A follow-up study of children born after intracytoplasmic sperm injection (ICSI) with epididymal and testicular spermatozoa and after replacement ...

A follow-up study of children born after
   intracytoplasmic sperm injection (ICSI) with epididymal
     and testicular spermatozoa and after replacement of
          cryopreserved embryos obtained after ICSI
          M.Bonduelle1'3, A.Wilikens1, A.Buysse1, E.Van Assche1, P.Devroey2,
                        A.C.Van Steirteghem2 and I.Liebaers1
      'Centre for Medical Genetics and 2Centre for Reproductive Medicine, Medical Campus, Dutch-speaking
                          Brussels Free University (Vrije Universiteit Brussel), Belgium

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                   To whom correspondence should be addressed at: Centre for Medical Genetics,
                    Academisch Ziekenhuis V.U.B., Laarbeeklaan 101, 1090 Brussels, Belgium

The aim of this prospective follow-up study                  malformations, defined as those causing func-
of children born after intracytoplasmic sperm                tional impairment or requiring surgical correc-
injection (ICSI) was to compile data on karyo-               tion, were observed in four children: two born
types, congenital malformations, growth para-                after ICSI with epididymal spermatozoa, one
meters and developmental milestones in order                 after ICSI with testicular spermatozoa and one
to evaluate the safety of this new technique. The            after ICSI and cryopreservation. No particular
study design included karyotyping of the parents             malformation was disproportionally frequent.
and their agreement to genetic counselling and               In the follow-up examinations at 2 months (107/
prenatal diagnosis and it was based on a physical            161 or 66.5%) and at 1 year (37/161 or 22.9%),
examination of the child at the Centre for                   no additional anomalies were observed. Lost for
Medical Genetics at the ages of 2 months, 1                  follow-up rate at 2 months was 33.5%. These
year and at 2 years, where major and minor                   observations on a limited number of children
malformations and psychomotor evolution are                  do not suggest a higher incidence of diseases
recorded. Here we describe the first 57 children             linked to imprinting, nor do they suggest a
born from 40 ICSI pregnancies with epididymal                higher incidence of congenital malformations.
spermatozoa (group 1), the first 50 children                 These observations are still limited in number
born from 34 ICSI pregnancies with testicular                and should be further completed by others
spermatozoa (group 2) and the first 58 children              and by collaborative efforts. In the meanwhile
born from 48 pregnancies after replacement of                patients should be told about the available data
cryopreserved ICSI embryos (group 3). Parental               before any treatment: there appears to be some
karyotypes were obtained from only 72/246                    risk of transmitted chromosomal aberrations, of
(29%) parents and were all normal. Prenatal                  de-novo, mainly sex-chromosomal aberrations
karyotypes were determined for a total of 70                 and of transmitting fertility problems to the
samples (40%): 21 in group 1, 15 in group 2                  offspring. Patients should also be reassured that
and 34 in group 3. In this last group 2 abnormal             until now there seems to be no higher incidence
47,XXY karyotypes (5.8%) and no structural                   of congenital malformations in children born
aberrations were found. This increase in de-                 after ICSI with epididymal or testicular sperma-
novo sex-chromosomal aberrations has already                 tozoa or after replacement of ICSI embryos.
been described with regard to the first 877                  Key words: children/congenital malformation/
children born after ICSI carried out at our                  epididymal spermatozoa/intracytoplasmic sperm
Centre and is probably linked directly to the                injection/pregnancy outcome/prenatal karyotypes/
characteristics of the infertile men treated                 testicular spermatozoa
rather than to the ICSI procedure itself. Major
196       © European Society for Human Reproduction & Embryology      Human Reproduction Volume 13 Supplement 1 1998
Children born after ICSI

                     Introduction                        1995a). In order to evaluate the safety of the ICSI
When assisted fertilization and intracytoplasmic         procedure we compared the data on karyotypes,
sperm injection (ICSI) were introduced, there was        congenital malformations, growth parameters and
major concern about the safety of this new tech-         developmental milestones in the two groups of
nique. ICSI is indeed a more invasive procedure,         children and could find no statistically significant
since one spermatozoon is injected through the           differences. We thus concluded from this limited
oocyte membrane and since fertilization can be           number of children that when ICSI was carried
obtained using spermatozoa which could never             out and compared with standard IVF procedure,
have been used previously in fertility treatment.        no additional risk was observed.
More questions arose and concern was expressed              In a subsequent article, we evaluated the safety
when ICSI with non-ejaculated spermatozoa, either        of the ICSI procedure by studying 877 children
epididymal or testicular, was introduced. Emphasis       born after ICSI and a minority of children born
was placed on the fact that the risk of chromosomal      after fertilization with epididymal and testicular

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aberration might be even higher in men with              spermatozoa as well as children born after ICSI
non-obstructive azoospermia. On the other hand,          in combination with cryopreservation (Bonduelle,
imprinting may be less complete at the time of           1996b). In this article we decribe the separate
fertilization if testicular spermatozoa are used. If     groups of children born after the use of epididymal
this were so it would be unlikely to impair fertiliza-   and testicular spermatozoa as well as children born
tion and early development, but anomalies might          after replacement of cryopreserved ICSI embryos;
become manifest only later in postnatal life.            these data are compared with the previous findings
                                                         in the ICSI group, so as to evaluate whether
   Experimentation in mice suggests that even
                                                         problems predicted from the literature are actu-
under normal conditions parental genomes continue
                                                         ally present.
to be modified after fertilization, presumably prior
to syngamy (Latham et al., 1995). The regulatory
factor must be present in one or both gametes at                      Materials and methods
the time of fertilization and may be deficient in        From the cohort of ICSI pregnancies obtained at
immature or in-vitro matured gametes. Bypassing          the Centre for Reproductive Medecine of the
gamete surface contact may decrease the ability of       Dutch-speaking Brussels Free University, three
the egg cytoplasm to carry out such modifications.       groups of children were studied (Table I): group 1
Unfortunately, there is no information on the mech-      consisted of 57 children, from the first 34 consecut-
anisms and exact timing of imprinting in the             ive pregnancies born after replacement of embryos
human embryo.                                            obtained after ICSI with epididymal spermatozoa;
   The safety of the ICSI procedure has been             group 2 consisted of 50 children, from the first 40
assessed carefully in previous publications (Van         consecutive pregnancies born after replacement
Steirteghem et al., 1993a,b,c; Bonduelle et al.,         of embryos obtained after ICSI with testicular
1994, 1995b, 1996a,b; Palermo et al, 1993, 1996;         spermatozoa; group 3 consisted of 58 children,
Devroey et al, 1995a,b). This series of articles         from the first 48 consecutive pregnancies born
failed to demonstrate any increased risk of major        after replacement of embryos obtained after
congenital malformations as compared to the              replacemnt of cryopreserved ICSI embryos.
general population, but did find an increased risk          The follow-up of these cohorts of children was
of chromosomal aberrations, mostly sex-chromo-           carried out at the Centre for Medical Genetics. In
somal aneuploidies. We also evaluated the results        a previous article by our group a number of these
of the ICSI procedure by comparing the first group       children were described as part of the cohort of
of 130 children born after ICSI with a control           children born after ICSI: 29 children after ICSI
group of 130 matched children born after in-vitro        using epididymal spermatozoa, 29 after ICSI using
fertilization (IVF) pregnancies in the same period       testicular spermatozoa and 22 after replacement
of time and after the same ovarian stimulation           of cryopreserved embryos obtained after ICSI
and in-vitro culture conditions (Bonduelle et ah,        (Bonduelle et al. 1996b).

M.Bonduelle et al.

Table I. Total number of pregnancies and number of children born after 20 weeks of pregnancy, after intracytoplasmic sperm
injection (ICSI) with epididymal or testicular spermatozoa and after replacement of cryopreserved ICSI embryos

                 Epididymal spermatozoa                Testicular spermatozoa               Cryopreservation
                 Pregnancies       Children            Pregnancies      Children            Pregnancies        Children

Singleton        26                26                  21               21                  38                 38 b
Twin             11                22                  10               20                  10                 20
Triplet              3              9                   3                9                  -                  -
Total            40                57                  34               50                  48                 58
Liveborn                           55 •                                 50                                     56
Two intrauterine deaths.
Two interruptions both for an abnormal 47,XXY karyotype.

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   Of the 58 children born after the replacement               about the risks of chromosomal abnormalities and
of cryopreserved ICSI embryos described here,                  the desirability of having a prenatal test (Bonduelle
three were born after the use of testicular spermato-          et al., 1996b). Pros and cons of the different
zoa and 14 after the use of epididymal spermatozoa,            types of prenatal diagnosis were discussed in detail
in the initial treatment cycle. These 17 children              at ~6-8 weeks of gestation; amniocentesis was
were counted twice into two groups: one taking                 suggested for singleton pregnancies, while
into account the origin of the spermatozoa and one             chorionic villus sampling was proposed for mul-
taking into account the cryopreservation procedure.            tiple pregnancies (De Catte et al., 1996). Chromo-
   The design of the prospective follow-up study               some preparations were obtained from cultured
was as follows. Before starting ICSI, couples were             amniocytes according to a modified technique by
asked to adhere to the follow-up conditions of our             Verma et al. (1989). Chromosome preparations
study. These conditions included genetic counsel-              from non-cultured and cultured chorionic villus
ling and agreement to prenatal karyotype analysis              cells were obtained by means of the technique
as well as participation in a prospective clinical             described by Gibas et al. (1987) and Yu et al.
follow-up study of the children. They also included            (1986) respectively. If indicated, prenatal tests for
completion of the standardized questionnaire as                other genetic diseases were planned.
described in the article by Wisanto et al. (1995),                The follow-up study of the expected child was
returning it to the research nurse and when possible           further explained: it was to consist of a visit to the
visiting the Centre for Medical Genetics with the              clinical geneticist at 2 months and at 12 months
child after birth.                                             of age, and then once a year.
   All couples referred for assisted fertilization                For all pregnancies, written data on pregnancy
were evaluated for possible genetic problems,                  outcome with regard to the babies were obtained
either before starting treatment in cases of maternal          from the gynaecologists in charge. Perinatal data,
age >35 years, positive family history or a chromo-            including gestational age, mode of delivery,
somal aberration carried by a parent, or at 6-8                birthweight, Apgar scores, presence or absence
weeks of pregnancy. A history, including a pedi-               of malformations and neonatal problems were
gree, was obtained in order to identify genetic                registered. If any problem was mentioned, detailed
risks or possible causes of congenital malforma-               information was also requested from the paediatri-
tions. This history included details of medication,            cian in charge.
alcohol abuse, environmental or occupational risk                 For babies born in our university hospital, a
factors and socio-economic status. A karyotype                 detailed physical examination was performed at
was routinely performed for the couple. In view                birth, which looked for major and minor malforma-
of possible risk factors due to the new techniques             tions and included evaluation of neurological and
of assisted fertilization, the couple was counselled           psychomotor development. For babies born else-

Children born after ICSI

where, written reports were obtained from gynaeco-
logists as well as from paediatricians, while a         Table II. Sex ratio of children born after 20 weeks of
                                                        pregnancy after intracytoplasmic sperm injection (ICSI) with
detailed morphological examination by a clinical        epididymal or testicular spermatozoa and replacement of
geneticist from our centre was carried out after 2      cryopreserved ICSI embryos
months whenever possible. Additional investi-
                                                                      Epididymal     Testicular     Cryopreservation
gations were carried out if the anamnestic data or                    spermatozoa    spermatozoa
the physical examination suggested them.
   At the follow-up examination at 12 months and 2      Girls         23             30             28
                                                        Boys          34             20             30
years, the physical, neurological and psychomotor       Boys/girls     1.47           0.66           1.07
assessments were repeated by the same team of
clinical geneticists. At ~2 years or more, a Bayley
test was programmed in order to quantify the
psychomotor evolution of the children. If parents       death 3=20 weeks or ^=400 g) and 55 were liveborn.
                                                        In group 2 (after use of testicular spermatozoa),

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did not spontaneously attend the follow-up consul-
tations, they were reminded by phone to make an         of the 50 children studied, 21 were singletons, 20
appointment. For parents living abroad, reminders       were from twin pregnancies and nine were from
were mailed.                                            triplet pregnancies. In group 3 (after replacement
                                                        of cryopreserved embryos obtained after ICSI), of
   A widely accepted definition of major malforma-
                                                        the 58 children studied, 38 were singletons and
tions was used, i.e. malformations that generally
                                                        20 were from twin pregnancies; two singleton
cause functional impairment or require surgical
                                                        pregnancies were interrupted for an abnormal pre-
correction. The remaining malformations were con-
                                                        natal karyotype (twice a 47,XXY karyotype) and
sidered minor. A minor malformation was distingu-
                                                        56 children were liveborn.
ished from normal variation by its occurrence in
=£4% of the infants of the same racial group.              The sex ratio in the different subgroups is shown
Malformations or anomalies were considered syn-         in Table II.
onymous with structural abnormality (Smith, 1975;          The mean maternal age as regards the children
Holmes, 1976).                                          born was 32.2 years (range 22.9^4-2.5) in group 1,
                                                        32.1 years (range 24.0-41) in group 2 and 31.9
                                                        (range 24.9^2.5) in group 3 (Table III). The mean
                       Results                          paternal age as regards the children born was 39.2
From the pregnancy cohort (positive serum human         years (range 30-60.4) in group 1, 37.2 years (range
chorionic gonadotrophin) of 73 pregnancies in           29.4-46.3) in group 2 and 34.0 (range 26.8^2.5)
group 1, 77 pregnancies in group 2, and 79              in group 3.
pregnancies in group 3 we obtained data for only           We obtained data from physical examination
94% of the ongoing pregnancies, even after several      at birth for all the children. We compiled this
attemps to obtain the information. In group 1 we        information from the medical records as well as
had complete data (partly from the paediatricians       from careful questioning of the parents during
and partly from the parents) for 57 children at         follow-up consultations. For the children living
birth and incomplete data for one child; in group       further away, or where the parents were no longer
2 we had complete data for 50 children and              willing to come to the clinic, detailed histories
incomplete data for nine children; in group 3 we        (except for one major malformation where we
had complete data for 58 children and incomplete        were given only the name of the malformation)
data for two (the same two as in the previous           were obtained from the paediatrician if any problem
groups). In group 1 (after use of freshly collected     was mentioned in answers to the questionnaire.
or frozen-thawed epididymal spermatozoa), of the           During the follow-up at 2 months, 33/55 children
57 children studied, 26 were singletons, 22 were        in group 1, 30/50 in group 2 and 44/56 children
from twin pregnancies and nine were from triplet        in group 3 were examined by one of the geneticists;
pregnancies; two twin children in separate pregnan-     for 11, 16 and nine children information was
cies suffered an intrauterine death (defined as fetal   obtained from letters from parents or paediatricians.

M.Bonduelle et al.

Table III. Parental age and genetic counselling to 122 parents of 146 children born after intracytoplasmic sperm injection
(ICSI) with epididymal or testicular spermatozoa and after replacement of cryopreserved ICSI embryos

                                    Epididymal spermatozoa       Testicular spermatozoa        Cryopreservation

Maternal age (years)
 range                              22.9^2.5                     24.0-41.0                     24.9-42.5
 mean                               32.2                         32.1                          31.9
 median                             33.0                         32.3                          30.6
 SD                                  4.2                          4.3                           4.2
 age 5=35 years                      7                            6                             8
Paternal age (years)
  range                             30.0-60.4                    29.4-46.3                    26.8^2.5
  mean                              39.2                         37.2                         34.0
  median                            35.6                         37.0                         34.1
  SD                                 8.8                           4.7                          3.8

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  age s?50 years                     2                           -                            -
Monogenic disorders
  CF carriers (CBAVD)                   9a                       -                                1
  CF homozygote                     -                                1                            1
Chromosomal disorders               -                            -                            -
Multifactorial condition            -                                1                        -
Consanguinity                           1                        -                                1
(3rd degree)

    in nine couples had a 1/4 risk for a cystic fibrosis (CF) child because both parents were carrier of a CF mutation.
CBAVD = congenital bilateral absence of the vas deferens.

At 2 months of age lost for follow-up rate was                   congenital bilateral absence of the vas deferens
thus respectively 40, 40 and 21%. So far 14/55,                  (CBAVD). After screening all the partners of
10/50 and 13/56 children have been examined a                    CBAVD patients we found one couple to have a
second time at 1 or 2 years of age. Most of the                  1/4 risk of cystic fibrosis (CF). For this couple a
children are still
Children born after ICSI

in group 3; amniocentesis was performed for 19                    including the births of 14 twins in group 1;
fetuses in group 1, 13 in group 2 and 26 in                       in group 2, five pregnancies ended prematurely,
group 3. No spontaneous interruptions after test                  involving the births of 10 twins; and in group 3,
procedures were noted.                                            three pregnancies ended prematurely involving the
                                                                  births of six twins. Birthweight 20 weeks of gestation in groups 1, 2
and 3 and birthweights for singletons, twins and
triplets are listed in Table V.                                           Major malformations (Table VI)
   Prematurity (birth before 37 weeks of preg-                    For one child in group 1, ureteral dilatation was
nancy) occurred in seven of the 40 pregnancies,                   detected before birth during ultrasound examina-

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Table IV. Prenatal diagnosis: abnormal results/number of tests for children born after intracytoplasmic sperm injection (ICSI)
with epididymal or testicular spermatozoa and after replacement of cryopreserved ICSI embryos

Prenatal test                Epididymal spermatozoa       Testicular spermatozoa        Cryopreservation             Total

CVS                          0/2                          0/2                           0/8                          0/12
Amniocentesis                0/19                         0/13                          2/26a                        2/58
Total 0/21                   0/15                         2/34a                         2/70
Failure                      _                            1                                                          1/70

"Abnormal results were 2X47,XXY.
CVS = chorionic villus sampling.

Table V. Neonatal measurements of liveborn children after 20 \veeks of pregnancy, after intracytoplasmic sperm injection
(ICSI) with epididymal or testicular spermatozoa and after replacement of cryopreserved ICSI embryos

                                    Epididymal spermatozoa        Testicular spermatozoa        Cryopreservation

Birthweight (g)
  range                             1280-4625                     1220-4911                      950-4030
  mean                              2822                          2740                          3006
  median                            2876                          2646                          2860
  SD                                 758                           799                           700
Length (cm)
  range                                39-56                        43-56                           39-53.5
  mean                                 48.1                         48.3                            48.5
  median                               49.3                         48.0                            49.0
  SD                                    3.8                          3.2                             3.59
Head circumference (cm)
  range                                28-37                        29-36                           29-37.5
  mean                                 33.4                         33.5                            34.4
  median                               33.8                         33.5                            34.5
  SD                                    2.4                          1.8                             1.8
Birthweight (g)
     mean                           3409                          3379                          3301
     median                         3400                          3200                          3390
     mean                           2562                          2518                          2418
     median                         2600                          2450                          2525
     mean                           1769                          1691                          -
     median                         1620                          1630                          -

M.Bonduelle et al.

tion at 29 weeks of pregnancy; this child needed                                     Surgery
urological surgery at 3 and at 12 months of age.                 Surgery was needed for one child with an inguinal
One other child in group 1 had a leg and hip                     hernia in group 1 and for the children in groups 1
malformation at birth. For one child in group 2,                 and 3 with major malformations.
cleft lip and palate was detected at 22 weeks of                   At the age of 2 months, no new major or minor
pregnancy; this child died at 2 months of age of a               malformations were found. Minor malformations
degenerative muscle disease. For one child in                    observed at birth did not cause any functional
group 3 severe intrauterine growth retardation was               impairment.
detected prenatally; this child was born after 30
weeks of pregnancy with a cleft lip and palate and                                   Follow-up
a unilateral hand and foot malformation.
                                                                 Follow-up at the age of 2 months was still limited
                                                                 up to the date 1 May 1996: 33/55 children of
        Minor malformations (Table VII)                          group 1, 30/50 children of group 2 and 44/56

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These were found in 4/55 children in group 1, 3/                 children of group 3. Twenty-two, 14 and 35 chil-
50 in group 2 and 7/56 in group 3. These children                dren came to the Centre while for the others we
did not have major anomalies.                                    received information about development from the
                                                                 paediatricians or the parents. At 1 year we saw
                                                                 14, 10 and 13 children respectively. In group 1,
                                                                 two children had a minor developmental problem;
                                                                 one child had an axial hypotonia at 2 months, but
Table VI. Major congenital malformations in liveborn
children after intracytoplasmic sperm injection (ICSI) with      was normal at 2 years and one child had a language
epididymal or testicular spermatozoa and after replacement of    delay at 2 years. No neurological problems were
cryopreserved ICSI embryos                                       encountered in the children during follow-up con-
              Epididymal    Testicular     Cryopreservation      sultations.
              spermatozoa   spermatozoa

Singleton     2             0              1
Twin          0             0              0                                             Discussion
Triplet       0             1              0                     From the beginning of our ICSI treatment, nearly
Total         2/55          1/50           1/56
                                                                 all patients have been seen at the Centre for Medical

Table VII. Minor malformation in liveborn children after intracytoplasmic sperm injection (ICSI) with epididymal or testicular
spermatozoa and after replacement of cryopreserved ICSI embryos

                                                                        Epididymal        Testicular        Cryopreservation
                                                                        spermatozoa       spermatozoa

Facial abnormality: broad nasal bridge                                  0                 0                 1
Cardiac anomaly: transient ductus arteriosus (+capp haemangioma)        0                 0                 1
   Minor preaxial polydactily                                           0                 1                 0
   Semian crease                                                        0                 1                 0
Dermatological abnormalities
   Congenital naevi                                                     2                 0                 0
   Small haemangioma                                                    0                 0                 2
   Mongolian spot                                                       0                 0                 2
   Xanthogranuloma                                                      0                 0                 1
Pilonidal sulcus                                                        2                 1                 0
Total                                                                   4/55              3/50              7/56

Children born after ICSI

 Genetics either before starting the treatment or       experience with ICSI patients (Bonduelle et al.,
 at 6-8 weeks of pregnancy. Since in the group           1996b; Van Assche et al., 1996) as well as from
treated with epididymal and testicular spermatozoa      the literature data (Chandley et al., 1979; Yoshida
many patients live outside Belgium, these tended        et al., 1995) there is a higher risk of chromosomal
to leave the country early and not to attend the        abnormality in male-fertility patients and we should
genetic counselling session. We have seen only          persuade gynaecologists to perform a karyotype
43% of the couples in group 1, 30% in group 2           before starting any treatment cycle. Yoshida found
and 55% in group 3. Apart from the maternal age         in a review of 1007 males with infertility, 14.6%
risk, the risk associated with CBAVD was most           chromosomal aberrations in the azoospermic males
frequent; in group 1, 9/55 or 16% of the children       and as high as 20.3% chromosomal aberrations in
had an increased risk of CF and of CBAVD.               the non-obstructive azoospermia cases. If we did
Screening for CF is therefore mandatory. One of         not see any anomaly in the chromosomes of these
the couples had a 1/4 risk of CF; for this couple       groups it is probably because only eight men were

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we proposed a preimplantation diagnosis, which          tested and we probably missed the opportunity to
was accepted. From the frequency of CF carriers         discuss a prenatal diagnosis in some higher-risk
in the populations with European ancestry we            situations.
expect a 1/4 risk for 4-5% of the couples (Collins         Since we also know that meiotic errors can
et al., 1992). We think that for couples with a         occur more frequently in azoospermic men (Pang
fertility problem requiring an ICSI treatment in        et al., 1994, 1995) and also in infertile men in
combination with a genetic high-risk situation,         general (Moosani et al., 1995), it would also be
preimplantation diagnosis should be offered when-       of help for future counselling if chromosome
ever feasible. In group 2 (and for the same patient     analysis of germ cells were to become possible on
who was present in group 3), a homozygous CF            a routine basis in order to discriminate some high-
patient was treated successfully. This patient's risk   risk situations.
of having a CF child was, when the wife had been           Although most patients were informed through
screened for a standard mutation set of eight           the informed consent procedure and from the oral
frequent mutations in our populaton, 1/150X1/2 =        information from gynaecologists and nurses about
 1/300. This inevitable risk was explained to the       the risks of chromosomal aberrations in ICSI
couple, who accepted it.                                pregnancies in general, only 21/57 (37%) fetuses
   During the genetic consultation we did not detect    in group 1, 15/50 (30%) in group 2 and 34/58
monogenic disease apart from the CF patient in          (58%) in group 3 were tested, more singletons (58/
groups 2 and 3, where the diagnosis was made            85 or 68%) than multiples (12/80 or 15%). More
during the investigations for his fertility problem.    parents of a multiple pregnancy were afraid of the
Even if we did not find many genetic diseases we        test procedure as we counselled them to have
are aware of the fact that a fertility problem may      chorionic villus sampling rather than amniocentesis
be one of the expressions of a more general disease.    and attributed a higher risk (of 1%) of miscarriage
As expressivity can vary a lot, as for example in       to the latter. Most of the couples withdrew from
myotonic dystrophy, it is important to draw a           testing once pregnant because the risk of miscar-
complete pedigree and to screen family members          riage after a fertility treatment was considered too
for other than reproductive problems. We also           high or because of ethical or religious considera-
think it necessary to continue to perform parental      tions, which were more frequent in groups 1 and
karyotypes, since for couples with a numerical          2 since some of these patients were practising Jews
abnormality of the chromosomes, a mosaicism or          and different from our previously described ICSI
a structural aberration, the global chances of suc-     population. We know from our experience with
cess of the treatment procedure can be reduced          ICSI that in this situation the risk of a de-novo
and the stronger indications for a prenatal test and    chromosomal aberration is no higher than the risk
the risks for the offspring should be discussed         to 37 year old women in the general population
(Hens et al., 1988). As we know from our own            [based on Ferguson-Smith's (1983) calculation at

M.Bonduelle et al.

 1.2%]. In these particular subgroups, however,        chromosomal aberrations at the time of prenatal
where testicular or epididymal spermatozoa were        diagnosis (Hook et al., 1977; Ferguson-Smith,
used, there could be a higher risk of de-novo           1983).
(sex) chromosomal and inherited chromosomal               In our previous group of ICSI patients, we
aberrations. As we did not put moral pressure on       described 1.2% family-based structural aberrations.
the patients if they did not wish to accept the        They were certainly not induced by the microinjec-
added risk from testing, we obtained karyotypes        tion technique, since they were all detected in the
for only 70 of the 165 (42%) fetuses.                  infertile males before their treatment. Statistically,
   Abnormal fetal karyotypes were not found in         family-based structural aberrations can lead to
groups 1 and 2. This is not what we expected from      normal karyotypes, to exactly the same structural
our experience with ICSI, where 1.2% aneuploidies      aberration as in the parent, or to 0-50% of non-
were found (Bonduelle et al, 1996b). Moreover,         balanced karyotypes. In this limited group of
for many patients we did not have the karyotype        parents, no father was found to carry a structural

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after birth. As we expect mainly sex-chromosomal       aberration and no fetuses with an unbalanced
aberrations, these could have been missed in a         karyotype were found.
simple clinical evaluation. We must therefore con-        The figure of 2.4% or four major malformations
clude that we do not have enough data in groups        out of 161 liveborn children is similar to most of
 1 and 2 to draw conclusions about the risk of         the general population national registries (Office
chromosomal anomalies in this limited sample.          of Population Censuses and Surveys, 1982-88,
   In the group with cryopreserved embryos              1987-88; National Perinatal Statistics Unit and
obtained after ICSI with ejaculated spermatozoa,       Fertility Society of Australia, 1992) and the assisted
we found that two of 34 tested fetuses (5.8 %)         reproduction surveys (Saunders et al., 1987; Cohen,
were abnormal. Both abnormalities consisted of         et al, 1988; Beral et al, 1990; Rizk and Dole,
the same sex-chromosomal aberration, a 47,XXY           1991; Friedler et al, 1992; Medical Research
karyotype or Klinefelter syndrome. Apart from the      International, 1992; Rufat et al, 1994, Bachelot et
fertility problem, the clinical picture can vary a     al, 1995, Lancaster et al, 1995). We here consid-
lot, and patients were informed as objectively as      ered the livebirth malformation rate as this is the
possible. Both parents opted for a second-trimester    most frequently used, rather than a more precise
abortion. Maternal age was 26 and 31 years and         calculation of the ratio, taking fetal deaths and
paternal age was 28 and 31 years. The origin of        interruptions of affected fetuses into account, which
the extra X chromosome was checked by DNA              is used in only very few malformation surveys.
polymorphism (DXS52) for one pregnancy and             However, the more intensive follow-up of ICSI
this was paternally derived.                           pregnancies and the frequent fetal karyotypes could
   We think that this higher incidence of abnormal     theoretically lead to an increase in the number of
fetal karyotypes in pregnancies after replacement      terminations artificially decreasing the observed
of cryopreserved ICSI embryos is probably only         rate of malformations at birth. In this survey two
a statistical variation of a higher risk of sex-       pregnancy interruptions for Klinefelter syndrome
chromosomal aberrations found in the ICSI preg-        were performed; if these children were born they
nancies (Bonduelle et al, 1996b) as there is no        would probably not have been counted in the figure
theoretical ground to suppose that the cryopreserv-    of major malformations. No malformations were
ation technique could induce chromosomal aberra-       observed by ultrasound and therefore there was no
tions of this type, which are present already before   need to consider other pregnancy interruptions.
the first mitotic division. The mean maternal age      National registries most often register the anomal-
of the mothers conceived in group 3 was 31.9           ies at birth or during the first week of life, while
years, and the mean paternal age was 34 years,         in this study the follow-up is carried through to 2
which does not explain the higher rate of chromo-      years. Moreover, risk figures in the national statist-
somal aberrations. For a mean maternal age of          ics will probably also be somewhat lower as it is
32 years we would expect a figure of ~0.3%             unlikely that malformations are generally searched

Children born after ICSI

for as carefully as in this survey. We found two       against physiologically or genetically abnormal
malformations in the first group: a unilateral hip     spermatozoa might be bypassed; abnormal oocytes
and leg malformation for which we do not know          might be fertilized; the altered environment or
whether a genetic or environmental mechanism           mechanical or chemical damage to the oocyte
was to blame, or the treatment procedure itself;       might lead to perturbations of meiosis and mitosis;
the other malformation was a urogenital malforma-      various chemical or environmental exposures might
tion, to which we know that there is often a genetic   also lead to point mutations resulting in genetic
basis besides an environmental factor.                 disease visible at birth or later in life. With the
   In the group receiving ICSI with testicular         use of immature testicular spermatozoa, diseases
spermatozoa, a child with a cleft lip and palate       linked to imprinting were expected to occur
was found. In this case, where the mother was not      more often.
exposed to drugs or medication during pregnancy           With normal morphology and normal develop-
and where no associated malformations were             mental processes observed so far, there seem to be

                                                                                                                Downloaded from http://humrep.oxfordjournals.org/ by guest on September 23, 2015
found, there was probably a multifactorial problem.    none of the expected problems. Caution is still
The fact that after 2 months the child died of a       needed, since many of the problems due to abnor-
degenerative muscle disease is probably a second       mal imprinting would be detectable only during
problem with a monogenetic basis. Unfortunately        later development.
we do not have the exact diagnosis for this child.
   In the third group a child with a cleft lip and
palate in combination with a unilateral hand and                           Conclusion
foot malformation and an intrauterine growth           In this follow-up study of children born after
retardation was found. As we do not have the           ICSI, a slight increase in de-novo chromosomal
exact diagnosis we do not know the mechanism           aberrations of 5.8% (2/34) on prenatal diagnosis
behind this malformation. Closure of lip and pala-     in the group of children born after replacement of
tum occurs before the 12th week of pregnancy. A        cryopreserved ICSI embryos is probably directly
karyotype analysis performed on cord blood was         linked to the characteristics of the infertile men
normal 46,XY.                                          treated rather than to the ICSI technique itself.
   As we saw only one-third of the children at 2          In the group of children born after ICSI with
months of age and as we know that the lost for         epididymal or testicular spermatozoa we failed to
follow-up data can vary considerably from the          find a higher risk in a limited sample of 36
collected data, firm conclusions on the rate of        prenatal tests.
major malformations cannot be drawn. As we                Major malformations were found in an expected
compare our data on ICSI children born afer            range of 2.4% (4/161), comparable to the figures
replacement of cryopreserved embryos with the          from other studies after assisted reproductive tech-
data of Olivennes et al. (1996), on children born      niques or in population registries, but the one-third
after IVF and cryopreservation, we found 1/56          of children lost for follow-up at the age of 2
major malformations where Olivennes et al. found       months must be mentioned.
1/89 major malformations. In this group the lost          These observations should be further by others
for follow-up rate was considerably better and         and by collaborative efforts. Meanwhile, patients
only 4.3%.                                             should be counselled on the basis of the available
   During follow-up consultations we have seen         data before any treatment: the higher risk of
37 children until now and we have not encountered      transmitted chromosomal aberrations, the risk of
any major developmental problem, apart from in         de-novo, mainly sex-chromosomal, aberrations and
those children with major malformations.               the risk of transmitting fertility problems to the
   A number of hypotheses exist to support the         offspring. They should also be reassured that there
idea of an additional risk due to the ICSI procedure   so far seems to be no higher incidence of congenital
and in particular due to the use of epididymal         malformation or developmental problems in chil-
or testicular spermatozoa. Selective mechanisms        dren born after ICSI with epididymal or testicular

M.Bonduelle et al.

spermatozoa or after replacement of frozen-thawed             Cohen, J., Mayaux, M.J. and Guihard-Moscato, L. (1988)
ICSI embryos.                                                    Pregnancy outcomes after in vitro fertilization. A
                                                                 collaborative study on 2342 pregnancies. Ann. NY
                                                                Acad. Sci., 541, 1-6.
                                                              Collins, F. S. (1992) Cystic fibrosis, molecular biology
                 Acknowledgements                                and therapeutic implications. Science, 256, 774-779.
We are indebted to many colleagues: the clinical, scient-     De Catte, L., Liebaers, I., Foulon, W. et al. (1996) First
ific, nursing and technical staff of the Centre for Medical      trimester chorion villus sampling in twin gestations.
Genetics and the Centre for Reproductive Medicine,              Am. J. Perinat., 13, 413-417.
especially Marleen Magnus, Johan Schietecatte and             Devroey, P., Liu, J., Nagy, Z. et al. (1995a) Normal
Hubert Joris for their efforts in collecting and computing       fertilization of human oocytes after testicular sperm
these data. Frank Winter of the Language Education               extraction and intracytoplasmic sperm injection.
Centre corrected the manuscript. Research grants from            Fertil. Steril., 62, 639-641.
the Belgian Fund for Medical Research and an uncondi-
                                                              Devroey, P., Liu, J., Nagy, Z. et al. (1995b) Pregnancies
tional educational grant from Organon International are          after testicular sperm extraction and intracytoplasmic
kindly acknowledged.
                                                                 sperm injection in non-obstructive azoospermia. Hum.

                                                                                                                              Downloaded from http://humrep.oxfordjournals.org/ by guest on September 23, 2015
                                                                Reprod., 10, 1457-1460.
                                                              Ferguson-Smith, M. (1983) Prenatal chromosomal
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