1 Division of Reproductive Medicine and Gynecologic Endocrinology, Department of Gynecology and Obstetrics, University Hospital, Ratzeburger Allee 160, 23538 Lübeck and 2 Institute of Cancer Epidemiology and Institute for Social Medicine, Medical University of Lübeck, Lübeck, Germany
3 To whom correspondence should be addressed. e-mail: Ludwig_M{at}t-online.de
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Abstract |
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Key words: follow-up study/ICSI/pregnancy course/sperm count/surgical sperm retrieval
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Introduction |
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However, criticism has been raised with regard to the safety of this procedure. Even if there seems to be no increased risk of major malformations after ICSI compared with conventional IVF (Bonduelle et al., 2002; Hansen et al., 2002
), there might be an increased risk compared with naturally conceived children. This risk was described limited to defined malformations like hypospadias (Wennerholm et al., 2000a
; Ericson and Kallen, 2001
) or increased overall (Hansen et al., 2002
; Ludwig and Katalinic, 2002
). Until now, it has not be clarified whether this risk is due to the procedure itself or to certain risk factors of the parents. The latter seems more probable, since the malformation risk between children born after conventional IVF and ICSI was similar (Hansen et al., 2002
; Bonduelle et al., 2002
; Ludwig and Diedrich, 2002
). On the other hand, up to now no prospective randomized trial has been performed to study the outcome of children born after these procedures.
More data are needed to exclude the possibility that there might be a certain group of patients who have an increased risk during pregnancy, or a certain high risk group of children born after ICSI. To our knowledge, only three studies are available that have analysed larger cohorts of pregnancies with respect to the source of sperm for an ICSI procedure (Aytoz et al., 1998; Bonduelle et al., 1998
; Wennerholm et al., 2000b
). These studies have shown no difference between the use of ejaculated, epididymal or testicular sperm. However, recent reports have suggested that there might be an increased risk following ICSI with the use of testicular sperm for pregnancy complications (Wang et al., 2002
) or for imprinting errors in children born (Arney et al., 2002
; Cox et al., 2002
).
In this study, data have also been compared with a group of spontaneously conceived children who were examined in a similar manner (Ludwig and Katalinic, 2002). This comparison resulted in a relative risk (RR) of 1.25 [95% confidence interval (CI) 1.111.40], showing an increased risk of major malformations for a cohort of children conceived by ICSI, and in that publication the distribution and localization of malformations are described (Ludwig and Katalinic, 2002
).
In the present paper we have analysed the same prospectively collected database with respect to pregnancies after ICSI and differences between those pregnancies established following the use of sperm from ejaculates, testicular biopsies or epididymal aspirates.
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Materials and methods |
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The study was done prospectively. The study protocol was approved by the ethical committee of the Medical University of Lübeck. A total of 3198 couples, pregnant after an ICSI procedure and the transfer of fresh embryos, was recruited for participation before the 16th week of gestation and gave written informed consent. Patients, who received either a transfer with previously cryopreserved oocytes at the pronuclear stage or embryos, as well as those who have had additionally a transfer of embryos from conventional IVF in the same treatment cycle, were excluded. Origin of sperm (ejaculatory, epididymal, testicular) was not an exclusion criterion.
Recruitment was done beginning August 1998 and went up to August 2000. All the recruited couples were then contacted by telephone from the study secretariat beginning in the 16th week of gestation, to clarify whether pregnancy was still ongoing. If an ongoing pregnancy was confirmed by the couple, the patients were included in the study and the first data assessment was done.
A second contact was started in the 26th week of gestation in the case of a multiple pregnancy, and 28th week of gestation in singleton pregnancies. From that time-point on, attempts were made to contact each patient every 24 weeks until delivery. During these contacts the same questions were asked and updated each time.
After delivery the patients were contacted by telephone for the last time, and the data for the second contact sheet were completed. A date was then arranged for an examination within the first 8 weeks of life by one of the 25 participating doctors.
If patients could not be contacted despite several telephone calls, a written invitation was send out to remind them to contact the study secretariat. If patients could not be contacted at any point during the study course, either by several telephone calls or by at least two written invitations, they were excluded from the study.
Patients were asked to inform the study secretariat about any abnormality during the course of pregnancy, or if they had any questions regarding pregnancy, delivery, prenatal diagnosis etc. This contact was possible for 7 days a week. All these phone calls were answered by the same person (M.L.).
Data collection for study cohort
Data assessment during second contacts (up to delivery)
During the second contacts, data regarding pregnancy course were collected concerning regularity of pregnancy examination by the obstetrician (at least once a month), hospital stays during pregnancy (time-period, causes), number of ultrasound examinations, invasive prenatal diagnosis procedures and pathologies detected during these procedures. Furthermore all complications during pregnancies as well as medication intake, each coded according to a catalogue, were recorded. For medication intake, the number of days of intake as well as the time-period (1st, 2nd or 3rd trimester) were documented. Intravenous and oral tocolytics were recorded separately as well as the periconceptional use of folic acid.
The data from this second contact were updated at each phone call until the telephone contact after delivery had taken place. This allowed a prospective assessment of all these data. Only data regrading complications were analysed.
Procedure for examination of children and data assessment
Children in the study cohort were examined by one of 25 examiners, experienced in neonatology and/or medical genetics. The examination procedure was the same as described for the control cohort. Parents were asked to bring their children for examination within the first 8 weeks of life.
If parents did not agree after birth to have their children examined according to the procedure outlined above, they were contacted by telephone again. If they could not be convinced to contact one of the examiners, they were asked to send copies of the first three paediatric examinations, which are performed in a standardized way in Germany at birth (U1), during day 310 of life (U2), and during the 4th6th week of life (U3). In addition they were asked to provide data on a sonography of hips and kidneys.
If children were in a hospital after birth for a longer time-period, the examination was done in the hospital itself according to the standardized procedure as outlined above.
In the case of a late abortion or a stillbirth, the parents were asked to allow posthumous examination in a specialized pathology. They were offered a free-of-charge examination in a specialized unit of Pediatric Pathology (Prof. Müntefering, Department of Pediatric Pathology, Johannes Gutenberg University, Mainz, Germany). If pathological examination was done at another place, written information was requested from the pathologists by the study secretariat. Karyotyping was requested in as many cases of terminated pregnancies, late abortions and stillbirths as possible.
Major malformations were defined as structural defects of the body and/or the organs, which affect viability and quality of life requiring medical intervention (International Clearinghouse of Birth Defects Monitoring Systems, 1991; EUROCAT Report 7, 1997). Major malformation rate included all late spontaneous abortions, induced abortions, stillbirths and liveborn children.
Statistics
Mean and SD of continuous variables such as maternal age, week of gestation etc. were assessed for the three subgroups of sperm origin. Differences between these subgroups were analysed with KruskalWallis test and MannWhitney U-test for unpaired samples. For qualitative variables we used contingency tables with 2-test and calculated RR with 95% CI intervals. Percentage values also were provided with 95% CI. Statistical significance was assumed at P < 0.05. Data was recorded within a Microsoft Access database and evaluated with the personal computer-based program SPSS 10.0.7 (Chicago, IL, USA)
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Results |
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The outcome of pregnancies with regard to livebirths, stillbirths, pregnancy terminations and spontaneous abortions after 16th week of gestation are shown in Table I. The data are analysed with respect to the origin of sperm. No statistically significant differences were apparent.
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Discussion |
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Our database is reliable because all data were collected prospectively. Pregnancies were included in this study at a time-point when the further course of pregnancy could not be foreseen, i.e. in the 16th week of gestation.
On the other hand, this makes it impossible for us to estimate the rate of early abortions or the rate of ectopic pregnancies in these three cohorts, as was done by others (Aytoz et al., 1998; Wennerholm et al., 2000b
). These authors, however, could not find a correlation between the method of sperm retrieval and this kind of complication.
The findings of both groups can be confirmed with regard to pregnancy course. As in other studies, we found no significant difference between any of the conditions analysed (Aytoz et al., 1998; Bonduelle et al., 1998
; Wennerholm et al., 2000b
).
We focused on the risk of pre-eclampsia in women who became pregnant after the use of testicular sperm. These pregnancies were reported to have an increased risk of pre-eclampsia and pregnancy-induced hypertension compared with those conceived naturally or following conventional IVF or ICSI using ejaculated sperm (Wang et al., 2002). These authors hypothesized that a partner-specific immune maladaptation may be involved in the pathogenesis of this complication. This maladaptation might be more prevalent in those women who have not been exposed to their partners sperm compared with those, who had. In fact Wang et al. found an odds ratio of 2.10 (95% CI 1.306.32) for the risk of gestational hypertension and of 3.10 (1.596.73) for the risk of pre-eclampsia in those pregnancies which were established after the use of surgically obtained sperm compared with IVF pregnancies. This risk was not present for ICSI pregnancies after the use of ejaculated sperm with an odds ratio of 1.03 (0.751.40) for the risk of gestational hypertension and 1.02 (0.621.81) for pre-eclampsia. The data presented here include a higher number of pregnancies after use of ejaculated (n = 2944), testicular (n = 229) and epidiymal sperm (n = 26) compared with the study of Wang et al. (2002
) with 464 pregnancies following ICSI with ejaculated sperm and 82 pregnancies with surgically retrieved sperm. Furthermore, whereas our own data were collected prospectively within a standardized study, the data from Wang et al. were taken retrospectively from registries. Therefore our own estimation might be somewhat more reliable.
Regarding the risk of fetuses from pregnancies after ICSI, it might be hypothesized that those resulting from the use of non-ejaculated sperm might have a higher risk due to sperm immaturity (Arney et al., 2002; Cox et al., 2002
). Theoretically, it is possible that by the use of sperm from ejaculates with lower sperm counts, a higher number of chromosome abnormalities is transmitted to the offspring (Egozcue et al., 1997
; Sbracia et al., 2002
), resulting in a higher rate of late abortions, stillbirths and major malformations. A higher rate of major malformations was be correlated with the indication for ICSI, the method of sperm retrieval or the number of sperm in the ejaculate on the day of ICSI. This is in agreement with the results of Bonduelle et al. (2002
), who described a risk of major malformation following the use of ejaculated sperm (84/2477; 3.39%), epididymal sperm (4/105; 3.80%) and testicular sperm (6/206; 2.91%) in the same range.
However, we only had a limited number of chromosome analyses performed in our own cohort (25.1%) (Ludwig and Katalinic, 2002). Therefore the estimation of this risk in fetuses and newborns is of limited value. Furthermore, imprinting errors are very rare events with an incidence in the range of
1:30 000. A much larger cohort of children and, since some of the imprinting errors are made manifest later in life such as nephroblastoma, a longer follow-up, would be necessary for a reliable evaluation. Thus case reports are not really helpful to estimate the risk of ICSI offspring. According to the presented data a substantial risk seems not to be present.
Overall, the risk of a major malformation was similar in all three groups (Table III). It neither depended on the indication for ICSI (Table IV) nor on the number of sperm in the ejaculate (Figure 1). This is in agreement with other studies on this topic (Aytoz et al., 1998; Bonduelle et al., 1998
; Wennerholm et al., 2000b
). A recent report from French registries, which has shown a significantly increased major malformation risk following the use of testicular sperm (6.48%) compared with the use of epididymal sperm (2.38%) or ICSI overall (3.17%) (P = 0.0001) in a retrospective analysis (Bajirova et al., 2001
), therefore cannot be confirmed.
Aytoz et al. (1998) were able to perform an analysis not only with respect to the number of sperm in the ejaculate, but also according to other factors. They differentiated between sperm with <20% progressive motility,
4% normal morphology according to Krugers strict criteria, and a sperm count of <5x106 sperm/ml. In those pregnancies established after the use of ejaculated sperm showing at least two of these abnormalities, the rate of intrauterine death was increased compared with the other groups (Aytoz et al., 1998
). Such an analysis could not be done in this study, since we only had data regarding the number of sperm available. However, we found no differences between the three groups in the rate of stillbirths (Table I) or in the rates of perinatal deaths (Table II).
The data for children born in the three groups were similar and not significantly different from each other. We could not confirm previous results of others, who had described a significantly higher rate of very low birthweight in the group of pregnancies achieved after use of epididymal sperm (Aytoz et al., 1998). However, this group was quite underrepresented in our cohort, since the use of testicular sperm is the most established method in cases of obstructive or non-obstructive azoospermia in Germany.
The data regarding pregnancy course and complications are reassuring in so far as sperm used for ICSI does not seem to have a negative influence. However, the rates of very low birthweight in 3, 10 and 45% in singletons, twins and triplets respectively illustrate the substantial risk which is carried by these multiple pregnancies. It underlines the higher risk of suffering from neurological sequelae following birth involving multiples and suggests that this may not be from the method of conception (Strömberg et al., 2002).
In conclusion, our prospectively collected data confirm previous studies in the field which could not find a risk associated with the use of epididymal or testicular sperm compared with ejaculated sperm. The course of pregnancy, pregnancy outcome and birth data of children born were not affected. For certain defined risks, however, larger databases with a similar quality would be necessary to be reliably evaluated and compared with naturally conceived children.
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Acknowledgements |
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The standardized examination of the children was done by 25 specialists in paediatrics and/or medical genetics (ordered by zip code): Professor Dr U.G.Froster, Institut for Human Genetics, University of Leipzig; Dr J.Sander, Department of Neonataloy, Clinic for Paediatrics, University of Jena; Professor Dr J.Kunze, Division of Medical Genetics, Department of Paediatrics, Charité, Berlin; Priv. Doz. Dr P.Meinecke, Department of Medical Genetics, Altonaer Kinderkrankenhaus, Hamburg; Dr M.Kohl, Department of Paediatrics, Medical University of Lübeck; Dr S.Spranger, Praxis for Medical Genetics, Bremen; Dr B.Bohnhorst, Paediatric Pulmonology and Neonatology, Medical University Hannover; Priv. Doz. Dr K.Harms and Priv. Doz. Dr E.Herting, Department of Paediatrics, University Hospital, Göttingen; Dr C.Rösch, Malformation Registry Sachsen-Anhalt, Medical Faculty of O.-v.-Guericke University, Magdeburg; Professor Dr F.Majewski, Institut for Human Genetics, HeinrichHeineUniversity, Düsseldorf; Priv. Doz. Dr G.Gillessen-Kaesbach, Institut for Human Genetics, University Clinic Essen; Dr D.Föll, Department of Paediatrics, University Hospital Münster; Dr N.Kau, Department of Neonatology, Paediatrics, University Hospital Bonn; Priv. Doz. Dr A.Queißer-Luft and Dr G.Stolz, Deparment of Paediatrics, Johannes-Gutenberg University, Mainz; Dr R.König, Institut for Human Genetics, Frankfurt; Professor Dr J.Mücke, St Ingbert; Prof. Dr G.Tariverdian, Department of Medical Genetics, RuprechtsKarlsUniversity, Heidelberg; Dr U.Mau, Department of Medical Genetics, University of Tübingen; Dr R.Hentschel, Department of Paediatrics, University Hospital, Freiburg; Dr I.Rost, Department of Medical Genetics, Paediatric Hospital, Dr von Haunersches Kinderspital, Munich; Dr Seidel, Department of Paediatric Genetics; LudwigMaximiliansUniversity, Munich; Dr E.Rossier, Department of Medical Genetics, University Hospital Ulm; Dr A.Rauch, Institute for Human Genetics, Friedrich-Alexander-University, Erlangen.
The main sponsor of the study was Serono GmbH. Additional sponsors were Organon GmbH, ASTA Medica AG, Ferring Arzneimittel GmbH, Gück Zellkulturbedarf GmbH, Takeda Pharma GmbH, Gynemed Medizinprodukte GmbH and Co. KG, MTG Medical Technology Vertriebs GmbH, and OCTAX Microscience GmBH. There was a personal grant to Dr Ludwig from the Rockstroh Stiftung of the Deutsche Gesellschaft für Gynäkologie und Geburtshilfe. The study was also sponsored to a substantial part by IVF centres in Germany themselves.
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References |
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Submitted on July 15, 2002; resubmitted on August 20, 2002; accepted on October 9, 2002.