A Danish national cohort of 730 infants born after intracytoplasmic sperm injection (ICSI) 1994–1997

A. Loft1,14, K. Petersen2,3, K. Erb4,5, A.L. Mikkelsen6, J. Grinsted7, F. Hald8, J. Hindkjær9, K.M. Nielsen10, P. Lundstrøm11, A. Gabrielsen2,3, S. Lenz12, P. Hornnes13, S. Ziebe1, H.B. Ejdrup1, A. Lindhard1, Y. Zhou1 and A. Nyboe Andersen1

1 The Fertility Clinic, University of Copenhagen, Rigshospitalet, 2100 Copenhagen Ø, 2 The Fertility Clinic, Ciconia V, 8270 Højbjerg, 3 The Fertility Clinic, Ciconia Ø, 2000 Frederiksberg, 4 The Fertility Clinic, Odense University Hospital, 5000 Odense, 5 The Fertility Clinic, Odense, 5000 Odense, 6 The Fertility Clinic, University Hospital of Copenhagen, Herlev County Hospital, 2730 Herlev, 7 The Fertility Clinic Trianglen, 2100 Copenhagen, 8 The Fertility Clinic, Brædstrup Hospital, 8740 Brædstrup, 9 The Fertility Clinic, Århus University Hospital, 8000 Århus C, 10 Danish Fertility Clinic, 2000 Frederiksberg, 11 The Fertility Clinic IVF, 2750 Ballerup, 12 The Fertility Clinic, The Private Hospital Hamlet, 2000 Frederiksberg, 13 The Fertility Clinic, City of Copenhagen, 1100 Copenhagen K, Denmark


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This national cohort study included all clinical pregnancies obtained after intracytoplasmic sperm injection (ICSI) registered in Denmark between January 1994 and July 1997 at five public and eight private fertility clinics. Laboratory and clinical data were obtained from the fertility clinics. The couples answered a questionnaire regarding the pregnancy and the health of the child (response rate 94%). Data validation was carried out through discharge charts. The mean age of the women was 32.1 years. In 84.2% of couples, male factor was the main reason for performing ICSI, and in 4.8% epididymal spermatozoa were used. The mean number of embryos replaced was 2.3 (range 1–3) and in 95% of cases fresh embryos were transferred. Only 183 women (28.5%) underwent prenatal diagnosis, resulting in 209 karyotypes with seven (3.3%) chromosome aberrations. Six major chromosomal abnormalities (2.9%) and one inherited structural chromosome aberration (0.5%) were found, but no sex chromosome aberrations. The frequency of multiple birth, Caesarean section rate, gestational age, preterm birth, and birth weight were comparable with previous studies. The perinatal mortality rate was 13.7 per 1000 children born with a gestational age of 24 weeks or more. In 2.2% (n = 16) of the liveborn infants, and in 2.7% (n = 20) of all infants, major birth defects were reported by the parents. Minor birth defects were found in nine liveborn infants (1.2%). In conclusion, the results of this study on outcome of ICSI pregnancies are in line with earlier reports, except that no sex chromosome abnormalities were found.

Key words: intracytoplasmic sperm injection/malformation/obstetric outcome/pregnancy outcome/prenatal karyotypes


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The first delivery of a healthy child after intracytoplasmic sperm injection (ICSI) was reported in 1992 (Palermo et al., 1992Go), and within a few years microfertilization was adopted as a routine treatment throughout the world. The obstetric outcome, the malformation rate and karyotype abnormalities of children born after ICSI have been reported in a number of studies (Bonduelle et al., 1995Go, 1996aGo,Bonduelle et al., bGo, 1998aGo,Bonduelle et al., bGo; In't Veld et al., 1995; Wisanto et al., 1995Go, 1996Go; Liebaers et al., 1996Go; Palermo et al., 1996Go; Wennerholm et al., 1996Go; Govaerts et al., 1998Go; Tarlatzis and Bili, 1998Go). In these reports it has been discussed whether children born after ICSI have a slightly increased risk of chromosome abnormalities, especially in relation to inherited structural aberrations and sex chromosome abnormalities. These studies found no different obstetric outcome after ICSI compared with conventional in-vitro fertilization (IVF), and concluded that there seems to be no higher incidence of congenital malformations in children born after ICSI. However, using a re-classification of the definition of what constitutes a major birth defect, the Belgian data (Bonduelle et al., 1995Go) were re-analysed (Kurinczuk and Bower, 1997Go), and results indicated that children born after ICSI were twice as likely to have major birth defects compared with Western Australian infants.

In contrast to earlier studies from selected centres with an extensive routine in ICSI, the present study from the Danish Fertility Society is a nationwide follow-up study. The aim of the study was to evaluate the pregnancy outcome and the health of all children born after ICSI performed in Denmark between January 1994 and July 1997.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The study included all five public and eight private fertility clinics in Denmark offering ICSI between 1st January 1994 and 30th June 1997. The indications were male subfertility and absent or low fertilization rate after conventional IVF. All clinical pregnancies with an intrauterine gestational sac identified by ultrasound were included in the study.

The clinics reported the following laboratory data: indication for ICSI, number and date of embryo replacement (fresh or thawed embryos), number of implantations and number of fetuses with fetal heart activity. Hormone stimulation, oocyte collection, embryo culture and replacement were carried out according to the standard of each clinic. No more than three embryos were replaced.

A questionnaire regarding clinical data (pregnancy outcome, prenatal diagnosis, hospitalization during pregnancy, mode of delivery and health of the child) was sent by the clinics to the parents. The couples were asked whether or not they allowed contact to the departments where the delivery took place or where the child eventually had been treated. Except for one couple, whose child had been treated for congenital hip luxation, all parents who reported a birth defect gave their permission. In two cases of abortion (trisomy 21 and 18), the couple did not want the hospitals to be contacted, but rather to be contacted themselves. The questionnaire was sent twice to non-responders. If the couple did not answer the second request, or if they did not want to participate in the study, data from the registration of the clinics were used. For ethical reasons some of the clinics did not wish to contact 17 couples, where early ultrasound examination at the clinic had shown a missed or spontaneous abortion. Eight couples returned the questionnaire, stating that they did not wish to participate in the study, and 34 couples did not reply. In total, 665 questionnaires were answered, and the response rate was 94%.

All infants born with a gestational age of 24 weeks or more were included in the analyses concerning the obstetric and paediatric outcome.

Definitions
These were as follows:

Gestational age: calculated from the day of aspiration, which was defined as day 14 of the cycle.

Abortion: fetus with a gestational age less than 24 weeks.

Pre-eclampsia: no specific definition. Information obtained from the questionnaire.

Preterm birth: delivery before 37 weeks gestation.

Perinatal mortality: number of intrauterine or intrapartum deaths and neonatal deaths <7 days per 1000 children born with a gestational age of 24 weeks or more.

Low birth weight: less than 2500 g at birth.

Very low birth weight: less than 1500 g at birth.

Malformations: conditions registered in the International Classification of Diseases and Health Related Problems, 10th Revision (ICD-10; Danish National Board of Health, 1993Go), as a congenital malformation or chromosome abnormality (ICD-10: Q00–Q99).

It should be noted that neonatal facial paralysis, pilonidal cyst and amniotic band syndrome do not have a specific ICD10 diagnosis, and so were classified as unspecified diagnosis.

The protocol was approved by the Central Ethical Committee in Denmark.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The indication and laboratory data for couples who answered the questionnaire (n = 665), compared with those who did not receive (n = 17) or did not answer (n = 42) the questionnaire are shown in Table IGo. The age of the women was calculated on the day of embryo replacement. Among the 665 clinical pregnancies where the couple returned a questionnaire, 600 (90.2%) resulted in a delivery with a gestational age of 24 weeks or more.


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Table I. Indication and laboratory data for all Danish clinical intracytoplasmic sperm injection (ICSI) pregnancies, January 1994 to July 1997
 
Prenatal diagnosis
Prenatal diagnosis was carried out in 183 women: these included 49 chorion villus samples (CVS) and 139 amniocenteses, though both CVS and amniocenteses were carried out in five cases. Due to multiple pregnancies, 209 karyotypes were analysed (Table IIGo). Of the 665 questionnaires returned, 22 reported a missed abortion, such that 643 pregnant women were eligible for prenatal diagnosis. As the exact date for all early abortions is not known, these are not excluded from the population, which constituted the possible candidates for prenatal diagnosis.


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Table II. Number of prenatal diagnosis and karyotypes in all ICSI pregnancies
 
Thus, 28.5% of the women with a living fetus at early ultrasound examination had a prenatal diagnosis, and of these 48.6% were aged >=35 years. Among those women who answered the questionnaire 29% were aged >=35 years. Amniocentesis was performed most frequently compared with CVS: 76% of singletons and 79% of twins, but in no cases of triplets.

Complications to prenatal diagnosis
Five women (2.7%) reported leakage of amniotic fluid after amniocentesis. In one twin gestation without malformations, continuous amniotic fluid leakage for 8 weeks was reported, and this resulted in intrauterine deaths in week 25. One singleton with trisomy 21 died 10 days after CVS, and in one triplet gestation intrauterine death occurred in two fetuses with normal karyotypes shortly after the CVS.

Abnormal karyotypes and major malformations (gestational age <24 weeks)
In all cases where an abnormal prenatal karyotype or inherited structural chromosome aberration was reported (Table IIIGo), ejaculated spermatozoa were used. The autopsy of the fetus with an unbalanced translocation demonstrated multiple malformations. One pregnancy where prenatal diagnosis had shown a normal female karyotype, was interrupted after repeated ultrasound examinations had demonstrated oligohydramnios, hypoplasia of the lungs and bilateral dilatation of the renal pelvis and the ureters. Besides slight dilatation of the renal pelvis, no other malformations were found at autopsy. In total, six cases with major malformations were observed in all interrupted pregnancies or intrauterine deaths with a gestational age <24 weeks: one trisomy 18, two trisomy 21, one triploidy, one unbalanced translocation, and one fetus with oligohydramnios.


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Table III. Abnormal prenatal karyotypes and inherited structural chromosome aberrations in 209 karyotypes
 
Fetal reduction
Four cases of triplets had fetal reduction carried out in weeks 10–12. In one case, two fetuses were reduced and the pregnancy later resulted in an abortion. In the other three cases, one fetus was reduced and three pairs of healthy twins were delivered.

Postnatal karyotyping (gestational age >=24 weeks)
Six postnatal karyotypings were performed; five of these were liveborn children with a gestational age of >=24 weeks, and the tests identified one case of deletion 13 and one case of trisomy 21. The age of these mothers were 30 and 34 years respectively.

Obstetric and neonatal data
Among the women who delivered, hospitalization due to haemorrhage and pre-eclampsia was reported in 17 and 54 cases respectively. The mode of delivery is shown in Table IVGo. Overall, Caesarean section was performed in 32.5% of all deliveries.


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Table IV. Mode of delivery of children born with a gestational age of 24 weeks or more
 
Gestational age and birth weight
The gestational age and birth weight for singletons, twins and triplets with a gestational age of >=24 weeks are shown in Table VGo. The frequency of multiple births was 20.7%, including 34.7% of the children. Gestational age or weight was unknown in two singletons and one twin gestation. The frequencies of preterm birth and low birth weight are also indicated in Table VGo. All triplets were born with a gestational age of >=32 weeks and a birth weight <2500 g.


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Table V. Gestational age and birth weight in 730 ICSI children born with a gestational age of 24 weeks or more (600 deliveries)
 
Perinatal mortality
Among singletons, two cases of intrauterine death (one fetus with narrowing of the umbilical cord died in week 25, and one with placental fibrosis and signs of infection died in week 30) and one intrapartum death (placental abruption at term) without malformations were reported. Among twins, six intrauterine deaths occurred; four children with malformations (Table VIGo) and two without (prolonged amnion leakage after amniocentesis). The frequency of intrauterine or intrapartum death in children with a gestational age of >=24 weeks was 12.3 per 1000 children born. The perinatal mortality rate was 13.7 per 1000 children born.


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Table VI. Major birth defects in intrauterine or intrapartum deaths and liveborn children with a gestational age of 24 weeks or more, reported by the couples
 
Malformations
If parents reported that a child was not healthy at birth, discharge reports from the relevant departments were received in order to verify the diagnosis. Major birth defects among intrauterine deaths or stillbirths, and in liveborn infants with a gestational age of >=24 weeks, are listed in Table VIGo. Intrauterine or intrapartum death occurred in three singletons, and none of these had major malformations. Six cases of intrauterine or intrapartum death occurred in twins, and in four cases major malformations were described. The child with amniotic band syndrome died soon after delivery, and the child with deletion 13 died when aged 4.5 months.

The minor birth defects in liveborn children, as reported by the parents, are listed in Table VIIGo. In the cases where epididymal spermatozoa had been used, no birth defects have been reported. The embryos of the infants with amniotic band syndrome and short tongue frenulum have been cryopreserved.


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Table VII. Minor birth defects in liveborn children with a gestational age of 24 weeks or more, reported by the couples
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This follow-up study included all the fertility clinics in Denmark who carried out ICSI during the study period. The response rate was as high as 94%. Although the indication and laboratory data were approximately similar for the 6% who did not participate in the study compared with those who responded, the outcome of non-responders may be different.

An interesting finding was that, although a prenatal test was recommended to most couples, only 28.5% of women in the present study underwent a prenatal diagnosis compared with 55% in the Belgian study (Bonduelle et al., 1996bGo, 1998aGo). The mean age of the women in the two studies was similar. In Denmark, prenatal diagnosis is offered to women if they have a familial disposition, or if they are aged 35 years or more at the time of conception. In 1995, 11.2% of all Danish women who delivered had a prenatal genetic examination (Cytogenetic Central Registry, 1997Go), and 2.4% had a chromosomal abnormality. The national data showed that 57.6% of all women aged 35 years or more underwent a prenatal test, compared with 48.5% in the present study. Without doubt, the slightly elevated risk of miscarriage in relation to prenatal test prevented many of these infertile couples from having the test performed.

An abnormal prenatal karyotype was found in seven out of 209 karyotypes (3.4%). Six were major chromosome aberrations (2.9%), and four were related to maternal age >=35 years. Only one inherited structural aberration (0.5%) was found. Recently published results (Bonduelle et al., 1998aGo) include 1082 prenatal diagnoses, among whom were observed 28 aberrations (2.5%). Of these, 18 [1.66%; confidence interval (CI) 1.0–2.7] were de-novo chromosomal aberrations, of which nine were sex chromosome abnormalities and the others were autosomal aberrations. A further 10 cases of inherited aberrations (0.92%; CI 0.4–1.7) were observed, of which one was unbalanced. Compared with the percentage of aberration described in the literature (Nielsen and Wohlert, 1991Go; Jacobs et al., 1992Go), these figures demonstrated a statistically significant increase in sex chromosome aberrations, and in structural de-novo and inherited aberrations. In contrast, no sex chromosome aberrations were identified among the 209 prenatal and six postnatal karyotypes in this study. However, this may be due to the relatively small number of prenatal tests carried out in this study.

Several studies have shown that the fertilization rate and pregnancy rate after ICSI are approximately the same as after conventional IVF (Palermo et al., 1996Go; Govaerts et al., 1998Go; Silber, 1998Go; Tarlatzis and Bili, 1998Go). However, the microfertilization technique implies that the natural selection of the spermatozoa which fertilize the ovum is omitted, and as a consequence the risk of introducing a spermatozoon with a chromosome abnormality into the oocyte may be increased. Recently published studies have indicated that men with oligozoospermia have an increased risk of chromosome aberrations (Wilkins-Haug et al., 1997Go; Johnson, 1998Go; Tuerlings et al., 1998Go). In the Belgian study (Bonduelle et al., 1996bGo), the couples routinely had a karyotype carried out, and 1.9% of the fathers demonstrated a structural aberration. This was higher than the expected value of 0.5% identified from previous surveys of the general population (Nielsen and Wohlert, 1991Go; Jacobs et al., 1992Go), but was in accord with studies of males with infertility due to severe oligozoospermia (Johnson, 1998Go; Lundin et al., 1998Go). Although in Denmark only a minority of the males—at least at the start of the study period—had undergone a chromosome analysis before ICSI, there were still no more structural inherited chromosome aberrations found compared with the Belgian group.

The gestational age for ICSI pregnancies was 39.8 weeks for singletons and 36.8 weeks for twins. This is in accord with results from another Scandinavian ICSI follow-up study (Wennerholm et al., 1996Go), though these authors only included births with a gestational age >28 weeks. In the present study, preterm birth occurred in only 6.1% of singletons compared with 7.6% to 9.3% in previous studies (Wennerholm et al., 1996Go; Wisanto et al., 1996Go; Govaerts et al., 1998Go). In 1995, 4.7% of all singletons born in Denmark were delivered before 37 weeks gestation (Danish National Board of Health, 1997Go). Multiple pregnancies, which in the present study were reported in 20.7% with 1% triplets, are associated with a higher risk of preterm birth, and the prematurity rate was 35% in twins and 100% in triplets. These data are comparable with the National data from 1995 (Danish National Board of Health, 1997Go), where preterm births were found with frequencies of 39.9% in twins and 97.4% in triplets.

The mean birth weight for singletons (3483 g) was less than for the general population (3513 g), and low birth weight (6.7%) was more common among ICSI pregnancies than in the general population (3.7%). The higher rate of prematurity and lower birth weight for ICSI singletons may, at least partly, be caused by a lower rate of previous parity, increased maternal age and the cause of the underlying infertility. Birth weights below 2500 g in ICSI singletons were reported to occur in 5% to 10.3% of cases (Govaerts et al., 1998Go). On the other hand, the birth weight of twins (2568 g) and triplets (1992 g) born after ICSI are even higher than those cited in the National data from 1995 (Danish National Board of Health, 1997Go) (2524 g and 1907 g respectively). Of all ICSI twins, 37.6% weighed <2500 g at birth, compared with 40.8% among the general population. This is consistent with the slightly lower frequency of preterm birth.

The frequency of Caesarean section was higher in ICSI deliveries (32.5%) compared with all deliveries in Denmark in 1995 (12.6%) (Danish National Board of Health, 1997Go). In ICSI pregnancies, as well as in the general population, the rate of Caesarean section increased according to the number of children born (25.6% versus 12.6% in singletons, and 58.9% versus 50.6% in multiple births). Meanwhile, other ICSI studies have reported a Caesarean section rate of 21–26% in singletons and 41–57% in twins (Wennerholm et al., 1996Go; Wisanto et al., 1996Go; Govaerts et al., 1998Go), and have suggested that the high rate of Caesarean section was linked to the stress of the obstetrician.

In this study the perinatal mortality—which included all intrauterine or intrapartum deaths and early neonatal deaths with a gestational age of >=24 weeks—was 13.7 per 1000 children born, compared with a reported value of 17.1 per 1000 (Wisanto et al., 1996Go); however, the latter report included all stillbirths with a gestational age of >=26 weeks, and early neonatal deaths.

Whether there is an increased risk of birth defects following microfertilization has been discussed since the introduction of the technique. In the present study, major congenital malformations are conditions registered in ICD-10 with a Q00–Q99 diagnosis. The Belgian group (Bonduelle et al., 1996bGo, 1998bGo) and others (Palermo et al., 1996Go; Wennerholm et al., 1996Go; Tarlatzis and Bili, 1998Go), defined major malformations as a condition requiring surgical correction, or causing functional repair, and they reported a frequency of major malformation of between 1.0% and 2.6%. Re-analysis of the Belgian data, which included 420 liveborn infants (Kurinczuk and Bower, 1997Go), revealed that infants born after ICSI were twice as likely to have major birth defects as Western Australian infants. Meanwhile, over-reporting in the Belgian study may explain the results of the re-analysis (Bonduelle et al., 1997Go; Mitchell, 1997Go). When a recalculation was made of the prevalence of major malformations without cases of atrial septal defect, and which were of no clinical importance (Bonduelle et al., 1997Go), a frequency of major malformations of 5.23% was found, this being not significantly higher than in the Western Australian population (3.78%; odds ratio 1.41; CI 0.91–2.16). As for a previous study (Kurinczuk and Bower, 1997Go), we have classified all reported atrial septal defects as being a major birth defect, and found the rate of major malformations to be 2.2%. As the results of the present study primarily rely on information provided by the couples, it is reasonable to believe that major malformations were reported with a high degree of certainty, whereas minor malformations may not have been perceived as such by the parents. It is well known that malformations are discovered not only at birth but also in early childhood. Thus, as the follow-up period of the present study ranges from 1 to about 30 months, some major malformations may not be included, and this may result in under-reporting compared with the Western Birth Defects Registry in Australia (Bower et al., 1996Go), which includes birth defects diagnosed in children aged up to 6 years.

The results of this study on outcome of ICSI pregnancies are in line with earlier reports, except those involving sex chromosome abnormalities, where none was found. Although many thousands of ICSI children have been born (Tarlatzis and Bili, 1998Go), the number of infants in reported outcome studies is still small. It remains necessary to collect data in order to obtain more precise estimates, especially with regard to the genetic risk, malformation rate and developmental outcome.


    Acknowledgments
 
The authors thank The Ciconia Foundation for financial support.


    Notes
 
14 To whom correspondence should be addressed at: The Fertility Clinic, Rigshospitalet, University Hospital of Copenhagen, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on February 1, 1999; accepted on May 5, 1999.