Full text of DOI:10.1007/BF03343797

J. Endocrinol. Invest. 23: 711-716, 2000
Preimplantation diagnosis after assisted reproduction
techniques for genetically-determined male infertility
L. Gianaroli, M.C. Magli, A.P. Ferraretti, and E. Iammarrone
S.I.S.ME.R., Reproductive Medicine Unit, Bologna, Italy
ABSTRACT. One hundred and thirty-six cycles with
a poor prognosis for full-term pregnancy under-
went preimplantation genetic diagnosis (PGD) of
aneuploidy. The mean maternal age was 31.8±±.ꢀ
years. Only patients younger than 36 years were in-
cluded in the study with the aim of evaluating
whether sperm indices have an effect on the chro-
mosomal constitution of preimplantation embryos.
No differences were detected in the percentage
of aneuploid embryos; however a higher incidence
of monosomies and trisomies was found in MESA-
TESE embryos compared to the group of normo-
spermic patients. In addition, an increase in the
proportion of gonosomal aneuploidy seemed to
be associated with the severity of the male factor
parameters. The rate of de-novo chromosomal ab-
normalities in embryos from patients with a nor-
mal karyotype suggested an increased frequency
proportional to the severity of the male factor con-
dition, the proportion of monosomic and trisomic
embryos, and the percentage of gonosomal aneu-
ploidy increased accordingly. In the case of cou-
ples with a male altered karyotype, comparable
frequency of chromosomally abnormal embryos,
and monosomy and trisomy were observed irre-
spective of semen indices, gonosomal aneuploidy
was only observed in one case where the patient
had a karyotype with gonosomal mosaicism. These
data confirm that the severe male infertility con-
dition determines an increase in the rate of de-no-
vo abnormalities, as anticipate by the follow-up of
the children born after ICSI.
(J. Endocrinol. Invest. 23: 711-716, ±000)
©
±000, Editrice Kurtis
INTRODUCTION
(
balanced translocations, gonosomal aneuploidy)
The intracytoplasmic sperm injection (ICSI) tech-
nique has made possible the successful treatment
of severe male factor patients. The few spermato-
zoa recovered from the ejaculate of extremely se-
vere oligoasthenoteratospermic samples, or ex-
tracted from testicular biopsies or epididymal flu-
ids give origin to pregnancies at a rate that is com-
parable to that characterized in normospermic pa-
tients. Following the initial euphoria for the results
obtained, the increasing number of reports re-
garding the association between inherited genetic
disorders and male infertility has disclosed a gen-
eral concern regarding the ICSI’s generation (1, 2).
An inverse relationship between sperm indices and
the prevalence of karyotype abnormalities in infer-
tile men has been demonstrated (3). More specifi-
cally, microdeletions in the long arm of the Y chro-
mosome and structural chromosomal abnormalities
seem to be more frequent in men with pathological
sperm compared with the general population (4).
In addition, recent studies on children born after
ICSI with ejaculated, epididymal and testicular sper-
matozoa reported a 1.66% frequency of de novo
chromosomal abnormalities (5, 6). This rate is sig-
nificantly higher in comparison with the incidence
generally observed after assisted reproductive tech-
niques (ART) with normospermic samples (7, 8).
Furthermore, gonosome aneuploidies are espe-
cially frequent in cases of de novo originated ab-
normal karyotypes; they derive mainly from the pa-
ternal side (2).
Overall these considerations suggest the possibili-
ty that non-disjunctional events or malsegregation
of balanced structural abnormalities can generate
gametes with an abnormal chromosome constitu-
tion. Failed implantation or recurrent miscarriages
are the potential consequences. Actually, chromo-
some abnormalities represent one of the major
causes of embryonic loss as detected by the aneu-
ploidy screening of preimplantation embryos (9). A
clear correlation has been assessed between ma-
ternal age and the higher frequency of aneuploid
Key-words: Chromosomal abnormalities, gonosomal aneuploidies, male
infertility, preimplantation genetic diagnosis.
Corresponcence: Dr. Luca Gianaroli, S.I.S.ME.R., Reproductive Medicine
Unit, Via Mazzini 12, 40138 Bologna, Italy.
711

L. Gianaroli, M.C. Magli, A.P. Ferraretti, et al.
events in women of advanced reproductive age
somally normal embryos were transferred by re-
placement into the uterine cavity, mainly on day 4
(12). Clinical pregnancies were confirmed by the
presence of a gestational sac with fetal heart beat.
The implantation rate was calculated by dividing
the number of gestational sacs with fetal heart beat
by the total number of embryos transferred.
(10). On the other hand, only a few data evaluate
the male contribution to the chromosomal status
of the generated embryos (11). The purpose of the
present study was to verify whether sperm indices
have an effect on the incidence of aneuploid con-
cepti. In vitro generated embryos from couples with
a poor prognosis of full term pregnancy underwent
preimplantation genetic diagnosis (PGD) of aneu-
ploidy for the simultaneous screening of the chro-
mosomes which are responsible for the most fre-
quent aneuploidies in the human. The results ob-
tained are described in the current report.
Embryo biopsy, blastomere fixation, and
fluorescence in situ hybridization (FISH) technique
Regularly cleaving embryos underwent blastomere
biopsy at 62-64 hours after insemination. Each em-
bryo was manipulated individually in Hepes-
buffered medium under pre-equilibrated oil. A
breach of 20-22 μm was opened in the zona pellu-
cida with acidic Tyrode’s solution, and a nucleated
blastomere gently aspirated with a glass polished
needle (inner diameter 40 μm approximately). After
incubation in hypotonic solution, the nucleus was
fixed on a glass slide with methanol: acetic acid.
For the multicolor FISH analysis, 9 probes were
used in a two-round protocol for the simultaneous
detection of chromosomes XY, 13, 14, 15, 16, 18,
21, and 22. From December 1998 the probe for
chromosome 14 was replaced by the probe specif-
ic for chromosome 17, and from March 1999 by the
probe detecting chromosome 1. These changes
were made in the effort of recognizing other chro-
mosomes beside those detected in spontaneous
abortions, which could be so important for embryo
development to cause embryonic death in case of
aneuploidy.
MATERIALS AND METHODS
Patients
From September 1996 to April 2000, 343 ART cy-
cles with a poor prognosis of full term pregnancy
underwent PGD of aneuploidy. In 136 of them, in-
dications to the treatment were: ꢀ3 previous ART
failures or ꢀ1 failure in combination with sponta-
neous abortion (78 cycles), an altered karyotype due
to gonosomal mosaicism or balanced translocations
(
39 cycles) MESA-TESE (19 cycles). The mean ma-
ternal age was 31.8±2.5. Only patients younger than
6 years were included in the study in order to elim-
3
inate the age factor. Following induction of multi-
ple follicular growth, oocyte retrieval was performed
at 36 hours post-HCG administration. Oocytes were
incubated in Earle’s medium supplemented with
10% heat inactivated maternal serum in a 5% CO2
humidified gas atmosphere at 37 C. The insemina-
tion techniques used were conventional IVF (in vi-
tro fertilization) or ICSI depending on the semen
sample parameters; ICSI was implemented in nor-
mospermic samples when low or zero fertilization
rates were obtained in previous cycles. The ICSI
samples were arbitrarily divided into 4 groups de-
pending on the characteristics of sperm indices: nor-
mospermic (group 1, 22 cycles), oligoasthenoter-
Statistical analysis
2
Data were analyzed by Student’s t test and χ anal-
ysis applying the Yates’ correction, 2x2 contingen-
cy tables.
RESULTS
As reported in Table 1, 731 embryos were biop-
sied and FISH analyzed. The procedure was infor-
mative for 721 of them (99%): 265 embryos ex-
hibited a normal chromosomal complement (37%)
whereas the remaining 456 were diagnosed as
aneuploid (63%). Monosomies and trisomies con-
tributed 35% of the detected abnormalities
(163/456); in 17 embryos (3.7% of total abnormal)
aneuploidy was due to sex chromosomes. In 32
cycles (23%) no euploid embryos resulted; conse-
quently embryo transfer was not performed. In the
remaining 104 cycles, an average of 2.1±0.9 FISH
normal embryos per cycle was transferred and
yielded 30 clinical pregnancies (29%). The im-
plantation rate was 19.2% and the incidence of
6
atospermic with ꢀ0.5x10 total motile count (TMC,
6
group 2, 15 cycles), <0.5x10 TMC (group 3, 38 cy-
cles), epididymal or testicular sperm (group 4, 19
cycles). Male patients with an altered karyotype
were: 4 with robertsonian translocations (5 cycles), 5
with reciprocal translocations (6 cycles), 1 with a 47
XYY karyotype, 1 with a 46 XY+Yqh karyotype and
1
with gonosomal mosaicism (2 cycles).
Assessment of fertilization and embryo evaluation
The presence of 2 pronuclei and 2 polar bodies
identified normally fertilized oocytes. They were cul-
tured individually and examined at regular intervals
(40 and 62 hours post insemination). Only chromo-
712

Preimplantation diagnosis and male infertility
Table 1 - Preimplantation genetic diagnosis of aneuploidy: FISH and clinical results according to the insemination technique imple-
mented.
IVF
42
ICSI
94
Total
136
No. cycles
Mean age (mean±SD)
31.8±2.1
31.8±2.6
31.8±2.4
No. diagnosed embryos
FISH normal (%)
234
487
721
84 (36)
150 (64)
49 (33)
4 (2.7)
181 (37)
306 (63)
114 (37)
13 (4.2)
265 (37)
456 (63)
163 (36)
17 (3.7)
FISH abnormal (%)
Monosomies and trisomies (%)
Gonosomal aneuploidies (%)
No. transferred cycles (%)
No. transferred embryos (%)
No. clinical pregnancies (%)
No. spontaneous abortions (%)
Implantation rate (%)
32 (76)
2.2±0.9
9 (28)
0
72 (77)
2.1±0.8
21 (29)
2 (9)
104 (76)
2.1±0.9
30 (29)
2 (7)
21.4
18.2
19.2
FISH: fluorescence in situ hybridization; ICSI: intracytoplasmic sperm injection; IVF: in vitro ferilization.
spontaneous abortions was 7%. When the results
were analyzed according to the insemination tech-
nique implemented (ICSI vs conventional IVF), no
differences were detected in either the FISH re-
sults or the clinical outcome.
The chromosomal constitution of the embryos gen-
erated by ICSI was evaluated in relationship to the
characteristics of the sperm samples. Table 2 rep-
resents the frequency of FISH abnormal embryos
that was similar among the 4 groups. A higher in-
cidence of monosomies and trisomies over total ab-
normalities resulted in group 4 vs group 1 (45% vs
chromosomally abnormal embryos along with the
severity of the male factor condition seemed to be
meaningful. In addition, the proportion of mono-
somic and trisomic embryos increased accordingly,
as well as the percentage of gonosomal aneuploi-
dies.
Table 4 represents the data derived from the
chromosomal analysis in those patients diag-
nosed with a male altered karyotype. Although
the maternal age was significantly lower in group
3, similar percentages of chromosomally abnor-
mal embryos resulted in the 4 groups with com-
parable frequency of monosomies and trisomies.
Gonosomal aneuploidy was only detected in
group 4, associated to a male karyotype with
gonosomal mosaicism.
2
7%, p<0.05). In addition, a tendency towards an
increase in the proportion of gonosomal aneuploidy
along with the severity of the male factor condition
was observed.
In order to evaluate the rate of de-novo chromo-
somal abnormalities, the FISH results derived from
patients with a normal karyotype are presented in
Table 3. Although statistically not significant, the
trend demonstrating an increased frequency of
DISCUSSION
The diagnostic relevance of investigating the chro-
mosomal status in preimplantation embryos has be-
Table 2 - Chromosomal abnormalities in ICSI embryos divided according to the severity of the male factor condition.
ICSI
Group 1
22
Group 2
15
Group 3
38
Group 4
19
No. cycles
Mean age (mean±SD)
31.7±1.8
32.2±2.4
30.9±2.8
31.6±2.7
No. diagnosed embryos
FISH abnormal (%)
Monosomies and trisomies (%)
Gonosomal aneuploidies (%)
131
81
194
81
78 (59)
21 (27)*
2 (2.6)
55 (68)
22 (40)
2 (3.6)
115 (59)
45 (39)
4 (3.5)
58 (72)
26 (45)*
5 (8.6)
*
p<0.05. FISH: fluorescence in situ hybridization; ICSI: intracytoplasmic sperm injection.
713

L. Gianaroli, M.C. Magli, A.P. Ferraretti, et al.
Table 3 - Chromosomal abnormalities in IVF and ICSI embryos generated by patients with a normal karyotype.
IVF
ICSI
Group 1
22
Group 2
9
Group 3
24
Group 4
13
No. cycles
29
Mean age (mean±SD)
32.0±2.4
31.7±1.8
32.5±2.4
31.4±2.8
31.2±2.8
No. diagnosed embryos
FISH abnormal (%)
Monosomies and trisomies (%)
Gonosomal aneuploidies (%)
155
131
45
127
53
98 (63)
27 (27)*
2 (2.0)
78 (59)
21 (27)*
2 (2.6)
28 (62)
9 (32)
1 (3.6)
76 (60)
29 (38)
4 (5.3)
38 (72)
18 (47)*
3 (7.9)
*
p<0.05. FISH: fluorescence in situ hybridization; ICSI: intracitoplasmic sperm injection; IVF: in vitro fertilization.
come evident in the treatment of patients with a
poor prognosis of full term pregnancy. In these cas-
es, the selection and transfer of euploid embryos
is associated to increased implantation rates and
reduced incidence of spontaneous abortions (13,
in the Y chromosome. Following ICSI, these disor-
ders are maintained in the population by making
possible pregnancies (and therefore the transmis-
sion of abnormal genetic traits to the offspring) in
individuals which otherwise were destined not to
conceive. Recent reports have demonstrated that
in cases of oligosthenoteratospermia or azoosper-
mia due to testicular failure, the frequency of ane-
uploid spermatozoa is higher compared to the nor-
mal population (15). Interestingly, most of the ane-
uploidies detected were accounted for sex chro-
mosomes (16). This seems to find a correspon-
dence with the results reported in the current study
where PGD of aneuploidy was implemented in
couples which are at higher reproductive risk com-
pared to the general population. In order to eval-
uate the male contribution to the chromosomal
status of in vitro generated embryos, only patients
with a maternal age lower than 35 years were in-
cluded. No differences were observed in the pro-
portion of aneuploid embryos in relation to the
characteristics of the semen samples. However,
1
4). Data on preimplantation embryos have con-
firmed maternal age as the main factor associated
to numerical chromosomal abnormalities, mostly
resulting from non-disjunction events during oo-
genesis. On the other hand, the analysis of aneu-
ploidy frequency in sperm suggested that the con-
tribution of the male gamete to the chromosomal
constitution of the generated concepti is generally
negligible. Nevertheless, the more and more wide-
spread treatment of extremely severe male factor
patients is starting to provide evidence of an in-
creased risk of inherited and de-novo chromoso-
mal abnormalities.
Male infertility is often associated with inherited
genetic disorders such as structural chromosomal
rearrangements, mutations in the genetic region
responsible for cystic fibrosis, and microdeletions
Table 4 - Chromosomal abnormalities and incidence of monosomies and trisomies in embryos generated by couples with a male al-
tered karyotype.
IVF
ICSI
Group 1
Group 2
5
Group 3
Group 4
2
No. cycles
3
0
-
5
,
,
Mean maternal age (mean±SD)
33.7±1.5**
34.0±2.2*
28.6±2.2* ** °
34.0±1.4°
Karyotype alterations
Robertsonian translocations
Reciprocal translocations
1
1
-
1
-
-
-
-
-
-
3
2
-
1
3
1
-
-
-
4
7 XYY
-
4
6 XY+Yqh
-
-
2
Gonosomal mosaicism
-
-
No. diagnosed embryos
FISH abnormal (%)
Monosomies and trisomies (%)
Gonosomal aneuploidies (%)
15
7 (63)
4 (57)
0
-
-
-
-
28
20(71)
8 (40)
0
22
14 (64)
6 (43)
0
11
5 (45)
2 (40)
2 (40)
*
p<0.004; **p<0.01; °p<0.02. FISH: fluorescence in situ hybridization; ICSI: intracytoplasmic sperm injection; IVF: in vitro fertilization
714

Preimplantation diagnosis and male infertility
monosomic and trisomic embryos were more com-
mon in the case of epididymal and testicular sperm
compared to normospermic samples. This could
be attributed to a higher frequency of aneuploid
gametes in cases of azoospermia, with gonosomes
giving an important contribution to the total ab-
normalities. In addition, when only patients pre-
senting with a normal karyotype were considered,
there was a more homogeneous trend demon-
strating a close association between aneuploidy
and severity of the male factor infertility (Table 3).
For instance, the IVF group and ICSI group 1,
where the sperm samples presented normal pa-
rameters according to the WHO criteria (17),
showed similar percentages of monosomic and tri-
somic embryos. This finding could support the hy-
pothesis that, provided the homogeneity of the fe-
male counterpart, the increased proportion of
monosomic and trisomic embryos, as well as gono-
somal aneuploid embryos, in the other groups de-
pends on the sperm sample condition. Interestin-
gly, patients undergoing PGD of aneuploidy be-
cause of repeated ART failures have complex ab-
normalities as the most frequent chromosomal al-
terations. Although more than 50% of their em-
bryos are chromosomally abnormal, the benefit as-
sociated to the transfer of those diagnosed as FISH
normal is not as significant as in other poor prog-
nosis categories of patients (13). This could be at-
tributed to different reasons. Among them, the
male gamete could play an important role, both
for the ploidy condition and the centriolar organi-
zation presiding the oocyte’ s mitotic divisions (18).
In view of these considerations, it is possible that
this poor prognosis group is heterogeneous and
includes different types of couples under the com-
mon denominator of repeated ART failures. The
current data provide an estimation of the de novo
numerical chromosomal abnormalities in in vitro
generated embryos. The similar rates of total ab-
normalities could be an expression of the hetero-
geneity of these patients’ category. On the other
hand, the association between monosomic and tri-
somic embryos with severe male factor infertility
strongly supports the hypothesis of an increased
proportion of aneuploid gametes in pathological
ejaculated and non-ejaculated spermatozoa.
Variation of gonosomes is especially involved. All
these findings are in agreement with the data gen-
erated by the post-natal examination of children
born after ICSI. It is important to underline that da-
ta on preimplantation embryos give a more pre-
cise evaluation of the abnormalities contributed by
the gametes compared to the results derived from
pre-natal diagnosis. In fact, no selection due to
spontaneous abortion occurred yet, taking into
consideration that the great majority of monosomic
embryos do not even implant and the high early
abortion rate associated to trisomic embryos; these
events make many aneuploidies undetected at the
time of pre-natal diagnosis.
The frequency of chromosomally abnormal em-
bryos generated by infertile men with an altered
karyotype does not seem to be different compared
to the general trend observed in poor prognosis
patients (Table 4). Furthermore, the contribution
of monosomy and trisomy to the total abnormali-
ties resembles the rate generally characterized in
couples with an altered karyotype, confirming that
balanced translocations and gonosomal mo-
saicisms represent a risk factor for reproduction
due to the high frequency of non-disjunction or
malsegregation. Although only a few data were
generated, it is interesting to note that only two
embryos aneuploid for sex chromosomes were ob-
served; both derived from a patient (the couple
performed 2 treatment cycles) diagnosed with
gonosomal mosaicism. Altogether these findings
suggest that an altered karyotype has a key role in
determining an increased reproductive risk, irre-
spective of its male or female origin. Conversely,
gonosomal aneuploidy does not appear to be af-
fected.
In conclusion, there is growing evidence that the
severe male infertility condition could determine an
increased reproductive risk due to the generation of
de novo chromosomal abnormalities. More data
are required to establish whether these findings are
confirmed with special regard to gonosomal aneu-
ploidies. For these patients, the need of imple-
menting an extensive genetic counseling is hope-
fully not a matter of discussion any more. The pos-
sibility of performing the aneuploidy screening on
the generated embryos could represent the pre-
vailing approach in the incoming future of repro-
ductive medicine.
REFERENCES
1
. Engel W., Murphy D., Schmidt M.
Are there genetic risks associated with microassist-
ed reproduction?
Hum. Reprod. 1996, 11: 2359-2370.
2
. Meschede D., Lemcke B., Exeler J.R., et al.
Chromosome abnormalities in 447 couples un-
dergoing intracytoplasmic sperm injection-preva-
lence, types, sex distribution and reproductive rel-
evance.
Hum. Reprod. 1998, 13: 576-582.
715

L. Gianaroli, M.C. Magli, A.P. Ferraretti, et al.
3
. De Braeckeleer M., Dao T.M.
Cytogenetic studies in male infertility: a review.
Hum. Reprod. 1991, 6: 245-250.
11. Gianaroli L., Munné S., Magli M.C., et al.
Preimplantation genetic diagnosis of aneuploidy and
male infertility.
Int. J. Androl. 1997, 20: 31-34.
4
. Reijo R., Alagappan R.K., Patrizio P., et al.
Severe oligospermia resulting from deletions of
azoospermia factor gene on Y chromosome.
Lancet 1996, 347: 1290-1293.
12. Gianaroli L., Magli M.C., Munné S., et al.
Advantages of day four embryo transfer in patients
undergoing preimplantation genetic diagnosis of
aneuploidy.
5. Bonduelle M., Willikens J., Buysse A.
J. Assist. Reprod. Genet. 1999, 16: 170-175.
Prospective study of 877 children born after intracyto-
plasmic injection with ejaculated, epididymal and tes-
ticular spermatozoa after replacement of cryopreserved
embryos obtained after ICSI.
13. Gianaroli L., Magli M.C., Ferraretti A.P. et al.
Preimplantation diagnosis for aneuploidies in pa-
tients undergoing in vitro fertilization with a poor
prognosis: identification of the categories for which
it should be proposed.
Hum. Reprod. 1996, 11 (Suppl. 4): 131-159.
6
. Bonduelle M., Aytoz A., Van Assche E., et al.
Incidence of chromosomal aberrations in children
born after assisted reproduction through intracyto-
plasmic sperm injection.
Fertil. Steril. 1999, 72: 837-844.
14. Munné S., Magli M.C., Cohen J., et al.
Positive outcome after preimplantation diagnosis of
aneuploidy in human embryos.
Hum. Reprod. 1999, 14: 2191-2199.
15. Levron J., Ayala A. Goldring A., et al.
Hum. Reprod. 1998, 13: 781-782.
7
. Martin R.H., Rademaker A.W., Long-Simpson L., et al.
Variation in the frequency and type of the sperm
chromosomal abnormalities among normal men.
Hum. Genet. 1987, 77: 108-111.
Studies on sperm chromosomes in patients with se-
vere male factor infertility undergoing assisted re-
productive technology treatment.
8
. Martin R.H., Spriggs E.L., Ko E. et al.
The relationship between paternal age, sex ratios
and aneuploidy frequencies in human sperm as as-
sessed by multicolour FISH.
Mol. Cell. Endocrinol. (Special Issue), 2000 in press.
16. Liebaers I., Bonduelle., Van Assche E., et al.
Sex chromosome abnormalities after intracytoplas-
mic sperm injection.
Lancet 1995, 346: 1095.
17. World Health Organization.
Am. J. Hum. Genet. 1995, 57: 1395-1399.
9
. Gianaroli L., Magli M.C., Munné S., et al.
Will preimplantation genetic diagnosis assist patients
with a poor prognosis to achieve pregnancy?
Hum. Reprod. 1997, 12: 1762-1767.
WHO laboratory manual for the examination of hu-
man semen and semen-cervical mucus interaction.
Cambridge University Press, Cambridge, 1999.
1
0. Munné S., Alikani M., Tomkin G., et al.
Embryo morphology, developmental rates, and
maternal age are correlated with chromosomal ab-
normalities.
18. Sathanathan H., Ratnam S.S., Ng S.C., et al.
The sperm centriole: its inheritance, replication and
perpetuation in early human embryos.
Fertil. Steril. 1995, 64: 382-391.
Hum. Reprod. 1996, 11: 345-356.
716