Autism spectrum disorders and fetal hypoxia in a population-based cohort: Accounting for missing exposures via Estimation-Maximization algorithm
© Burstyn et al; licensee BioMed Central Ltd. 2011
Received: 19 July 2010
Accepted: 5 January 2011
Published: 5 January 2011
Autism spectrum disorders (ASD) are associated with complications of pregnancy that implicate fetal hypoxia (FH); the excess of ASD in male gender is poorly understood. We tested the hypothesis that risk of ASD is related to fetal hypoxia and investigated whether this effect is greater among males.
Provincial delivery records (PDR) identified the cohort of all 218,890 singleton live births in the province of Alberta, Canada, between 01-01-98 and 12-31-04. These were followed-up for ASD via ICD-9 diagnostic codes assigned by physician billing until 03-31-08. Maternal and obstetric risk factors, including FH determined from blood tests of acidity (pH), were extracted from PDR. The binary FH status was missing in approximately half of subjects. Assuming that characteristics of mothers and pregnancies would be correlated with FH, we used an Estimation-Maximization algorithm to estimate HF-ASD association, allowing for both missing-at-random (MAR) and specific not-missing-at-random (NMAR) mechanisms.
Data indicated that there was excess risk of ASD among males who were hypoxic at birth, not materially affected by adjustment for potential confounding due to birth year and socio-economic status: OR 1.13, 95%CI: 0.96, 1.33 (MAR assumption). Limiting analysis to full-term males, the adjusted OR under specific NMAR assumptions spanned 95%CI of 1.0 to 1.6.
Our results are consistent with a weak effect of fetal hypoxia on risk of ASD among males. E-M algorithm is an efficient and flexible tool for modeling missing data in the studied setting.
The autism spectrum disorders (ASD) comprise a group of neurodevelopmental conditions that are associated with impaired verbal and non-verbal communication and social interaction, and restricted and repetitive patterns of behavior, which typically manifest before age of 3 years. Although relatively rare[2–7], ASD can have a devastating effect on the quality of life of entire families and is associated with considerable societal economic burden. There is some evidence that ASD prevalence is on the increase, although it remains unclear as to whether this increase can be completely accounted for by changes in case definition and improved ascertainment. While some genetic risk factors for ASD are known, the potential contribution of environmental factors has not been excluded and mediations involving gene-environment interactions would contribute to the observed genetic complexity in ASD. A recent review suggests that advanced parental age and fetal growth restriction place children at an elevated risk of developing ASD. However, a recent meta-analysis concluded it is premature to implicate specific pregnancy complications in etiology of ASD. Nonetheless, Kolevzon et al., on the basis of epidemiological evidence, advanced a hypothesis that neonatal or fetal hypoxia are implicated in ASD, even though hypoxia was not measured in any of the reviewed studies. Chronically high maternal levels of dopamine have also been suggested by Previc as the underlying cause of ASD, which would be expected to correlate with occurrence of fetal hypoxia. More recently Mueller and Bale argued, based on studies of stress sensitivity in juvenile mice following prenatal hypoxic insults, that males are more susceptible to development of maladaptive stress responses associated with many neurodevelopmental disorders such as ASD. They suggested that this vulnerability is determined by sex-specific differences in placental physiology. Excess of ASD among males is well-documented. If fetal hypoxia were equally likely among boys and girls, but boys were more susceptible to its effect on ASD risk, this would contribute to explaining male predominance among ASD patients.
Parallel line of research suggests that neonatal encephalopathy caused by perinatal asphyxia/hypoxia in infants born at term is associated with neurological impairment later in life. Therefore, it may well be that foetal hypoxia is simply an upstream cause of neonatal encephalopathy that then relates to risk of ASD. Indeed, the most recent review of the contribution of neonatal encephalopathy to infant neurological development concluded that even moderate post-asphyxia neonatal encephalopathy appears to contribute to cognitive and sensory-motor impairments, although heterogeneity in published results was noted.
In fact, one study of 239 children who survived neonatal encephalopathy reported that 4.2% were diagnosed with an ASD. This represents, after adjustment for confounders, about a 6-fold increase in ASD risk compared to ASD rates among 563 children who did not have neonatal encephalopathy (0.9%). The small sample size here led to render results of this report vulnerable to observing "significant" findings purely due to chance and the 95% confidence intervals of the relative risk estimate (2 to 17) is extremely wide, casting doubts about the reproducibility of the finding.
None of the epidemiological studies conducted to date studied direct effects of hypoxia at birth on ASD. Therefore, we tested, in a population-based birth cohort, the hypothesis that risk of ASD is related to fetal hypoxia and investigated whether this effect is greater among males.
This cohort and analysis of association of perinatal risk factors with risk of ASD are described elsewhere, without consideration of fetal hypoxia, defined as fetal scalp pH < 7.25, or umbilical artery pH < 7.20, or venous pH < 7.28. Delivery records held by the Alberta Perinatal Health Program (APHP) identified the cohort of singleton live births in the province of Alberta, Canada, between January 1, 1998 and December 31, 2004. The APHP provided information regarding relevant ante- and peri-natal risk factors. Information on the risk factors was collected on admission to hospital for delivery, as part of routine clinical care. They are considered to be accurate and any internal inconsistency in the records is scrutinized by APHP; if apparent error in the delivery records cannot be resolved, APHP records a missing value for a given variable. The unique Personal Health Number (PHN) of mother and child along with child's gender and date of birth in APHP file were used to follow-up the cohort through records held by the Alberta Health and Wellness (AHW). In the universal healthcare insurance system of Alberta, all residents are served by physicians who bill the government for their services, linked to specific diagnostic codes, International Classification of Diseases 9th edition (ICD-9). Children matched in APHP file to AHW records were followed-up by the investigators until March 31, 2008 for (a) ICD-9 codes indicating ASD (299.0 or 299.8) listed with the physician billing record, (b) child's residency and mortality (in a given fiscal year ending on March 31) and (c) mothers' socioeconomic status. Outcome was defined using most liberal definition from our previous work: at least one ASD claim by any physician because such case definition showed good sensitivity and specificity and did not affect associations between studied risk factors and ASD. Such case definition for ASD is analogous to the one used in another, very similar Canadian study, where it was shown to have acceptable sensitivity and specificity relative to gold standard diagnostic assessment in a specialty clinic. Study protocol was approved by the University of Alberta Health Ethics Research Board and the custodians of the data.
Ascertainment of Fetal Hypoxia1 Among Neonates
All tests positive
At least one test positive
Number of subjects
Number of subjects
All three tests
Fetal scalp and umbilical arterial blood pH
Umbilical arterial and venous pH
Fetal scalp and umbilical venous blood pH
Fetal scalp pH
Umbilical arterial blood pH
Venous umbilical blood pH
Any of the three tests
There were 273,343 singleton live births in Alberta according to APHP records between 1998 and 2004, among whom 25,970 children could not be identified by AHW and 28,421 either died, or lost residence during follow-up; a further 62 had missing gender, leaving 218,890 children for analysis. There were no systematic differences on observable data among births that were included and excluded (details not shown). In 48% (105,636/218,890) of births, fetal hypoxia tests via blood pH were performed. In a minority of births multiple tests were performed (Table 1). A total of 17,083 tests (14%) of the 120,836 that were preformed satisfied our definition of fetal hypoxia: positive on at least one of the (multiple) tests. Umbilical arterial blood pH was the most common test used either on its own (n = 75,775) or in combination with venous blood pH (n = 41,245). As expected, there was some disagreement among tests on the same neonate. However, overall prevalence of fetal hypoxia among those tested appeared to be similar whether all tests were required to be congruent (10%) or only one had to be positive (14%). Measurement of fetal scalp pH, although relatively infrequent, appears to be associated with elevated likelihood of the overall positive test result, perhaps because not of the inherent superior sensitivity of the test, but because it is administered only when there is considerable suspicion that the fetus may be hypoxic. There were 200,557 full-terms births in the cohort and gestational age at birth was missing for 444 neonates. 1,011 of full-terms neonates were diagnosed with ASD during the follow-up.
Summary of Data on Reported Fetal Hypoxia and Diagnosis of Autism Spectrum Disorders in the Same Child (All Gestational Ages at Birth)1
Autism Spectrum Disorders
Among full-term births the pattern of observed rates is similar to that seem among all births in Table 2. The observed ASD rate among full-terms births was higher among those who tested positive for hypoxia (0.62%, 96/15,557) compared to those who tested negative for hypoxia (0.58%, 557/95,352). The test for hypoxia was not performed among 105,205 members of this sub-cohort who had an even lower observed rate of ASD (454, 0.43% affected). 480 of 49,165 full-term male births with data on hypoxia were diagnosed with ASD during the follow-up (observed rate 0.98%). Among 8,286 males hypoxic at birth, the observed rate of ASD was 1.03% (85 cases), but among males not hypoxic at birth the observed rate of ASD was slightly lower: 0.97% (395 cases). In this male sub-cohort, when information on hypoxia was missing, the observed rate of ASD was even lower 0.69% (364 cases). 77 of 46,187 full-term female births with data on hypoxia were diagnosed with ASD during the follow-up (observed rate 0.17%). Among 7,271 females hypoxic at birth, the observed rate of ASD was 0.15% (11 cases), but among females not hypoxic at birth the observed rate of ASD was higher: 0.17% (66 cases). In this female sub-cohort, when information on hypoxia was missing, the observed rate of ASD was the same as among girls who tested as not hypoxic at birth: 0.17% (90 cases).
Association of Fetal Hypoxia at Birth1 and Diagnosis of Autism Spectrum Disorders2: Results of Logistic Regression Fitted with EM Algorithm for Handling Unknown Exposures That are Modeled From Maternal and Birth Characteristics Under MAR Assumption (r = 1) Stratified on Child's Gender
All gestational ages at birth
Odds ratio estimate
95% confidence interval
Full-term births only
Odds ratio estimate
95% confidence interval
Association of Fetal Hypoxia at Birth1 and Diagnosis of Autism Spectrum Disorders2: Results of Logistic Regression Fitted with EM Algorithm for Handling Unknown Exposures That are Modeled From Maternal and Birth Characteristics Under Specific NMAR Assumption Among Full-Term Male Births
Deviation from MAR3
Odds ratio estimate
95% confidence interval
There are many difficulties with these data beyond missing-ness: both exposures and outcomes are suspected of being misclassified, which would bias apparent OR estimates, most likely towards the null if both misclassification mechanisms are non-differential. Covariates from perinatal database are known to be recorded accurately [Ms N. Bott of APHP, personal communications] because they use data essential to medical care, not just billing, even though some information (such as maternal smoking used to model risk of fetal hypoxia) is known to be collected with error. Of course error in variables can bias our results, but we lack information on the extent and direction of such bias. Our analysis is also limited by small number of girls, so that our failure to detect an association among them may simply be due to low power. Short follow-up for some members of the cohort born in 2004 may have introduced error due to incomplete ASD ascertainment, since they were be only 4 at the end of follow-up, but our previous results suggest that in this population peak age of diagnosis was between ages of 3 and 4 , therefore our data cannot address this potential limitation. Nonetheless, our data are valuable because they enable the first test of association of fetal hypoxia with risk of ASD in a population-based birth cohort. Innovative statistical methodology that we used to meet challenged posed by the data should prove to be helpful to researchers facing similar complications.
We observed that males who were tested for foetal hypoxia were more likely to develop ASD. This supports the generally establish contention that ASD patients tend to have complicated pregnancies, which often involve, upon suspicion, testing for foetal hypoxia. Presence of hypoxia itself, under the causal models that we considered, appears to only affect males. This finding is consistent with our a priori specified hypothesis about greater sensitivity of males to hypoxia-induced insults during gestation.
We conducted most of our analyses under a strong assumption that the majority of observed pregnancy and birth characteristics only affected risk of ASD through fetal hypoxia. In doing so, we reflected on discussion by Newschaffer and Cole of testing alternative causal models in studies of perinatal risk factors in ASD. Since our entire analysis is predicated on apparent association of hypoxia with pregnancy and birth characteristics (Z), we can reject the "etiologic heterogeneity" model: if hypoxic status was independent of Z we would not have been able to implement analysis that relies on EM-algorithm. We tested a prior justified effect modification by gender and report non-multiplicative interaction on the basis of stratified analysis. Then we have to consider "epiphenomena" models . "Epiphenomena model 2" is in fact represented by Figure 1 and was estimated here directly. With respect to "epiphenomena model 1", it is currently unreasonable to suspect that ASD causes fetal hypoxia, since that would postulate a fetus that is both destined to have ASD and able to affects its own supply of oxygen. This leaves the possibility that "shared risk factor" model applies and there may be confounding by direct effect of elements of Z on risk of ASD if model in Figure 1 is fitted to the data. We considered confounding by prematurity: in a stratum of full-term infants for who we had sufficient sample size to obtain meaningful estimates (92% of all subjects) we found little evidence that prematurity induced association between hypoxia and ASD. None of the other elements of Z appeared to be likely candidates for examining the direct effect on risk of ASD. Of course given how little we know about causes of ASD, it is possible that we failed to postulate and test true causal model. If that is the case, then our estimates may in fact be biased by latent confounding or effect modification, which is of course true of most observational research into subject matters, such as ASD, where uncertainty abounds about what the actual causes may be.
Missing values are common in public health research. Understanding the missing mechanism is the key for a proper missing data analysis. Relaxing unrealistic MAR assumptions changed the point estimate of the disease-exposure odds ratio and sifted confidence intervals (P-values) towards the region conventionally thought of as "significant". Thus, the use of EM-algorithm for dealing with the special missing value problem in this project in conjunction with sensitivity analysis using the specified NMAR assumption altered statistical inference and was therefore useful.
Our results are consistent with a weak effect of fetal hypoxia on risk of ASD that is confined to males. It may well be that some latent indication or an unknown correlated of testing for hypoxia at birth are responsible for the increased risk of ASD among males and therefore investigation of role of hypoxia at birth in ASD remains a fruitful area for research.
Igor Burstyn and Lonnie Zwaigenbaum were supported, respectively, by the Population Health Investigator and Health Scholar salary awards from the Alberta Heritage Foundation for Medical Research. This study is based on data supplied by Alberta Health and Wellness (AHW) and the Alberta Perinatal health Program (APHP). The interpretation and conclusions contained herein are those of the researchers and do not necessarily represent the views of the Government of Alberta or APHP. Neither the Government, nor AHW, nor the APHP expresses any opinion in relation to this study. We are grateful to AHW and APHP for supplying and linking the data; special thanks go to Alex Marinov, Wendy Mitchell, Nancy Piche, Nancy Bott and Sharon Zhang. Craig Newschaffer provided invaluable comments on the final draft of the manuscript.
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