No increase in autism-associated genetic events in children conceived by assisted reproduction.

As shown in Table 1 , in the sample of 1,994 individuals with ASD, 122 were the product of assisted reproduction. Demographics are not listed in the table but are as follows: 264 female, 1,730 male; 1,567 white, 84 Asian, 76 African American, 5 Native American, 157 more than one race, 90 other, 15 not specified. The percentage of autism-associated CNVs in ASD patients exposed to ART was 9.8%, compared with 10.3% in ASD patients not exposed to assisted reproduction, a nonsignificant difference [chi-square (1, N = 1994) = .02, P <.88]. Of the 424 patients with available gene mutation status, 24 were the result of assisted reproduction. The percentage of autism-associated gene-disrupting events in ASD patients exposed to assisted reproduction was 58.3%, compared with 51.3% in ASD patients not exposed to assisted reproduction, here again indicating no statistically significant differences in mutation rate [chi-square (1, N = 219)=.45, P <.50]. Despite the statistically nonsignificant results, given the 7% difference of autism-associated gene-disrupting events in ASD patients exposed to assisted reproduction versus ASD patients not exposed to assisted reproduction, a power analysis was run. Using G*Power 3.13 for a 1 degree of freedom test and assuming an effect size of w = 0.14 (as determined by the 7% difference in the observed sample) with alpha = 0.05 an observed power of 0.83 was calculated to detect this difference using the chi-square analysis. Thus, there was adequate power to have detected a significant difference had there been one. Importantly, because assisted reproduction is a broad category of interventions, Table 1 also presents numbers of autism-associated CNVs and gene-disrupting events according to the following types of assisted reproduction: IVF (including with cryopreservation), GIFT, ZIFT, ICSI, artificial insemination, and fertility medication only. These numbers did not in general reach sufficient power for statistical analysis, but the genetic mutations appeared to be evenly distributed throughout the types of assisted reproduction. Tables 2 and 3 present detailed information regarding all autism-associated genetic events identified in the ASD group exposed to assisted reproduction, along with descriptors such as parental ages, pregnancy number, and infertility reason. In Table 2 , out of 12 patients with identified CNVs, 16 different regions were affected–none more than once–across 11 different chromosomes. In Table 3 , out of 14 patients with genetic events, 23 genes were affected–none more than once–across 13 chromosomes. Results from the ANOVA in the subsample of individuals with CNV status indicated that assisted reproduction usage was associated with increased paternal ( P <.000017) and maternal ( P <.000014) age. Results from the ANOVA in the subsample of individuals with gene mutation status indicated a somewhat different pattern. No relation between assisted reproduction usage and paternal age was observed ( P =.13) in this subsample, but a trend was observed suggesting that mothers of children with autism-associated gene-disrupting events using assisted reproduction were older than mothers not using assisted reproduction ( P =.06). Results from the logistic regression indicated that CNV status was not associated with paternal ( P =.97) or maternal age ( P =.73). However, results from the logistic regression in the subsample of individuals with gene mutation status indicated a different pattern. Although paternal age again did not reach statistical significance ( P =.56), mothers of children with autism-associated gene-disrupting events were older than mothers of children without autism-associated gene-disrupting events ( P =.03). This association is illustrated in Figure 1 , with use of assisted reproduction included in the figure so as to demonstrate the impact of maternal age regardless of assisted reproduction.

Our study is the first to examine the genotype of patients with ASD who were conceived by assisted reproduction versus a control group in a relatively large sample. The purpose of this study was to examine the relations between assisted reproduction and genetic abnormalities in patients with ASD. Our results did not show an increased rate of autism-associated CNVs or autism-associated gene-disrupting events in children with ASD who were conceived by assisted reproduction versus children with ASD who had no contact with assisted reproduction. Further, autism-associated CNVs and autism-associated gene-disrupting events did not appear overrepresented within any particular subtype of assisted reproduction. Additionally, CNVs and gene mutations were seen across a wide variety of genomic loci. If assisted reproduction was a significant independent risk factor for autism-associated genetic events, it would be expected that there would be a higher rate of these events in the group of children with ASD who were exposed to assisted reproduction. Additionally, it might be expected that if assisted reproduction leads to genetic events, there would be a specific type or pattern of genetic event that was seen. Neither was the case. Increased paternal and maternal age was statistically significantly associated with use of assisted reproduction in the CNV group. In the subsample of individuals with gene mutation status, maternal age trended toward a significant association with use of assisted reproduction and was found to be a potential contributor. This variable reached statistical significance when comparing maternal age and presence of autism-associated gene-disrupting events in the ASD group exposed to assisted reproduction. As alluded to previously, it is possible that the association between assisted reproduction and ASD found in other reports is related to characteristics of the parents using assisted reproduction that increases ASD risk and not assisted reproduction itself. Specific to this report, it is possible that parental age–not assisted reproduction–is associated with an increased risk of autism-associated gene disruptions. It was somewhat unexpected to see maternal age associated with gene mutations, given the already mentioned studies linking paternal age to autism-associated de novo CNV. However, the population studied in this report is more likely to have maternal age as a primary driver of the use of assisted reproduction. Indeed, the results above showed that in the group with gene mutation status no relation between assisted reproduction usage and paternal age was seen but a trend was observed suggesting that mothers using assisted reproduction were older than mothers not using assisted reproduction. There are limitations to the conclusions that can be reached from these data. It is possible that some patients with ASD and assisted reproduction may not have developed ASD or any type of autism-associated genetic event had they not been exposed to assisted reproduction. Phrased differently, it is possible that ASD is an oligogenic disorder and that the group exposed to assisted reproduction will not have a different genotype than the group not exposed to assisted reproduction, but that in the group exposed to assisted reproduction it was assisted reproduction that caused the last mutation necessary to reach a pathological threshold. With that in mind, it would have been helpful to have a typically developing control group in this study. Additionally, although all currently known autism-associated genetic events were considered, there are likely to be more of these events discovered. Furthermore, although the study was sufficiently powered to look at assisted reproduction as a broad category of risk, it was not sufficiently powered to look at any subtype of assisted reproduction. With that acknowledged, as presented in Table 1 , no type of assisted reproduction appeared grossly different from another in terms of CNV or mutation rates. Finally, this retrospective study did not incorporate medical records to verify parent report, which would have been ideal. This study adds valuable information because it is the first to compare the genotype of patients with ASD who have also been exposed to assisted reproduction with patients with ASD who have not been exposed to assisted reproduction. In doing so, no association was found between rates of autism-associated genetic events and assisted reproduction, and no a major genotypic difference were found between patients with ASD exposed to assisted reproduction as compared to patients with ASD not exposed to assisted reproduction.

In this large sample, there was no increased rate of ASD-associated genetic events in ASD patients conceived by assisted reproduction compared with ASD patients conceived without assisted reproduction. Additionally, maternal age was identified as a potential contributor to ASD associated genetic events in the context of assisted reproduction. This study is the first on the subject to concurrently examine the genotype of individuals with ASD in relation to their exposure to assisted reproduction versus spontaneous conception and therefore adds valuable, reassuring evidence to the argument that assisted reproduction does not increase the risk of ASD.

Participants were 2,760 4- to 18-year-old children with ASD and their families who previously had participated in the Simons Simplex Collection (SSC). The SSC, a project funded by the Simons Foundation Autism Research Initiative to identify de novo genetic variants related to ASD, includes 12 collaborating data collection sites across North America (for a description of the ascertainment, data collection, and validation procedures for the sample, see http://sfari.org ) ( 40 ). Approval was obtained from each local institutional review board. All participants completed informed consent before participation in the study. As part of SSC study participation, experienced clinicians confirmed the ASD diagnosis using the Autism Diagnostic Interview, Autism Diagnostic Observation Schedule, and expert clinical judgment according to Diagnostic and Statistical Manual of Mental Disorders, 4th Edition (DSM-IV) criteria ( 41 ). Information regarding assisted reproduction was collected through a clinician-administered, semistructured caregiver interview designed to gather data regarding early medical history for children and families participating in the SSC. Clinicians administering the interview were primarily specialists in mental health, not obstetrics. Specific to this study, caregivers were asked whether assisted reproduction had been used to initiate the pregnancy of the proband or siblings. If the caregiver responded yes, data regarding what type of assisted reproduction was collected. The options were oral fertility medications, injected fertility medications, artificial insemination, IVF, assisted hatching, frozen embryo, frozen eggs, sperm donor, egg donor, ICSI, GIFT, ZIFT, surrogacy, unknown, and other. Additionally, the maternal and paternal age at the time of conception was gathered. Genotypic data regarding each participating family were collated from previously published studies identifying autism-associated CNV and putative causal ASD gene mutations ( 4 , 42 – 44 ). For the purposes of this study, two categories of genetic data were examined, autism-associated CNV and autism-associated gene-disrupting events. Autism-associated gene-disrupting events were defined as gene-truncating loss-of-function mutations identified through exome sequencing. In the sample of 2,760 SSC families with ASD, information regarding assisted reproduction history was available for 2,418 (87.6%) of the children with ASD. Of this subsample, 1,994 had published CNV information available, and 424 had published gene mutation status yielded through exome sequencing. Analyses were conducted with the subset of individuals with CNV and gene mutation data separately. Genetic data was not available for all patients primarily due to the limits of the cost of the genetic analysis. The participants were selected for genetic analysis out of the larger pool by authors who had previously published their results with the SSC data set. These authors used a variety of criteria to select the patients they believed would most likely provide valuable genetic information. The criteria included low nonverbal intelligence, multiple unaffected siblings, and known multigenic CNV ( 43 ). Some patients were selected randomly as well. A chi-square goodness of fit test was used to compare the rates of autism-associated genetic events in children with ASD conceived via assisted reproduction versus those conceived spontaneously. Analysis of variance (ANOVA) was used to examine relations between assisted reproduction use and genetic events (either CNV or gene mutation). Logistic regression was used to examine relations between genetic events and parental age.