Symptoms of Common Maternal Infections in Pregnancy and Risk of Islet Autoimmunity in Early Childhood

From January 1994 to January 2003, >27,800 cord blood samples from children born at St. Joseph’s Hospital in Denver have been screened for diabetes-associated HLA genotypes (10). We excluded families in which parents had difficulties understanding English or whose infant had a severe congenital malformation or disease. Eighty-six percent of families approached gave informed consent to genetic screening. Samples of whole blood in EDTA were sent to the HLA typing laboratory at Roche Molecular Systems (Alameda, CA) for PCR-based class II genotyping, as previously described (10). Children were categorized into three groups defined by the odds of developing type 1 diabetes by the age of 20 years. The high-risk group (odds 1:16) was DRB1*04-DQB1*0302/DRB1*0301-DQB1*0201, the moderate risk genotypes (odds 1:75 in non-Hispanic whites and 1:230 in Hispanics) were DRB1*04-DQB1*0302/DRB1*04-DQB1*0302 or DRB1*0301/*0301 or DRB1*04-DQB1*0302/X (in which X does not include DRB1*04, DQB1*0302, DRB1*0301, DQB1*0602, or DR2 [DRB1*15 or 16]). All other genotypes were classified as low risk. For the present analysis, we grouped low- and moderate-risk categories together. Children with at least one sibling or parent with type 1 diabetes were referred from clinics in the Denver metropolitan area and recruited regardless of their HLA genotype. Participants were recruited soon after birth and scheduled for clinic visits at ages 9, 15, and 24 months and annually thereafter. Venous blood was collected at each clinic visit, and children who tested positive for one or more islet autoantibodies were scheduled for more frequent visits. Informed consent was obtained from parents of all children in the study, and the study was approved by the Colorado Multiple Institutional Review Board. Between January 1994 and February 2003, 1,431 children were seen in the clinic at age ≤15 months. Of these, the families of 871 children without and 391 children with a parent or sibling with type 1 diabetes had completed interviews about environmental factors relating to the mother during pregnancy and to the child at ages 3–15 months.

All measures of autoantibodies in blood were performed in the laboratory of Dr. George Eisenbarth at Barbara Davis Center (Denver, CO). We used radioimmunoassays for insulin, GAD₆₅, and IA-2 autoantibodies. Insulin autoantibodies are measured by a micro-IAA assay with sensitivity of 58%, specificity of 99%, and interassay coefficient of variation 11% (13). The combined anti-GAD and -IA-2 radioassay is performed in duplicate on a 96-well filtration plate, and radioactivity is counted on a TopCount 96-well plate β-counter using a modification of a previously reported method. The levels of both antibodies are expressed as an index (sample cpm − negative control cpm)/(positive control cpm − negative control cpm) (14). In the 1995 Immunology of Diabetes Society Workshop, the GADA assay had 82% sensitivity and 99% specificity using sera from new-onset diabetic patients aged <30 years. The interassay coefficient of variation was 6%. The IA-2 assay had 73% sensitivity and 100% specificity, and the interassay coefficient of variation was 10% (15). All samples with IAA, GADA, or IA-2 levels exceeding the 99th percentile and a random 10% of the remaining samples are retested in a blinded manner for quality assurance. The 99th percentile based on testing 198 nondiabetic control subjects aged 0.4–67 years was 0.01 for IAA, 0.032 for GADA, and 0.049 for IA-2. The single highest value for IA-2 among control subjects (0.07) was used as a cutoff for positivity for this assay. For GADA and IAA, we used the 99th percentile as the cutoff for positivity. The definition of islet autoimmunity was positivity for one or more of the three islet autoantibodies at two or more consecutive clinic visits at 1- to 6-month intervals (of the 52 that satisfied this definition, 16 have been negative at one or more subsequent visits and 14 have developed clinical type 1 diabetes up to 28 February 2003). The time of onset of islet autoimmunity was defined as the midpoint between the dates of the two clinic visits when the child was last negative and first positive for at least one autoantibody.

Soon after recruitment, the child’s mother completed a mailed questionnaire on relevant factors. Mothers were asked: “When you were pregnant with the study child, did you have any of the following conditions,” with precoded categories: “bad cold or influenza,” “sore throat or tonsillitis,” “sinus infection,” “diarrhea/gastroenteritis,” “kidney or urine infection,” “bronchitis,” “pneumonia,” and “other infection or fever.” Any infection during pregnancy versus none was the primary variable. For neonatal conditions, we considered any versus none of the following: “respiratory problems,” “cold or runny nose,” “meningitis,” “pneumonia,” “diarrhea,” and “other infections or fever.” When the child was 3, 6, 9, 12, and 15 months old, families were asked whether the child had attended “daycare (church, gym, family daycare home, or center)” on a regular basis in the preceding 3 months, the number of children attending, and the number of days per week the child attended. We considered children exposed to daycare if they had attended daycare with at least one other child at least once a week in any 3-month period up to 15 months of age. When the child was 6 months old, families were asked how many of several specified animals the child had been exposed to, only counting animals that were at least occasionally inside the home. Few children were exposed to animals other than cats and dogs. We also asked: “When the child was 6 months old, how many people lived in your household, and how many rooms were there in your home (count the kitchen but not the bathrooms).” At each interview, mothers were asked about breast-feeding.

With the cohort defined as above, we calculated based on the logistic model that we could detect a relative risk just under 0.5 with 80% power for exposures with 50–70% prevalence (two-sided tests, 5% significance level). We used Cox regression analysis to estimate the hazard ratio (HR) with 95% CI for the relation between study variables and future development of islet autoimmunity with time from birth to islet autoimmunity or the last clinic visit when the child tested negative as the main time variable (16). We decided a priori to adjust for family history (first-degree relative with type 1 diabetes or not), HLA risk category, and ethnic group. Further adjustment by maternal education, maternal age at delivery, and other variables considered relevant was also done. We investigated whether the HRs were of similar magnitude for children with and without first-degree relatives with type 1 diabetes, for the HLA categories, and for boys and girls by stratified analyses and testing the respective interactions. Unless reported otherwise, results were similar in these subgroups. The robustness of the analyses against deviations from the proportional hazard assumption and against interval censoring of the outcome was assessed by running the same analyses using logistic regression. All results were essentially the same whether we used Cox or logistic regression. Outcome and exposures were defined before inspecting the relationships between them. A two-sided P value <0.05 or a 95% CI for the HR not overlapping 1.00 was regarded statistically significant.

During the current follow-up, 52 children developed islet autoimmunity, of which 14 subsequently developed clinical type 1 diabetes (Table 1).

Children whose mother reported at least one symptom of infection (mostly respiratory or gastrointestinal) during pregnancy had approximately one-half the risk of islet autoimmunity compared with other children (Table 2). This significant association was essentially unchanged after further adjustment for maternal age at delivery, maternal education, month of birth, or duration of breast-feeding. Maternal respiratory infections and gastrointestinal infections both tended to predict lower risk of islet autoimmunity, although not significantly so when considered separately. Other infections were not related to islet autoimmunity (Table 2). There was no evidence of a dose-response pattern with increasing number of maternal symptoms of infection in pregnancy. However, the association between maternal symptoms of infection and lower risk of islet autoimmunity among children was strong and significant among girls (HR 0.21; 95% CI 0.09–0.48), but not significant among boys (1.09; 0.47–2.51). The test for interaction between maternal infections and sex of the child gave a P value of 0.005 (not shown).

Presence of at least one symptom of neonatal infection (mostly respiratory) and daycare attendance were not related to islet autoimmunity (Table 3). Further analysis of number of other children attending or age at start in daycare did not convey any important additional information. Exposure to cats, dogs, or a combination of the two and household crowding in early life were also not related to islet autoimmunity (Table 3).

Maternal symptoms of common infections during pregnancy predicted a significantly lower risk of developing islet autoimmunity in the young girls in this prospective study.

A role of Th1/Th2 balance has been suggested to explain the lower risk of atopic disorders associated with markers of infections (4,5). Many intracellular infections promote Th1-dominated immune responses (e.g., secretion interferon-γ), which may protect against typically Th2-biased atopic disorders. This concept is probably too simplistic and does not account for associations between parasitic infections, which usually induce predominantly Th2 immune responses, and lower risk of atopic disorders (5). Furthermore, the Th1/Th2 paradigm in its simplest form cannot accommodate a relation between general infectious exposure and lower risk of organ-specific autoimmune diseases, which are thought to be Th1 biased (5,17). Thus, the idea has been proposed that stimulation of the immune system by natural infections, as opposed to vaccinations and a “clean” environment, can prevent development of both atopic and autoimmune diseases (5). Bach (5) has recently summarized potential mechanisms of relevance for autoimmune diseases in this regard, including a role of regulatory T-cells and cytokines (e.g., interleukin 10 and transforming growth factor-β) induced by many types of infections. Infectious agents seem to induce changes in the immune system beyond that of producing antibodies to the specific agent. Another possible mechanism, although not well defined, is “antigenic competition,” whereby immune responses to one antigen are reduced by immune responses to other, unrelated antigens. A protective effect of maternal infections in pregnancy rather than of postnatal infections may also point toward a possible role of maternal antibodies (18,19), but our data do not implicate a role of specific infections. Perhaps general infections in the mother during pregnancy also confer protection against related or unrelated diabetogenic postnatal infections in the child. The stronger association between maternal symptoms of infections and lower risk of islet autoimmunity among girls compared with boys detected while checking the data for consistency was not expected, and the explanations for this sex difference are unknown.

The few previous studies of general symptoms of maternal infections during pregnancy and risk of type 1 diabetes in children have not found any significant association (20,21), but these case-control studies were prone to recall or selection bias. Specific markers of maternal infections in pregnancy with enterovirus have been associated with increased risk of type 1 diabetes in the children (2), but this could not be confirmed in a recent larger study (22). Infections and respiratory problems in the neonatal period have been associated with increased risk of type 1 diabetes in two case-control studies (21,23). However, some case-control studies have found associations between symptoms of infections in childhood and lower risk of type 1 diabetes (7,9). Indicators of general exposure to microbial and infectious agents, such as daycare attendance and exposure to pets, have been extensively studied in relation to atopic disorders (4) but relatively rarely in relation to type 1 diabetes. A recent meta-analysis concluded that there is some evidence for a lower risk of type 1 diabetes among children who attended daycare centers early in life, although the amount of heterogeneity between studies makes it difficult to draw strong conclusions (24). The present study does not support a role of early daycare attendance in protecting against islet autoimmunity. Children exposed to household pets are likely to be more exposed to a variety of microbial and infectious agents, and such exposure has been associated with lower risk of atopic disorders, although the evidence is not conclusive. The only previous study of exposure to pets and risk of type 1 diabetes did not find any significant association (25), which is consistent with our study of islet autoimmunity.

Strengths of the current study include that it is prospective with serial follow-up and assessment of exposure in interviews before development of outcome. The fact that symptoms of infections were self-reported and may be difficult to define for mothers is a limitation. However, any possible misclassification is likely to attenuate the association with islet autoimmunity. Many common infections are asymptomatic. It is important to note that our data relates to infectious symptoms and does not answer whether asymptomatic infections are relevant in our context. The infectious symptoms in this study may have been caused by a large number of different pathogens, and it would be impossible to collect blood samples during the symptomatic period for the pregnant women given the design of this study. Future studies may identify and investigate suitable biomarkers of common infectious exposure.

Although we adjusted for a number of factors in the analyses, we cannot rule out the possibility of unmeasured confounding factors. Symptoms of infections in pregnancy may be markers of general hygiene or perhaps of some specific infections. Our results apply to islet autoimmunity and not necessarily to clinical type 1 diabetes, but nearly all children who develop type 1 diabetes first go through the state of islet autoimmunity. Because the cohort consists of children selected to be at genetically increased risk for type 1 diabetes (because they have a sibling or parent with type 1 diabetes or carry HLA alleles conferring increased risk for type 1 diabetes), the results are not necessarily directly applicable to the general population. However, because the majority of children developing type 1 diabetes are at genetically increased risk, and because we did not find evidence for a different association depending on family history or HLA risk category, the present results are likely to be of high relevance at the population level.

In conclusion, symptoms of common maternal infections during pregnancy predicted a significantly lower risk of islet autoimmunity in young girls at genetically increased risk of type 1 diabetes, suggesting a protective effect of such infections.

This study was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-32493 DK-32083, the Diabetes Endocrinology Research Center (DERC) Clinical Investigation & Bioinformatics Core Grant P30-DK-57516, and the Children’s Diabetes Foundation. L.C.S. was supported by a Postdoctoral fellowship grant from the National Research Council of Norway (148359/330).

We thank the Diabetes Autoimmunity Study in the Young and Barbara Davis Center staff for their help, Liping Yu for running the autoantibody assays, and Dory Bugawan for help with the HLA genotyping.