Seminars in Fetal & Neonatal Medicine xxx (2014) 1e6
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Review
Birth defects and assisted reproductive technologies Joe Leigh Simpson* March of Dimes, 1275 Mamaroneck Avenue, White Plains, NY 10605, USA
s u m m a r y Keywords: Assisted reproductive technologies (ART) Birth defects Hypospadias Imprinting Intracytoplasmic sperm injection (ICSI) In-vitro fertilization (IVF)
Assisted reproductive technologies (ART) using in-vitro fertilization (IVF) account for w1% of births in the USA and as much as 3e4% in Europe or Australia. Initially studies involved infants prospectively examined in an early cohort of US births, with salutary results. Later studies began to show the frequency of birth defects to be increased. In meta-analysis, odds ratio was >1.0, with the 95% confidence limit not extending to 1.0, with the 95% CI not extending across the 1.0 isobar [6e15]. Thus, there evolved the present consensus that increased birth defects are indeed positively associated with ART. The major question at present is whether this increase is due to ART protocols per se or merely reflective of the biological perturbations that generated the infertility that necessitated ART to achieve pregnancy. In this communication, we shall first consider the frequency of congenital anomalies in offspring of ART pregnancies, stratified by specific subgroups depending on technique. We shall also comment on the pitfalls that make difficult definitive conclusions concerning the explanation for anomalies detected.
2. Frequency of anomalies in ART pregnancies Almost 50 cohort studies have addressed the question of anomalies in ART pregnancies, as referenced elsewhere [16e19]. Initial studies naturally focused on IVF alone, because not until the mid-1990s could male infertility be managed by intracytoplasmic sperm injection (ICSI) followed by traditional IVF. In the 1990s and early 2000s, one could confidently conclude the overall risk was not, say, a two- or three-fold increase above the accepted population baseline of 2e3%. However, more precise statements could not be made. Notwithstanding general reassurance, this author and others pointed out pitfalls that could sway opinions on safety in either
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J.L. Simpson / Seminars in Fetal & Neonatal Medicine xxx (2014) 1e6
Table 1 Earlier population-based studies of anomalies in assisted reproductive technology pregnancies. Study
Years of sample accrual
Adjusted OR (95% CI)
Statistical significance
Country
Dhont et al. [4] Westergaard et al. [3] Anthony et al. [5]
1986e2002 1994e1995 1995e1996
1.25 (0.96e1.64) 1.04 (0.78e1.39) 1.03 (0.86e1.23)
No No No
Belgium Sweden Netherlands
OR, odds ratio; CI, confidence interval. The initial population-based studies depend on data in registries. Ascertainment varied between studies with respect to duration of time in which anomalies were sought and definition of major defects. The interval of accumulated ART registry cases does not in any report correspond to the extant laboratory and ovulation stimulation protocols used in 2014.
direction [20,21]. It was in particular appreciated that power to detect a significantly increased frequency of anomalies was not possible due to small sample size. Table 2 shows later population-based studies on which this altered conclusion began to be based [6e15]. All were based on registries that recorded cohorts of births, from which those with anomalies could be stratified. A caveat is that the definition of birth defects among registries varies, usually involving International Classification of Diseases (ICD)-based diagnosis codes not well suited to distinguish major from minor anomalies. ICD codes at birth are not robust in detecting subtle anomalies not externally visible, nor in excluding inherited syndromes or chromosomal anomalies. Neither is plausibly due to ART. Not always followed is the pragmatic, accepted, definition of a major birth defect as one that causes death, functional impairment or (if structural) requires surgery. Reviewing several studies will suffice as illustrative. In 2005 Klemetti et al. [7] published Finnish registry of IVF and ICSI cases delivered in 1996e1999, finding the adjusted OR for all birth defects to be 1.31 (95% CI: 1.10e1.57), when comparing 4459 cases and 27 078 controls. In 2007, Pinborg et al. [9] used Danish registry data to derive an OR of 1.24 (95% CI: 1.09e1.43). In 2005 Kallen et al. [6,12] published an analysis of Swedish registries (1982e1999) data and in 2010 analysed cases for the years 2001e2007. The latter study showed OR of 1.25 (95% CI: 1.5e1.37) based on 15 570 cases [12]. Outside of Scandinavia, the region generating greatest attention is Australia, befitting the country’s sentinel role in developing ART. In 2002 Hansen et al. reported Western Australia cases, reprising this in 2012 [14,22]. Their 2012 report of Western Australia registry data involved ART cases delivered 1994e2002, an interval of significance given many changes having since occurred in laboratory methodology. This study was laudatory, however, in trying to take into account anomalies in pregnancy terminations. Both the 2911 ART cases and the 210 997 non-ART cases were followed for 6 years. A ‘major birth defect’ was found in 8.7% of ART cases and 5.4
of non-ART cases (OR: 1.53; 95% CI: 1.30e1.79). Rates were not different in unlike sex twins (obligatory dizygosity) (OR: 1.08; 95% CI: 0.77e1.51). The 5.4% birth defects in the control group is at odds with general acceptance, absent comprehensive laboratory assessments such as array comparative genomic hybridization. These authors concluded also that there has been a decrease in the prevalence of birth defects compared with their earlier cohort (1994e1998) [14,23]. One explanation may simply be greater appreciation between major and minor anomalies. Plausible scientific reasons include changes in culture media, better regulation of heat and CO2 in incubators, and differential ovulation stimulation regimes. A second Australian group extending interval of ascertainment to 5 years was that of Davies et al. [13], who used registry data of 308 974 births in South Australia (Adelaide). Minor defects were excluded unless they required treatment or were ‘disfiguring’. Among 6163 ART offspring were 513 with anomalies (8.4%), compared with 5.8% in non-ART births. The adjusted OR was 1.28 (95% CI: 1.16e1.41). OR was 1.26 for IVF alone, and 1.77 for ICSI/IVF. It would be of interest to know the temporal sequence in which the cumulative absolute frequency of birth defects occurred, i.e. ascertainment at birth versus later. Of special interest to US readers is the report of Kelley-Quon et al. [15], who used the California Patient Discharge Linked Birth Cohort Database that lists anomalies by ICD-9 codes. This study involved 4795 infants born in 2006e2007 after ART, compared with 46 025 naturally conceived in the same interval. The overall rate of ‘major congenital abnormalities’ was 9.0% vs 6.6% (OR: 1.25; 95% CI: 1.12e1.39; P < 0.001). Incidence of birth defects this high bespeaks inclusion of many minor anomalies. In this author’s opinion, this alone casts doubt on conclusions that ORs were significantly increased for certain organ-specific anomalies: eye, head and neck, heart and genitourinary track. These limitations notwithstanding, meta-analyses reached similar arithmetic conclusions: Rimm et al. [16]: 1.29 (95% CI: 1.01e1.67); Hansen et al. [17,19], risk ratio: 1.32 (95% CI: 1.24e
Table 2 Later population-based studies on anomalies in assisted reproductive technology (ART) pregnancies (2005e2013). Study
Years of sample accrual
Adjusted OR (95% CI)
Statistical significance
Country
Kallen et al. [6] Davies et al. [13] Halliday et al. [11] Hansen et al. [14] Pinborg et al. [9] Klemetti et al. [7] Ombolet et al. [8] Kallen et al. [12] Kelley-Quon et al. [15] Fuji et al. [10]
1982e2001 1986e2002 1991e2004 1994e2002 1995e2000 1996e1999 1997e2003 2001e2007 2006e2007 2006
1.44 1.24 1.36 1.53 1.24 1.31 1.11 1.25 1.25 1.17
Yes Yes Yes Yes No Yes No Yes Yes No
Sweden Australia (Adelaide) Australia (Parkville) Australia (Perth) Denmark Finland Belgium Sweden USA (California) Japan
(1.32e1.57) (1.09e1.41) (1.19e1.55) (1.30e1.79) (0.97e1.58) (1.10e1.57) (0.08e1.58) (1.15e1.37) (1.21e1.39) (0.81e1.69)
OR, odds ratio; CI, confidence interval. These population-based studies depend on data in registries. Ascertainment varied with respect to duration of time in which anomalies were sought and definition of major defects. The interval of accumulated ART registry cases does not in any report correspond to the extant laboratory and ovulation stimulation protocols used in 2013.
Please cite this article in press as: Simpson JL, Birth defects and assisted reproductive technologies, Seminars in Fetal & Neonatal Medicine (2014), http://dx.doi.org/10.1016/j.siny.2014.01.001
J.L. Simpson / Seminars in Fetal & Neonatal Medicine xxx (2014) 1e6 Table 3 Reported meta-analyses on birth defects and assisted reproductive technologies. Study
Year
Studies accepted
OR (95% CI)
Rimm et al. [16] Hansen et al. [17] Wen et al. [18] Hansen et al. [19]
2004 2005 2012 2013
19 25a 46 45a
1.29 1.29 1.37 1.32
(1.01e1.67) (1.21e1.37) (1.26e1.48) (1.24e1.42)
OR, odds ratio; CI, confidence interval. a Many studies considered acceptable in the 2005 review by Hansen et al. [17] were no longer considered acceptable in their 2013 report [19].
1.42); Wen et al. [18]: 1.37 (95% CI: 1.26e1.48) (Table 3). Similar results are not surprising because the same studies were generally analyzed. 3. Anomalies by organ system Because of sample size limitations, individual anomalies have generally been assessed only by pooling anomalies by organ system. Yet within a given system there is a plethora of different anomalies of difficult etiologies e genetic and otherwise. Collapsing all such anomalies to assess a single organ system is convenient for generating statistical power, but this does not obviate the methodological flow. Hypospadias is the only organ system in which OR has been consistently shown to be increased. An odds ratio of 3.0 (95% CI: 1.09e6.50) was reported by Wennerholm et al. [23] and 1.5 (95% CI: 1.0e2.1) by Ericson and Kallen [24]. Klemetti et al. [7] also showed increased hypospadias. A potential explanation for increased hypospadias in ART is heritable low testosterone, also adversely affecting spermatogenesis. Recurrence risk for hypospadias is 2e5% in first degree relatives (sons). This could lead to transmission of a polygenic trait (hypospadias). There was no significant difference in the metaanalysis by Wen et al. [18] One confounder is that indications for ICSI are now much less stringent than when ICSI was initially introduced, including many cases analyzed by Davies (1986e2002) [13]. ICSI now constitutes half of all ART cases in the USA and even more in some venues. The only other organ system seriously implicated is cardiac. Sala et al. [25] found a significant increase, but in general other authors have found no increase. Special pitfalls exist in assessing cardiac anomalies. A patent ductus arteriosus is normal at birth, but soon closes in full-term infants; in premature infants persistence is normal, and surgical ligation is routine. Ventricular septal defects are not usually evident at birth but manifest about one week after birth when cardiac circulation is adapted for postnatal life. Taking these temporal changes into account is not facile, and beyond the charge of most if not all registries recording birth defects. 4. Methods of data analysis Determining whether anomalies are increased in ART could be based on data from a single center or from a registry. It is fashionable for many to use pooled data (systematic reviews or metaanalysis), and consider this approach superior. Meta-analysis is based mostly on obvious strength of sample size compared with single center studies. Center-specific studies are thus marginalized. However, the latter can utilize checklist comparisons by a limited pool of examiners, an approach not possible in population registries. Indeed, it is studies with less systematic assessments that more often showed no increase in
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birth defects. Meta-analysis has other limitations, namely lack of consensus on inclusion criteria. In meta-analysis the axiom is that all studies are comparable, and thus suitable for pooling. Consider, however, the laudatory reports of Hansen et al. [17,19] The latter exhaustive report excluded non-English reports, reports in which no birth defects were found in either cases or controls, and reports in which birth defects ascertainment extended to a later age. Numbers of studies deemed suitable were 45, similar to Wen et al. (n ¼ 46) [18]. Yet Rimm et al. [16] accepted only 19 studies. Although the validity of meta-analysis in many circumstances is strong, this is more arguable in ART and birth defects. In ART virtually no units utilize similar protocols. Yet many different laboratories contribute to a single registry, to say nothing of differences between registries. Different authors must reach their own opinion as to whether preset criteria are met. Decisions on inclusion criteria may be discrepant at different points in time, even by the same author. For example, upon reassessment 8 years later Hansen et al. [17,19] concluded that many studies accepted as valid in their 2006 study were no longer acceptable. One widely used decision tree is to include or exclude series in which follow-up extended beyond the newborn period. (Separate analysis would be more appropriate.) Thus excluded is the report of Davies et al. [13], despite being an excellent population-based study that extended to 5 years of age and contained ascertainment from multiple sources. Another study excluded for the same reason has been Bonduelle et al. [27] who showed increased sex chromosomal abnormalities in ICSI. These cannot be detected in registries because physical abnormalities are usually not evident at birth. In some meta-analyses it is sometimes decided that certain studies are given higher ‘quality scores’ and, hence, greater weight. For example, Hansen et al. [19] assigned a ‘quality 1’ score (best) to 11 of 13 registry-based studies, and only seven population-based studies were not scored quality 1. Among 32 ‘clinic’ samples, only one was assigned quality 1. Many of the latter were indeed of insufficient sample size, but numbers alone should not be the sole criterion for high quality because quality of clinical examination is less than ideal in registries. Consistent clinical acumen is not possible among all examiners, some of whom lack familiarity in distinguishing major from minor anomalies. It is difficult but not impossible to form a collaborative network of clinical centers to assess anomalies consistently among centers, but not impossible (see the reports from the South American Registry ECLAMC [27]). Another problem with registry data is long interval during which cases were accumulated. Anomalies were initially probably more vigorously sought in ART cases than controls [20,21]. This bias is probably less so now, but the earlier bias is still relevant because registry data being used for currently published meta-analyses are often based on deliveries even extending as far back as the 1980s. Paradoxically, the larger the study (greater power), the more ‘ancient’ and now ‘atypical’ cases included. The above criticisms are not meant to denigrate work pursued to unravel a difficult problem, but to remind the reader that crisp data suitable for analysis do not exist.
5. Pitfalls in assessing presence of birth defects This author has expressed concern on pitfalls precluding data accumulated and used for comparison of ART outcomes [20,21]. Box 1 lists common pitfalls so far discussed, a few aspects of which are highlighted in the following subsections.
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Box 1 Pitfalls in experimental design and interpretation in assessing birth defects in assisted reproductive technology (ART) offspring.
pregnancies analyzed and publication data makes generalizability to women undergoing ART in 2014 arguable. 5.4. Interval for anomaly surveillance after birth
A. Ascertainment Including or not including anomalies in stillborns. Including or not including anomalies in terminated fetuses. Including or not including anomalies detectable on ultrasound but not externally visible at birth. Varying duration of surveillance. Varying aggressiveness of surveillance. B. Definition of anomalies ‘Minor’ anomalies versus ‘major’ anomalies. Failure to utilize pre-set definitions and check lists (i.e. minimizing individual observer variability). Determining when a ‘minor’ anomaly becomes ‘major’ (e.g. requiring surgery). C. Methodology Failure to stratify analyses into isolated anomalies versus multiple anomalies (syndromic) disorders. Failure to recognize (exclude) heritable disorders whose etiology is not plausibly ART-related. Generalization of conclusions to a contemporary population when data were derived in a prior era that used now-outdated protocols. Application of meta-analysis when studies are of dissimilar design (unavoidable given lack of standardized protocols).
Another problem is the varying length of surveillance after birth. Birth defects registries traditionally are restricted to anomalies ascertained in the neonatal period (before hospital discharge). However, several excellent single-center studies extend to 5 years, providing information not possible with registries (e.g. sex chromosomal abnormalities) [13,26]. Certain organ-specific ART-related anomalies could be manifested only in the newborn period and other only later. Data analyzed in meta-analysis should ideally be stratified by length of surveillance. 5.5. Lack of true control group What is the control group? In theory, the proper control should consist of women who require ART but who become pregnant without ART, ideally within the time defined as normal to become pregnant (12 months). Obviously, this group does not exist. Thus, no ‘gold standard’ randomized clinical trials or caseecontrol study can be conducted. Instead, published reports compare ART outcomes to naturally conceived offspring. The closest to a proper control group seems to be a subfertile couple (inability to conceive by 12 months) who conceive naturally while awaiting ART. Other options include same-sex couples using a surrogate, but sample sizes are inadequate.
5.1. Definition of anomalies
6. Anomalies in singleton versus twin gestation
The major source of confusion is probably inconsistent definition of an anomaly (birth defect). How precisely does one distinguish major from minor anomalies? Should one rely on studies in which minor anomalies are collapsed into a single category with major anomalies? In fact, criteria for distinguishing major from minor anomalies are well known to geneticists and well codified, but generally not familiar to clinicians outside medical genetics [28]. Rigor of physical examination is an issue because geneticists record more minor anomalies than non-genetic pediatricians. Relying upon pooled data when the primary outcome (birth defects) is not assessed identically is inherently hazardous. Examiners not surprisingly have variable skills, training and interest; thus, criteria cannot be assumed to be comparable across centers, studies, or countries. This becomes immediately evident by noting variable rates of anomalies in controls, namely signaled by rates considerably higher than the accepted 2e4% major birth defects at birth and shown in checklist-based studies [29].
Consensus exists that overall birth defects are increased in singletons, but the same cannot be said for multiple gestations of which twins constitute by far the largest group. When adjustment was made for zygosity by Hansen et al. [19], OR was 1.26 (95% CI: 0.9e1.60). Anomaly rates were not different between ART and nonART twins. In unlike sex twins (obligatory dizygosity), OR was 1.08 (95% CI: 0.77e1.51). Recall that twins naturally conceived have long been known to show increased birth defects, particularly if monozygotic. Prior to the era of ART, half of all like-sexed twins were monozygotic. Now the percentage should be lower. Interestingly, preterm birth in ART offspring is considered to be increased in singleton births but not in twin gestations [30,31]. Perhaps infertility (and, hence, likelihood of embryo implantation) is greater in women whose embryos are more likely to be abnormal and, hence, less likely to sustain two embryos. Or, perhaps, singletons had a cotwin who underwent unrecognized demise (vanishing twin) that nonetheless exerted a persistent deleterious effect, perhaps involving the decidua [32]. In conclusion, anomalies in both non-ART twins and in ART twins are increased compared with singleton controls, but an additive risk in ART is surprisingly unproven at present.
5.2. ART protocols There is a lack of conformity in ovulation stimulation regimens, a plethora of different culture media, and multiple embryo incubation techniques. Standardization does not exist, and commercially utilized media list only components and not concentrations. 5.3. Interval of pregnancy ascertainment Especially egregious is, as noted, assuming that results derived from methods used long ago are still generalizable to the contemporary patient, a concern raised earlier. The most contemporary study involves 2006e2007 cycles, well behind currently used techniques like vitrification [15]. The long interval between
7. Anomalies in ICSI/IVF versus IVF alone Initially only IVF per se was assessed for association with birth defects. As stated earlier, no statistically significant association was reported during the 1990s. With utilization in the 1990s of ICSI (i.e. ART using ICSI followed by traditional IVF), increased birth defects began to be reported. With ICSI the frequency of sex chromosomal abnormalities appears increased in prenatal samples (amniocenteses) as well as hypospadias [7,23,24]. Otherwise, anomaly rates have appeared similar in ICSI and non-ICSI IVF, rendering overall anomaly rates not significantly different. The exception was Davies
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J.L. Simpson / Seminars in Fetal & Neonatal Medicine xxx (2014) 1e6
et al. [13], showing increased risk only in ICSI offspring (OR: 1.47; 95% CI: 1.15e1.89). ICSI couples have the confounder of having increased frequencies of balanced translocations, not only in males (2.05%) but female (1.38%) sex chromosomal abnormalities, and inversions were also found not infrequently. Thus, two subfertile individuals presumably constitute a single infertile couple. The frequency of balanced translocations in men undergoing ICSI is higher in oligospermic (4%) than in azoospermic men (1%). By analogy the same principle may hold for single mutant genes, certain men undergoing ICSI having a pleiotropic mutation one component of which is infertility and another predisposition to a structural anomaly. Chromosomal abnormalities as assessed by preimplantation genetic diagnosis are increased in ICSI-assisted embryos [32]. In conclusion, sex chromosomal abnormalities and hypospadias are considered increased in ICSI compared with IVF alone. 8. Cryopreservation Data are limited concerning whether cryopreservation affects birth defects in liveborns, but at present data are reassuring. Loss of cell(s) from cleavage stage biopsy is clearly deleterious to embryo survival, as shown by decreased pregnancy rates for embryos that had loss of one cell upon thawing [33]. Similarly, removal of more than two cells from cleavage stage embryos reduced the pregnancy rate [34]. These statements are not applicable for trophectoderm biopsy where a dozen or more cells are removed. Fortunately, loss of cells at either cleavage stage or blastocyst stage does not result in liveborn anomalies because the ‘all or none’ effect exists. Embryos surviving to live birth are not affected long term because when loss of a single cell occurs at this stage of embryogenesis the function of that cell can be subsumed by another because all cells are totipotential. In their own cohort Hansen et al. [22] claimed to observe a trend favoring fresh rather than frozen singleton IVF; however, this was not found in twins or ICSI pregnancies. Davies et al. [13] found no differences at all. This author counsels that neither benefit nor harm exists for cryopreservation with respect to birth defects. 9. Is imprinting an explanation for increased birth defects? The biological basis for increased frequency of birth defects in ART is unclear. Heritable single gene or polygenic factors conferring infertility could have a pleiotropic effect on offspring, but this cannot explain the magnitude of the increase. There also seems little reason to postulate de-novo chromosomal arrangements in vitro or accumulation of the requisite multiple mutations at multiple loci that would constitute a polygenic/multifactorial explanation for isolated structural anomalies. A plethora of variables in ART technology (vicissitudes of culture media; ovulation stimulation) could, however, perturb embryonic differentiation through altered gene expression. Given lack of obvious explanations, the biological mechanism most often invoked is perturbations of imprinting. A myriad of growth factors are included, and these alone give any biologists pause. A disturbance in methylation precluding scheduled transcription and translation would yield differences in gene products. Some genes would be expressed that were not originally scheduled to do so, and the converse. In 2002, de Braun et al. [35] reported that seven cases with the autosomal dominant BeckwitheWiedeman syndrome (BWS) were associated with either IVF alone (n ¼ 2) or ART (n ¼ 5). In BWS the usual molecular basis lies in overexpression of the paternal allele; the maternal allele is ordinarily not expressed in BWS or normal children. This usually occurs as result of uniparental (paternal) disomy. In
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ART-related cases studied by de Braun et al. [35], however, perturbation was usually due not to increased paternal expression due to uniparental disomy (UPD), but rather to unscheduled expression of the maternal allele normally quiescent (hypomethylation of LIT1). A second study from the UK generated not dissimilar findings [36]. Other data have been reviewed by Vermeiden et al. [37]. Hansen et al. [14] gathered data in a cohort of pooled known imprinting disorders: BWS, RusseleSilver syndrome, PradereWilli syndrome, pseudohypoparathroidism, UPD for chromosome 14, maternal hypomethylation syndrome. There were four occurrences in ART cases (0.2%) versus 30 in non-ART (0.01%); OR was 8.21 (95% CI: 2.81e24.00). On the other hand, a study of Danish registries, for selecting a list of purported imprinting disorders, found no increase among ART cases [38]. This argues against imprinting as an omnibus explanation for increased OR in ART offspring. In conclusion, invoking imprinting due to ART protocols is a popular and plausible explanation for the imprinting disorders listed alone. However, imprinting seems an implausible explanation for isolated structural anomalies (e.g. facial clefts, cardiac). Moreover, if ART perturbs imprinting only in selected disorders, the absolute increase will be low. Given an OR of 8, there would be little arithmetic (absolute) risk in ART offspring, compared with the general population. Thus, if the sole explanation for the increased rate of birth defects in ART offspring is imprinting, this is either disturbed in disorders not yet appreciated or the phenomenon acts in ways not appreciated (e.g. enhancers in non-coding regions). 10. Subfertility, infertility and birth defects The likely cause of increased frequency of birth defects in ART is underlying subfertility [39]. The sentinel observation is that the frequency of birth defects in infertile couples who belatedly (>12 months unprotected intercourse) achieve pregnancy without ART is almost the same as that in couples requiring ART [40,41]. In the study by Zhu et al. [40], OR was 1.17 (95% CI: 1.0e1.36) for infertile couples who conceived naturally while awaiting ART. Criteria consisted of requiring more than 12 months of unprotected sex to succeed. Davies et al. [13] likewise reported increased birth defects in offspring of women who were subfertile but who conceived without ART (OR: 1.29; 95% CI: 0.99e1.68). Of special note, women who had prior offspring with ART but who conceived spontaneously in a later cycle had offspring with increased birth defects (OR: 1.25; 95% CI: 1.01e1.56). In conclusion subfertility is a major factor in, if not the sole explanation for, ART-related birth defects. Rimm et al. [41] believe that almost all the increased risk observed in ART offspring reflects selection bias of subfertile couples, who, without medical assistance, would never achieve pregnancy. Hansen et al. [19] disagree, and indeed one must continue to ferret out potential technologyrelated causes. However, like Pinborg et al. [9] and Rimm et al. [41], this writer believes that selection bias seems more likely. 11. Conclusion Assisted reproductive technology is associated with a small (OR: 1.3) increase in birth defects. This should be communicated to patients prior to undergoing ART. The counselor may also wish to communicate concern over pitfalls in data used to derive these opinions, but must realize that perfection cannot be achieved in experimental design because the ideal control group cannot be constructed. It is often instructive to remind all couples contemplating pregnancy that the baseline anomaly rate is 2e3%, compared with 3e4% in ART. The consensus to be communicated is that both traditional IVF as well as ICSI/IVF show the same increased risk, except for increased sex chromosome abnormalities
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and hypospadias in ICSI. Otherwise, no particular organ system seems disproportionately affected. No additive risk seems to exist in ART twins compared with non-ART twins, nor in embryos previously cryopreserved. Overall, the increased risk observed e irrespective of etiology e seems unlikely to dissuade couples from attempting to have their own children. Conflict of interest statement None declared. Funding sources None. References [1] Morin NC, Wirth FH, Johnson DH, Frank LM, Presburg HJ, Van de Water VL, et al. Congenital malformations and psychosocial development in children conceived by in vitro fertilization. J Pediatr 1989;115:222e7. [2] Lancaster PA. Registers of in-vitro fertilization and assisted conception. Hum Reprod 1996;11(Suppl. 4):89e104. discussion 105e9. [3] 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;14:1896e902. [4] Dhont M, De Sutter P, Ruyssink G, Martens G, Bekaert A. Perinatal outcome of pregnancies after assisted reproduction: a caseecontrol study. Am J Obstet Gynecol 1999;181:688e95. [5] Anthony S, Buitendijk SE, Dorrepaal CA, Lindner K, Braat DD, den Ouden AL. Congenital malformations in 4224 children conceived after IVF. Hum Reprod 2002;17:2089e95. [6] Kallen B, Finnstrom O, Lindam A, Nygren KG, Olausson PO. In vitro fertilization (IVF) in Sweden: risk for congenital malformations after different IVF methods. Birth Defects Res A Clin Mol Teratol 2005;73:162e9. [7] Klemetti R, Gissler M, Sevon T, Koivurova S, Ritvanen A, Hemminki E. Children born after assisted fertilization have an increased rate of major congenital anomalies. Fertil Steril 2005;84:1300e7. [8] Ombolet W, Peeraer K, DeSutter P, Gerris J, Bosmans E, Martens G, et al. Perinatal outcomes of ICSI pregnancies compared with a matched group of natural conception pregnancies in Flanders (Belgium): a cohort study. Reprod Biomed Online 2005;11:244e53. [9] Pinborg A, Henningsen AK, Malchau SS, Loft A. Congenital anomalies after assisted reproductive technology. Fertil Steril 2013;99:327e32. [10] Fujii M, Matsouka R, Bergel E, van der Poel S, Okai T. Perinatal risk in singleton pregnancies after in vitro fertilization. Fertil Steril 2010;94:2113e7. [11] Halliday JL, Ukoumunne OC, Baker HW, Brehany S, Jacques AM, Garrett C, et al. Increased risk of blastogenesis birth defects, arising in the first 4 weeks of pregnancy, after assisted reproductive technologies. Hum Reprod 2010;25: 59e65. [12] Kallen B, Finnstrom O, Lindam A, Nilsson E, Nygren KG, Otterblad PO. Congenital malformations in infants born after in vitro fertilization in Sweden. Birth Defects Res A Clin Mol Teratol 2010;88:137e43. [13] 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;366:1803e13. [14] Hansen M, Kurinczuk JJ, de Klerk N, Burton P, Bower C. Assisted reproductive technology and major birth defects in Western Australia. Obstet Gynecol 2012;120:852e63. [15] Kelley-Quon LI, Tseng CH, Janzen C, Shew SB. Congenital malformations associated with assisted reproductive technology: a California statewide analysis. J Pediatr Surg 2013;48:1218e24. [16] 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;21:437e43. [17] Hansen M, Bower C, Milne E, de Klerk N, Kurinczuk JJ. Assisted reproductive technologies and the risk of birth defects e a systematic review. Hum Reprod 2005;20:328e38.
[18] Wen J, Jiang J, Ding C, Dai J, Liu Y, Xia Y, et al. Birth defects in children conceived by in vitro fertilization and intracytoplasmic sperm injection: a meta-analysis. Fertil Steril 2012;97:1331e7. e1e4. [19] Hansen M, Kurinczuk JJ, Milne E, de Klerk N, Bower C. Assisted reproductive technology and birth defects: a systematic review and meta-analysis. Hum Reprod Update 2013;19:330e53. [20] Simpson JL. Registration of congenital anomalies in ART populations: pitfalls. Hum Reprod 1996;11(Suppl. 4):81e8. [21] Simpson JL, Liebaers I. Assessing congenital anomalies after preimplantation genetic diagnosis. J Assist Reprod Genet 1996;13:170e6. [22] 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;346:725e30. [23] Wennerholm UB, Bergh C, Hamberger L, Lundin K, Nilsson L, Wikland M, et al. Incidence of congenital malformations in children born after ICSI. Hum Reprod 2000;15:944e8. [24] Ericson A, Kallen B. Congenital malformations in infants born after IVF: a population based study. Hum Reprod 2001;16:504e9. [25] Sala P, Ferrero S, Buffi D, Pastorino D, Bertoldi S, Vaccari L, et al. Congenital defects in assisted reproductive technology pregnancies. Minerva Ginecol 2011;63:227e35. [26] Bonduelle M, Wennerholm UB, Loft A, Tarlatzis BC, Peters C, Henriet S, 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;20:413e9. [27] Castilla EE, Oriolo IM. ECLAMC: the Latin-American collaborative study of congenital malformations. Community Genet 2004;7:76e94. [28] Jones KL. Smith’s recognizable patterns of human malformations. 7th ed. Philadelphia: Saunders; 2013. [29] Mills J, Knopp RH, Simpson JL, Javanovich-Peterson L, Metzger BE, Holmes LB, et al. Lack of relation of increased malformation rates in infants of diabetic mothers to glycemic control during organogenesis. N Engl J Med 1988;318: 671e6. [30] Helmerhorst FM, Perquin DA, Donker D, Keirse MJ. Perinatal outcomes in singletons and twins after assisted conception: a systematic review of controlled studies. BMJ 2004;328:261. [31] Jackson RA, Gibson KA, Wu YW, Croughan MS. Perinatal outcomes in singletons following in vitro fertilization: a meta-analysis. Obstet Gynecol 2004;103:551e63. [32] Ponjaert-Kristoffersen I, Bonduelle M, Barnes J, Nekkebroeck J, Loft A, Wennerholm UB, et al. International collaborative study of intracytoplasmic sperm injection-conceived, in vitro fertilization-conceived, and naturally conceived 5-year-old child outcomes: cognitive and motor assessments. Pediatrics 2005;115:e283e9. [33] Cohen J, Wells D, Musse S. Removal of 2 cells from cleavage stage embryos is likely to reduce the efficacy of chromosomal tests that are used to enhance implantation rates. Fertil Steril 2008;87:496e503. [34] De Vos A, Staessen C, De Rycke M, Verpoest W, Haentjens P, Devroey P, et al. Impact of cleavage-stage embryo biopsy in view of PGD on human blastocyst implantation: a prospective cohort of single embryo transfer. Hum Reprod 2009;24:2988e96. [35] DeBaun MR, Niemtz EL, Feinberg AP. Association of in vitro fertilization with BeckwitheWiedemann syndrome and epigenetic alterations of LIT1 and H19. Am J Hum Genet 2003;72:156e60. [36] Maher ER, Brueton LA, Bowdin SC, Luharia A, Cooper W, Cole TR, et al. BeckwitheWiedemann syndrome and assisted reproduction technology (ART). J Med Genet 2003;40:62e4. [37] Vermeiden JP, Bernardus RE. Are imprinting disorders more prevalent after human in vitro fertilization or intracytoplasmic sperm injection? Fertil Steril 2013;99:642e51. [38] Lidegaard O, Pinborg A, Andersen AN. Imprinting diseases and IVF: Danish National IVF cohort study. Hum Reprod 2005;20:950e4. [39] Zhu JL, Basso O, Obel C, Bille C, Olsen J. Infertility, infertility treatment, and congenital malformations: Danish national birth cohort. BMJ 2006;333:679. [40] Jaques AM, Amor DJ, Baker HW, Healy DL, Ukoumunne OC, Breheny S, et al. Adverse obstetric and perinatal outcomes in subfertile women conceiving without assisted reproductive technologies. Fertil Steril 2010;94:2674e9. [41] Rimm AA, Katayama AC, Katayama KP. A meta-analysis of the impact of IVF and ICSI on major malformations after adjusting for the effect of subfertility. J Assist Reprod Genet 2011;28:699e705.
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Review
Birth defects and assisted reproductive technologies Joe Leigh Simpson* March of Dimes, 1275 Mamaroneck Avenue, White Plains, NY 10605, USA
s u m m a r y Keywords: Assisted reproductive technologies (ART) Birth defects Hypospadias Imprinting Intracytoplasmic sperm injection (ICSI) In-vitro fertilization (IVF)
Assisted reproductive technologies (ART) using in-vitro fertilization (IVF) account for w1% of births in the USA and as much as 3e4% in Europe or Australia. Initially studies involved infants prospectively examined in an early cohort of US births, with salutary results. Later studies began to show the frequency of birth defects to be increased. In meta-analysis, odds ratio was >1.0, with the 95% confidence limit not extending to 1.0, with the 95% CI not extending across the 1.0 isobar [6e15]. Thus, there evolved the present consensus that increased birth defects are indeed positively associated with ART. The major question at present is whether this increase is due to ART protocols per se or merely reflective of the biological perturbations that generated the infertility that necessitated ART to achieve pregnancy. In this communication, we shall first consider the frequency of congenital anomalies in offspring of ART pregnancies, stratified by specific subgroups depending on technique. We shall also comment on the pitfalls that make difficult definitive conclusions concerning the explanation for anomalies detected.
2. Frequency of anomalies in ART pregnancies Almost 50 cohort studies have addressed the question of anomalies in ART pregnancies, as referenced elsewhere [16e19]. Initial studies naturally focused on IVF alone, because not until the mid-1990s could male infertility be managed by intracytoplasmic sperm injection (ICSI) followed by traditional IVF. In the 1990s and early 2000s, one could confidently conclude the overall risk was not, say, a two- or three-fold increase above the accepted population baseline of 2e3%. However, more precise statements could not be made. Notwithstanding general reassurance, this author and others pointed out pitfalls that could sway opinions on safety in either
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Please cite this article in press as: Simpson JL, Birth defects and assisted reproductive technologies, Seminars in Fetal & Neonatal Medicine (2014), http://dx.doi.org/10.1016/j.siny.2014.01.001
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J.L. Simpson / Seminars in Fetal & Neonatal Medicine xxx (2014) 1e6
Table 1 Earlier population-based studies of anomalies in assisted reproductive technology pregnancies. Study
Years of sample accrual
Adjusted OR (95% CI)
Statistical significance
Country
Dhont et al. [4] Westergaard et al. [3] Anthony et al. [5]
1986e2002 1994e1995 1995e1996
1.25 (0.96e1.64) 1.04 (0.78e1.39) 1.03 (0.86e1.23)
No No No
Belgium Sweden Netherlands
OR, odds ratio; CI, confidence interval. The initial population-based studies depend on data in registries. Ascertainment varied between studies with respect to duration of time in which anomalies were sought and definition of major defects. The interval of accumulated ART registry cases does not in any report correspond to the extant laboratory and ovulation stimulation protocols used in 2014.
direction [20,21]. It was in particular appreciated that power to detect a significantly increased frequency of anomalies was not possible due to small sample size. Table 2 shows later population-based studies on which this altered conclusion began to be based [6e15]. All were based on registries that recorded cohorts of births, from which those with anomalies could be stratified. A caveat is that the definition of birth defects among registries varies, usually involving International Classification of Diseases (ICD)-based diagnosis codes not well suited to distinguish major from minor anomalies. ICD codes at birth are not robust in detecting subtle anomalies not externally visible, nor in excluding inherited syndromes or chromosomal anomalies. Neither is plausibly due to ART. Not always followed is the pragmatic, accepted, definition of a major birth defect as one that causes death, functional impairment or (if structural) requires surgery. Reviewing several studies will suffice as illustrative. In 2005 Klemetti et al. [7] published Finnish registry of IVF and ICSI cases delivered in 1996e1999, finding the adjusted OR for all birth defects to be 1.31 (95% CI: 1.10e1.57), when comparing 4459 cases and 27 078 controls. In 2007, Pinborg et al. [9] used Danish registry data to derive an OR of 1.24 (95% CI: 1.09e1.43). In 2005 Kallen et al. [6,12] published an analysis of Swedish registries (1982e1999) data and in 2010 analysed cases for the years 2001e2007. The latter study showed OR of 1.25 (95% CI: 1.5e1.37) based on 15 570 cases [12]. Outside of Scandinavia, the region generating greatest attention is Australia, befitting the country’s sentinel role in developing ART. In 2002 Hansen et al. reported Western Australia cases, reprising this in 2012 [14,22]. Their 2012 report of Western Australia registry data involved ART cases delivered 1994e2002, an interval of significance given many changes having since occurred in laboratory methodology. This study was laudatory, however, in trying to take into account anomalies in pregnancy terminations. Both the 2911 ART cases and the 210 997 non-ART cases were followed for 6 years. A ‘major birth defect’ was found in 8.7% of ART cases and 5.4
of non-ART cases (OR: 1.53; 95% CI: 1.30e1.79). Rates were not different in unlike sex twins (obligatory dizygosity) (OR: 1.08; 95% CI: 0.77e1.51). The 5.4% birth defects in the control group is at odds with general acceptance, absent comprehensive laboratory assessments such as array comparative genomic hybridization. These authors concluded also that there has been a decrease in the prevalence of birth defects compared with their earlier cohort (1994e1998) [14,23]. One explanation may simply be greater appreciation between major and minor anomalies. Plausible scientific reasons include changes in culture media, better regulation of heat and CO2 in incubators, and differential ovulation stimulation regimes. A second Australian group extending interval of ascertainment to 5 years was that of Davies et al. [13], who used registry data of 308 974 births in South Australia (Adelaide). Minor defects were excluded unless they required treatment or were ‘disfiguring’. Among 6163 ART offspring were 513 with anomalies (8.4%), compared with 5.8% in non-ART births. The adjusted OR was 1.28 (95% CI: 1.16e1.41). OR was 1.26 for IVF alone, and 1.77 for ICSI/IVF. It would be of interest to know the temporal sequence in which the cumulative absolute frequency of birth defects occurred, i.e. ascertainment at birth versus later. Of special interest to US readers is the report of Kelley-Quon et al. [15], who used the California Patient Discharge Linked Birth Cohort Database that lists anomalies by ICD-9 codes. This study involved 4795 infants born in 2006e2007 after ART, compared with 46 025 naturally conceived in the same interval. The overall rate of ‘major congenital abnormalities’ was 9.0% vs 6.6% (OR: 1.25; 95% CI: 1.12e1.39; P < 0.001). Incidence of birth defects this high bespeaks inclusion of many minor anomalies. In this author’s opinion, this alone casts doubt on conclusions that ORs were significantly increased for certain organ-specific anomalies: eye, head and neck, heart and genitourinary track. These limitations notwithstanding, meta-analyses reached similar arithmetic conclusions: Rimm et al. [16]: 1.29 (95% CI: 1.01e1.67); Hansen et al. [17,19], risk ratio: 1.32 (95% CI: 1.24e
Table 2 Later population-based studies on anomalies in assisted reproductive technology (ART) pregnancies (2005e2013). Study
Years of sample accrual
Adjusted OR (95% CI)
Statistical significance
Country
Kallen et al. [6] Davies et al. [13] Halliday et al. [11] Hansen et al. [14] Pinborg et al. [9] Klemetti et al. [7] Ombolet et al. [8] Kallen et al. [12] Kelley-Quon et al. [15] Fuji et al. [10]
1982e2001 1986e2002 1991e2004 1994e2002 1995e2000 1996e1999 1997e2003 2001e2007 2006e2007 2006
1.44 1.24 1.36 1.53 1.24 1.31 1.11 1.25 1.25 1.17
Yes Yes Yes Yes No Yes No Yes Yes No
Sweden Australia (Adelaide) Australia (Parkville) Australia (Perth) Denmark Finland Belgium Sweden USA (California) Japan
(1.32e1.57) (1.09e1.41) (1.19e1.55) (1.30e1.79) (0.97e1.58) (1.10e1.57) (0.08e1.58) (1.15e1.37) (1.21e1.39) (0.81e1.69)
OR, odds ratio; CI, confidence interval. These population-based studies depend on data in registries. Ascertainment varied with respect to duration of time in which anomalies were sought and definition of major defects. The interval of accumulated ART registry cases does not in any report correspond to the extant laboratory and ovulation stimulation protocols used in 2013.
Please cite this article in press as: Simpson JL, Birth defects and assisted reproductive technologies, Seminars in Fetal & Neonatal Medicine (2014), http://dx.doi.org/10.1016/j.siny.2014.01.001
J.L. Simpson / Seminars in Fetal & Neonatal Medicine xxx (2014) 1e6 Table 3 Reported meta-analyses on birth defects and assisted reproductive technologies. Study
Year
Studies accepted
OR (95% CI)
Rimm et al. [16] Hansen et al. [17] Wen et al. [18] Hansen et al. [19]
2004 2005 2012 2013
19 25a 46 45a
1.29 1.29 1.37 1.32
(1.01e1.67) (1.21e1.37) (1.26e1.48) (1.24e1.42)
OR, odds ratio; CI, confidence interval. a Many studies considered acceptable in the 2005 review by Hansen et al. [17] were no longer considered acceptable in their 2013 report [19].
1.42); Wen et al. [18]: 1.37 (95% CI: 1.26e1.48) (Table 3). Similar results are not surprising because the same studies were generally analyzed. 3. Anomalies by organ system Because of sample size limitations, individual anomalies have generally been assessed only by pooling anomalies by organ system. Yet within a given system there is a plethora of different anomalies of difficult etiologies e genetic and otherwise. Collapsing all such anomalies to assess a single organ system is convenient for generating statistical power, but this does not obviate the methodological flow. Hypospadias is the only organ system in which OR has been consistently shown to be increased. An odds ratio of 3.0 (95% CI: 1.09e6.50) was reported by Wennerholm et al. [23] and 1.5 (95% CI: 1.0e2.1) by Ericson and Kallen [24]. Klemetti et al. [7] also showed increased hypospadias. A potential explanation for increased hypospadias in ART is heritable low testosterone, also adversely affecting spermatogenesis. Recurrence risk for hypospadias is 2e5% in first degree relatives (sons). This could lead to transmission of a polygenic trait (hypospadias). There was no significant difference in the metaanalysis by Wen et al. [18] One confounder is that indications for ICSI are now much less stringent than when ICSI was initially introduced, including many cases analyzed by Davies (1986e2002) [13]. ICSI now constitutes half of all ART cases in the USA and even more in some venues. The only other organ system seriously implicated is cardiac. Sala et al. [25] found a significant increase, but in general other authors have found no increase. Special pitfalls exist in assessing cardiac anomalies. A patent ductus arteriosus is normal at birth, but soon closes in full-term infants; in premature infants persistence is normal, and surgical ligation is routine. Ventricular septal defects are not usually evident at birth but manifest about one week after birth when cardiac circulation is adapted for postnatal life. Taking these temporal changes into account is not facile, and beyond the charge of most if not all registries recording birth defects. 4. Methods of data analysis Determining whether anomalies are increased in ART could be based on data from a single center or from a registry. It is fashionable for many to use pooled data (systematic reviews or metaanalysis), and consider this approach superior. Meta-analysis is based mostly on obvious strength of sample size compared with single center studies. Center-specific studies are thus marginalized. However, the latter can utilize checklist comparisons by a limited pool of examiners, an approach not possible in population registries. Indeed, it is studies with less systematic assessments that more often showed no increase in
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birth defects. Meta-analysis has other limitations, namely lack of consensus on inclusion criteria. In meta-analysis the axiom is that all studies are comparable, and thus suitable for pooling. Consider, however, the laudatory reports of Hansen et al. [17,19] The latter exhaustive report excluded non-English reports, reports in which no birth defects were found in either cases or controls, and reports in which birth defects ascertainment extended to a later age. Numbers of studies deemed suitable were 45, similar to Wen et al. (n ¼ 46) [18]. Yet Rimm et al. [16] accepted only 19 studies. Although the validity of meta-analysis in many circumstances is strong, this is more arguable in ART and birth defects. In ART virtually no units utilize similar protocols. Yet many different laboratories contribute to a single registry, to say nothing of differences between registries. Different authors must reach their own opinion as to whether preset criteria are met. Decisions on inclusion criteria may be discrepant at different points in time, even by the same author. For example, upon reassessment 8 years later Hansen et al. [17,19] concluded that many studies accepted as valid in their 2006 study were no longer acceptable. One widely used decision tree is to include or exclude series in which follow-up extended beyond the newborn period. (Separate analysis would be more appropriate.) Thus excluded is the report of Davies et al. [13], despite being an excellent population-based study that extended to 5 years of age and contained ascertainment from multiple sources. Another study excluded for the same reason has been Bonduelle et al. [27] who showed increased sex chromosomal abnormalities in ICSI. These cannot be detected in registries because physical abnormalities are usually not evident at birth. In some meta-analyses it is sometimes decided that certain studies are given higher ‘quality scores’ and, hence, greater weight. For example, Hansen et al. [19] assigned a ‘quality 1’ score (best) to 11 of 13 registry-based studies, and only seven population-based studies were not scored quality 1. Among 32 ‘clinic’ samples, only one was assigned quality 1. Many of the latter were indeed of insufficient sample size, but numbers alone should not be the sole criterion for high quality because quality of clinical examination is less than ideal in registries. Consistent clinical acumen is not possible among all examiners, some of whom lack familiarity in distinguishing major from minor anomalies. It is difficult but not impossible to form a collaborative network of clinical centers to assess anomalies consistently among centers, but not impossible (see the reports from the South American Registry ECLAMC [27]). Another problem with registry data is long interval during which cases were accumulated. Anomalies were initially probably more vigorously sought in ART cases than controls [20,21]. This bias is probably less so now, but the earlier bias is still relevant because registry data being used for currently published meta-analyses are often based on deliveries even extending as far back as the 1980s. Paradoxically, the larger the study (greater power), the more ‘ancient’ and now ‘atypical’ cases included. The above criticisms are not meant to denigrate work pursued to unravel a difficult problem, but to remind the reader that crisp data suitable for analysis do not exist.
5. Pitfalls in assessing presence of birth defects This author has expressed concern on pitfalls precluding data accumulated and used for comparison of ART outcomes [20,21]. Box 1 lists common pitfalls so far discussed, a few aspects of which are highlighted in the following subsections.
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Box 1 Pitfalls in experimental design and interpretation in assessing birth defects in assisted reproductive technology (ART) offspring.
pregnancies analyzed and publication data makes generalizability to women undergoing ART in 2014 arguable. 5.4. Interval for anomaly surveillance after birth
A. Ascertainment Including or not including anomalies in stillborns. Including or not including anomalies in terminated fetuses. Including or not including anomalies detectable on ultrasound but not externally visible at birth. Varying duration of surveillance. Varying aggressiveness of surveillance. B. Definition of anomalies ‘Minor’ anomalies versus ‘major’ anomalies. Failure to utilize pre-set definitions and check lists (i.e. minimizing individual observer variability). Determining when a ‘minor’ anomaly becomes ‘major’ (e.g. requiring surgery). C. Methodology Failure to stratify analyses into isolated anomalies versus multiple anomalies (syndromic) disorders. Failure to recognize (exclude) heritable disorders whose etiology is not plausibly ART-related. Generalization of conclusions to a contemporary population when data were derived in a prior era that used now-outdated protocols. Application of meta-analysis when studies are of dissimilar design (unavoidable given lack of standardized protocols).
Another problem is the varying length of surveillance after birth. Birth defects registries traditionally are restricted to anomalies ascertained in the neonatal period (before hospital discharge). However, several excellent single-center studies extend to 5 years, providing information not possible with registries (e.g. sex chromosomal abnormalities) [13,26]. Certain organ-specific ART-related anomalies could be manifested only in the newborn period and other only later. Data analyzed in meta-analysis should ideally be stratified by length of surveillance. 5.5. Lack of true control group What is the control group? In theory, the proper control should consist of women who require ART but who become pregnant without ART, ideally within the time defined as normal to become pregnant (12 months). Obviously, this group does not exist. Thus, no ‘gold standard’ randomized clinical trials or caseecontrol study can be conducted. Instead, published reports compare ART outcomes to naturally conceived offspring. The closest to a proper control group seems to be a subfertile couple (inability to conceive by 12 months) who conceive naturally while awaiting ART. Other options include same-sex couples using a surrogate, but sample sizes are inadequate.
5.1. Definition of anomalies
6. Anomalies in singleton versus twin gestation
The major source of confusion is probably inconsistent definition of an anomaly (birth defect). How precisely does one distinguish major from minor anomalies? Should one rely on studies in which minor anomalies are collapsed into a single category with major anomalies? In fact, criteria for distinguishing major from minor anomalies are well known to geneticists and well codified, but generally not familiar to clinicians outside medical genetics [28]. Rigor of physical examination is an issue because geneticists record more minor anomalies than non-genetic pediatricians. Relying upon pooled data when the primary outcome (birth defects) is not assessed identically is inherently hazardous. Examiners not surprisingly have variable skills, training and interest; thus, criteria cannot be assumed to be comparable across centers, studies, or countries. This becomes immediately evident by noting variable rates of anomalies in controls, namely signaled by rates considerably higher than the accepted 2e4% major birth defects at birth and shown in checklist-based studies [29].
Consensus exists that overall birth defects are increased in singletons, but the same cannot be said for multiple gestations of which twins constitute by far the largest group. When adjustment was made for zygosity by Hansen et al. [19], OR was 1.26 (95% CI: 0.9e1.60). Anomaly rates were not different between ART and nonART twins. In unlike sex twins (obligatory dizygosity), OR was 1.08 (95% CI: 0.77e1.51). Recall that twins naturally conceived have long been known to show increased birth defects, particularly if monozygotic. Prior to the era of ART, half of all like-sexed twins were monozygotic. Now the percentage should be lower. Interestingly, preterm birth in ART offspring is considered to be increased in singleton births but not in twin gestations [30,31]. Perhaps infertility (and, hence, likelihood of embryo implantation) is greater in women whose embryos are more likely to be abnormal and, hence, less likely to sustain two embryos. Or, perhaps, singletons had a cotwin who underwent unrecognized demise (vanishing twin) that nonetheless exerted a persistent deleterious effect, perhaps involving the decidua [32]. In conclusion, anomalies in both non-ART twins and in ART twins are increased compared with singleton controls, but an additive risk in ART is surprisingly unproven at present.
5.2. ART protocols There is a lack of conformity in ovulation stimulation regimens, a plethora of different culture media, and multiple embryo incubation techniques. Standardization does not exist, and commercially utilized media list only components and not concentrations. 5.3. Interval of pregnancy ascertainment Especially egregious is, as noted, assuming that results derived from methods used long ago are still generalizable to the contemporary patient, a concern raised earlier. The most contemporary study involves 2006e2007 cycles, well behind currently used techniques like vitrification [15]. The long interval between
7. Anomalies in ICSI/IVF versus IVF alone Initially only IVF per se was assessed for association with birth defects. As stated earlier, no statistically significant association was reported during the 1990s. With utilization in the 1990s of ICSI (i.e. ART using ICSI followed by traditional IVF), increased birth defects began to be reported. With ICSI the frequency of sex chromosomal abnormalities appears increased in prenatal samples (amniocenteses) as well as hypospadias [7,23,24]. Otherwise, anomaly rates have appeared similar in ICSI and non-ICSI IVF, rendering overall anomaly rates not significantly different. The exception was Davies
Please cite this article in press as: Simpson JL, Birth defects and assisted reproductive technologies, Seminars in Fetal & Neonatal Medicine (2014), http://dx.doi.org/10.1016/j.siny.2014.01.001
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et al. [13], showing increased risk only in ICSI offspring (OR: 1.47; 95% CI: 1.15e1.89). ICSI couples have the confounder of having increased frequencies of balanced translocations, not only in males (2.05%) but female (1.38%) sex chromosomal abnormalities, and inversions were also found not infrequently. Thus, two subfertile individuals presumably constitute a single infertile couple. The frequency of balanced translocations in men undergoing ICSI is higher in oligospermic (4%) than in azoospermic men (1%). By analogy the same principle may hold for single mutant genes, certain men undergoing ICSI having a pleiotropic mutation one component of which is infertility and another predisposition to a structural anomaly. Chromosomal abnormalities as assessed by preimplantation genetic diagnosis are increased in ICSI-assisted embryos [32]. In conclusion, sex chromosomal abnormalities and hypospadias are considered increased in ICSI compared with IVF alone. 8. Cryopreservation Data are limited concerning whether cryopreservation affects birth defects in liveborns, but at present data are reassuring. Loss of cell(s) from cleavage stage biopsy is clearly deleterious to embryo survival, as shown by decreased pregnancy rates for embryos that had loss of one cell upon thawing [33]. Similarly, removal of more than two cells from cleavage stage embryos reduced the pregnancy rate [34]. These statements are not applicable for trophectoderm biopsy where a dozen or more cells are removed. Fortunately, loss of cells at either cleavage stage or blastocyst stage does not result in liveborn anomalies because the ‘all or none’ effect exists. Embryos surviving to live birth are not affected long term because when loss of a single cell occurs at this stage of embryogenesis the function of that cell can be subsumed by another because all cells are totipotential. In their own cohort Hansen et al. [22] claimed to observe a trend favoring fresh rather than frozen singleton IVF; however, this was not found in twins or ICSI pregnancies. Davies et al. [13] found no differences at all. This author counsels that neither benefit nor harm exists for cryopreservation with respect to birth defects. 9. Is imprinting an explanation for increased birth defects? The biological basis for increased frequency of birth defects in ART is unclear. Heritable single gene or polygenic factors conferring infertility could have a pleiotropic effect on offspring, but this cannot explain the magnitude of the increase. There also seems little reason to postulate de-novo chromosomal arrangements in vitro or accumulation of the requisite multiple mutations at multiple loci that would constitute a polygenic/multifactorial explanation for isolated structural anomalies. A plethora of variables in ART technology (vicissitudes of culture media; ovulation stimulation) could, however, perturb embryonic differentiation through altered gene expression. Given lack of obvious explanations, the biological mechanism most often invoked is perturbations of imprinting. A myriad of growth factors are included, and these alone give any biologists pause. A disturbance in methylation precluding scheduled transcription and translation would yield differences in gene products. Some genes would be expressed that were not originally scheduled to do so, and the converse. In 2002, de Braun et al. [35] reported that seven cases with the autosomal dominant BeckwitheWiedeman syndrome (BWS) were associated with either IVF alone (n ¼ 2) or ART (n ¼ 5). In BWS the usual molecular basis lies in overexpression of the paternal allele; the maternal allele is ordinarily not expressed in BWS or normal children. This usually occurs as result of uniparental (paternal) disomy. In
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ART-related cases studied by de Braun et al. [35], however, perturbation was usually due not to increased paternal expression due to uniparental disomy (UPD), but rather to unscheduled expression of the maternal allele normally quiescent (hypomethylation of LIT1). A second study from the UK generated not dissimilar findings [36]. Other data have been reviewed by Vermeiden et al. [37]. Hansen et al. [14] gathered data in a cohort of pooled known imprinting disorders: BWS, RusseleSilver syndrome, PradereWilli syndrome, pseudohypoparathroidism, UPD for chromosome 14, maternal hypomethylation syndrome. There were four occurrences in ART cases (0.2%) versus 30 in non-ART (0.01%); OR was 8.21 (95% CI: 2.81e24.00). On the other hand, a study of Danish registries, for selecting a list of purported imprinting disorders, found no increase among ART cases [38]. This argues against imprinting as an omnibus explanation for increased OR in ART offspring. In conclusion, invoking imprinting due to ART protocols is a popular and plausible explanation for the imprinting disorders listed alone. However, imprinting seems an implausible explanation for isolated structural anomalies (e.g. facial clefts, cardiac). Moreover, if ART perturbs imprinting only in selected disorders, the absolute increase will be low. Given an OR of 8, there would be little arithmetic (absolute) risk in ART offspring, compared with the general population. Thus, if the sole explanation for the increased rate of birth defects in ART offspring is imprinting, this is either disturbed in disorders not yet appreciated or the phenomenon acts in ways not appreciated (e.g. enhancers in non-coding regions). 10. Subfertility, infertility and birth defects The likely cause of increased frequency of birth defects in ART is underlying subfertility [39]. The sentinel observation is that the frequency of birth defects in infertile couples who belatedly (>12 months unprotected intercourse) achieve pregnancy without ART is almost the same as that in couples requiring ART [40,41]. In the study by Zhu et al. [40], OR was 1.17 (95% CI: 1.0e1.36) for infertile couples who conceived naturally while awaiting ART. Criteria consisted of requiring more than 12 months of unprotected sex to succeed. Davies et al. [13] likewise reported increased birth defects in offspring of women who were subfertile but who conceived without ART (OR: 1.29; 95% CI: 0.99e1.68). Of special note, women who had prior offspring with ART but who conceived spontaneously in a later cycle had offspring with increased birth defects (OR: 1.25; 95% CI: 1.01e1.56). In conclusion subfertility is a major factor in, if not the sole explanation for, ART-related birth defects. Rimm et al. [41] believe that almost all the increased risk observed in ART offspring reflects selection bias of subfertile couples, who, without medical assistance, would never achieve pregnancy. Hansen et al. [19] disagree, and indeed one must continue to ferret out potential technologyrelated causes. However, like Pinborg et al. [9] and Rimm et al. [41], this writer believes that selection bias seems more likely. 11. Conclusion Assisted reproductive technology is associated with a small (OR: 1.3) increase in birth defects. This should be communicated to patients prior to undergoing ART. The counselor may also wish to communicate concern over pitfalls in data used to derive these opinions, but must realize that perfection cannot be achieved in experimental design because the ideal control group cannot be constructed. It is often instructive to remind all couples contemplating pregnancy that the baseline anomaly rate is 2e3%, compared with 3e4% in ART. The consensus to be communicated is that both traditional IVF as well as ICSI/IVF show the same increased risk, except for increased sex chromosome abnormalities
Please cite this article in press as: Simpson JL, Birth defects and assisted reproductive technologies, Seminars in Fetal & Neonatal Medicine (2014), http://dx.doi.org/10.1016/j.siny.2014.01.001
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and hypospadias in ICSI. Otherwise, no particular organ system seems disproportionately affected. No additive risk seems to exist in ART twins compared with non-ART twins, nor in embryos previously cryopreserved. Overall, the increased risk observed e irrespective of etiology e seems unlikely to dissuade couples from attempting to have their own children. Conflict of interest statement None declared. Funding sources None. References [1] Morin NC, Wirth FH, Johnson DH, Frank LM, Presburg HJ, Van de Water VL, et al. Congenital malformations and psychosocial development in children conceived by in vitro fertilization. J Pediatr 1989;115:222e7. [2] Lancaster PA. Registers of in-vitro fertilization and assisted conception. Hum Reprod 1996;11(Suppl. 4):89e104. discussion 105e9. [3] 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;14:1896e902. [4] Dhont M, De Sutter P, Ruyssink G, Martens G, Bekaert A. Perinatal outcome of pregnancies after assisted reproduction: a caseecontrol study. Am J Obstet Gynecol 1999;181:688e95. [5] Anthony S, Buitendijk SE, Dorrepaal CA, Lindner K, Braat DD, den Ouden AL. Congenital malformations in 4224 children conceived after IVF. Hum Reprod 2002;17:2089e95. [6] Kallen B, Finnstrom O, Lindam A, Nygren KG, Olausson PO. In vitro fertilization (IVF) in Sweden: risk for congenital malformations after different IVF methods. Birth Defects Res A Clin Mol Teratol 2005;73:162e9. [7] Klemetti R, Gissler M, Sevon T, Koivurova S, Ritvanen A, Hemminki E. Children born after assisted fertilization have an increased rate of major congenital anomalies. Fertil Steril 2005;84:1300e7. [8] Ombolet W, Peeraer K, DeSutter P, Gerris J, Bosmans E, Martens G, et al. Perinatal outcomes of ICSI pregnancies compared with a matched group of natural conception pregnancies in Flanders (Belgium): a cohort study. Reprod Biomed Online 2005;11:244e53. [9] Pinborg A, Henningsen AK, Malchau SS, Loft A. Congenital anomalies after assisted reproductive technology. Fertil Steril 2013;99:327e32. [10] Fujii M, Matsouka R, Bergel E, van der Poel S, Okai T. Perinatal risk in singleton pregnancies after in vitro fertilization. Fertil Steril 2010;94:2113e7. [11] Halliday JL, Ukoumunne OC, Baker HW, Brehany S, Jacques AM, Garrett C, et al. Increased risk of blastogenesis birth defects, arising in the first 4 weeks of pregnancy, after assisted reproductive technologies. Hum Reprod 2010;25: 59e65. [12] Kallen B, Finnstrom O, Lindam A, Nilsson E, Nygren KG, Otterblad PO. Congenital malformations in infants born after in vitro fertilization in Sweden. Birth Defects Res A Clin Mol Teratol 2010;88:137e43. [13] 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;366:1803e13. [14] Hansen M, Kurinczuk JJ, de Klerk N, Burton P, Bower C. Assisted reproductive technology and major birth defects in Western Australia. Obstet Gynecol 2012;120:852e63. [15] Kelley-Quon LI, Tseng CH, Janzen C, Shew SB. Congenital malformations associated with assisted reproductive technology: a California statewide analysis. J Pediatr Surg 2013;48:1218e24. [16] 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;21:437e43. [17] Hansen M, Bower C, Milne E, de Klerk N, Kurinczuk JJ. Assisted reproductive technologies and the risk of birth defects e a systematic review. Hum Reprod 2005;20:328e38.
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Please cite this article in press as: Simpson JL, Birth defects and assisted reproductive technologies, Seminars in Fetal & Neonatal Medicine (2014), http://dx.doi.org/10.1016/j.siny.2014.01.001
