Human Reproduction Update, Vol.9, No.4 pp. 319±330, 2003
DOI: 10.1093/humupd/dmg029
PGD in 47,XXY Klinefelter's syndrome patients
C.Staessen1,3, H.Tournaye1, E.Van Assche2, A.Michiels2, L.Van Landuyt1, P.Devroey1,
I.Liebaers2 and A.Van Steirteghem1
1
Centre for Reproductive Medicine and 2Centre for Medical Genetics, University Hospital, Dutch-speaking Free University of
Brussels (Vrije Universiteit Brussel), Brussels, Belgium
3
The use of ICSI has been a major breakthrough in the treatment of male infertility. Even azoospermic patients with
focal spermatogenesis in the testis, may bene®t from the ICSI technique in order to father a child. As ICSI use has
become more common, centres have introduced infertility treatment for Klinefelter patients. To date, 34 healthy
children have been born using ICSI without PGD, and the conception of one 47,XXY fetus has been reported. In
view of the possible risk of an increased gonosome number in the spermatozoa of Klinefelter patients, a safer
approachÐoffering these couples ICSI combined with PGDÐhas been used, and has resulted in the birth of three
healthy children. Couples in which the male suffered from Klinefelter's syndrome were ®rst treated in 1995; these
patients were offered ICSI + PGD using FISH technology, notably to enumerate the X and Y chromosomes. ICSI +
PGD was performed in 32 cycles of 20 couples with spermatozoa originating from a fresh ejaculate (n = 1), testicular
biopsy (n = 21) or frozen±thawed testicular biopsy (n = 10). Normal fertilization occurred in 56.0 6 22.4% of the
successfully injected oocytes. On day 3 of development, 119 embryos from 29 cycles were of suf®cient quality to
undergo biopsy and subsequent PGD; a positive result was obtained in 113 embryos. Embryos were available for
transfer in 26 cycles, with a mean of 1.6 6 0.6 embryos per transfer. Eight pregnancies were obtained, and ®ve
resulted in a delivery. A total of 113 embryos from couples with Klinefelter's syndrome was compared with 578
embryos from control couples with X-linked disease where PGD was used to determine gender. A signi®cant fall
occurred in the rate of normal embryos for couples with Klinefelter's syndrome (54.0%) compared with controls
(77.2%). Moreover, a signi®cantly increased risk of abnormalities was observed for sex chromosomes and autosomes; for each autosome separately, this reached signi®cance level for chromosomes 18 and 21 only. Hence, a cautious approach is warranted in advising couples with non-mosaic Klinefelter's syndrome. Moreover, the use of ICSI
+ PGD or prenatal diagnosis should be carefully considered.
Key words: FISH/ICSI/Klinefelter syndrome/PGD/testicular sperm
Introduction
In 1942, Klinefelter and colleagues ®rst described a syndrome
characterized by gynaecomastia, aspermatogenesis, without aleydigism and increased excretion of FSH (Klinefelter et al., 1942),
and some 17 years later this syndrome was related to a 47,XXY
karyotype (Jacobs and Strong, 1959).
Although most Klinefelter's syndrome patients are considered
to have a non-mosaic 47,XXY karyotype, a mosaic 47,XXY/
46,XY karyotype is found in about 10% of cases (Wilson and
Grif®n, 1987). Within the general population, the incidence of
47,XXY Klinefelter's syndrome is 0.1% (Nielsen and Wohlert,
1991), and this value reaches 3.1% in the infertile male population
(Guichaoua et al., 1993). Although a few cases with proven
paternity (Laron et al., 1982; Terzoli et al., 1992) have been
reported, non-mosaic 47,XXY Klinefelter patients are in general
considered to be sterile. The introduction of ICSI led to a change in
the clinical approach towards fertility treatment in these patients.
In cases with a 46,XY/47,XXY mosaic karyotype, spermatozoa
can usually be recovered from the ejaculate, and fertilization has
ensued after ICSI (Harari et al., 1995; Bielanska et al., 2000). Nonmosaic Klinefelter patients (47,XXY) are generally azoospermic
owing to primary testicular failure, although in some cases focal
spermatogenesis has been present and testicular spermatozoa have
been recovered and successfully used for ICSI (Staessen et al.,
1996; Tournaye et al., 1996).
Investigations on the meiotic products of non-mosaic
Klinefelter patients by direct analysis of the ejaculated or testicular
spermatozoa with the ¯uorescent in-situ hybridization (FISH)
Human Reproduction Update 9(4) ã European Society of Human Reproduction and Embryology 2003; all rights reserved
319
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To whom correspondence should be addressed at: Centre for Reproductive Medicine, Academisch Ziekenhuis, Vrije Universiteit
Brussel, Laarbeeklaan 101, B-1090 Brussels-Belgium. E-mail: [email protected]
C.Staessen et al.
Table I. Overview of reported pregnancies obtained after ICSI treatment of patients with apparent non-mosaic Klinefelter's syndrome
PGDperformed
No. of
cycles (n)a
Outcome of pregnancy
Karyotype (n)b
No
No
No
Case report
Case report
2 (2)
Abortion tissue: 46,XX
46,XX (1)
No information
Tachdjian et al. (2003)
47,XXY: frozen±thawed ejaculated spermatozoa
Bourne et al. (1997)
47,XXY: fresh testicular spermatozoa
No
Case report
Singleton: miscarriage at 9 weeks
Singleton pregnancy
1 biochemical pregnancy and 1 miscarriage
at 8 weeks
Twin pregnancy
No
Case report
Twin pregnancy
46,XX (1)46,XY (1)
Staessen et al. (1996); Tournaye et al. (1997)
Palermo et al. (1998)
Reubinoff et al. (1998)
Levron et al. (2000)
Poulakis et al. (2001)
Friedler et al. (2001)
Nodar et al. (1999)
Kitamura et al. (2000)
Greco et al. (2001)
Yamamoto et al. (2002)
47,XXY: frozen±thawed testicular spermatozoa
Friedler et al. (2001)
Rosenlund et al. (2002)
Yes
No
Yes
No
No
No
No
No
No
No
3 (3); 7 (5)
3 (2)
5 (4)
Not known (8)
2 (2)
5 (5)
Case report
1 (1)
Case report
12 (9)
1 biochemical and 2 singleton pregnancies
Singleton and twin pregnancy
Singleton pregnancy
1 triplet, 1 twin and 2 singleton pregnancies
2 singleton pregnancies
2 singleton and 1 twinc pregnancy
Twin pregnancy
Singleton pregnancy
Twin pregnancy
1 twin and 3 singleton pregnancies
2 healthy neonates
46,XY (2)46,XX (1)
46,XY (1)
46,XY (4)46,XX (3)
46,XY (1)46,XX (1)
4 healthy neonates
46,XY (2)
46,XX (1)
46,XY (2)
46,XY (2)46,XX (3)
Twin pregnancy
Singleton pregnancy
Both healthy males
46,XY (1)
Reference
47,XXY: ejaculated spermatozoa
Hinney et al. (1997)
CruÈger et al. (2001)
Kitamura et al. (2000)
5 (5)
Case report
aNumber
of couples in parentheses.
of children in parentheses.
cTriplet with 47,XXY fetus reduced in the 14th gestational week.
bNumber
technique have shown varying incidences of normal spermatozoa
ranging from 50.0 to 93.7% (Guttenbach et al., 1997; Estop et al.,
1998; Levron et al., 2000) and an increased incidence of 24,XX
and 24,XY hyperhaploid spermatozoa as compared with a control
population (Foresta et al., 1998; Hennebicq et al., 2001;
Yamamoto et al., 2002), while the incidence of 24,YY hyperploid
spermatozoa was similar (Guttenbach et al., 1997) and disturbed
23,X/23,Y ratios (Guttenbach et al., 1997). These different studies
in fact con®rm that, in Klinefelter patients, there is an increased
incidence of genetically imbalanced spermatozoa, though two
different hypotheses have been formulated. The ®rst hypothesis is
that 47,XXY spermatogonia may undergo meiosis to produce
hyperhaploid spermatozoa (Guttenbach et al., 1997; Estop et al.,
1998; Foresta et al., 1998; Bielanska et al., 2000; Hennebicq et al.,
2001; Yamamoto et al., 2002); the second hypothesis is that the
rare break-through patches of spermatogenesis in XXY males are
due to the presence of normal XY germ cells but, as a result of the
compromised testicular environment, these 46,XY germ cells are
susceptible to meiotic abnormalities (Mroz et al., 1998).
As the use of ICSI has become increasingly widespread,
different centres have introduced infertility treatment for
Klinefelter patients. Their reportsÐthe majority of which are
case reportsÐrelate the centres' experience with fertility treatment
of Klinefelter patients with ICSI but without the use of PGD, and
this has resulted in the birth of healthy children (Table I). With the
use of freshly ejaculated sperm, the birth of three healthy children
has been reported, while two healthy children have been born after
320
the use of frozen±thawed ejaculated sperm, and 26 healthy
children have been born after the use of fresh testicular sperm.
Finally, three healthy children were born following the use of
cryopreserved-thawed testicular spermatozoa. One group (Ron-El
et al., 2000a) reported a triplet gestation, one of which (a 47,XXY
fetus) was reduced, while others (Kyono et al., 2001) reported a
pregnancy following ICSI with fresh testicular sperm from a man
with non-mosaic Klinefelter's syndrome and spinal cord injury.
The outcome of this pregnancy was not documented however, as at
the time of this writing the pregnancy was ongoing.
In view of the possible risk of an increased number of
gonosomes in their spermatozoa, anotherÐmore safeÐapproach
is to offer these couples ICSI in combination with PGD using FISH
technology, in order to enumerate the X and Y chromosomes in
particular (Staessen et al., 1996; Reubinoff et al., 1998). To date,
by using this approach of combining ICSI with fresh testicular
sperm and PGD, three healthy children have been born (Table I).
However, since these ®rst reports were made (Staessen et al.,
1996; Tournaye et al., 1997; Reubinoff et al., 1998), a large
number of non-mosaic Klinefelter patients have been treated with
ICSI in combination with PGD at the authors' centre. The aim of
the present review was to describe the fertility treatment in
Klinefelter patients, and also to discuss the use of ICSI treatment
with and without PGD. Several couples with non-mosaic
Klinefelter's syndrome were treated with ICSI combined with
PGD, and the genetic constitution of the embryos produced was
compared with that of embryos obtained from control couples who
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No
No
46,XX (1)46,XY (1)
PGD after ICSI for 47,XXY patients
underwent ICSI plus PGD in order to determine fetal gender with
relation to sex-linked disease.
Assisted reproduction techniques, PGD procedures and
results
Patients
Sperm retrieval
In general, spermatozoa were retrievable in ~50% of
Klinefelter patients (Tournaye et al., 1996). The only predictive factor for successful sperm recovery in these patients was
the histopathological ®nding, as for all other parametersÐ
including ultrasonography and frequency of 46,XY in lymphocytes and buccal cellsÐno signi®cant difference was found
between Klinefelter patients with successful sperm recovery
and those in whom sperm recovery failed (Westlander et al.,
2001). Therefore, sperm recovery is a crucial step in the
fertility treatment of Klinefelter patients. Before patients were
scheduled for infertility treatment, one or two of their sperm
ejaculate samples were carefully evaluated for the presence of
sperm cells by using high-speed centrifugation. If the presence
of sperm cells was con®rmed, the stimulation was planned.
However, if no sperm cells were observed in the ejaculate, a
testicular biopsy was scheduled (Tournaye et al., 1996; 1997).
In cases where no spermatozoa were found after mechanical
mincing of the biopsies, enzymatic treatment of the testicular
tissue with collagenase type IV was employed (Crabbe et al.,
1998) in order to maximize the chance of sperm retrieval.
Attempts to freeze testicular sperm cells from non-obstructive
males were successful (Verheyen et al., 1997); consequently, it
was possible to freeze testicular sperm obtained from a
Oocyte retrieval and ICSI procedure
Ovarian stimulation was carried out by pituitary desensitizing
with GnRH analogues (Buserelin, Suprefact; Hoechst,
Brussels, Belgium) combined with hMG (Humegon;
Organon, Oss, The Netherlands) or recombinant FSH (GonalF; Serono, Brussels, Belgium or Puregon; Organon) and hCG
(Pregnyl, Organon; Profasi, Serono). Oocyte retrieval was
carried out using ultrasound-guided puncture at 36 h after hCG
administration. The cumulus±corona±oocyte complexes were
placed in 25 ml droplets of medium and covered by lightweight
paraf®n oil. Before ICSI was carried out, the cumulus and
corona cells were removed and nuclear maturation was
assessed under an inverted microscope.
The ICSI procedure was carried out as described previously
(Van Steirteghem et al., 1998). The testicular spermatozoa
were frequently found to be embedded in Sertoli cell
cytoplasm, and had to be carefully extracted. For the injection,
a motile sperm cell (if present) was aspirated into the injection
pipette and injected into a metaphase II (MII) oocyte which had
extruded the ®rst polar body. Injected oocytes were washed and
incubated in 25 ml droplets of medium in a Petri dish at 37°C in
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Between November 1994 and March 2002, sperm was obtained
from 20 couples of which the husband had a 47,XXY
karyotype. The subsequent 32 treatment ICSI cycles which
were obtained, together with the data obtained from them, form
the basis of the present report. The mean (6 SD) age of the
female partners was 29.5 6 4.1 years (maximum age 38 years),
and all women had a normal fertility work-up. The non-mosaic
47,XXY karyotype in the patients was proven by an analysis of
15 to 30 metaphases with G-banding and/or an analysis of 50±
100 interphases with FISH analysis in peripheral blood
preparations and/or ®broblasts. A weak mosaicism of 5±8%
was detected after FISH analysis of 200 interphase lymphocytes in three patients; all of these patients consented to
undergo PGD by FISH in order to determine the number of sex
chromosomes if embryos were subsequently available for
transfer.
Another group of patients who underwent PGD to determine
fetal gender with regard to sex-linked diseases was selected for
comparison of the aneuploidy rate. This latter (control) group
comprised 43 couples of which the female partner was aged
<38 years (mean age 30.6 6 3.5 years) and included 88
treatment cycles. The results of the latter patient group were
published in part previously (Staessen et al., 1999).
diagnostic biopsy before a treatment cycle could be commenced.
Two options exist in scheduling of the testicular biopsy.
With the ®rst approach, the biopsy can be scheduled before
starting hyperstimulation treatment, with subsequent freezing
of the retrieved spermatozoa. Although this approach prevents
the woman from being needlessly hyperstimulated, there is an
estimated risk of 19.4% (Verheyen et al., 2002) that the
retrieved sperm cells will not survive the freeze±thawing
procedure. To counteract this risk, the patient should be
scheduled for a back-up fresh biopsy sperm retrieval on the day
of oocyte retrieval. The cryopreservation of testicular tissue
obtained after diagnostic testicular biopsy is a routine procedure option at the authors' institution. Following an unsuccessful ®rst ICSI cycle with either fresh or frozen testicular
spermatozoa, repeated testicular surgery for subsequent ICSI
cycles may be avoided by using frozen specimens. This is
important, as repeated biopsy retrieval may be dif®cult in
Klinefelter patients.
The second approach option is to perform an initial testicular
biopsy during a hyperstimulation treatment cycle. As sperm is
generally recovered in only 50% of Klinefelter patients, this
option could be proposed to couples agreeing to the use of
donor sperm.
ICSI was performed in 32 cycles of 20 couples, with sperm
originating from a fresh ejaculate in one cycle, from a testicular
biopsy in 21 cycles, and from a frozen±thawed testicular
biopsy in 10 cycles (Table II). In two cycles, insuf®cient
spermatozoa were obtained after thawing of the testicular
biopsy specimens in order to inject all the oocytes, and the two
couples decided to have the remaining oocytes injected with
donor spermatozoa rather than undergo repeat testicular
biopsy. The results obtained from oocytes injected with
donor sperm were excluded from the data analysis.
C.Staessen et al.
Table II. Results of fertility treatment of Klinefelter patients
Origin of sperm
cells (no. of cycles;
no. of transfers)
OCC retrieved
(mean 6 SD
per cycle)
MII injected
(mean 6 SD
per cycle)
2PN
(mean 6 SD
per cycle)a
Embryos
biopsied (with
diagnosis)
Normal XX
or XY (%)
Embryos
transferred
(mean 6 SD
per transfer)
Positive hCG
(with FHB)
No. of
deliveries
14
9
7 (77.8)
5 (4)
3 (75)
2
1 (1)
1
261
(12.4 6 6.1)
89
(8.9 6 4.5)
364 (11.4 6 5.8)
236
(11.2 6 5.2)
65
(6.5 6 4.0)b
310 (9.7 6 5.0)
130
(55.2 6 21.2)
35
(55.6 6 25.9)
172 (56.0 6 22.4)
90 (85)
47 (55.3)
5 (3)
3
24 (24)
11 (45.8)
31
(1.7 6 0.7)
8 (1.1 6 0.4)
2 (1)
1c
119 (113)
61 (54.0)
41 (1.6 6 0.6)
8 (5)
5
aMean
fertilization rate (%) per cycle.
one patient only six of 12 oocytes were injected, and from one patient only one of 10 oocytes was injected with sperm from the Klinefelter patient and
included in the data analysis.
cChild liveborn preterm at 23 weeks, but did not survive.
FHB = fetal heart beats; MII = metaphase II; OCC = oocyte±cumulus complex; 2PN = 2 pronuclei; TESE = testicular sperm extraction.
bFrom
an incubator under an atmosphere of 5% CO2, 5% O2 and 90%
N2. The medium used for oocyte and embryo culture was
subject to changes during the period of treatment, though since
1999 a sequential medium (G1-G2; Scandinavian IVF,
Vitrolife, GoÈteborg, Sweden) has been used for all patients
undergoing ICSI in combination with PGD.
In total, 364 oocyte±cumulus complexes (OCCs) were
retrieved, with a mean of 11.4 6 5.8 OCC per cycle
(Table II). Among these OCCs, 325 (89.3%) were at MII
stage, and 310 of them were injected with sperm from
Klinefelter patients. As mentioned earlier, in two patients
insuf®cient sperm was retrieved to inject all oocytes; in one of
these patients only six of 12 MII oocytes were injected, while
in the other patient only one of 10 MII oocytes was injected
with sperm from the Klinefelter husband. The other oocytes
were injected with donor sperm and excluded from further
consideration in this report. Although when selecting sperm for
microinjection the preference was to use a spermatozoon that
had shown signs of motility, suf®cient motile spermatozoa
could not always be found for the oocytes retrieved; consequently, in some cycles ICSI was carried out with non-motile
testicular spermatozoa. In three cycles, all the oocytes were
injected with non-motile sperm cells, and in seven cycles some
of the oocytes were injected with non-motile sperm cells due to
a scarcity of motile sperm cells. In total, 247 (79.7%) oocytes
were injected with a motile sperm cell, and 63 (20.3%) with a
non-motile sperm cell. Fertilization was checked at 16±18 h
after injection (Staessen et al., 1995). Normal fertilization was
con®rmed by the presence of two distinct pronuclei and polar
bodies using microscopy (inverted microscope, original magni®cation 3400).
A mean fertilization rate of 56.0 6 22.4% per injected MII
oocyte was obtained in all the cycles. There was no difference
322
in fertilization rate when either fresh sperm or sperm originating from frozen testicular-extraction (TESE) were used (see
Table II). In three cycles, only non-motile sperm were injected;
of the 26 injected MII oocytes, a total of 15 (57.7%) was
fertilized (i.e. a mean of 51.6 6 19.1% for the three cycles).
Among a total of 64 oocytes in seven cycles, 27 were injected
with motile spermatozoa, and 15 (55.6%) were fertilized (i.e.
mean 56.1 6 30.7% per patient); among 37 oocytes injected
with non-motile sperm, 15 (40.5%) were fertilized (mean 38.3
6 27.7% per patient). By comparing the fertilization rate of
motile versus non-motile sperm cells in sibling oocytes (n = 7
cycles), a tendency towards a higher fertilization rate in the
group of oocytes injected with motile sperm cells was observed
(Fisher's exact test, P = 0.3; NS). The likely reason for
fertilization rates not being signi®cantly different in this group
was that not enough cases were involved. These data demonstrate that the selection of motile testicular spermatozoa is
always preferable for ICSI, although as sperm production in
non-mosaic Klinefelter patients is at best scarce, it is worthwhile using non-motile sperm cells for injecting the oocytes.
These results were in agreement with fertilization rates
obtained after the injection of motile (65%) versus non-motile
(45%) testicular sperm cells in patients with complete
azoospermia and normal karyotype (Nagy et al., 1998). The
same group (Nagy et al., 1998) also showed that embryo
quality at 2 days after ICSI was independent of either motile or
non-motile testicular spermatozoa being microinjected; this
indicated that when normal fertilization has occurred, the
quality of the resulting embryos is generally independent of
sperm factors (Nagy et al., 1995). Likewise, among 30
fertilized oocytes injected with non-motile sperm cells, 20
developed into embryos suitable for biopsy; a diagnosis was
obtained in 15 of these, and four were normal and transferred.
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Fresh ejaculate
(n = 1; 1)
Fresh TESE
(n = 21; 18)
Frozen TESE
(n = 10; 7)
Total (n = 32; 26)
Number of:
PGD after ICSI for 47,XXY patients
Table III. Number and quality of embryos for biopsy in patients with Klinefelter syndrome in relation to origin of the sperm, and in control patients
Origin of sperm
cells (no. of cycles; no.
of cycles with biopsy)
*Fisher's
No. of embryos
for biopsy (% A)
<5-cell stage (n)
6- to 8-cell stage (n)
Type A-B (%)
Type C (%)
Type A-B (%)
Type C (%)
7
130
35
172
5
90
24
119 (69.2)*
0 (0)
12 (13.3)
7 (29.2)
19 (16.0)
2 (40)
15 (16.7)
3 (12.5)
20 (16.8)
1 (20)
54 (60.0)
12 (50.0)
67 (56.3)
2 (40)
9 (10.0)
2 (8.3)
13 (10.9)
761
604 (79.4)*
125 (20.7)
59 (9.8)
368 (60.9)
52 (8.6)
exact test: P < 0.01.
As a consequence there was one clinical pregnancy. It was also
reported that, for a small number of oocytes, quite similar
fertilization rates were achieved regardless of whether motile
or non-motile spermatozoa were injected (Ron-El et al.,
2000b); among 14 oocytes injected with immotile spermatozoa, seven were fertilized and six cleaved, whereas among the
eight oocytes injected with motile spermatozoa, seven were
fertilized and cleaved. Both sets of embryos led to the
establishment of a pregnancy.
Embryo development and biopsy
Oocytes with two pronuclei (2PN) were assessed on days 2 and
3 after injection for embryonic development, and embryos of
grade A, B or C reaching at least the 5-cell stage on the
morning of day 3 were biopsied. Type A embryos were de®ned
as those in which all the blastomeres were of an approximately
equal size, without anucleate fragments. Type B embryos had
blastomeres of equal or non-equal size and with anucleate
fragments comprising a maximum 20% of the embryo volume,
while type C embryos contained anucleate fragments as 20±
50% of their volume. Grade A, B, or C embryos with at least
four blastomeres were biopsied on the morning of day 3 after
microinjection. The selection criteria for embryo biopsy were
similar to those used to decide whether an embryo was
transferable on day 3 in the regular ICSI programme without
PGD. Those embryos not suitable for biopsy were discarded.
The same policy was applied as with embryos not considered
for transfer. Before biopsy, the blastomeres were checked for
the presence of nuclei. From the 6-cell stage onward, two
blastomeres per embryo were removed (Van de Velde et al.,
2000; De Vos and Van Steirteghem et al., 2001).
Among 172 oocytes showing 2PN, 119 (69.2%) developed
into embryos suitable for blastomere biopsy, and the remaining
zygotes were arrested or severely delayed on day 3. In three
cycles, no embryos suitable for biopsy were obtained, despite
fertilization. In two cycles using fresh testicular sperm, ®ve and
two zygotes were obtained respectively, but none of these
could be considered for biopsy. In one cycle the only oocyte
injected with frozen±thawed testicular sperm was fertilized,
but fragmented on day 3 of development. Finally, among 29 of
the 32 cycles, embryos were available for biopsy; cell stages
and embryo quality for biopsy as de®ned by morphological
grade are summarized in Table III. Of these embryos, 32.8%
had less than six cells, and 67.2% had at least six cells. As
mentioned earlier, one group of patients undergoing PGD for
sex-linked disease were added retrospectively (as controls) for
comparison with the study group. In the control group (see
Table III), a signi®cantly higher proportion of zygotes (79.4%)
developed into embryos suitable for biopsy as compared with
the Klinefelter patients (69.2%; Fisher's exact test, P < 0.01),
although the distribution of the different grades of embryos for
biopsy was not signi®cantly different between the control and
Klinefelter subjects.
One patient with a mosaic Klinefelter karyotype was
reported to have a normal fertilization rate after ICSI; however,
there was a high incidence of cleavage failure (44%) after
pronuclei were observed (Bourne et al., 1995). These results
further con®rmed that spermatozoa recovered either from the
ejaculate or by testicular biopsy from non-mosaic 47,XXY
males can induce normal fertilization and cleavage following
ICSI, though the cleavage rate may be impaired.
FISH procedure
The individually biopsied blastomeres were spread onto a
Superfrost Plus glass slide (Kindler GmbH, Freiburg,
Germany) using 0.01 mol/l HC1/0.1% Tween 20 solution
(Coonen et al., 1994; Staessen et al., 1996). Both blastomeres
from the same embryo were ®xed on the same slide, in very
close proximity.
The FISH procedure was used as described previously
(Staessen et al., 1996) and, over a period of time, has included
the use of several different probe mixtures. For the ®rst three
PGD cycles, double target FISH was performed using directly
labelled DNA probes speci®c for chromosomes X (Alpha
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Klinefelter patients
Fresh ejaculate (n = 1; 1)
Fresh TESE (n = 21; 19)
Frozen TESE (n = 10; 9)
Total Klinefelter patients
(n = 32; 29)
Control patients
(n = 88)
No. of
2PN (A)
C.Staessen et al.
Table IV. Chromosomal abnormalities in embryos of couples with Klinefelter syndrome and control group in relation to the chromosomes analysed
Variable
aP
XY
XY, 18
XY, 18, 21
XY, 13, 18, 21
XY, 13, 16, 18, 21, 22
Control
Age <38 years
4
4 (100)
66
35 (53.0)
Not done
Not done
40
21 (52.5)
3
1 (33.3)
113
61 (54.0)a
Not done
Not done
315
241 (76.5)
54
43 (79.6)
161
133 (82.6)
48
29 (60.4)
578
446 (77.2)a
< 0.01 (contingency table).
Satellite DNA probe, Spectrum Green; Vysis, Inc., Downers
Grove, IL, USA) and Y (Alpha Satellite DNA probe, Spectrum
Orange; Vysis, Inc.) (Staessen et al., 1996). For the next 13
PGD cycles, a triple-target FISH technique was performed
using directly labelled DNA probes speci®c for chromosomes
X (Alpha Satellite DNA, Spectrum Green; Vysis, Inc.), Y
[Alpha Satellite III DNA (DYZ1 locus), Spectrum Orange;
Vysis, Inc.] and 18 (Alpha Satellite DNA, 1:1 mixture Green/
Orange spectrum; Vysis, Inc.). In the remaining 15 cycles,
probes for chromosomes XY, 13, 18 and 21 (DXZ1, Spectrum
Blue; DYZ3, Spectrum Gold; LSI13, Spectrum Red; D18Z1,
Spectrum Aqua; LSI21 Spectrum Green; Multivision PGT
Probe Panel; Vysis, Inc.) were applied.
An aliquot (0.2 ml) of the probe solution was added to the
nuclei, covered with a round coverslip (4 mm diameter),
denatured for 5 min at 73°C and left to hybridize for between 4
h and overnight at 37°C in a moist chamber. After washing in
0.43 standard saline citrate (SSC) at 71°C for 2 min, antifade
solution (Vectashield) was added and ¯uorescence signals
were evaluated. The nuclei were then examined using a Zeiss
Axioskop ¯uorescence microscope with the appropriate ®lter
sets. The FISH images were captured with a computerized
system, and the results interpreted by two independent
observers.
In one cycle, a two-round FISH procedure was performed
which allowed the detection of chromosomes XY, 13, 18, 21
(round 1) and 16, 22 (round 2).
Following the analysis of the ®rst set of probes, the
coverslips were gently removed and the slides rinsed in 13
phosphate-buffered saline at room temperature, denatured in
0.06253 SSC for 7 min at 75°C, and then dehydrated (70, 90,
100 and 100% ethanol at ±18°C, 40 s each). The second
hybridization solution was prepared by mixing a probe for
chromosome 16 (Satellite II DNA/D16Z3 probe, Spectrum
Orange; Vysis, Inc.) and a probe for chromosome 22 (LSI 22,
22q11.2, Spectrum Green; Vysis, Inc.). The probes were
denatured separately in a hot waterbath at 75°C for 5 min. An
324
aliquot (0.2 ml) of the probe solution was then added to the
nuclei, covered with a round coverslip (4 mm diameter), sealed
with rubber cement and then hybridized overnight in a water
bath at 37°C. Finally, the slides were washed for 2 min in 0.43
SSC at 73°C and 23 SSC/0.1% Nonidet P40 for 60 s at room
temperature. The washed slides were then mounted with DAPI
in antifade solution, and analysed.
Following biopsy and FISH procedure, a FISH result was
obtained in 113 of 119 (95.0%) embryos available. A normal
result was obtained for the chromosomes analysed in 61 of the
113 embryos (54.0%). Three out of four (75%) embryos
obtained with fresh ejaculate were normal, 47 out of 85
(55.3%) embryos obtained with fresh testicular sperm were
normal, and 11 out of 24 (45.8%) embryos obtained with
frozen testicular sperm cells were normal. Although the groups
were very small, there was no inter-group difference with
regard to the percentage of normal embryos according to the
different origins of sperm used.
Embryo transfer and outcome
During the study period, the policy was changed with regard to
the day of transfer, from day 3 to day 5. This allowed the FISH
procedure to be performed with a greater number of probes in
two consecutive FISH rounds.
The number of embryos transferred was also in line with a
policy to transfer a maximum of two embryos in patients aged
<37 years if the embryo quality was good. Transfer on day 5
also allowed the selection of those embryos which reached the
compacting or blastocyst stage on day 5. Spare, genetically
normal embryos of suf®cient morphological quality were
cryopreserved (Van den Abbeel et al., 1997).
Implantation was con®rmed when two hCG concentrations
of >10 U/l were measured at least 10 days after embryo
transfer. Clinical pregnancy was de®ned by an intrauterine
gestational sac with a fetal heartbeat seen by vaginal ultrasound
at least 6 weeks after embryo transfer. PGD is a novel
technique, and it is recommended that in the case of pregnancy
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No. of embryos analysed for chromosomes
No. of normal embryos (%)
No. of embryos analysed for chromosomes
No. of normal embryos (%)
No. of embryos analysed for chromosomes
No. of normal embryos (%)
No. of embryos analysed for chromosomes
No. of normal embryos (%)
No. of embryos analysed for chromosomes
No. of normal embryos (%)
Total number of embryos analysed
Total number of normal embryos (%)
Klinefelter syndrome
Age <38 years
PGD after ICSI for 47,XXY patients
Table V. Frequency of abnormalities for each chromosome for patients with Klinefelter syndrome and for controls
Klinefelter(age <38 years)
Control(age <38 years)
I. Sex chromosomal abnormalities
No. of sex chromosome abnormalities/no. of embryos analysed for this chromosome (%)
Monosomy XO
Monosomy OY
Trisomy XYY
Trisomy XXX
Trisomy XXY
Mosaic XY/XXY
Mosaic XY/XYY
Mosaic XX/XO
15/113a (13.2)
7*
2
2
1
0
2
1
0
18/578a (3.1)
10
0
1
1
4
1
0
1
II. Autosomal abnormalities
No. of chromosome 13 abnormalities/no.
Monosomy 13
Trisomy 13
No. of chromosome 16 abnormalities/no.
Monosomy 16
Trisomy 16
No. of chromosome 18 abnormalities/no.
Monosomy 18
Trisomy 18
Mosaic monosomy 18/disomy 18
No. of chromosome 21 abnormalities/no.
Monosomy 21
Trisomy 21
Mosaic disomy 21/trisomy 21
No. of chromosome 22 abnormalities/no.
Monosomy 22
Trisomy 22
3/43 (7.0)
1
2**
0/3
0
0
8/109b (7.3)
4
4**
0
5/43c (11.6)
3*
1
1
1/3 (33.3)
1
0
5/209 (2.4)
3
2
4/48 (8.3)
2
2
12/578b (2.1)
10
1
1
8/263c (3.0)
6
2
0
1/48 (2.1)
1
0
III. Ploidy status abnormalities
No. of haploid embryos/total no. of embryos analysed (%)
No. of triploid embryos/total no. of embryos analysed (%)
No. of tetraploid embryos/total no. of embryos analysed (%)
6/113d (5.3)
4/113 (3.5)
2/113 (1.7)
4/578d (0.7)
9/578 (1.6)
12/578 (2.1)
IV. Combined abnormalities
No. of embryos with combined abnormalities/ total no. of embryos analysed (%)
10/113 (8.8)
59/578 (10.2)
of embryos analysed for this chromosome (%)
of embryos analysed for this chromosome (%)
of embryos analysed for this chromosome (%)
of embryos analysed for this chromosome (%)
of embryos analysed for this chromosome (%)
*Including the double monosomy X-21: in fact counted twice.
**Including the double trisomy 13±18: in fact counted twice.
a, b, c, d: Fisher's exact test: P < 0.01.
the couple should undergo prenatal diagnosis in order to
con®rm the PGD diagnosis, either by chorionic villus sampling
or by amniocentesis. Among the pregnancies obtained to date,
only one couple effectively decided to perform amniocentesis;
the fetus was normal and the PGD result con®rmed. Clearly, as
experience with PGD is increased and accurate estimates of
error risks become available, the attitude to prenatal diagnosis
will be reconsidered. The introduction of techniques such as
comparative genomic hybridization, which enable all chromosomes to be investigated, will remove the risk of all chromosome imbalances.
A total of 41 embryos was transferred (see Table II) in 26
transfer procedures (mean 1.6 6 0.6 embryos per transfer).
Embryo transfer could not be performed in three patients, this
being due to an absence of normal embryos in two cases, and of
poor embryo quality (but genetic normality) in one case. A
total of eight cycles with positive hCG was obtained from the
26 transfers; three of these were preclinical abortions and ®ve
were ongoing. This provided an ongoing pregnancy rate of
15.6% per cycle, or 19.2% per transfer. The implantation rate
with fetal heart activity was 12.8% per transferred embryo. One
patient delivered prematurely at 23 weeks gestation, most
likely due to chorioamnionitis; the child was stillborn at
delivery. The four other pregnant patients delivered healthy
babies (two girls, two boys) at term. To date, among the 20
couples in which sperm were found, four of them delivered
(live birth rate 20%).
Which genetic abnormalities are found at diagnosis?
As mentioned earlier, 54.0% of the embryos from Klinefelter
patients were found to be normal, with an almost equal sex ratio
(32/61 female; 29/61 male). The numbers of embryos analysed
with the different probe mixtures, together with details of embryos
325
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Abnormality
C.Staessen et al.
Table VI. Results of embryos at biopsy and reanalysis of the non-transferred or non-cryopreserved embryos
At biopsy
FISH results
No. of embryos
Blastomere 1
32
29
6
Reanalysed embryos
FISH results
normal female
normal male
monosomy X0
4
6
5
1
2
2
double monosomy X-21
monosomy 0Y
trisomy XYY
1
2
2
1
2
1
1
1
4
trisomy XXX
XY
XY
monosomy 13
trisomy 13
monosomy 18
3
1
2
1
1
1
4
2
4
2
10
trisomy 18
double trisomy 13 ±18
monosomy 21
trisomy 21
disomy 21
monosomy 22
haploid X
haploid Y
triploid
tetraploid
combined abnormalities
4:
6:
3:
1:
1:
1:
2:
1:
1:
1:
1:
1:
1:
1:
2:
2:
1:
1:
1:
1:
1:
normal female
normal male
monosomy X0
disomy XX*
chaotic
double monosomy X-21
monosomy 0Y
trisomy XYY
mosaic XYYY/XY/XYY
mosaic: monosomy X0 /trisomy XXX
mosaic XY/XXY
mosaic XY1818 / XYY1818
monosomy 13
trisomy 13
monosomy 18
mosaic: disomy 18 / monosomy 18
double trisomy 18-XYY
double trisomy 13±18
mosaic monosomy 21/disomy 21/ trisomy 21/
trisomy 21
mosaic monosomy 21/disomy 21
2:
2:
4:
1:
2:
mosaic XX diploid/X haploid; 2: chaotic
chaotic
triploid
tetraploid
normal diploid*; 4: chaotic
XXY
XYY
monosomy 21
1
1
1
1
1
4
1
1
1
1
1
0
4
2
4
1
6
considered as not con®rmed.
found to be normal for the chromosomes tested are listed in
Table IV. Abnormalities related to the gain or loss of a speci®c
chromosome, and to the ploidy status of the embryo (haploid,
triploid or tetraploid) are detailed in Table V. An abnormality of
the gonosomes was apparent in 15 of the 113 (13.2%) embryos
tested, and of the autosomes in 17 of 109 (15.6%). Likewise, 12 of
113 (10.6%) embryos showed an abnormality of ploidy status, and
10 of 113 (8.8%) showed combined abnormalities.
Are embryos generated by Klinefelter patients at
increased genetic risk?
In comparison with controls, Klinefelter patients showed a
signi®cantly higher percentage of abnormal embryos (Fisher's
exact test; P < 0.01). The prevalence of sex chromosome
abnormalities was only 3.1% in FISH for sex-linked disease
group, compared with 13.2% in Klinefelter patients (Fisher's exact
test; P < 0.01), while the prevalence of autosomes was 30 of 578
embryos (5.2%) in controls and 17 of 109 (15.6%) in Klinefelter
patients (Fisher's exact test; P < 0.01). If data for each autosome
chromosome are compared separately, then a signi®cantly
increased risk was observed for chromosomes 18 and 21
(Fisher's exact test; P < 0.01). Moreover, among the control
subjects 25 of 578 embryos (4.3%) had abnormalities of ploidy
status compared with 12 of 113 (10.6%) (P < 0.01) in Klinefelter
326
patients. Finally, in the control group 59 of 578 embryos (10.2%)
had combined abnormalities compared with 10 of 113 (8.8%) in
the Klinefelter group.
One group (Magli et al., 2002) reported their experience with
PGD in couples with an altered karyotype due to gonosomal
mosaicism, and focused on the chromosomal condition of embryos
generated. Some 63% of the embryos were chromosomally
abnormal, and the incidence of aneuploid mosaics was higher as
compared with PGD patients with normal karyotype of the same
age. This suggested that constitutional carriers of sex chromosome
mosaicism are predisposed to autosomal mosaicism of embryos.
Although few data were generated, the incidence of chromosomally abnormal embryos was slightly higher when the woman was
the carrier. These authors concluded that their ®ndings support a
predisposition in patients with gonosomal mosaicism to give rise
to different cell lines, possibly due to disturbances and malfunctioning of the mechanisms entering cell division.
Analysis of non-transferred and non-cryopreserved
embryos
After PGD and embryo transfer, cells from embryos which were
classi®ed as genetically normal but morphologically unsuitable for
transfer or cryopreservation (n = 10) or as genetically abnormal (n
= 52) were spread as a whole as described previously (Staessen and
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No. of embryos reanalysed
*Embryos
Blastomere 2
PGD after ICSI for 47,XXY patients
Spreading of the remainder of the cells from the above two
embryos revealed nuclei with various X and Y patterns, consistent
with chaotic cell division. In contrast with the results of the present
study, none of the embryos analysed in either study showed a
XXY1818 or XXX1818 pattern, but of course both studies were
dealing with a limited number of analysed embryos.
The present data and that from both other reports on embryos
from Klinefelter patients demonstrate that the incidence of
chromosomal mosaicism in in-vitro-fertilized human embryos is
substantial. When PGD is attempted, the patients should be
consulted regarding the limitations of aneuploidy detection by this
experimental technique. In some cases, blastomere analysis at the
cleavage stage will not be representative of the whole embryo, due
to the high frequency of chromosomal mosaicism in human
embryos (Munne and Cohen, 1993; Harper et al., 1995). However,
the present data show that on the basis of the results at diagnosis
and after PGD reanalysis, the initial diagnosis could not be
con®rmed in only three of 51 (5.9%) embryos and that, on the basis
of the PGD result, the embryos which could have been transferred
were actually rejected for transfer.
Conclusion
It is generally accepted that the introduction of ICSI has
constituted a breakthrough in the treatment of male infertility.
Even patients considered azoospermic but with focal spermatogenesis in the testis, such as those with non-mosaic 47,XXY
Klinefelter syndrome, have been able to bene®t from the ICSI
technique. Furthermore, ICSI of the patient's spermatozoa induced
fertilization, produced a high percentage of cleavage-stage
embryos, and led to implantation and ongoing pregnancy. All
neonates that have been reported as being born after ICSI without
PGD using spermatozoa from patients with non-mosaic Klinefelter
syndrome are normal. One fetus conceived by spermatozoa
recovered from a man with non-mosaic Klinefelter syndrome
was of 47,XXY karyotype, and the embryo was reduced at week
14 (Ron-El et al., 2000a).
The FISH method with directly labelled DNA probes speci®c
for the X and Y chromosomes constitutes an ef®cient and rapid
procedure to determine the exact number of sex chromosomes
present in one or two blastomeres of a preimplantation embryo.
Since PGD by FISH technology was available at the present
authors' institution, a policy was adopted which proposed that
patients should combine ICSI treatment with PGD, particularly in
order to enumerate the sex chromosomes before transfer (Staessen
et al., 1996).
The experience of combining ICSI with PGD indicates that
there was a signi®cant fall in the rate of normal embryos for
couples with Klinefelter's syndrome compared with control
patients undergoing PGD for gender determination and using
ejaculated sperm. Indeed, a signi®cantly increased risk of abnormalities for the sex chromosomes as well as for the autosomes was
observed; for each autosome separately, this reached signi®cance
level only for chromosomes 18 and 21.
A much higher frequency of disomy 21 was found in the
spermatozoa of a Klinefelter patient than in those of control
patients (Hennebicq et al., 2001). Any child conceived by ICSI
using this sperm will thus have a proportionally higher risk of
trisomy 21. Hennebicq explained this by suggesting that, ``The
327
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Van Steirteghem, 1997), and FISH was carried out using the same
probe mixture as was used for the diagnosis. All patients involved
consented for further research to be carried out on these embryos.
The results were compared with the one-cell diagnosis in order to
verify the accuracy of the technique. In total, 62 embryos were
spread for reanalysis, of which 527 blastomeres were counted. A
FISH result was obtained for 426 blastomeres from 51 embryos
(Table VI).
The remaining normal embryos (n = 10) which could not be
frozen due to poor quality were reanalysed. In all 10 embryos the
normal status was con®rmed in most of the nuclei. A reanalysis
was obtained for ®ve of the six embryos diagnosed as monosomy
X0; in three cases a uniform X0 embryo was con®rmed, one
embryo was found to be XX diploid (FISH diagnosis not
con®rmed), and one was chaotic. The diagnosis was con®rmed
in one embryo with a double monosomy for X and 21. Both 0Y
diploid embryos were found to be uniform 0Y diploid embryos. A
reanalysis was obtained of both XYY embryos; one embryo was
con®rmed to be XYY uniformly, while in the other embryo XYY
was found to originate from a XYY conception followed by postzygotic non-disjunction resulting in a XYYY and a XY cell line.
The trisomic XXX may have arisen from an XX embryo at
conception followed by post-zygotic non-disjunction leading to
X0 and XXX cell lines. In two embryos, one of the two
blastomeres analysed showed a normal XY pattern, and the
other an abnormal XXY pattern; both embryos were spread. FISH
results were obtained from one embryo only. The XY/XXY
mosaic embryo was most likely the result of a XXY conception
followed by post-zygotic loss of an X chromosome in one cell line.
An analogous situation pertained to the XY/XYY mosaic embryo.
Here, a conception of an XYY embryo with subsequent loss of a Y
chromosome in one cell might have occurred, or an XY embryo at
conception followed by post-zygotic non-disjunction involving a
Y chromosome leading to X0/XY/XYY cell lines from which the
X0 cell line was lost during the procedure. Intracytoplasmic
fertilization of oocytes with a Klinefelter male's 24,XY- or 24,XXbearing spermatozoa, could theoretically produce a 47,XXY or a
47,XXX embryo. These results indicate that indeed, 47,XXY or
47,XXX embryos were formed. The results were given for the
autosomal abnormalities and explanations similar to those for the
sex chromosome abnormalities are valid.
To the present authors' knowledge, only two other groups have
reported FISH analysis on embryos of 46,XY/47,XXY or 47,XXY
Klinefelter patients. In the ®rst study (Bielanska et al., 2000),
results were reported on FISH analysis with DNA probes speci®c
for chromosomes X, Y and 18 on 10 spare embryos from fertility
treatment of a 46,XY/47,XXY Klinefelter patient. The data
revealed that only three of 10 embryos were normal for the
chromosomes testedÐone embryo with an XX1818 and two
embryos with an XY1818 pattern. The abnormalities detected
included ®ve chaotic mosaic embryos and two diploid mosaic
embryos with the majority of blastomeres normal for the
chromosomes tested. The second study (Reubinoff et al., 1998)
reported on PGD with probes for chromosomes X, Y and 18 in
three embryos from two patients. One embryo was diagnosed as
normal and transferred; this resulted in the birth of a karyotypically
normal neonate. The biopsied blastomere from the second embryo
had an abnormal XX18 constitution. The blastomere from the third
embryo was also found to contain an abnormal pattern, XXY1818.
C.Staessen et al.
328
autosomal disomy was the most frequent in ejaculated sperm.
These ®ndings point to a higher incidence of sperm chromosomal
abnormalities in sperm of non-obstructive azoospermic men,
where sex chromosome aneuploidy appears to be the most
common. The question arising here is whether the increased
abnormalities in embryos from Klinefelter patients are related to
the 47,XXY karyotype, or to the use of testicular sperm of
azoospermic patients. For Klinefelter patients in particular this
would imply that the rare break-through patches of spermatogenesis in XXY males may be due to the presence of normal XY germ
cells, but as a result of the compromised testicular environment
these 46,XY germ cells are susceptible to meiotic abnormalities,
including non-disjunction of the sex chromosomes (Mroz et al.,
1998) and the autosomes (Hennebicq et al., 2001). A comparative
analysis of the incidence and type of chromosomal abnormalities
between Klinefelter patients and non-obstructive azoospermic
males will provide more information regarding possible mechanisms at the origin of chromosomal abnormalities.
In conclusion, at the present authors' institution, broad genetic
counselling is offered to couples with non-mosaic Klinefelter's
syndrome, and the use of ICSI in combination with PGD is
recommended. Ultimately, however, the ®nal decision must be
made by the couples concerned.
Acknowledgements
The authors thank the clinical, scienti®c, nursing, and technical staff
of the Centre for Reproductive Medicine, and especially the
colleagues of the microinjection and IVF laboratory. They are also
very grateful to Mrs Marleen CarleÂ, Sylvie Mertens and Griet
Meersdom for technical assistance with the FISH studies, and to Mrs
Ines Devolder of the University Language Centre for correcting the
English text. Grants from the Belgian Fund for Medical Research are
kindly acknowledged.
References
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most likely reason for the high rate of disomy 21 in our XXY
patient is that the abnormal sex chromosome content disturbs the
meiotic segregation of some chromosomal pairs, possibly via the
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chromosome 21 with one or both sex chromosomes during meiosis
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The high proportion of normal fetuses that develop from the
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It is clear that there is an increased risk of sex and autosomal
chromosomal abnormal embryos obtained in Klinefelter patients
when compared to patients with normal karyotype and normal
spermatogenesis. A possible approach is to transfer all embryos
available in Klinefelter patients, assuming that only the best ones
will implant. Although this approach is reasonable, it is in
contradiction with the general policy of limiting the numbers of
embryos for transfer, and in any case does not remove the risk of
producing a child with a chromosome abnormality. Without PGD,
the chance of selecting and transferring abnormal embryos is high
because even embryos with normal morphology have a distinct
percentage of abnormalities (Harper et al., 1995; Munne et al.,
1995). Moreover, in case of pregnancy there is an indication for
prenatal diagnosis with the potential risk of procedure-related
miscarriage and, in the case of an abnormal result, termination of
the pregnancy. Finally, by transferring genetically abnormal
embryos, genetically normal ones will be frozen; as a consequence, since only 50% of embryos will survive thawing, the
patients' chances are reduced yet again. With PGD, the identi®cation of abnormalities in a cohort of morphologically goodquality embryos prevents the transfer of those embryos that are
destined either not to implant or to abort spontaneously. One
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might affect the viability of these precious embryos which have
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would demonstrate a possible dramatic effect of biopsy on the
implantation potency of the embryo.
The incidence of chromosomal abnormalities in epididymal and
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aneuploidy rate, analysed by FISH for chromosomes XY, 18 and
21, of 11.4% in men with non-obstructive azoospermia was
signi®cantly higher than the rate of 1.8% detected in epididymal
sperm from men with obstructive azoospermia, and also higher
than the rate of 1.5% found in ejaculated sperm. No signi®cant
difference was found between the epididymal and ejaculated
samples. When the chromosomal abnormalities were analysed,
gonosomal disomy was the most recurrent abnormality in both
obstructive and non-obstructive azoospermic patients, while
PGD after ICSI for 47,XXY patients
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Rosenwaks, Z. (2002) Chromosome analysis of epididymal and
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