DÄ internationalArchive1-2/2008The Risk of Malformation Following Assisted Reproduction

Original article

The Risk of Malformation Following Assisted Reproduction

Dtsch Arztebl Int 2008; 105(1-2): 11-7. DOI: 10.3238/arztebl.2008.0011

Bertelsmann, H; Gomes, H d C; Mund, M; Bauer, S; Matthias, K

Introduction: Intracytoplasmatic sperm injection (ICSI) is currently the most frequently used human reproductive technology in Germany. ICSI was introduced as routine, insurance-funded medical care in 2002 by the Federal Joint Committee. A re-evaluation of published literature on malformation rates in children born of ICSI pregnancies within a period of 3 years formed part of the committee's decision. The analysis investigated whether ICSI increases the risk of malformation in the offspring, compared to in vitro fertilization (IVF) and natural conception.
Methods: Systematic literature review.
Results: 929 studies were identified. 3 meta-analyses, 15 studies investigating malformations, and 12 studies analyzing imprinting disorders were included. The risk of malformation was not significantly different in 9 studies comparing ICSI versus IVF. 2 meta-analyses and 3 of 8 cohort studies and retrospective analysis showed significantly more severe malformations after assisted reproduction than after natural conception. The remaining 5 studies displayed no significant results. Current evidence does not show a higher risk of major malformations in the offspring resulting from the use of ICSI compared to IVF. However, there is evidence that both techniques increase the risk for major malformations considerably, compared to natural conception, and further research is needed. The validity of the results is low since the studies were heterogeneous and the cohorts used in the studies had limited comparability. Dtsch Arztebl Int 2008; 105(1–2): 11–7
DOI: 10.3238/arztebl.2008.0011

Key words: assisted reproduction, congenital malformation, intracytoplasmic sperm injection, in vitro fertilization, literature review
LNSLNS It is estimated that 10% to 15% of all couples in Europe are childless despite a desire to have children (1). Some of these couples can achieve pregnancy only with the aid of artificial fertilization. Intracytoplasmic sperm injection (ICSI) has been performed in Germany since the early 1990's: this technique increases the chance that a couple will be able to have a child, particularly if it is the male partner that is infertile. It involves the injection of a single spermatozoon through a needle into the oocyte.

Artificial fertilization techniques have been included in the catalog of procedures covered by statutory health insurance in Germany (gesetzliche Kran­ken­ver­siche­rung, GKV) since 1990. The German Joint Federal Committee (JFC) has been given the task of writing guidelines regarding the medical details of these techniques in reproductive medicine, including the preconditions for their use and the manner and extent of the interventions to be performed. ICSI was incorporated into the GKV procedures catalog in 2002 (diagram 1 gif ppt).

ICSI became the most common method of artificial fertilization shortly after it was introduced; other methods include artificial insemination and in vitro fertilization (IVF). From 2004 onward, couples have been required to pay half of the cost of artificial fertilization themselves. Diagram 2 (gif ppt) shows the falloff in case numbers after the 50% co-payment rule was introduced. The introduction of ICSI gave rise to concern that this procedure might cause a higher rate of congenital malformations than in vitro fertilization or spontaneous (natural) conception. There are essentially three factors associated with assisted reproductive methods that might influence the malformation rate:

- chromosomal anomalies in the gametes used,
- medications used to stimulate fertility,
- and the injection of the sperm cell into the egg cell in ICSI.

Injection of a sperm cell into an egg cell circumvents natural selection at the egg cell membrane, damages the egg cell itself, and introduces foreign material (e.g. culture medium) into the cell.

There are only a few reports in the literature about the potentially damaging effects of stimulation on mature egg cells; the connection between spermiogram parameters and the occurrence of chromosomal anomalies is better documented (2). In recent years, a number of so-called imprinting defects have been diagnosed in a small number of children conceived by ICSI. Imprinting defects arise through a disturbance of epigenetic processes during gametogenesis and/or the preimplantation phase of the fertilized egg (3).

Congenital malformations are inborn structural or functional abnormalities. They can be of mono- or polygenic origin or they may be due to external influences such as teratogenic medications or ionizing radiation. They may also be of multifactorial (genetic and external) origin. In most cases, the cause remains unknown. International classifications distinguish major from minor malformations. There is a problem, however, in that the definitions currently in use are not uniformly applied. A possible solution is provided by the classification system of the European Register of Congenital Anomalies (EUROCAT), in which major malformations are defined as physical abnormalities that are incompatible with life, require surgical correction, and/or cause functional impairment. Such malformations can place major burdens on the affected children and their parents. One-quarter of all deaths in childhood are associated with major congenital malformations (6).

In view of these facts, the JFC stipulated, when ICSI was introduced, that the rate of congenital malformations among children conceived by this method should be assessed three years after its introduction. Thus, the question whether children conceived by ICSI have more congenital malformations than those conceived naturally or by IVF was investigated by a systematic evaluation of current data.

Methods
Scientific publications in the PubMed, Cancerlit, and Embase databases were systematically searched in October 2005, and the search was updated in May 2006. The search strategy consisted of search terms related to intracytoplasmic sperm injection, in vitro fertilization, and major malformations. A particular difficulty complicating the task of assessing the risks of these types of treatment is the impossibility of performing randomized, controlled studies. The search was therefore restricted to cohort and case-control studies.

The relevant publications were identified by a two-step search by two assessors working independently – all of the authors of this article participated in the evaluation – and then filtered according to previously determined inclusion and exclusion criteria. Studies were included that compared the rate of malformations in a cohort of ICSI children with that in a cohort of IVF or naturally conceived children. As a further requirement for inclusion the odds ratio (OR) for major malformations had to be either directly stated in the study or else calculable from the reported study data. Independently of these criteria, studies reporting imprinting defects were also included.

Data were systematically extracted from the sources selected in this way (for the manner of data extraction, see www.g-ba.de/informationen/abschlussberichte). To identify systematic distortions in these publications, we looked in each publication for information about the use of prenatal diagnosis and about selective and spontaneous abortions and stillbirths, and we assessed the degree of comparability of the cohorts in each study (diagram 3 gif ppt).

Findings of the systematic literature review
The literature search yielded three meta-analyses (57) and 15 primary studies (822) from which the OR and the 95% confidence interval (CI) (box 1 gif ppt) could be determined for the comparison of ICSI versus IVF and/or ICSI versus natural conception. These figures were either directly stated in the publications or could be calculated from the reported data. All of these studies were cohort studies. 12 further primary studies (e1e12) dealt with the subject of diseases traceable to imprinting defects.

Meta-analyses
Among the three meta-analyses that were found, only the one by Lie et al. (5) compared the rate of malformations among children conceived by ICSI with that among children conceived by IVF, while the meta-analyses by Rimm et al. (6) (including 7 studies) and by Hansen et al. (7) (including 25 studies) compared the rates of malformations in IVF- and ICSI-children with the rates in naturally conceived children. In both of the latter two studies, the malformation rates in IVF- and ICSI-children were significantly higher than that among naturally conceived children, with odds ratios of 1.4 (95% CI: 1.28 to 1.53) (7) and 1.29 (95% CI: 1.01 to 1.67), respectively. The meta-analysis by Lie et al. (5) involved four systematically selected clinical studies comparing ICSI cohorts with IVF cohorts with respect to the rate of major malformations, including a total of 5 395 children conceived by ICSI and 13 085 children conceived by IVF. There was no significant elevation of risk for ICSI as compared to IVF, with a relative risk of 1.12 (95% CI: 0.97 to 1.28).

A common weakness of all three meta-analyses is the lack of information about the classification of malformations used in the studies that they included, or about the method of clinical examination of the children. The individual studies included in the analyses of Hansen et al. (7) and Rimm et al. (6) were quite heterogeneous with respect to study design and results. Nor can it be excluded that various sources of error in the individual studies led to distorted results in the meta-analyses.

Having found that the meta-analyses failed to answer the question adequately, we also examined the relevant primary studies directly. Six of the primary studies that we selected had been included in at least one of the meta-analyses.

Primary studies
Study characteristics and assessment of the evidence
We identified a total of 15 studies that allowed a comparison of the rates of major malformations in a cohort of children conceived by ICSI and in another cohort of children conceived either by IVF (9 studies) or naturally (8 studies) (comparisons were made with both types of cohort in two of these studies). Kuwata et al.’s small study (14) was performed exclusively on twins and is therefore not shown in the diagrams together with the other studies (see table gif ppt).

There was a notable degree of variation across studies in the rate of malformations in each type of cohort – ICSI, IVF, and naturally conceived children. The malformation rate for the ICSI cohorts ranged from 1% to 13%, for the IVF cohorts from 2% to 9%, and for naturally conceived children from 2% to 13%. The mean baseline risk in the naturally conceived cohorts was 4.9% (standard deviation ±2.9%). The malformation rates in the individual studies can be found in the table. Because of this wide heterogeneity in both study design and study results, we refrained from performing a meta-analysis for this article, instead choosing to compare the odds ratios with each other descriptively.

The risk of malformations in children conceived by ICSI versus IVF
Nine of the 15 primary studies addressed this question. In four of them, the ICSI and IVF cohorts were both observed prospectively; in two of them, a prospective ICSI cohort was compared with a retrospective IVF control cohort. Six of the 15 primary studies were purely retrospective cohort studies, and the remaining three exclusively involved registry data and were therefore classified as retrospective studies with secondary data. The informational value of these primary studies was limited because of their mostly small size, imperfect comparability of the cohorts, and multiple testing. Diagram 4 (gif ppt) shows the OR and CI as a function of the size of the cohorts (depicted on a logarithmic scale); the studies were numbered in decreasing order of size.
As seen in diagram 4, the confidence interval for the odds ratio includes 1.0 in all studies, i.e., no significant difference was found in any study. Six studies showed an odds ratio less than 1.0, i.e., a lower risk of malformations for children conceived by ICSI compared with those conceived by IVF. In three studies, however, the odds ratio was greater than 1.0, corresponding to a higher risk of malformations after ICSI than after IVF. Two studies that were larger by far than the remaining 13 studies, each involving more than 1000 children conceived by ICSI, both belonged to the first group and yielded very similar results: the odds ratios in these two studies were 0.84 and 0.89, i.e., values very near 1.0 indicating no significant differences between the two groups. There is thus no evidence for a higher rate of malformations among children conceived by ICSI compared to children conceived by IVF.

The risk of malformations in children conceived by ICSI versus naturally conceived children
Eight of the 15 primary studies addressed this question. Diagram 5 (gif ppt) shows the maximally adjusted odds ratios with their 95% confidence intervals as a function of the size of the ICSI cohort (depicted on a logarithmic scale). While the ICSI cohort of most studies was observed prospectively, the cohorts of naturally conceived children were often obtained retrospectively and sometimes drawn from secondary data sources. Thus, it is highly likely that the cohorts differed from each other not just in the mode of conception, but also in other relevant risk factors for major congenital malformations. Only a few of the studies provided adequate data to control for known risk factors such as maternal age, malformations in siblings, or maternal alcohol use during gestation (see table) and the validity of the results is therefore questionable (diagram 5). The results with regard to the question of ICSI versus natural conception are much more heterogeneous than those for the comparison of ICSI with IVF; this is presumably due to a large degree of distortion of the results by other risk factors for malformations that are differently distributed in normally fertile couples and in infertile couples trying to have children.

In three of the eight studies, the risk of major malformations was found to be significantly higher in children conceived by ICSI than in naturally conceived children. The study with by far the largest prospective cohort, compared to a retrospective control group, was among the three studies showing a significant elevation of risk (13). In this largest study, the malformation rate was 8.7% in the ICSI cohort and 6.1% in the control group. After adjustment for other factors, the odds ratio was found to be 1.24 with a 95% confidence interval of 1.02 to 1.5. Among the five studies in which statistically insignificant differences were found, the rate of malformations in the ICSI cohort was higher than that in the control group in three, and lower in two. All in all, these results point toward a possibly higher risk among children conceived by ICSI, but no firm conclusions can be drawn because of the heterogeneity of the findings and the high likelihood that they have been distorted by selection and confounding variables.

Imprinting defects
Imprinting is the process of activation and deactivation of parental genes during the development and fusion of germ cells. In recent years, a number of reports have been published with initial evidence of a possible association between artificial fertilization and an increased frequency of certain rare diseases that might be due to imprinting defects.

The diseases that are most commonly mentioned in this context are Beckwith-Wiedemann syndrome (BWS) and Angelman syndrome (AS); others are Prader-Willi syndrome, retinoblastoma, and further rare syndromes such as Silver-Russell syndrome (box 2 gif ppt).

The 12 publications that we found on imprinting defects (e1e12) mostly dealt with uncontrolled, retrospective studies and single case reports or case series. Because of their small size and their liability to distortions of many different kinds, these types of study are not suitable for a demonstration of causation, yet they may provide clues toward possible associations and thus enable the formulation of hypotheses to be tested. These 12 publications provided no evidence of a difference between ICSI and IVF with regard to malformation rates, but they do arouse suspicion of a possible connection between artificial fertilization and BWS, AS, and retinoblastoma. This question will need to be addressed in future studies.

Discussion
The variability of the reported malformation rates was notably high. Reported malformation rates depend on the system of classification used as well as on the time at which the children are examined, the examination method (clinical alone or with ancillary testing), and the qualifications of the examiner. The reported study results seem to depend largely on the degree of care with which procedures susceptible to selection bias were reported, the application of definitions, and the methods of calculating the malformation rate from the data that were obtained. The sources differ considerably from one another in all of these respects. The malformation rate can be heavily influenced, e.g., by selection before embryo transfer, as well as by the induction of abortion after pathological findings are made in prenatal diagnosis.

The rate of malformations after assisted fertilization is low in any case in all of the studies that were analyzed. It follows that differing designations of various types of malformation as major or minor can bring about vast differences in the final results. Furthermore, most studies reported the malformation rates as a fraction of live births only, while failing to consider malformations among stillbirths and induced abortions as recommended by EUROCAT. No final conclusion about malformation rates after artificial fertilization can be drawn until large prospective cohort studies are performed whose procedures and definitions are transparently reported.

Conclusion
After our review of the relevant literature, it seems unlikely that conception by ICSI significantly increases the risk of malformations compared to conception by IVF. This, in turn, should lessen concern about the possible induction of congenital malformations by manipulation of the oocyte, which is a risk inherent to the technique of ICSI. We cannot rule out a possible elevation of the risk of major malformations by ICSI as compared to natural conception, but this concern applies equally to IVF. Our review of the meta-analyses on this question yielded fairly consistent findings of an elevation of the risk by about 30%, with odds ratios of 1.4 (95% CI, 1.28 to 1.53) (7) and 1.29 (95% CI, 1.01 to 1.67) (6). These figures should be interpreted with caution, however, because biases of various types in the primary studies cannot be excluded. These results might reflect a real, technique-dependent risk due to gamete manipulation, but they might also reflect differences in the primary risks of the parents. More research is needed in this area. These findings imply that, when prospective parents are counseled about assisted reproductive techniques, they should be informed that a possible elevation of the rate of congenital malformations as compared to natural conception is of some concern not just for ICSI, but for IVF as well.

No firm conclusion can be drawn from the sparse publications in recent years that have addressed the question of possible associations between ICSI and/or IVF and certain rare diseases that are thought to be due to so-called imprinting defects. A possible elevation of risk by artificial fertilization cannot be excluded at present. Therefore, the occurrence of these imprinting defects should be observed further. The Health Technology Assessment report "Fehlbildungsrisko der mit der Methode der ICSI gezeugten Kinder in Vergleich zu IVF- beziehungsweise natürlich konzipierten Kindern" (The risk of malformations in children conceived by ICSI compared to those conceived naturally or by IVF) will be released by the Joint Federal Committee and will be available at www.g-ba.de/informationen/abschlussberichte.

Conflict of interest statement
The authors state that no conflict of interest exists as defined by the guidelines of the International Committee of Medical Journal Editors.

Manuscript received on 5 April 2007; revised version accepted on 9 October 2007.

Translated from the original German by Ethan Taub, M.D.


Corresponding author
Gemeinsamer Bundes­aus­schuss
Prof. Dr. rer. nat. Hilke Bertelsmann
Auf dem Seidenberg 3a
53721 Siegburg, Germany
hilke.bertelsmann@g-ba.de


For e-references please refer to:
www.aerzteblatt-international.de/ref0108
1.
Schirren C et al.: Unerfüllter Kinderwunsch – Leitfaden Reproduktionsmedizin für die Praxis. Köln: Deutscher Ärzte-Verlag 2003; 3. Auflage.
2.
Seifer I, Amat S, Delgado-Viscogliosi P, Boucher D, Bignon YJ: Screening for microdeletions on the long arm of chromosome Y in 53 infertile men. Int J Androl. 1999; 22:148–54. MEDLINE
3.
De Rycke M, Liebaers I, Van Steirteghem A: Epigenetic risks related to assisted reproductive technologies. Hum Reprod 2002; 17: 2487–94. MEDLINE
4.
Queißer-Luft A, Spranger J: Fehlbildungen bei Neugeborenen. Dtsch Arztebl 2006; 103(38): A 2464–71. VOLLTEXT
5.
Lie RT, Lyngstadaas A, Orstavik KH, Bakketeig LS, Jacobsen G, Tanbo T: Birth defects in children conceived by ICSI compared with children conceived by other IVF-methods; a meta-analysis. Int J Epidemiol 2005; 34: 696–701. MEDLINE
6.
Rimm AA, Katayama AC, Diaz M, Katayama KP: A meta-analysis of controlled studies comparing major malformation rates in IVF and ICSI infants with naturally conceived children. J Assist Reprod Genet 2004 ; 21: 437–43. MEDLINE
7.
Hansen M, Bower C, Milne E, de KN, Kurinczuk JJ: Assisted reproductive technologies and the risk of birth defects – A systematic review. Hum Reprod 2005; 20: 328–38. MEDLINE
8.
Bonduelle M, Liebaers I, Deketelaere V et al.: Neonatal data on a cohort of 2 889 infants born after (1991–1999) and of 2 995 infants born after IVF (1983–1999). Hum Reprod 2002; 17: 671–94. MEDLINE
9.
Bonduelle M, Bergh C, Niklasson A, Palermo GD, Wennerholm UB: Medical follow-up study of 5-year-old children. Reprod Biomed Online 2004; 9: 91–101. MEDLINE
10.
Bonduelle M, Wennerholm UB, Loft A et al.: A multi-centre cohort study of the physical health of 5-year-old children conceived after intracytoplasmic sperm injection, in vitro fertilization and natural conception. Hum Reprod 2005; 20: 413–9. MEDLINE
11.
Bowen JR, Gibson FL, Leslie GI, Saunders DM: Medical and developmental outcome at 1 year for children conceived by intracytoplasmic sperm injection. Lancet 1998; 351: 1529–34. MEDLINE
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Hansen M, Kurinczuk JJ, Bower C, Webb S: The risk of major birth defects after intracytoplasmic sperm injection and in vitro fertilization. N Engl J Med 2002; 346: 725–30. MEDLINE
13.
Katalinic A, Rosch C, Ludwig M: Pregnancy course and outcome after intracytoplasmic sperm injection: a controlled, prospective cohort study. Fertil Steril 2004; 81: 1604–16. MEDLINE
14.
Kuwata T, Matsubara S, Ohkuchi A et al.: The risk of birth defects in dichorionic twins conceived by assisted reproductive technology. Twin Res 2004; 7: 223–7. MEDLINE
15.
Ludwig M, Katalinic A: Malformation rate in fetuses and children conceived after ICSI: results of a prospective cohort study. Reprod Biomed Online 2002; 5: 171–8. MEDLINE
16.
Olson CK: In vitro fertilization is associated with an increase in major birth defects. Fertil Steril 2005; 84: 1308–15. MEDLINE
17.
Palermo GD, Neri QV, Hariprashad JJ, Davis OK, Veeck LL, Rosenwaks Z: ICSI and its outcome. Semin Reprod Med 2000; 18: 161–9. MEDLINE
18.
Place I, Englert Y: A prospective longitudinal study of the physical, psychomotor, and intellectual development of singleton children up to 5 years who were conceived by intracytoplasmic sperm injection compared with children conceived spontaneously and by in vitro fertilization. Fertil Steril 2003; 80: 1388–97. MEDLINE
19.
Sutcliffe AG, Taylor B, Saunders K, Thornton S, Lieberman BA, Grudzinskas JG: Outcome in the second year of life after in-vitro fertilisation by intracytoplasmic sperm injection: a UK case-control study. Lancet 2001; 357: 2080–4. MEDLINE
20.
Sutcliffe AG, Saunders K, McLachlan R et al.: A retrospective case-control study of developmental and other outcomes in a cohort of Australian children conceived by intracytoplasmic sperm injection compared with a similar group in the United Kingdom. Fertil Steril 2003; 79: 512–6. MEDLINE
21.
Van Golde R, Boada M, Veiga A, Evers J, Geraedts J, Barri P: A retrospective follow-up study on intracytoplasmic sperm injection. J Assist Reprod Genet 1999; 16: 227–32. MEDLINE
22.
Westergaard HB, Johansen AM, Erb K, Andersen AN: Danish national in-vitro fertilization registry 1994 and 1995: a controlled study of births, malformations and cytogenetic findings. Hum Reprod 1999; 14: 1896–902 MEDLINE
e1.
Chang AS, Moley KH, Wangler M, Feinberg AP, Debaun MR: Association between Beckwith-Wiedemann syndrome and assisted reproductive technology: a case series of 19 patients. Fertil Steril 2005; 83: 349–54. MEDLINE
e2.
Cox GF, Bürger J, Lip V et al.: Intracytoplasmic sperm injection may increase the risk of imprinting defects. Am J Hum Genet 2002; 71: 162–4. MEDLINE
e3.
DeBaun MR, Niemitz EL, Feinberg AP: Association of in vitro fertilization with Beckwith-Wiedemann syndrome and epigenetic alterations of LIT1 and H19. Am J Hum Genet 2003; 72: 156–60. MEDLINE
e4.
Gicquel C, Gaston V, Mandelbaum J, Siffroi JP, Flahault A, Le Bouc Y: In vitro fertilization may increase the risk of Beckwith-Wiedemann syndrome related to the abnormal imprinting of the KCNQ1OT gene. Am J Hum Genet 2003; 72: 1338–40. MEDLINE
e5.
Halliday J, Oki K, Breheny S, Algar E, Amor DJ: Beckwith-Wiedemann-syndrome and IVF: A case control study. Am J Hum Genet 2004; 75: 526–8. MEDLINE
e6.
Lidegaard O, Pinborg A, Andersen AN: Imprinting diseases and IVF: Danish National IVF cohort study. Hum Reprod 2005; 20: 950–4. MEDLINE
e7.
Ludwig M, Katalinic A, Groß S, Sutcliffe A, Varon R, Horsthemke B: Increased prevalence of imprinting defects in patients with Angelman syndrome born to subfertile couples. J Med Genet 2005; 42: 289–91. MEDLINE
e8.
Maher ER, Afnan M, Barratt CL: Epigenetic risks related to assisted reproductive technologies: Epigenetics, imprinting, ART and icebergs? Hum Reprod 2003; 18: 2508–11. MEDLINE
e9.
Manning M, Lissens W, Bonduelle M et al.: Study of DNA-methylation patterns at chromosome 15q11-q13 in children born after ICSI reveals no imprinting defects. Mol Hum Reprod 2000; 6: 1049–53. MEDLINE
e10.
Moll AC, Imhof SM, Cruysberg JRM, Schouten-van Meeteren AYN, Boers M, Van Leeuwen FE: Incidence of retinoblastoma in children born after in-vitro fertilisation. Lancet 2003; 361: 309–10. MEDLINE
e11.
Orstavik KH, Eiklid K, van der Hagen CB et al.: Another case of im-printing defect in a girl with Angelman syndrome who was conceived by intracytoplasmic sperm injection. Am J Hum Genet 2003; 72: 218–9. MEDLINE
e12.
Yoon G, Beischel LS, Johnson JP, Jones MC: Dizygotic twin pregnancy conceived with assisted reproductive technology associated with chromosomal anomaly, imprinting disorder, and monochorionic placentation. J Pediatr 2005; 146: 565–7. MEDLINE
Gemeinsamer Bundes­aus­schuss, Siegburg: Prof. Dr. rer. nat. Bertelsmann
Kassenärztliche Bundesvereinigung, Berlin: Dr. med. de Carvalho Gomes, Dr. med. Mund
Medizinischer Dienst der Spitzenverbände der Krankenkassen, Essen: Dr. med. Bauer
AOK-Bundesverband, Bonn: Dr. med. Matthias
1. Schirren C et al.: Unerfüllter Kinderwunsch – Leitfaden Reproduktionsmedizin für die Praxis. Köln: Deutscher Ärzte-Verlag 2003; 3. Auflage.
2. Seifer I, Amat S, Delgado-Viscogliosi P, Boucher D, Bignon YJ: Screening for microdeletions on the long arm of chromosome Y in 53 infertile men. Int J Androl. 1999; 22:148–54. MEDLINE
3. De Rycke M, Liebaers I, Van Steirteghem A: Epigenetic risks related to assisted reproductive technologies. Hum Reprod 2002; 17: 2487–94. MEDLINE
4. Queißer-Luft A, Spranger J: Fehlbildungen bei Neugeborenen. Dtsch Arztebl 2006; 103(38): A 2464–71. VOLLTEXT
5. Lie RT, Lyngstadaas A, Orstavik KH, Bakketeig LS, Jacobsen G, Tanbo T: Birth defects in children conceived by ICSI compared with children conceived by other IVF-methods; a meta-analysis. Int J Epidemiol 2005; 34: 696–701. MEDLINE
6. Rimm AA, Katayama AC, Diaz M, Katayama KP: A meta-analysis of controlled studies comparing major malformation rates in IVF and ICSI infants with naturally conceived children. J Assist Reprod Genet 2004 ; 21: 437–43. MEDLINE
7. Hansen M, Bower C, Milne E, de KN, Kurinczuk JJ: Assisted reproductive technologies and the risk of birth defects – A systematic review. Hum Reprod 2005; 20: 328–38. MEDLINE
8. Bonduelle M, Liebaers I, Deketelaere V et al.: Neonatal data on a cohort of 2 889 infants born after (1991–1999) and of 2 995 infants born after IVF (1983–1999). Hum Reprod 2002; 17: 671–94. MEDLINE
9. Bonduelle M, Bergh C, Niklasson A, Palermo GD, Wennerholm UB: Medical follow-up study of 5-year-old children. Reprod Biomed Online 2004; 9: 91–101. MEDLINE
10. Bonduelle M, Wennerholm UB, Loft A et al.: A multi-centre cohort study of the physical health of 5-year-old children conceived after intracytoplasmic sperm injection, in vitro fertilization and natural conception. Hum Reprod 2005; 20: 413–9. MEDLINE
11. Bowen JR, Gibson FL, Leslie GI, Saunders DM: Medical and developmental outcome at 1 year for children conceived by intracytoplasmic sperm injection. Lancet 1998; 351: 1529–34. MEDLINE
12. Hansen M, Kurinczuk JJ, Bower C, Webb S: The risk of major birth defects after intracytoplasmic sperm injection and in vitro fertilization. N Engl J Med 2002; 346: 725–30. MEDLINE
13. Katalinic A, Rosch C, Ludwig M: Pregnancy course and outcome after intracytoplasmic sperm injection: a controlled, prospective cohort study. Fertil Steril 2004; 81: 1604–16. MEDLINE
14. Kuwata T, Matsubara S, Ohkuchi A et al.: The risk of birth defects in dichorionic twins conceived by assisted reproductive technology. Twin Res 2004; 7: 223–7. MEDLINE
15. Ludwig M, Katalinic A: Malformation rate in fetuses and children conceived after ICSI: results of a prospective cohort study. Reprod Biomed Online 2002; 5: 171–8. MEDLINE
16. Olson CK: In vitro fertilization is associated with an increase in major birth defects. Fertil Steril 2005; 84: 1308–15. MEDLINE
17. Palermo GD, Neri QV, Hariprashad JJ, Davis OK, Veeck LL, Rosenwaks Z: ICSI and its outcome. Semin Reprod Med 2000; 18: 161–9. MEDLINE
18. Place I, Englert Y: A prospective longitudinal study of the physical, psychomotor, and intellectual development of singleton children up to 5 years who were conceived by intracytoplasmic sperm injection compared with children conceived spontaneously and by in vitro fertilization. Fertil Steril 2003; 80: 1388–97. MEDLINE
19. Sutcliffe AG, Taylor B, Saunders K, Thornton S, Lieberman BA, Grudzinskas JG: Outcome in the second year of life after in-vitro fertilisation by intracytoplasmic sperm injection: a UK case-control study. Lancet 2001; 357: 2080–4. MEDLINE
20. Sutcliffe AG, Saunders K, McLachlan R et al.: A retrospective case-control study of developmental and other outcomes in a cohort of Australian children conceived by intracytoplasmic sperm injection compared with a similar group in the United Kingdom. Fertil Steril 2003; 79: 512–6. MEDLINE
21. Van Golde R, Boada M, Veiga A, Evers J, Geraedts J, Barri P: A retrospective follow-up study on intracytoplasmic sperm injection. J Assist Reprod Genet 1999; 16: 227–32. MEDLINE
22. Westergaard HB, Johansen AM, Erb K, Andersen AN: Danish national in-vitro fertilization registry 1994 and 1995: a controlled study of births, malformations and cytogenetic findings. Hum Reprod 1999; 14: 1896–902 MEDLINE
e1. Chang AS, Moley KH, Wangler M, Feinberg AP, Debaun MR: Association between Beckwith-Wiedemann syndrome and assisted reproductive technology: a case series of 19 patients. Fertil Steril 2005; 83: 349–54. MEDLINE
e2. Cox GF, Bürger J, Lip V et al.: Intracytoplasmic sperm injection may increase the risk of imprinting defects. Am J Hum Genet 2002; 71: 162–4. MEDLINE
e3. DeBaun MR, Niemitz EL, Feinberg AP: Association of in vitro fertilization with Beckwith-Wiedemann syndrome and epigenetic alterations of LIT1 and H19. Am J Hum Genet 2003; 72: 156–60. MEDLINE
e4. Gicquel C, Gaston V, Mandelbaum J, Siffroi JP, Flahault A, Le Bouc Y: In vitro fertilization may increase the risk of Beckwith-Wiedemann syndrome related to the abnormal imprinting of the KCNQ1OT gene. Am J Hum Genet 2003; 72: 1338–40. MEDLINE
e5. Halliday J, Oki K, Breheny S, Algar E, Amor DJ: Beckwith-Wiedemann-syndrome and IVF: A case control study. Am J Hum Genet 2004; 75: 526–8. MEDLINE
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