Certain genetic factors have been associated with type-1 diabetes; but the observed changes in incidence over time cannot be attributable to susceptible genes alone, only in combination with environmental factors. Functions of isolated genes include insulin production and metabolism, protection from β-cell apoptosis, immunity and other unknown factors [3]. The role of inflammation in insulitis as a biological response to infection and β-cell loss has been studied in animal models. This process is not clearly understood but it must be induced early in life, where some combination of genetic and environmental factors operate to activate both endogenous and exogenous ligands of pattern-recognition receptors that induce islet inflammation and death of pancreatic β-cells. This is followed by a second process where insulitis is amplified through interplay between immune cells and β-cells, and third process where inflammatory mediators contribute to prolonged suppression and death of β-cells, or promotion, survival and proliferation of β cells [4].

Information on birth outcomes, such as gestational age at birth and birth weight, were obtained from the Danish Medical Birth Registry which has been computerized since 1973. Annual information on maternal education, residence, and income was retrieved from the Fertility Database at Statistics Denmark, available since 1979. The date of conception was estimated by the date of birth minus gestational age, and the prenatal exposure period included from 12 months before conception until the birth of the child. We categorized children as exposed to bereavement during prenatal life if their mothers lost a child, husband, sibling or parent during the prenatal period, ascertained via the death registry; all the remaining children were included in the unexposed cohort. Cohort members were followed from birth until first diagnosis of diabetes, death, emigration, or December 31^st 2004, whichever came first.

Diabetic status of children and their parents was ascertained from the National Hospital Registers, which holds information on all discharges from Danish hospitals since 1977; outpatients have been included in the register since 1995. Diagnostic information was based on the Danish version of the International Classification of Disease (ICD) 8^th revision, from 1979 to 1993 (249) and 10^th revision, from 1994 onwards (E10). We also looked for additional cases from the Pharmaco-epidemiological Prescription Database, in operation since 1995, using the ATC codes for insulin analog drugs (A10A), though there was almost 100% overlap with these cases and those found in the hospital register. If an individual has a diagnosis for both type-1 and type-2 diabetes, they were classified as type-1 since the later is more often misclassified than the former. Also, type-1 diabetes is a more serious disease than type-2 and therefore probably better diagnosed.

We estimated incidence rate ratios (IRRs) from birth using log-linear poisson regression models and used person-years as the offset variable. The analysis was performed using PROC GENMOD in SAS version 9.1.3. Unadjusted and adjusted models were generated. Adjusted estimates of IRRs (aIRR) included maternal age (≤18, 19–34, 35–40, 41+); residence (Copenhagen, cities with over 100,000 inhabitants, and other); income (1^st, 2^nd, 3^rd, and 4^th quartile); maternal education (primary, secondary, high); parental diabetes status (any hospitalization diabetes ICD code); marital status (married, not married); gestational age at birth (<32 weeks, 32–36 weeks, 37+ weeks); birth weight (<1500 grams, 1500–2500 grams, 2500+ grams); sibling order (1,2,3,4+); calendar year (1979–1989, 1990–1999, 2000–2004) and offspring's sex (male, female). Age, residence, calendar period, maternal education, maternal income and parental marital status were treated as time-dependent variables as they were extracted from the birth year of the offspring.

We found that children exposed to bereavement during their prenatal life were more likely to be hospitalized with a type-1 diabetes diagnosis later in life (see table 2). This association was more pronounced when deaths were caused by a traumatic event and most significant for bereavement caused by death of a father or sibling (aIRR: 2.03, 1.22–3.38); the association was mainly seen for girls (aIRR: 2.91, 1.61–5.26). We did not detect significant effect measure modification for timing of exposure. We did attempt a matched sibling-pair analysis but the number of discordant type-1 diabetes pairs were too small to provide significant data for meaningful analysis.

The main strengths of our study are the large cohort size, long follow-up time and high quality of data. Completeness of type-1 diabetes has been demonstrated in previous studies [13]. This study was made possible due to the vast information available in the Danish National Registers. Information in the CRS has been made available for research purposes by Danish legislation [14]. Outpatient data has been collected since 1995, though type-1 diabetes cases are most likely to be found via the in-patient hospital register. This is due to the disease having a sudden and aggressive onset and needing a thorough medical investigation. Today parental links for individuals born in Denmark since 1969 are considered accurate; Danish legislation requires all legal address changes be submitted to the CRS shortly after the move [15]. The main limitation of our study is the lack of inclusion of other sources of stressors and baseline measures of biological stress responses and measures of allostatic load. To get these data longitudinal biological sampling would be needed but it will at present be impossible for ethical and economic reasons. Another limitation in our study is the possibility of uncontrolled confounding. While we were able to control for sociodemographic factors such as education and income, as well as parents' history of diabetes, it is possible that other factors may confound the relationship between prenatal bereavement and development of type-1 diabetes. Lifestyle and background factors such as socioeconomic status may be partially controlled, but factors possibly related to type-1 diabetes such as smoking, maternal or child BMI may not. However, it is unclear clear why these factors should be related to bereavement, and our data indicate that in this population death of a close family member affect all segments of the population equally.

The etiology of type-1 diabetes and the timing of a causal window is not clearly understood, though the disease is postulated to have a multiple stages. It is possible that bereavement is one of the environmental factors that when combined with a genetic factor initiates the disease process shortly after conception. Type-1 diabetes is a rare disease in childhood and if stress exposure can cause the disease one would not expect common stressors to play a role. The disease could on the other hand be caused by a common cause if that cause produces susceptibility for a second hit later in life; should this be the case one would expect low to moderate association for the prenatal exposure if data were not stratified on the second set of causes. The association between prenatal bereavement and type-1 diabetes was observed mainly among female children, but not among male children, exposed to traumatic father or sibling deaths. Previous studies have demonstrated important sex differences in glucocorticoid programming of the HPA axis and metabolic physiology, but how these differences contribute to differential risk for type-1 diabetes is unknown [16], [17].

This study supports the view that type-1 diabetes may have causes that operate in fetal life. We have focused on stress as one of many possible exposures but are aware that stress may just be an upstream cause of other factors of importance such as infections, or changes in dietary habits. Stress exposure may impact the immune system and thus increase the susceptibility of infections. If stress is not a direct cause of diabetes it would explain the rather weak association we find.

While one cannot avoid bereavement as a potential stressor during pregnancy one may be able to avoid other sources of stress, or mitigate the effects via therapy. This study and other studies suggest that exposure to severe stress during the critical period that surrounds pregnancy may have life-long effects on the offspring. We did not find a strong link between bereavement and diabetes when the death was non-traumatic or that of a grandparent or maternal sibling, suggesting that only the most severe stress exposures have a programming effect. A small sample human study with a positive finding has been published on markers of glucose metabolism also suggested a link between stress and diabetes [18].