 13/14 and 14/21
Robertsonian translocations.
13 at PND; however, our study indicates that female carriers are
more likely to produce aneuploid embryos than male carriers, and,
in conjunction with recent evidence showing that female carriers
have an increased risk of miscarriage compared with the general population background risk (Engels et al., 2008), PGD should be considered
as a reproductive option for carrier females in fertile couples with a
history of recurrent miscarriages. However, other contributing
factors should also be investigated and we recommend a uterine
cavity assessment and testing for antiphospholipid syndrome and
Lupus anticoagulant. It is our opinion that PGD for fertile male carriers
of 13/14 Robertsonian is unlikely to be indicated. It is worth noting
that in an index-control study of carriers of Robertsonian translocations with a history of two or more miscarriages, in a 2 year follow-up
period there was no significant difference in subsequent pregnancy
outcome between these carriers and couples with normal karyotypes;
82 and 84% of couples had one or more healthy children while 34 and
30% had one or more miscarriage, respectively (Franssen et al., 2006).
Our experience with PGD for fertile Robertsonian couples is that 70%
had a healthy live birth pregnancy and 30% had a failed pregnancy.
In their study of 76 couples with Robertsonian translocations, Keymolen et al. (2009) concluded that, based on a live birth rate of 33%
per couple, PGD is a good reproductive option for such couples,
especially when there is also a fertility problem; however, that study
did not examine the predictive accuracy of the testing, nor did it
present the prevalence of abnormal findings in the different groups
represented in their study.
For more than a decade, FISH on the fixed nuclei of biopsied cells
with target-specific DNA probes has been the technique of choice
to detect chromosome imbalance associated with chromosome
rearrangements (Conn et al., 1998; Munné et al., 1998a; Pierce
et al., 1998; Harper et al., 2010). Testing single cells using the
FISH technique has inherent technical difficulties associated with preparing a single nucleus, the stage of the mitotic cell cycle at the time
of spreading, variable binding efficiency of the probes, and the arbitrary nature of scoring of FISH signals in interphase nuclei (Munné,
2002; Wilton et al., 2009; Treff et al., 2010a). Testing is also confounded by the nature of the early embryo, where the sample
1583
Segregation of Robertsonian translocations
may not be genetically representative of the whole embryo due to
errors associated with fertilization, cell mitosis and nucleus packaging
(Munné and Cohen, 1998c). Recent studies using PCR-based PGD
for Robertsonian and reciprocal translocations (Fiorentino et al.,
2010; Traversa et al., 2010) offer the promise of an improved
alternative to the laborious and inherently subjective FISH technique,
and microarray-based comparative genomic hybridization and single
nucleotide polymorphism quantitative and genotype analysis techniques have the potential for accurate testing of all 23 pairs of
chromosomes (Wells et al., 2008; Gutiérrez-Mateo et al., 2010;
Handyside et al., 2010; Treff et al., 2010b,c), with the important
caveat that the sample tested is truly representative of the
embryo. Our study presents a comprehensive evaluation of PGD
at a single centre using the FISH technique for carriers of Robertsonian translocations, which we hope will be of value in helping us to
evaluate existing and new approaches to PGD.
In conclusion, our findings show that although alternate segregation is favoured for both female and male carriers of 13/14
and 14/21 Robertsonian translocations, female carriers are
approximately four times more likely to produce embryos with
unbalanced translocation products; this will apply regardless of
which technology is used for testing. Following appropriate
genetic counselling, PGD using the FISH technique should be considered as an alternative to PND for female carriers of 14/21
Robertsonian translocations to reduce the significant risk of
Down syndrome and possibly miscarriage, and could be indicated
to reduce the risk of miscarriage for female carriers of 13/14
Robertsonian translocations.
PGD for fertile male carriers of 13/14 and 14/21 Robertsonian
translocations is unlikely to be indicated; however, revising our previous recommendation (Scriven et al., 2001), we conclude that,
where assisted conception is indicated, embryo transfer without
PGD should be considered a reasonable option in the case of
male carriers. We believe that couples who are unlikely to
benefit from PGD (because treatment will not increase their
chance of achieving a successful, chromosomally balanced pregnancy) should not undergo an expensive and invasive intervention
unnecessarily.
Authors’ roles
S.M.B. conducted the literature review and checked the data collation
and analysis; C.M.O. was the consultant Clinical Scientist and reviewed
the study design and checked the data interpretation; F.A.F. was the
consultant Clinical Geneticist involved in counselling the patients;
Y.K. was the consultant Gynaecologist and advised on assisted reproduction procedures; P.N.S. designed the study, interpreted and collated the data and performed the calculations. All the authors
contributed to the writing of the manuscript.
Acknowledgements
We are grateful to Prof. Peter Riven Braude for his comments on this
manuscript, and for his advice and guidance over the decade that it has
taken us to complete this study.
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