Preimplantation genetic diagnosis for aneuploidy

screening in patients with unexplained recurrent

miscarriages
Peter Platteau, M.D.,a Catherine Staessen, Ph.D.,a,b An Michiels, M.Sc.,b
Andre Van Steirteghem, Ph.D.,a Inge Liebaers, Ph.D.,b and Paul Devroey, Ph.D.a
a
Center for Reproductive Medicine, and b Center for Medical Genetics, University Hospital, Dutch-speaking Brussels Free
University (Vrije Universiteit Brussel), Brussels, Belgium

Objective: To determine the aneuploidy rate in embryos of women with idiopathic recurrent miscarriages and to
evaluate whether preimplantation genetic diagnosis for aneuploidy screening could be a feasible approach to
improve the possibility of successful pregnancy in these couples.
Design: Prospective cohort study.
Setting: Tertiary university referral center.
Patient(s): Women (n ⫽ 49) with recurrent idiopathic miscarriages.
Intervention(s): In vitro fertilization with preimplantation genetic diagnosis for aneuploidy screening.
Main Outcome Measure(s): Ongoing pregnancy rate (PR) and aneuploidy rate.
Result(s): The aneuploidy rate was, respectively, 43.85% and 66.95% in the younger and older group. The
ongoing PR per cycle was 25.71% in the younger and 2.94% in the older patients.
Conclusion(s): There is no therapeutic evidence to prescribe IVF with or without preimplantation genetic
diagnosis for aneuploidy screening for this heterogeneous group of patients. (Fertil Steril威 2005;83:393–7. ©2005
by American Society for Reproductive Medicine.)
Key Words: Chromosomal aneuploidy, fluorescence in situ hybridization, preimplantation genetic diagnosis,
recurrent miscarriages

Miscarriage is defined as the loss of a pregnancy before
viability, which is according to the definition of the World
Health Organization, the expulsion of an embryo or fetus
weighing 500 g or less from its mother. This corresponds to
a gestational age of 20 –22 weeks, the irreducible age for
fetal viability. Recurrent miscarriage is defined as the loss of
three or more consecutive pregnancies (1). Some clinicians
favor changing the definition of recurrent miscarriage to two
or more consecutive losses, but the efficacy of commencing
investigations after two losses has not been established.
Furthermore, including patients with only two miscarriages
dilutes this already heterogeneous group and renders litera-
ture reviews on this subject inconclusive.
Between 12% and 15% of clinically recognized pregnan-
cies miscarry (1). The expected chance of three consecutive
pregnancy losses is therefore (15%)3 ⫽ 0.34%. However,
1% of couples suffer from recurrent miscarriage (2). The fact
that the observed frequency of recurrent miscarriage is sig-
nificantly higher than that expected by chance alone implies
that in some women there is a persistent underlying cause to
account for their pregnancy losses.
Received March 1, 2004; revised and accepted June 30, 2004.
Reprint requests: Peter Platteau, M.D., Center for Reproductive Medicine,
University Hospital, Dutch-speaking Brussels Free University (Vrije Uni-
versiteit Brussel), Laarbeeklaan 101, 1090 Brussels, Belgium (FAX:
00-32-2-4776649; E-mail: peter.platteau@az.vub.ac.be).
Balasch et al. (3) suggested that IVF, by replacing several
embryos into the uterus, may be a therapeutic approach for
young women (mean age, 31 years) with unexplained recur-
rent miscarriages. They observed a 66.6% ongoing preg-
nancy rate (PR) in 12 patients (with a mean of 4.91 previous
miscarriages). They hypothesized that by replacing three to
four embryos into the uterus, it would be possible to poten-
tiate the maternal recognition of pregnancy, by altering the
maternal immune response. They believed, in addition, that
the replacement of this high number of embryos would also
increase the chance of becoming pregnant with a chromo-
somally normal embryo. However, Raziel et al. (4) found no
advantage of IVF in older women (mean age, 38 years); 42 IVF
cycles in 20 habitual aborters were retrospectively investigated:
the pregnancy was 32% per cycle with a 50% miscarriage rate.
Recently some investigators (5– 8) hypothesized that non-
inheritable de novo abnormalities arising from random errors
produced from gametogenesis through to embryonic devel-
opment could be an important etiology in unexplained re-
current abortions. This was based on cytogenetic evaluations
of first trimester spontaneous abortions, revealing an overall
incidence of 50%–70% chromosomal abnormalities (9 –13).
Their results, in a limited group of patients younger than 37
years, indicated that a large proportion of the embryos were
chromosomally abnormal and that preimplantation genetic
diagnosis for aneuploidy screening (PGD-AS) could be a
feasible approach to improve the possibility of successful
pregnancy in these couples (with an already good prognosis).

0015-0282/05/$30.00 Fertility and Sterility姞 Vol. 83, No. 2, February 2005 393
doi:10.1016/j.fertnstert.2004.06.071 Copyright ©2005 American Society for Reproductive Medicine, Published by Elsevier Inc.
 We conducted a prospective cohort study in two groups of
patients (women ⬍37 years and women ⱖ37 years) to con-
firm these findings and to try to explain the conflicting
results from Balasch and Raziel and their colleagues (3, 4).
MATERIALS AND METHODS
Study Population
Forty-nine patients aged between 26 and 44 years with a
history of unexplained recurrent abortion were included in
this study. All were screened according to the standard
work-up for recurrent miscarriage, which comprises periph-
eral blood karyotyping in both partners, a pelvic ultrasound
scan to assess ovarian morphology and uterine cavity, and
screening tests for antiphospholipid antibodies (both lupus
anticoagulant and anticardiolipin antibodies) performed on
two occasions at least 6 weeks apart. Institutional Review
Board approval was obtained. All patients gave their written
consent before entering the study.
Ovarian Stimulation and Intracytoplasmic Sperm Injection
Procedure
All female partners were superovulated using a GnRH analogue
suppression protocol (short or long) (14) or a GnRH antagonist
protocol (15) and hMG or recombinant FSH. Oocyte– cumulus
complexes were recovered 36 hours after the administration of
10,000 IU of hCG. Then the surrounding cumulus and corona
cells were removed and the nuclear maturation of the oocytes
was assessed under an inverted microscope. Only metaphase II
oocytes were injected with morphologically normal motile
spermatozoa into the ooplasm. These procedures have been
described previously (16, 17).
Assessment of Fertilization, Embryo Development, and
Biopsy
Additional culture of injected oocytes was performed in
25-␮L microdrops of culture medium under lightweight par-
affin oil. Fertilization was confirmed after 16 –18 hours by
the observation of two distinct pronuclei (2PN). Oocytes
with 2PN were assessed on day 2 and day 3 after injection
for embryonic development, and the embryos reaching at
least the 5-cell stage on day 3 of development were biopsied.
The selection criteria for embryo biopsy were similar to
those used to decide whether an embryo was transferable on
day 3 in the regular intracytoplasmic sperm injection (ICSI)
program without PGD. Before biopsy, the blastomeres were
checked for the presence of a nucleus. From the 6-cell stage
onward, two blastomeres per embryo were removed (18, 19).
Fluorescence In Situ Hybridization Procedure
The individually biopsied blastomeres were spread onto a
Superfrost Plus glass slide (Kindler GmbH, Freiburg,
Germany) using 0.01 N HCl/0.l% Tween 20 solution (20,
21). Both blastomeres from the same embryo were fixed on
the same slide in very close proximity.
394 Platteau et al. Aneuploidy rate after recurrent miscarriages
A two-round fluorescence in situ hybridization (FISH) pro-
cedure, as described previously (22), allowed us to detect the
chromosomes X, Y, 13, 18, 21 (round 1) and 16, 22 (round 2).
In short, an aliquot (0.2 ␮L) of the probe solution (DXZ1,
Spectrum Blue; DYZ3, SpectrumGold; LSI13, SpectrumRed;
D18Z1, SpectrumAqua; LSI21 SpectrumGreen; Multivision
PGT Probe Panel; Vysis Inc., Downers Grove, IL) was added to
the nuclei, covered with a round coverslip (4 mm in diameter),
denatured for 5 minutes at 75°C and left to hybridize for
between 4 hours and overnight at 37°C in a moist chamber.
After washing in 0.4 ⫻ standard saline citrate solution (SSC)/
0.3% Nonidet P40 at 73°C for 5 minutes and 2 ⫻ SSC/0.1%
Nonidet P40 for 60 seconds at room temperature. Antifade
solution (Vectashield, Burlingame, CA) was added and fluo-
rescence signals were evaluated. The nuclei were then exam-
ined using a Zeiss Axioskop fluorescence microscope (Zeiss;
Oberkochen, Germany) with the appropriate filter sets. The
FISH images were captured with a computerized system.
After the analysis of the first set of probes, the coverslips
were gently removed and the slides rinsed in 1⫻ phosphate-
buffered saline (PBS) at room temperature, denaturated in
0.0625⫻ SSC for 7 minutes at 75°C, and then dehydrated
(70%, 90%, 100%, and 100% ethanol at ⫺18°C, 60 seconds
each). The second hybridization solution was prepared by mix-
ing a probe for chromosome 16 (Satellite II DNA/D16Z3 probe,
spectrum Orange, Vysis, Inc.) and a probe for chromosome 22
(LSI 22, 22q11.2, SpectrumGreen, Vysis, Inc.). The probes
were denaturated separately in a hot water bath at 75°C for 5
minutes. An aliquot (0.2 ␮L) of the probe solution was then
added to the nucleus, covered with a round coverslip (4 mm in
diameter), sealed with rubber cement and then hybridized over-
night in a water bath at 37°C. Finally, the slides were washed
for 2 minutes in 0.4⫻ SSC solution at 73°C and 2⫻ SSC/0.1%
Nonidet P40 for 60 seconds at room temperature. The washed
slides were then mounted with 6-diamino-2-phenylindole
(DAPI) in antifade solution, and analyzed and the results inter-
preted by two independent observers.
Embryo Scoring and Selection for Transfer
The subsequent classification of embryos based on the bi-
opsy result of two blastomeres was followed: [1] when both
blastomeres had two copies of each chromosome analyzed,
the embryo was classified as normal; [2] when both blas-
tomeres had one or two chromosomes with an abnormal
number of copies, the embryo was classified as aneuploid;
[3] when both blastomeres had one, three, or more copies of
each chromosome, the embryo was classified as haploid or
polyploidy; [4] when one blastomere was normal and the
second blastomere had one or two chromosomes with an
abnormal number of copies, the embryo was classified as
mosaic; and [5] when at least one blastomere had more than
two chromosomes with an abnormal number of copies, the
embryo was classified as complex abnormal. Only chromo-
somally normal blastocysts were transferred on day 5.
Vol. 83, No. 2, February 2005
 TABLE 1
Characteristics of the patients, stimulation, and FISH results (values are means ⴞ SD).

Patients <37 Patients ≥ 37

years years
(35 cycles) (34 cycles)

Mean age (y) 32.48 ⫾ 2.72 yrs 40.15 ⫾ 2.48 yrs

Mean no. of miscarriages 4.46 ⫾ 2.10 4.93 ⫾ 2.41
Mean no. of COC 13.84 ⫾ 7.00 10.43 ⫾ 5.92
Mean no. of MII 11.70 ⫾ 5.83 8.88 ⫾ 5.14
Mean no. of 2PN 8.69 ⫾ 4.93 6.95 ⫾ 3.89
No. embryos biopsied 240 173
Diagnosis
% Normal 54.20% ⫾ 21.74% 28.40% ⫾ 28.69%
% Abnormal 43.85% ⫾ 22.50% 66.95% ⫾ 29.27%
% No diagnosis 1.94% ⫾ 5.04% 4.63% ⫾ 15.59%
Note: COC ⫽ combined oocyte complexes; MII ⫽ Metaphase II.
Platteau. Aneuploidy rate after recurrent miscarriages. Fertil Steril 2005.

Definition of End Point
An ongoing pregnancy was defined if at least one fetus with
a positive heartbeat was revealed by vaginal ultrasound after
12 weeks of gestation. The implantation rate was defined as
the number of viable fetuses, as assessed by ultrasound at 7
weeks gestation, divided by the number of embryos trans-
ferred for each subject.
Statistical Analysis
The mean age at the time of oocyte retrieval of each cycle
and the mean number of retrieved oocytes, metaphase II
oocytes, normally fertilized oocytes, and the mean number
of abnormal embryos of each cycle was first calculated per
couple. In a second step the mean of all previous parameters
of all the couples was calculated. The comparison of the
variables was performed by means of two-way ANOVA,
with Bonferroni t test to perform pairwise comparison of the
two groups. P⬍.05 was considered statistically significant.
RESULTS
There were 49 patients (after screening) included in this
study, divided (arbitrarily) in 25 patients younger than 37
years (35 cycles) and 24 women ⱖ37 years (34 cycles). The
results are summarized in Tables 1, 2, and 3.
The younger patients were on average 32.48 ⫾ 2.72 years
old with a mean of 4.46 ⫾ 2.10 previous miscarriages. The
older ones were on average 40.15 ⫾ 2.48 years old with a
mean of 4.93 ⫾ 2.41 previous miscarriages. After the re-
trieval of 13.84 ⫾ 7.00 and 10.43 ⫾ 5.92 oocytes, 11.70 ⫾
5.83 and 8.88 ⫾ 5.14 metaphase II oocytes, and 8.69 ⫾ 4.93
and 6.95 ⫾ 3.89 normally fertilized oocytes, 240 and 173
embryo biopsies could be taken in each age group.

TABLE 2
Chromosomal constitution of blastomeres derived from couples with idiopathic recurrent abortions
(values are means ⴞ SD).

<37 years ≥37 years

% Normal 54.20 ⫾ 21.74 28.40 ⫾ 28.69

% Abnormal 43.85 ⫾ 22.50 66.95 ⫾ 29.27*
Aneuploid 39.91 ⫾ 31.54 47.04 ⫾ 26.79
Complex 26.53 ⫾ 28.95 34.16 ⫾ 30.12
Haploid/polyploid 5.03 ⫾ 10.60 3.47 ⫾ 11.12
Mosaic 28.03 ⫾ 33.33 15.31 ⫾ 23.28
% No diagnosis 1.94 ⫾ 5.04 4.63 ⫾ 15.59
*P⫽.0032.
Platteau. Aneuploidy rate after recurrent miscarriages. Fertil Steril 2005.

Fertility and Sterility姞 395

 TABLE 3
Pregnancy outcome and implantation rates.
Mean no. of embryos transferred
% No transfer
No. of clinical pregnancies
% Ongoing pregnancy/cycle
% Ongoing pregnancy/embryo transfer
Implantation rate
Platteau. Aneuploidy rate after recurrent miscarriages. Fertil Steril 2005.
Sixty-three chromosomally normal embryos were trans-
ferred in 31 cycles of patients younger than 37 years, result-
ing in one biochemical and nine ongoing pregnancies (all
singleton, except one twin pregnancy) with an implantation
rate of 16.66% ⫾ 30.32%. In the older age group, 37
chromosomally normal embryos were transferred in 18 cy-
cles, resulting in two biochemical pregnancies, two miscar-
riages, and one ongoing pregnancy. There were four young
patients who did not have transfer: one because of severe
hyperstimulation syndrome (all embryos were frozen), one
patient had only chromosomally abnormal embryos, and the
embryos of two patients were of poor quality and not trans-
ferable. Sixteen older patients did not have embryo transfer,
as 12 of them had only chromosomally abnormal embryos
and four had no transfer because of poor embryo quality.
The overall percentage of chromosomal abnormal embryos
in the younger and older group was, respectively, 43.85% ⫾
22.50% and 66.95% ⫾ 29.27% (P⫽.0032). The classification
of the abnormal embryos (in aneuploid, complex abnormal,
haploid or polyploidy, and mosaic [see materials and methods])
in the two groups did not reveal any differences.
DISCUSSION
The aneuploidy rate in the group of young patients (43.85%)
is similar to the results (41%–53%) of previous smaller
studies (5– 8). To our knowledge, our series is the largest
study ever published, evaluating PGD-AS in patients with
idiopathic recurrent miscarriages. A similar study (23) found
even higher aneuploidy rates, especially in the younger pa-
tient group. The researchers, however, extended in this latter
study the definition of recurrent aborters up to two miscar-
riages. It is therefore difficult to compare the two studies.
The PGD-AS does not seem to improve the PR in the
group of young patients compared to our general IVF pop-
ulation. Although the aneuploidy rate is relatively high in
this group (43.85%), PGD-AS did not improve the selection
of the transferred embryos, resulting in a 29% ongoing PR
per transfer. Without any treatment the expected live birth
rate in this group of patients would have been ⬎60% (24 –
26). For one (young) patient this PGD-AS cycle might have
396 Platteau et al. Aneuploidy rate after recurrent miscarriages
<37 years ≥ 37 years
2.03 ⫾ 0.79 2.05 ⫾ 0.87
11.42% (4/35) 47.00% (16/34)
9 1
25.71% 2.94%
29.03% 5.55%
16.66% ⫾ 30.32% 2.77% ⫾ 11.78%
been diagnostic, as all her embryos were abnormal, making
decisions (as oocyte donation) easier.
The pregnancy results in the older group were disappoint-
ing. It seems that these patients are near the end of their
reproductive life. They first have several spontaneous preg-
nancies, sadly all resulting in miscarriages. They then have
no pregnancies at all anymore, which makes them consult an
assisted reproductive technology (ART) clinic sooner or
later, in view of their older age. Half of these patients had no
ET, as all their embryos were chromosomally abnormal; the
other half had a very poor pregnancy outcome. Therefore, it
seems that in these patients, PGD-AS does not bring any
benefit, apart from being diagnostic and redirecting treat-
ment to other options.
Our results confirm the data from both previously described
articles (3, 4). The Spanish group achieved very good ongoing
PRs by doing IVF in young patients; their results (58.3%
ongoing PR) were even better than ours, probably due to the
high number of embryos transferred (3 to 4), with a similar
implantation rate (17.6%). The ongoing PR was, however, the
same as the expected live birth rate, if no treatment would have
been done. The Israeli group had the same disappointing IVF
results as we had in our group of older patients. Therefore, it
seems that the mean age of the patients with recurrent miscar-
riages predicts their future obstetric outcome.
Our PGD-AS results are based on seven chromosomes. It
is possible that in the future with new techniques (compar-
ative genomic hybridization, microarray), we will be able to
screen for all chromosomes, which might influence our re-
sults as there is a possibility that we missed some chromo-
somal abnormalities. The addition of more FISH probes
decreases the efficiency of the procedure (27).
The PGD-AS might be beneficial in a subgroup of patients
with recurrent miscarriages, who have proven aneuploied
concepti by cytogenetic analysis. In our center, however,
only a minority of patients have these results. A lot of fetal
material is lost due to the unforeseen and dramatic circum-
stances and we believe that some clinicians find that the
usefulness of cytogenetic investigation of fetal tissues is
rather limited as it seldom influences clinical practice. The
Vol. 83, No. 2, February 2005
technique itself has also some problems, including external
contamination, culture failure, and selective growth of ma-
ternal cells (12). Other more sophisticated techniques, such
as comparative genomic hybridization (28) or M-FISH (29),
may overcome these problems in the future. A cytogenetic
diagnosis allows the patient, however, to be given a more
accurate diagnosis and more useful prognostic information
regarding consequent pregnancy outcome as patients with
karyotypically abnormal fetuses have a good reproductive
prognosis (30). At a scientific level it provides a more
accurate basis for judging various treatment forms.
In conclusion, on the basis of our data there seems no
therapeutic evidence to prescribe IVF with or without
PGD-AS for this heterogeneous group of patients, but more
sophisticated fetal cytogenetic testing should be performed,
whenever possible. The young patients can be reassured that
the prognosis for future pregnancy is ⬎60% without any
treatment. The future obstetric outcome for the older patients
remains grim, whatever treatment is done.
Acknowledgments: The authors thank the clinical, paramedical, and labora-
tory staff of the Center for Reproductive Medicine and Medical Genetics,
especially Marleen Carlé, Sylvie Mertens, and Griet Meersdom who work in
the FISH laboratory, Anick Devos, Lisbet Van Landuyt, Ronny Janssens,
and Hubert Joris, who did the biopsies. Furthermore, we are very grateful to
Dr. Marie-Paule Derde for statistical help.
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