Congenital anorectal malformations are major anomalies of the gastrointestinal system, with a prevalence of approximately 1 in 3000 births worldwide.1 Anorectal malformations are characterized by disrupted embryonic development of the anus and rectum in the 4th to 8th week after conception—often also involving other organs, such as the bladder or vagina. Although the physical and psychosocial impact is large for patients with anorectal malformations and their parents,2 knowledge about the potential risk factors is still limited.
In the past decade, the risks of congenital malformations following assisted reproductive techniques (ART), including in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), received large amounts of scientific attention. Most studies showed an increased prevalence of congenital malformations after ART, but they investigated only heterogeneous patient groups consisting of broad categories of congenital malformations, such as all gastrointestinal malformations.3,4 Four studies, specifically focused on anorectal malformations, found ART to increase the risk of anorectal malformations >3 times compared with spontaneous conceptions,5–8 whereas 2 studies did not.9,10 Clearly, increased risks of anorectal malformations were not found when all fertility treatments, also including intrauterine insemination (IUI) and ovulation induction, were analyzed together.10,11 However, 2 studies showed an association between anorectal malformations and ovulation induction without ICSI or IVF,10,12 whereas 1 study did not.13 The role of parental subfertility was investigated in only 1 study, which suggested an association between anorectal malformations and paternal but not maternal subfertility.11
Limitations of the previous studies, however, were low case numbers, moderately to poorly characterized cases, nonspecific or pooled exposure groups, lack of information on potential confounders, and/or different methods of data collection among cases and controls. Furthermore, none of these studies combined the effects of parental subfertility, prolonged time to pregnancy, and types of fertility treatment within 1 study. Therefore, we aimed to elucidate whether the excess risk of anorectal malformations is attributable to the underlying parental subfertility, the hormones used for ovulation induction, or the ART procedure itself. We performed a case-control study to investigate the role of parental subfertility and prolonged time to pregnancy without fertility treatment or with a range of fertility treatments in the etiology of nonsyndromic anorectal malformations. We also assessed these effects in subgroups of isolated anorectal malformations and anorectal malformations with one or more other major congenital malformations as etiologic heterogeneity was suggested previously.10,14
Aetiologic research into Genetic and Occupational/environmental Risk factors for Anomalies in children (AGORA) is a large data- and biobank with clinical and questionnaire data and DNA samples from children with congenital malformations or childhood cancer, population-based control children, and their parents (www.AGORAproject.nl). In total, 448 cases with anorectal malformations were derived from the AGORA databank. These cases were diagnosed shortly after birth and treated at the departments of Pediatric Surgery of the Radboud university medical center in Nijmegen, the Sophia Children’s Hospital–Erasmus Medical Centre in Rotterdam or the University Medical Center Groningen in the Netherlands. They were born between August 1988 and August 2012 and recruited from 2007 onward. Pediatric surgeons, clinical geneticists, and researchers reviewed the medical records to obtain information on anorectal malformation phenotypes and additional congenital malformations.
Phenotypes of anorectal malformations were classified according to the international Krickenbeck criteria,15 and additional congenital malformations were divided into major and minor malformations according to the EUROCAT classification.16 Cases with syndromic forms of anorectal malformations, chromosomal abnormalities, or cloacal exstrophy were excluded from the study (n = 41, 9%). The remaining cases were divided in isolated anorectal malformations with or without minor congenital malformations and anorectal malformations with one or more additional major congenital malformations. The latter subgroup also included cases with the VACTERL association (Vertebral, Anal, Cardiac, Tracheo-Esophageal, Renal, and Limb defects), defined as cases with anorectal malformations and the presence of at least 2 other features of this association.17 Eighty of the cases with anorectal malformations in this study (21%) were also included in our previous study, in which a wide range of potential risk factors for anorectal malformations were evaluated, such as parental lifestyle factors, occupational exposures, and pooled exposure groups of fertility-related issues.11
Control children were also derived from the AGORA databank (n = 2,196). They were born between January 1990 and March 2011 and randomly sampled and recruited in 2010–2011 via 39 municipalities in geographical areas that covered the referral areas of the 3 hospitals where the cases were treated. Control children with major congenital malformations, chromosomal abnormalities, or syndromes were excluded based on the questionnaire data (n = 56, 3%). In addition, 7 cases and 2 controls were excluded because a sibling was already included in the study, leaving 400 cases and 2138 controls.
Ethical approval for the study protocol of the AGORA data- and biobank was granted by the regional Committee on Research involving Human Subjects. All parents of cases and controls gave informed consent for the study.
Data Collection and Definitions
Cases and controls were all between 0 and 22 years of age. Their parents were invited to participate in AGORA. Both parents were asked to fill in a questionnaire with questions about demographic factors, family history of congenital malformations, and health and lifestyle before and during pregnancy. We used these questionnaires to obtain information about fertility issues and characteristics of cases and controls. Mothers were asked whether their pregnancies were planned, whether they or their partners had received any type of fertility treatment, and about the number of months between the start of trying to conceive and conception. Both mothers and fathers were asked whether they were ever diagnosed with subfertility by a physician. For this study, subfertile parents were defined as parents that had a time to pregnancy of >12 months, were diagnosed with either maternal or paternal subfertility, or both. We assumed that parents with an unplanned pregnancy were fertile. Parents were excluded from the analyses when information on time to pregnancy or parental subfertility was unknown (20 cases [5%] and 165 controls [8%]), leaving 380 cases and 1973 controls for the final analyses. In addition, the following types of fertility treatment were evaluated: ICSI, IVF, IUI (with or without donor), ovulation induction only, and surgical (eg, removal of adhesions in fallopian tube) or other treatment. Parents were classified according to the above priority sequence if >1 treatment was reported. In the Netherlands, treatment for subfertility typically starts after 12 months of trying to conceive.
Infant and parental characteristics included sex and year of birth of the child, maternal and paternal ethnicity (Dutch vs. other), maternal and paternal age at delivery, maternal and paternal education, anorectal malformations in first-degree relatives of the child, multiple pregnancy (vs. singleton pregnancy), parity (nulliparity vs. multiparity), periconceptional folic acid use (any use of folic acid supplements or multivitamins containing folic acid in 4 weeks before through 10 weeks after conception), maternal pre-existing and gestational diabetes, and maternal prepregnancy body mass index (BMI). The latter was calculated using reported weight (in kg) and height (in m) before conception and categorized in 4 groups (<18.5, 18.5–24.9, 25.0–29.9, and ≥30.0 kg/m2).
Statistical analyses were performed using SPSS 20.0 for Windows (IBM SPSS, Chicago, IL, USA). Logistic regression analyses were used to calculate crude and adjusted odds ratios (ORs) with 95% confidence intervals (CIs) for associations between anorectal malformations and parental fertility-related issues. The parents were divided into 3 groups: fertile parents who conceived spontaneously (reference), subfertile parents who conceived without treatment, and subfertile parents who conceived after any type of fertility treatment. We also distinguished among the types of fertility treatment and performed subgroup analyses that included singletons and nulliparous women only. The latter was done because multiple pregnancy seemed to be associated with both anorectal malformations and fertility treatment and because a previous study showed divergent results for the association between anorectal malformations and fertility treatment in singleton compared with multiple pregnancies.7 In addition, associations between anorectal malformations and classes of time to pregnancy (1–3, 4–6, 7–12, and >12 months) were assessed in singletons conceived spontaneously as a measure of fecundity, to study whether increased risks of anorectal malformations occurred with increasing time to pregnancy. Potential confounders included year of birth of the child (continuous), maternal age at delivery, maternal education, pre-existing diabetes mellitus, and prepregnancy BMI because these factors were previously identified in the literature as risk factors or found to be associated with anorectal malformations in this study. Potential confounding factors that changed the ORs in bivariable analyses were included in the multivariable models, from which they were excluded when the OR did not change >10% upon removal. Only maternal age at delivery and BMI, in 2 analyses, were found to be true confounders. We repeated the analyses for isolated anorectal malformations and anorectal malformations with one or more other major congenital malformations. Finally, we performed sensitivity analyses by including and excluding unplanned pregnancies and infants with affected first-degree relatives.
Response rates were 57% among cases and 34% among controls. After applying the exclusion criteria, maternal questionnaire data were available for 380 cases and 1973 controls and paternal questionnaire data for 350 cases and 1762 controls. In total, 230 cases had isolated anorectal malformations (61%) and 150 cases had anorectal malformations with one or more additional major congenital malformations (39%). Among the latter, 40 cases fulfilled the criteria for VACTERL. Vertebral (14%), cardiac (11%), and renal malformations (10%) were the most commonly associated major congenital malformations. The majority of cases were diagnosed with perineal fistulas (56%), followed by rectourethral fistulas (14%) in boys and vestibular fistulas (13%) in girls. We did not observe substantial differences between cases and controls in sex of the child, maternal ethnicity, multiple pregnancy, and nulliparity (Table 1). However, cases as well as their mothers were slightly younger and more often had a first-degree relative with anorectal malformations compared with controls. In addition, low education, periconceptional folic acid use, pre-existing or gestational diabetes, and prepregnancy overweight or obesity were more common among case mothers than among control mothers. The distributions of paternal characteristics, including age at delivery, ethnicity, and education, were similar to those of the maternal characteristics.
Approximately 20% of all parents reported parental subfertility with or without fertility treatment, ranging from 18% in control parents to 21% in case parents. The adjusted ORs for anorectal malformations among children of subfertile parents with and without fertility treatment for all births and for singleton births only are shown in Table 2. The risk of having a child with anorectal malformations seemed to be slightly increased for subfertile parents who conceived without treatment (OR = 1.3 [95% CI = 0.9–1.8]) compared with fertile parents. This increased risk was apparent in nulliparous women only (1.6 [0.9–2.7]). Subfertile parents included parents with a time to pregnancy >12 months and parents who ever had a diagnosis of parental subfertility with a normal time to pregnancy (≤12 months), unplanned pregnancy, or unknown time to pregnancy. No association with anorectal malformations was observed among the latter group of parents (1.2 [0.7–2.1]). One-third of these parents reported that the reason for subfertility was unknown (33%), whereas the reported reasons included polycystic ovary syndrome (11%) and irregular menstrual cycles (9%) as the main groups. In subanalyses stratifying on mutually exclusive groups of maternal (13 cases and 60 controls), paternal (9 cases and 27 controls), and couple subfertility without fertility treatment (1 case and 5 controls), only the OR for subfertility of the father without treatment deviated from unity (1.8 [0.8–3.9]).
Approximately 8% of all infants were born after any type of fertility treatment. These births seemed to be associated with a slightly increased risk of anorectal malformations compared with births among fertile parents (1.3 [0.9–1.8]). Ovulation induction was the most common treatment among controls, whereas parents of cases most often underwent IVF treatment. Surgical or other fertility treatments were not reported by case parents. When we restricted the analyses to ICSI, IVF, IUI, and ovulation induction, a slightly higher OR was found for any fertility treatment (1.4 [1.0–2.1]). This increased risk was due only to the relatively strong associations between anorectal malformations and ICSI or IVF treatments (2.0 [0.9–4.5] and 2.7 [1.4–5.4], respectively), however, because no associations were observed between anorectal malformations and treatments that did not involve gamete manipulation, including IUI and ovulation induction. Most risk estimates were stronger for singleton births only, especially the estimates for ICSI and IVF treatments compared with fertile parents (2.4 [1.0–5.9] and 4.2 [1.9–8.9], respectively).
For methodological reasons explained earlier, the further analyses were performed among singletons only. The adjusted ORs for isolated anorectal malformations and anorectal malformations with one or more additional major congenital malformations among children of subfertile parents with and without fertility treatment are presented in Table 3. No associations were observed between isolated anorectal malformations and parental subfertility without treatment or with any type of fertility treatment in singletons, except for IVF treatment compared with fertile parents (2.6 [1.0–7.3]). In contrast to isolated anorectal malformations, the risk of anorectal malformations with one or more additional malformations was increased for parental subfertility without fertility treatment (2.0 [1.3–3.3]), especially for subfertile parents with a time to pregnancy longer than 12 months (2.3 [1.3–3.9]). For ICSI or IVF treatments, the risks of anorectal malformations with other congenital malformations were increased 5- to 8-fold. The risk estimates for anorectal malformations with additional major congenital malformations were similar or slightly stronger when cases with the VACTERL association were excluded. Sensitivity analyses performed by restricting the abovementioned analyses to planned pregnancies or to infants without first-degree relatives with anorectal malformations only did not yield different results.
The risks of anorectal malformations and subgroups of anorectal malformations among children of subfertile parents who conceived with fertility treatment were also compared with subfertile parents who conceived without treatment to estimate the risk of treatment beyond the underlying subfertility. Overall, smaller risk estimates were observed when we used subfertile parents without treatment as the reference group (Table 4) compared with using fertile parents as reference group (Table 3). Interestingly, however, the risk of anorectal malformations was still increased for subfertile parents who underwent IVF when they were compared with subfertile parents who conceived without treatment (3.2 [1.4–7.2]), whereas a lower OR with a broader CI was found after ICSI treatment (1.8 [0.7–4.8]). Comparable risk estimates were observed for subgroups of anorectal malformations, including isolated anorectal malformations and anorectal malformations with other congenital malformations.
To investigate whether fecundity plays a role in the occurrence of anorectal malformations, we assessed whether increased risks of anorectal malformations with increasing classes of time to pregnancy could be observed in singletons conceived spontaneously, excluding unplanned pregnancies and pregnancies with unknown time to pregnancy (Table 5). For anorectal malformations as a group, a pattern of increasing risks of anorectal malformations with increasing time to pregnancy seemed to be present, but this may completely be due to the increased risk of anorectal malformations with additional congenital malformations after a time to pregnancy longer than 12 months, indicating parental subfertility. No pattern of increasing risks of isolated anorectal malformations with increasing time to pregnancy was observed.
Previous studies found indications for a role of parental fertility-related issues in the etiology of anorectal malformations.5–8,10–12 This large case-control study provided additional evidence that these factors are associated with an increased risk of anorectal malformations in offspring, including both isolated anorectal malformations and anorectal malformations with one or more additional congenital malformations. ICSI and IVF treatments, in particular, seemed to play a role in the etiology of anorectal malformations compared both with fertile parents and with subfertile parents who conceived without treatment. Although much smaller than the impact of ICSI and IVF treatments, subfertility itself seems to have an effect on the occurrence of anorectal malformations, but only for anorectal malformations with additional congenital malformations.
Our study population consisted of large numbers of well-characterized cases and population-based controls. The prevalence of associated congenital malformations in 39% of the cases in our study was slightly lower than that in previous studies, but these included syndromic and chromosomal forms of anorectal malformations and/or terminations of pregnancies as well.18–20 The relatively large number of cases enabled subanalyses on isolated anorectal malformations and anorectal malformations with additional congenital malformations separately. Unfortunately, we could not perform separate analyses on more specific phenotypes of anorectal malformations, VACTERL, multiple births, or first pregnancies only as the numbers of case parents who underwent fertility treatment in these subgroups were too small for reliable analyses. Population-based controls were a representative group as they were recruited via municipalities in comparable geographical areas as the cases and were of similar age.
Another strength of our study was the combination of information on several fertility-related issues, including the diagnosis of parental subfertility, time to pregnancy, and type of fertility treatment, within one study. Furthermore, information on several potential maternal and paternal confounders was used in our analyses. These data were more comprehensive than in previous studies on anorectal malformations and assisted conception.6–8 Additional analyses on the underlying reasons for parental subfertility were not feasible as this information was often unknown.
Despite the relatively low response rate, we do not think that selection bias was a major issue because the parents were probably unaware of the potential association between fertility issues and anorectal malformations because the questionnaires covered many pregnancy-related topics, such as pregnancy complications and lifestyle during pregnancy. Moreover, the rates of fertility treatment in the control group were representative for the Dutch general population,21 which probably excludes selection due to a higher or lower participation rate among control parents who underwent fertility treatment. A limitation of this study was the possibility of misclassification due to the self-reported nature of the data with the concomitant potential for recall problems, especially because the range of the time interval between childbirth and filling out the questionnaires was 0 to 22 years. However, this range was similar for cases and controls, and we do not expect parental subfertility and fertility treatment to be highly susceptible to recall errors, as these are major life events. In addition, the assumption that parents with an unplanned pregnancy were fertile may have resulted in nondifferential misclassification, but this seemed negligible as sensitivity analyses showed that the results were similar when unplanned pregnancies were excluded. In many studies on congenital malformations, the effects of fertility treatment may be underestimated when only live-born cases and controls are included in the study, as subfertile parents who receive fertility treatment may be more likely to be offered prenatal screening, possibly resulting in the termination of pregnancy. However, anorectal malformations can hardly be diagnosed prenatally.22 Therefore, the potential for underestimation due to inclusion of live-borns only is limited in the analyses on isolated anorectal malformations. However, the results on anorectal malformations with other congenital malformations may be underestimated if pregnancies were terminated because of severe associated malformations.
We found increased risks of anorectal malformations for subfertile parents who received ICSI or IVF treatment compared with fertile parents who conceived spontaneously, with even larger effects in singletons only, which is consistent with previous studies.7,8 Interestingly, ICSI and IVF treatments were still associated with increased occurrences of anorectal malformations when the analyses were restricted to subfertile parents with or without treatment, which has never been done before. This may point to an added effect of gamete manipulation, including culture medium and timing of fertilization, and/or of the hormones involved in ovulation induction as part of ICSI and IVF treatments, over and above possible adverse effects of the underlying parental subfertility. However, no association was found between anorectal malformations and the use of ovulation induction hormones without ICSI or IVF. This is in-line with a large population-based study13 but not with 2 register-based studies.10,12 Furthermore, the associations may still be confounded by the indication for ICSI or IVF treatment, as some of the subfertile parents who conceived after ICSI or IVF may be more severely subfertile than those who conceived without treatment. This indicates that although subfertile parents who conceived without treatment were used as the reference group, the underlying parental subfertility and/or its causal factors could still play a role. This was confirmed by our finding that subfertility without treatment led to an increased risk of anorectal malformations with additional major congenital malformations. Disturbed epigenetic processes as a consequence of ICSI or IVF and/or male subfertility may also be explanations for their involvement in the etiology of anorectal malformations,23,24 as was shown for the Beckwith-Wiedemann syndrome.25
Previously, increased risks of anorectal malformations after ICSI or IVF were found in singletons, whereas no associations were found in multiple births.7 Due to small numbers, we could not calculate risk estimates for multiple births, but we did find higher ORs in singleton births only than in singleton and multiple births combined. We did not find indications for changes over time in the risk of anorectal malformations after fertility treatment as the risk estimates for IVF treatment were similar when the analyses were stratified into before and after 2000. We were unable to stratify the analyses for more time windows or for ICSI treatment due to small numbers. The association between anorectal malformations and IVF was observed among isolated anorectal malformations but was much more pronounced for anorectal malformations with one or more additional congenital malformations, which was also shown by Zwink et al.8 Furthermore, subfertile parents who conceived without treatment were found to have an increased risk of anorectal malformations with additional major congenital malformations but not isolated anorectal malformations. Cases with additional congenital malformations more often have severe forms of anorectal malformations, such as rectourethral fistulas or cloacas, which may imply stronger associations of parental subfertility and IVF with severe forms of anorectal malformations.
Parental subfertility without treatment doubled the risk of anorectal malformations with additional congenital malformations. Subfertile parents were defined as those who had a time to pregnancy >12 months and/or were ever diagnosed with maternal and/or paternal subfertility. In some parents in the latter group, however, fertility might have been restored before this pregnancy, which could have led to underestimation of the effects due to nondifferential misclassification. Therefore, we performed separate analyses for these 2 groups and observed stronger associations between anorectal malformations and a time to pregnancy >12 months, which we considered most reliable. In addition, we found only an association between anorectal malformations and parental subfertility without treatment in nulliparous women, which may point to a difference in the severity of parental subfertility between nulliparous and multiparous women and their partners in this study. This was not in-line with a previous study on heterogeneous patient groups with congenital malformations that found similar results in nulliparous women as in the total group of women, including multiparous women as well.3 In our previous small study, we evaluated the role of parental subfertility in the etiology of anorectal malformations; we found paternal subfertility to potentially increase the risk of anorectal malformations but not maternal subfertility.11 This is in-line with the finding for subfertility of the father in the present study, in which only 21% of the cases were derived from the previous study. In the latter, however, the subfertile groups included parents with and without fertility treatment, and no analyses on subgroups of anorectal malformations were performed. In our time to pregnancy analyses, we found weak indications that fertile parents who conceive within 3 months have the smallest risk of having a child with anorectal malformations because the ORs for almost all other classes of time to pregnancy were above unity.
In conclusion, we found evidence for a role of ICSI and IVF treatments in the etiology of anorectal malformations, even compared with subfertile parents who conceived without treatment. We also found indications for an independent role of subfertility in the etiology of anorectal malformations with other congenital malformations. Future studies are needed to unravel the underlying biological mechanisms through which the development of the anorectal channel may be disrupted by these factors. The findings of this study on anorectal malformations, in combination with the existing literature on ART and other congenital malformations, may also provide guidance to counseling of subfertile couples.
We thank the surgical staff members Mariëtte van der Vorle, Marlaine Hammen, Herjan van der Steeg, and Bas Verhoeven of the Department of Pediatric Surgery of the Radboud University Medical Center and the participating municipalities in The Netherlands for their collaboration in collecting data from cases and controls. Finally, we are grateful to the children and their parents who participated in this study.
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