Newborns with congenital

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1 Congenital anomalies after assisted reproductive technology Anja Pinborg, M.D., D.M.Sci., Anna-Karina Aaris Henningsen, M.D., Sara Sofie Malchau, M.D., and Anne Loft, M.D. Fertility Clinic, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark Worldwide, more than 5 million children have been born after assisted reproductive technology (ART), and in many developed countries ART infants represent more than 1% of the birth cohorts. It is well known that ART children are at increased risk of congenital malformations even after adjustment for known confounders such as maternal age. The proportion of ART children is not negligible, and knowledge about the causes of the higher risk of congenital malformations is crucial to develop prevention strategies to reduce the future risk in ART children. The aim of this review is to summarize the literature on the association between ART and congenital anomalies with respect to subfertility, fertility treatment other than ART, and different ART methods including intracytoplasmic sperm injection, blastocyst culture, and cryotechniques. Trends over time in ART and congenital anomalies will also be discussed. (Fertil Steril Ò 2013;99: Ó2013 by American Society for Reproductive Medicine.) Key Words: Assisted reproductive technology, IVF, ICSI, congenital anomalies, malformation Discuss: You can discuss this article with its authors and with other ASRM members at fertstertforum.com/pinborga-congenital-anomalies-after-art// Use your smartphone to scan this QR code and connect to the discussion forum for this article now.* * Download a free QR code scanner by searching for QR scanner in your smartphone s app store or app marketplace. Newborns with congenital anomalies are observed at a low incidence in all populations. The incidence varies over time and by place and is, apart from genetic causes, influenced by, for example, medical drugs, with one of the most serious examples being the Thalidomide case. Since the first child conceived by assisted reproductive technology (ART) was born, concern has risen as to the safety of the methods. From the literature it is wellknown that ART children are at increased risk of congenital malformations even after adjustment for known confounders such as maternal age. This has been shown in larger national population-based studies in the United States, Australia, and the Nordic countries (1 3), as well as in several meta-analyses (4 8). Some of the Received November 5, 2012; revised December 1, 2012; accepted December 3, 2012; published online January 3, A.P. has nothing to disclose. A.-K.A.H. has nothing to disclose. S.S.M. has nothing to disclose. A.L. has nothing to disclose. Reprint requests: Anja Pinborg, M.D., D.M.Sci., Fertility Clinic, Section 4071, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, DK-2100 Copenhagen, Denmark ( apinborg@rh. regionh.dk). Fertility and Sterility Vol. 99, No. 2, February /$36.00 Copyright 2013 American Society for Reproductive Medicine, Published by Elsevier Inc. malformations are related to preterm birth, for example, cardiac septum defects and patent ductus arteriosus, but even after adjusting for prematurity, ART children are at a slightly increased risk of malformations compared with their spontaneously conceived counterparts. Worldwide, five million children have been born after ART (9). In2005, ART infants represented >1% of the total U.S. birth cohort (10), and in 2008 the proportion of children born after ART ranged from 0.8% to 4.9% of the national birth cohorts, with more than 1.5% being ART children overall in Europe (11). Certainly, the proportion of ART children is not negligible, and knowledge about the causes of the higher risk of congenital malformations is crucial to develop prevention strategies to reduce the future risk in ART children. The scientific literature on the possible causal pathways is limited, and the causality of the association of ART and congenital anomalies is probably multifactorial. The major concern is still whether the ART procedures themselves, embryo culture media, culture time, and advanced procedures such as intracytoplasmic sperm injection (ICSI) and cryopreservation of embryos, have a possible negative impact on the ART offspring. Moreover, the ovarian stimulation resulting in altered endocrine profiles with several corpora lutea in early pregnancy may influence implantation and early fetal and placental development. Concerns of epigenetic changes related to the ART procedures have also been raised with increasing evidence of a higher risk of epigenetic diseases such as Bechwith-Wiedemann and Angelman syndrome (12, 13). Finally, subfertility or the parental characteristics per se have been shown to affect the perinatal outcome including the risk of congenital anomalies (14). One obstacle in the literature on ART and congenital malformations is the heterogeneous classification of malformations, as some studies include both major and minor malformations, VOL. 99 NO. 2 / FEBRUARY

2 VIEWS AND REVIEWS whereas others only include major birth defects. Further, some studies report singletons and twins together, while others show the results for the two groups separately. In most of these studies, twins born after ART show no increased prevalence of congenital malformation compared with twins born after spontaneous conception (2 4, 15, 16). Finally, the definition of a major malformation varies from study to study, and some include analyses on other subsets of malformations such as weeded and relatively severe malformations, thereby excluding predefined less severe conditions (3, 17, 18). Weeded malformations were identified by excluding some conditions that are relatively common, variable in registration, and sometimes associated with preterm birth and low birth weight. The remaining malformations were classified as weeded and will contain mainly major malformations, but also some less severe conditions (17). Another obstacle is ascertainment bias, with children conceived by ART being more carefully examined and thus having higher malformation rates. Even in the large national controlled register studies this is possible as ART children are hospitalized more and therefore have more malformations recognized. This review will focus on birth defects in ART singletons, and if singleton data are not available, then on the general ART population. The aim of this review is to summarize the literature on the association between ART and congenital anomalies with respect to subfertility, fertility treatment other than ART, and different ART methods including ICSI, blastocyst culture, and cryotechniques. Subfertility (Time-to-Pregnancy >1 Year) Maternal age increases with longer time to pregnancy (TTP), therefore shortening of the TTP reduces the patient's age at time of conception. Population-based studies from the United States have described the impact of maternal age on birth defects with an incidence of 2.7% at 26 years of age and 3.4% at 37 years of age (19). Thus adjustment for maternal age is pivotal when analyzing the influence of subfertility on ART outcomes. Based on the Danish national birth cohort, Zhu et al. showed that singletons born of subfertile couples who conceived spontaneously had a higher risk of congenital anomalies (hazard ratio [HR], 1.20; 95% confidence interval [CI], ) than singletons born of fertile couples with TTP <1 year (14). Further, the study revealed that the overall prevalence of congenital malformations increased with increasing TTP. The children born to those women who received fertility treatment had an even higher prevalence of congenital malformations than singletons of fertile couples with TTP <1 year (HR, 1.39; 95% CI, ). On the basis of their findings, the investigators concluded that the increased malformation rate after ART was partly due to the underlying infertility or its determinants (14). One weakness of the Zhu et al. study is that TTP was categorized into 0 2, 3 5, 6 12, and >12 months with no categories above 12 months. Since prolonged TTP is associated with several adverse outcomes in the offspring, it should be noticed that on the TTP spectrum, ART patients are well beyond 1 year. In fact, 3 5 years of infertility is generally the norm (20 22). Hence it is assumed that ART couples, as they receive the most extensive fertility treatment, also are less reproductively healthy than subfertile couples with shorter TTP who conceive either spontaneously or by less invasive fertility treatments (22). In a recent report, Rimm et al. adjusted their previous finding from a meta-analysis on congenital malformations and found that the adjusted risk of malformations after ART was reduced from an HR of 1.29 (95% CI, ) to an HR of 1.01 (95% CI, ), when taking into account that 40% of major malformations were due to subfertility (4, 22). These results should be interpreted with caution as the results in the Rimm et al. study were based on two independent studies: first the study by Zhu et al. was used to calculate the magnitude of the influence of subfertility, and then this estimate was applied to the pooled risk estimate of congenital malformations after ART from the earlier meta-analysis. In a national Swedish register study, approximately 5% of all infants born after ART had a relatively severe malformation with an increased risk of an adjusted odds ratio (AOR) of 1.44 (95% CI, ) after adjustment for year of birth, maternal age, and parity (17). When additional adjustment was made for year of birth, maternal age, parity, years of known childlessness, and smoking, the increased risk disappeared (AOR, 1.12; 95% CI, ). Thus, increased risk was mainly due to maternal characteristics, with the most important factor being subfertility, which was estimated as years of known childlessness (17). Subfertility or intrinsic parental characteristics play a significant role in the association between ART and congenital anomalies. To separate the effect of intrinsic parental factors and the effect of the ART methods themselves on the development of congenital anomalies, it is necessary to either adjust for years of unwanted childlessness or to use subfertile populations conceiving spontaneously as controls to ART populations. In the vast majority of studies, normal fertile populations are used as controls and there is no correction for subfertility. In these studies it is not possible to distinguish between parental factors and the influence of the ART treatments per se. Fertility Treatment Other than ART (Ovulation Induction and IUI) On most health indicators, children born after ovulation induction (OI) and/or IUI seem to perform worse compared with spontaneously conceived children (23). Three smaller studies reported no significant difference in the occurrence of congenital anomalies in OI/IUI versus control children (24 26), while recent larger studies have shown an increased risk of malformations after OI/IUI (27 29). Finnish populationbased data have shown a significantly higher prevalence of major malformations among 4,467 children born after OI/IUI (3.52%) than among control children (2.85%) but still lower than after ART (4.27%) (27). The AOR for OI/IUI versus controls was 1.21 (95% CI, ), while for ART versus controls, the AOR was 1.31 (95% CI ) (27). In 790 Canadian infants born after medical assisted reproduction including ART (n ¼ 319), IUI/OI (n ¼ 173), and 328 VOL. 99 NO. 2 / FEBRUARY 2013

3 Fertility and Sterility OI (n ¼ 298), the overall risk of birth defects was increased (AOR, 1.55; 95% CI, ) after adjustments for maternal age, parity, smoking, and sex of baby (28). The prevalence was highest in the ART group (3.45%) compared with 2.89% after IUI/OI, 2.35% after OI, and 1.86% after natural conception; probably owing to a limited sample size no adjusted analyses were made for the subgroups. A recent French cohort study revealed a prevalence of major malformations of 3.6% in 445 IUI versus 4.2% in 903 ART and 2.1% among 4,044 controls (29). Children born after IUI were twice as likely to have a major birth defect as children from the background population (AOR, 2.0; 95% CI, ), but the risk in IUI children was similar to the risk in the ART group. From the available literature is seems as though infants conceived by fertility treatments other than ART also carry a higher risk of congenital anomalies and that this risk may be increasing with increasing intensity of the treatment or with the severity of the parental reproductive morbidity. However, future research is needed to confirm this. Clomiphene Citrate The use of clomiphene citrate (CC) is widespread, and in the United States it is estimated that 1.6% of pregnancies are conceived with the use of CC (30). In 1986, Mikkelson et al. found that an isomer of CC, the more active zuclomiphene, accumulates over consecutive cycles and can still be detected in plasma 1 month after administration (31). Thus there is a biological plausibility of the association between CC and birth defects, although CC is taken before conception. Two types of birth defects have been commonly reported with the use of CC: neural tube defects and hypospadias (32 35). In a recent U.S. national case-control study, associations were shown between the use of CC and birth defects, but there was some inconsistency between the findings and previous reports (36).A major limitation of the study was that the effect of CC could not be distinguished from the effect of subfertility (36). On the basis of the current literature, no firm conclusions can be drawn regarding the association between CC and birth defects. ART Meta-analyses have reported an increased prevalence of congenital malformations with an excess risk of 30% 40% in singletons born after ART (4 7). One meta-analysis observed a 67% increased risk (8). These results are consistent with the most recent population-based studies on congenital anomalies in ART children from Sweden and Australia (2, 3). K allen et al. showed in ART children (n ¼ 31,850) that the prevalence of congenital anomalies after ART declined over time, although a slightly increased risk of 15% remained in the youngest ART cohorts (3). In the youngest cohort of Swedish ART children born from 2001 to 2006 (n ¼ 15,570, including 9,372 after ICSI), the crude total malformation rate was 5.3% versus 4.4% in the general population (3), while in the earlier cohort (born , n ¼ 16,280 ART infants) the crude malformation rate was 5.0% in ART versus 4.0% in the general population (17). Only malformations diagnosed in the neonatal period were included in these figures, while in the first study the investigators aimed to include diagnoses from the Swedish Hospital Discharge register as well and found that by including malformations diagnosed later in life, there was a total malformation rate of 8.3% in ART children; unfortunately, no corresponding figure was available for the background population (17). In the cohort of Swedish ART infants born from 2001 to 2007, the adjusted risk of congenital anomalies in ART infants versus the background population was 1.15 (95% CI, ). The adjusted risk was lower in the younger ART generations (born ) compared with in the older ART generations (AOR, 1.33; 95% CI, ]) (3, 17). In a large Australian cohort of 6,163 ART infants (n ¼ 4,756 after IVF and n ¼ 1,407 after ICSI) born from 1986 to 2002, Davies et al. included malformations diagnoses from gestational week 20 with a 5-year follow-up period (2). The crude total malformation rate in ART children was 8.3% versus 5.8% in the background population, with an overall increased AOR of birth defects for ART versus the background population of 1.28 (95% CI, ) (2). However, in singleton newborns from fresh embryo IVF compared with the background population, the AOR was 1.06 (95% CI, ), thus showing no increased overall risk of birth defects after IVF (2). IVF Versus ICSI The incidence of Y-chromosome deletions and other chromosomal anomalies is increased in males with poor semen quality (37). Several studies have shown that children conceived by ICSI are at increased risk of inherent and de novo chromosomal aberrations (38 40). Further, a higher rate of urogenital malformations, primarily hypospadias, which has been related to paternal infertility, has been reported (3, 18). Zhu et al. found that the adjusted risk of genital organ malformations in ART children was significantly higher than in singletons born of subfertile couples conceiving spontaneously (HR, 2.32; 95% CI, ) (14). In the large Swedish study, the increased risk of hypospadias found in the older ICSI cohorts disappeared over time and was not present in the younger cohorts of ICSI children (3). One explanation for this declining incidence of hypospadias in ICSI boys over time may be a dilution effect, that is, the indications for ICSI nowadays are much wider and therefore severe male factor infertility in the father is less frequent in the younger generations of ICSI boys. However, improvement in the use of the ICSI technique may also have an influence. Two meta-analyses have raised the question of whether birth defects are more common in ICSI versus IVF infants (7, 41). Lie et al. included four studies and reached a pooled estimate of 1.12 (95% CI, ) (41). Wen et al. reported a pooled estimate of 1.05 in IVF versus ICSI after including 24 studies and a reverse pooled estimate of 0.95 (95% CI, ) in ICSI compared with in IVF infants (7). The Swedish study demonstrated no apparent risk difference according to ART method, with an adjusted risk of birth defects in ICSI versus IVF ( ) 0.90 (95% CI, ) (3). VOL. 99 NO. 2 / FEBRUARY

4 VIEWS AND REVIEWS In contrast, the Davies et al. group demonstrated a raised risk (AOR, 1.55; 95% CI, ) in ICSI but not in IVF singletons versus a fertile population with no use of assisted reproduction after adjustments for parental factors (2). Chromosomal anomalies may be one of the explanations for the higher birth defect risk in ICSI singletons observed in the Australian data. Moreover the Australian study included medical termination of pregnancies due to birth defects after week 20, while the Swedish study only included births after week 20. Finally, the Australian study included nonacquired cerebral palsy as a birth defect, but the prevalence of cerebral palsy in IVF and ICSI children was not reported separately. It should be emphasized that the youngest generations of ART infants seem to have a lower prevalence of congenital anomalies. This may be due to changes in the subfertile patient populations previously explained as the dilution effect, with the parents being less reproductively ill than one or two decades ago. The declining malformation rates may also be due to improvements in the ART stimulation regimens and ART laboratory procedures. Haliday et al. explored the risk of blastogenesis birth defects, which correspond to birth defects occurring in the first weeks of embryo development before organogenesis and tend to affect the formation of the midline and mesoderm (42). These early developed congenital anomalies include neural tube defects, abdominal wall defects, esophageal and anal atresia, and monozygotic twinning. In all, 6,946 IVF or ICSI singleton pregnancies were linked to birth defects occurring from 1991 to 2004 in Victoria, Australia. Because of the study period, it is expected that the vast majority of ETs were cleavage stage, but this is not clarified in the paper (42). Haliday et al. found that overall birth defects increased after IVF or ICSI with an AOR of 1.36 (95% CI, ) and with blastogenesis birth defects markedly increased (AOR, 2.80; 95% CI, ). The investigators suggested that a mechanism initiated about the time of implantation affecting early embryo development in ART pregnancies was responsible. The increase in blastogenesis defects appeared greater for fresh ET than for thawed ET, with the risk increase for fresh ET relative to controls being more than threefold. The authors claimed that cryopreservation/thawing may act as a selection gate for more viable embryos; alternatively, the ovarian stimulation in the fresh cycles could have a variety of adverse effects on implantation and very early pregnancy. An abnormal hormonal milieu was not present before cryopreservation as only frozen ET (FET) performed in a natural cycle was included. It is possible that the endometrial receptivity is compromised in the presence of the high hormone levels that persist beyond the time of oocyte collection. In contrast to FET, fresh ET is associated with lower levels of pregnancy-associated plasma protein A (PAPP-A) (43, 44), which has also been shown to be associated with preeclampsia and fetal growth retardation in spontaneously conceived pregnancies (45, 46). PAPP-A is known to play a key role in angiogenesis and placental development throughout pregnancy (47). ART children are at an increased risk of congenital anomalies of 15% 40%; whether ICSI children have an added risk compared with IVF children is still not clear. The younger generations of ART children seem to have a lower risk of congenital anomalies compared with the older ART generations. Blastogenesis birth defects should be embedded in future research. Blastocyst Culture Only two reports have examined the risk of congenital anomalies in singletons after blastocyst versus cleavage-stage embryo culture (48, 49). In 1,311 singletons born after blastocyst culture, not, however, stratified for fresh or frozen ET, the risk of congenital malformations was significantly increased to 1.43 (95% CI, ) for all congenital anomalies and to 1.33 (95% CI, ) for relatively severe congenital anomalies in infants born after blastocyst versus cleavagestage transfer (48). In a smaller one-center trial on 103 infants born after vitrified blastocyst transfer, 199 after fresh blastocyst transfer, and 194 after slow freeze, no increased risk of malformations was seen, however, the study was not powered to look at rare outcomes (49). The risk of congenital anomalies after blastocyst culture should be continuously monitored and incorporated in the large national ART registers to follow the prevalence of congenital malformations in these children. FET Slow freezing. On the basis of the existing literature, children born after slow freeze have the same prevalence of malformations as children born after the transfer of fresh embryos (50 52), although some obstetric outcomes seem to be even better in children after FET (53, 54). One study has found that ICSI children born after cryopreservation carry a higher risk of congenital anomalies (55). However, this finding has not been verified by other investigators (3, 51, 54). Vitrification. Only one review on children born after vitrification is available (56). The investigators constructed a database including all live-born infants after vitrification (n ¼ 936), out of which 12 (1.3%) were noted to have birth defects. Compared with spontaneously conceived infants, no difference was found. Only one report with sufficient sample size has looked at birth defects after vitrification of cleavage-stage embryos or blastocysts (57). The total birth defect rates were 2.4% versus 1.9% after vitrified and fresh ET, respectively, and the AOR was 1.41 (95% CI, ) after adjustments for maternal age, body mass index (BMI), parity, type of stimulation, ICSI method, blastocyst culture, and infant sex. In general, it seems that cryopreservation of embryos protects against some of the adverse obstetric outcomes after ART but not when it comes to congenital anomalies, where the prevalence apart from blastogenesis defects seems to be as high as after fresh ET. It is far too early to give an opinion about the risk of birth defects after vitrification. Perspectives ART children have a slightly increased risk of congenital anomalies of 15% 40% compared with spontaneously 330 VOL. 99 NO. 2 / FEBRUARY 2013

5 Fertility and Sterility conceived children after adjustments for relevant confounders. From recent Swedish cohort studies, the overall risk is not increasing over time as there is a tendency for a decline in the prevalence of congenital anomalies in the youngest ART generations. This may be due to a decreased reproductive morbidity caused by healthier patient populations, as the accessibility to ART has increased over time. The increased risk of congenital anomalies in children born after fertility treatment can partly be explained by the subfertility of the couples. However, longitudinal studies focusing on the causal factors including parental characteristics, that is, causes for infertility, endocrine diseases including polycystic ovarian syndrome and semen quality, and lifestyle factors such as smoking and BMI, are highly warranted. Further, the risk of congenital anomalies after different ovarian stimulation exposures, that is, CC versus gonodotropins and new laboratory procedures such as blastocyst culture and vitrification, needs continuous surveillance. The ongoing debate regarding the influence of different culture media on epigenetic changes and birth weight should also be related to the risk of birth defects in the offspring. On the basis of the current literature, it is not possible to distinguish the effect of parental subfertility, the effect of ovarian stimulation, and the ART laboratory techniques on the risk of congenital anomalies, but it has clearly been shown that children conceived without fertility treatment in subfertile couples also carry a higher risk of congenital anomalies compared with singletons conceived by fertile couples. Future research should be devoted to finding the mechanisms of the association between ART and congenital anomalies. This requires continuously updated large national ART registers or databases, which also include congenital anomalies in fetuses diagnosed by prenatal screening procedures resulting in miscarriages or induced abortions in the first or second trimester. New ART techniques should rapidly be incorporated into these registers, making as expeditious as possible continuous surveillance from the introduction of the procedure. Finally, detailed information about parental characteristics, ovarian stimulation, treatment modality, BMI, and smoking should also be recorded. This is the only way to explore the causal pathways and hence develop prevention strategies to reduce congenital malformations in ART children in the future. Because they represent >1% of the birth cohorts in many developed countries, much attention is being paid to the health and well-being of ART children. New research in the field raises a lot of media attention and may cause severe concerns in the couples achieving a long-awaited ART pregnancy. Thus it is pivotal that new results are presented meticulously and in the context of the existing literature. However, as a final remark, it is important to emphasize that the adjusted risk of congenital malformations is significantly increased but seems to decline over time and that the vast majority of ART infants are healthy. REFERENCES 1. Schieve LA, Rasmussen SA, Reefhuis J. Risk of birth defects among children conceived with assisted reproductive technology: providing an epidemiologic context to the data. Fertil Steril 2005;84: Davies MJ, Moore VM, Willson KJ, Van Essen P, Priest K, Scott H, et al. Reproductive technologies and the risk of birth defects. N Engl J Med 2012;366: K allen B, Finnstro om O, Lindam A, Nilsson E, Nygren K-G, Otterblad PO. Congenital malformations in infants born after in vitro fertilization in Sweden. 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