<title>1. INTRODUCTION</title> <p>Type 1 diabetes develops in genetically susceptible individuals as a result of
progressive autoimmune destruction of <italic>β</italic>-cells in the pancreas. The incidence of
type 1 diabetes has increased worldwide in recent decades [##REF##11724413##1##–##REF##16911623##4##]. The peak age at
onset is at 10 to 14 years of age, and while there is no difference in the
incidence between boys and girls [##REF##16911623##4##, ##REF##1428492##5##], several studies
have shown a gender difference after 15 years of age with a
male:female ratio of approximately 3:2 [##REF##1428492##5##–##REF##1541382##7##]. The reason for
this gender difference is not known but it cannot be excluded that susceptibility
to environmental factors may contribute.</p> <p>Several studies support the hypothesis that pre- and
perinatal exposures to environmental risk factors are significant in the development
of type 1 diabetes. Advanced maternal age at delivery, complications during
delivery, delivery by cesarean section, and blood group incompatibility are
related to increased risk of type 1 diabetes in childhood [##REF##1644246##8##–##REF##15229111##12##]. It is also thought that congenital
rubella increases the risk of type 1 diabetes later in life, especially in the
second and third decades [##UREF##0##13##–##UREF##2##15##]. Enterovirus
infections are one of the main candidates for an environmental trigger of type
1 diabetes [##REF##2988099##16##–##REF##12436483##20##], and maternal enterovirus infections during pregnancy have
also been associated with increased risk of offspring developing type 1
diabetes during childhood in the age group 0–14 years [##REF##7789630##21##–##REF##10333962##24##].</p> <p>We have previously reported that
cord blood islet autoantibodies did not affect the risk for type 1 diabetes in 15–30-year-old
subjects [##REF##14563016##25##].
Therefore, in this study, we
examined whether intrauterine exposure to enterovirus infection in the 15–30-year-old ages
was associated with an increased risk of offspring developing type 1 diabetes,
also with particular reference to possible gender difference.</p>
<title>2. MATERIALS AND METHODS</title> <title>2.1. Study population</title> <p>The city of Malmö, Sweden, has 270 000 inhabitants who are served by Malmö University 
Hospital (U-MAS), its only hospital. The vast majority of deliveries in Malmö take place in the Department
of Obstetrics at this hospital. Since 1970, umbilical cord blood serum has been taken at delivery from the majority
of children born at Malmö University Hospital and stored, together with a maternal serum sample similarly taken at delivery, at –20°C. Among
those children born from April 1970 to July 1984, 38 later developed type 1
diabetes between the ages of 15 and 30. They were identified using the Diabetes
Incidence Study in Sweden (DISS) Registry and classification of type 1 diabetes in the offspring
was done by the physicians, using clinical data and laboratory findings as
detailed in the DISS-study [##REF##3812028##26##]. Of the group,
32 had cord serum and a corresponding maternal serum sample saved from the time
of birth. Two of the thirty-two mothers (6%) had type 1 diabetes and were excluded, since only 3% of new onset type 1
diabetes patients have a mother with the disease. Consequently,
the study was comprised of 30 mothers whose offspring (14 males and 16 females)
developed type 1 diabetes at a median age of 18 years (range 15.2–25.5).
The median age of the mothers at delivery was 25.8 years (range 19.7–34.8).
Information about the incidence of type 1 diabetes among the fathers was not
available.</p> <p>The control group consisted of 90 maternal serum samples, including three control
mothers for each of the 30 case mothers, matched by date of delivery. The
median age of the control group was 25.9 years (range 19.4–40.1). Three of
the control mothers, although they were matched for month and day, gave birth
in the following year. Altogether, there were 52 males and 38 females born to
the control mothers. None of these children developed type 1 diabetes during
the follow-up time.</p> <title>2.2. Enterovirus antibodies</title> <p>All sera were subjected to blind analysis. Immunoglobulin M (IgM) was measured
using a capture enzyme immunoassay (EIA), as previously described [##REF##10330298##27##]. A cocktail of
heat-treated Coxsackievirus B3, Coxsackievirus A16, and echovirus 11 was used
as the antigen in this assay, making it broadly reactive to different
enterovirus serotypes. Biotinylated immunoglobulin G (IgG) class antibodies,
purified from the serum of rabbits immunized with sucrose-gradient purified
viruses (Coxsackievirus B3, Coxsackievirus A16, and echovirus 11), were used as
detection antibodies in the laboratory. After washing, streptavidin-peroxidase
conjugate 9534A (Bethesda Research Laboratories, Gaithersburg, Md, USA)
was added. The level of IgM was expressed as positive (+) or negative (−). The cutoff
limit for IgM positivity was three multiples of the background, which is the
optic density (OD) value when the serum layer is replaced by phosphate-buffered
saline [##REF##10923631##28##]. Values
exceeding the background by four times or more were considered highly positive.</p> <title>2.3. Enterovirus RT-PCR</title> <p>RNA was extracted according to the manufacturer's protocol by a QIAamp viral RNA
kit (Qiagen, Hilden, Germany) from 140 <italic>μ</italic>L of the serum. Enterovirus
RNA was detected by reverse transcription-polymerase chain reaction (RT-PCR)
and a subsequent hybridization step that detects practically all enterovirus
serotypes, as described elsewhere [##REF##10502272##29##]. All positive samples were confirmed by
repeated RT-PCR.</p> <title>2.4. Statistical analysis</title> <p>Nonparametric methods were applied. Group comparisons were performed using the Mann-Whitney test.
Four field tables displaying the frequencies of study groups were analyzed by
means of Fisher's exact test. Logistic regression analyses were also performed.
A two-tailed <italic>P</italic> value &lt;.05 was
considered statistically significant.
The statistical analyses were carried out with the standard statistical
package (SPSS) for Windows, V15.0 (SPSS Inc, Chicago, Ill, USA).</p>
<title>3. RESULTS</title> <p>The prevalence of enterovirus IgM in sera taken at delivery from mothers of
children who developed type 1 diabetes and from control mothers is given in
##TAB##0##Table 1##. There was no significant difference in ages between the groups of
mothers. The prevalence value of enterovirus IgM was higher in the mothers whose offspring developed type
1 diabetes, as compared to control mothers, but the difference did not reach
statistical significance (<italic>P</italic> &lt; .11)
(##TAB##0##Table 1##). Among the mothers of offspring with diabetes, 9/30 (30%)
were enterovirus IgM-positive, and 5/30 (17%) had high IgM titers. None was
positive for enterovirus RNA. In the control group, 14/90 (16%) were
enterovirus IgM-positive, 8/90 (9%) had high titers, and 4/90 (4%) were
positive for enterovirus RNA. No significant differences were found between the
groups (##TAB##0##Table 1##).</p> <p>The gender of the child did influence the risk of diabetes following maternal enterovirus
infection (##TAB##1##Table 2##). In logistic regression controlling for mother's age and
the interaction between IgM-positivity and gender we found that boys born to
IgM-positive mothers showed an increased risk of developing type 1 diabetes (odds
ratio [OR] 4.63; 95% confidence interval [CI] 1.22–17.6; <italic>P</italic> &lt; .025), as compared to boys of
IgM-negative mothers. No such increased risk was found in girls born to
IgM-positive mothers (OR 0.21; 95% CI 0.03–1.56). Mother's
age was included but was not a significant predictor of developing type 1
diabetes. The results were similar when logistic regression was done without
controlling for maternal age. The frequency of maternal enterovirus IgM with
regard to gender of the offspring is given in ##TAB##2##Table 3##.</p>
<title>4. DISCUSSION</title> <p>This study analyzed the correlation of
maternal enterovirus infections during pregnancy and the future risk of type 1
diabetes occurring in the offspring. The presence of enterovirus antibodies
(IgM) and enterovirus genome (RNA) was analyzed by means of stored serum
samples obtained at delivery from 30 mothers without diabetes whose offspring subsequently
developed type 1 diabetes during adolescence or young adulthood. Comparable samples were taken from 90 matched
control mothers. The mothers
of the offspring who later developed type 1 diabetes were carefully matched to
control mothers and the two groups were expected to be exposed to a similar
infectious environment during pregnancy. The strength of our study is that the
countywide DISS registry [##REF##3812028##26##] made it possible to identify not only the offsprings
who developed diabetes but to ensure that none of the children of the control
mothers had acquired the disease. The
study is explorative due to the limited study cohort, but it is unique as the
serum samples used were obtained from mothers who delivered their children as
long as 30 years ago in the same hospital.</p> <p>We observed a difference in the presence
of enterovirus IgM between the patient and control groups, although it did not
reach statistical significance. However, our study indicated that maternal
enterovirus infection was a significant risk factor for the development of
diabetes in boys, but not in girls. This finding suggests that boys may
be more susceptible to the diabetogenic effect of enteroviruses than girls
during the prenatal period. Prospective studies such as the ongoing DiPiS [##REF##15991024##30##] and TEDDY [##REF##17130573##31##] studies will be
needed to fully establish if maternal enterovirus infections contribute to the
gender difference in 15–25 year old type
1 diabetes patients.</p> <p>Enterovirus RNA was only observed in a few control mothers. PCR of enterovirus is known to be positive in
serum only for a period between a few days and 1-2 weeks during
viremia. Therefore, a larger number of mothers who gave birth to children developing type 1 diabetes
as 15–30 year olds would be needed to fully explore the possible role of
gestational infections in this age group. However, while PCR
analysis can reflect an infection in its acute stage, IgM antibodies persist
much longer, allowing one to detect an infection for a few months. In addition,
the extended storage of the samples, coupled with the fact that those of the patient
mothers were exposed to an additional thawing, may have caused a bias in the PCR analysis. It has also been
suggested that cellular elements in blood sequester enteroviruses [##REF##8043865##32##] and that whole
blood might be better for PCR analysis.</p> <p>Some earlier reports have shown an association between maternal virus infection
during pregnancy and diabetes later in life. Congenital rubella increased the
risk of diabetes in the second and third decades of life [##UREF##0##13##–##UREF##2##15##], indicating the
possibility of an extensive time lag. Other studies have suggested that
gestational enterovirus infections may be a precursor of diabetes in young children
[##REF##7789630##21##–##REF##7698508##23##]. In one of these
studies, the levels of enterovirus antibodies were found to be elevated in the mothers
of children who developed type 1 diabetes before the age of three years [##REF##7789630##21##]. In another
study tending to corroborate these results, mothers whose children developed
type 1 diabetes before age 15 showed an elevated number of enterovirus
infections during pregnancy, compared to controls [##REF##10333962##24##]. A case of
neonatal diabetes with evidence of maternal enterovirus infection during
pregnancy has also been reported [##REF##11079741##33##]. It is known that enterovirus infections show seasonal
variation [##REF##12378375##19##]. In an analysis such as ours it is possible that
controls matched for time of sampling also have been affected. Their virus
antibodies may therefore rather mask a relationship between gestational enterovirus
infection and development of type 1 diabetes.</p> <p>A research project using a larger cohort than those cited above tested for the
presence of enterovirus IgM in more than 900 mothers of children who developed
type 1 diabetes. Control mothers were carefully matched by the same method employed
in our study [##REF##12145172##34##]. No significant
difference appeared between case and control groups. However, the samples in that
study were taken at the end of the first trimester, thus revealing only
infections that occurred during the initial three months of pregnancy. This differs
from our study, where samples were taken at delivery, enabling an IgM assay to
detect infections arising during the last two trimesters of pregnancy. In
addition, the present study included children who were diagnosed with diabetes
at an older age than previously investigated. One report including only 16
mothers, most of whom already had type 1 diabetes from the German Multicenter BABY-DIAB
study [##REF##11771958##35##], did not support
the hypothesis of enterovirus infections during pregnancy causing type 1
diabetes in offspring.</p> <p>We found an increased risk of developing type 1 diabetes for boys born to IgM-positive
mothers. A similar male proclivity towards risk for <italic>β</italic>-cell damage in
enterovirus-induced diabetes was found earlier in mice and in prospective
studies evaluating the risk effect of postnatal enterovirus infections [##REF##2073786##36##, ##REF##9041325##37##]. Other studies
have also shown that boys might be more susceptible to enterovirus infections, possibly
due to having a weaker immune system [##UREF##3##38##]. HLA-DR alleles,
which mediate increased risk of type 1 diabetes (DR3 and DR4), have been associated with a stronger humoral
response to enterovirus antigens [##REF##12845430##39##] compared to HLA-DR2. In our study, HLA
genotypes were not available. This might be a confounding factor, as patient and
control subjects ideally should be matched for both sex and HLA type. It is
possible that the observed difference between patient and control subjects as
the present study noted (also reflected in approximately half of the previous
studies cited) indicates a genuine risk effect, particularly in boys, which is later
modulated by several postnatal factors.</p>
<title>5. CONCLUSIONS</title> <p>Taken together with previous studies, the present findings suggest that maternal enterovirus
infections during pregnancy may affect the risk of type 1 diabetes in
offspring. Our data suggest that the risk effect is not pronounced and may be
relevant to boys in particular. The risk may also be modulated by factors such as
when the infection occurs (early or late in pregnancy), the gender of the child,
as well as HLA and other susceptibility genes in the child and the mother.
Accordingly, large-scale studies covering the entire period of pregnancy and taking
into account children of all ages with type 1 diabetes are warranted.</p>
<p>Recommended by Rodica Pop-Busui</p> <p>Maternal enterovirus infections during pregnancy may increase the risk of offspring developing type 1 diabetes during childhood. The aim of this study was to investigate whether gestational enterovirus infections increase the offspring's risk of type 1 diabetes later in life. Serum samples from 30 mothers without diabetes whose offspring developed type 1 diabetes between 15 and 25 years of age were analyzed for enterovirus-specific immunoglobulin M (IgM) antibodies and enterovirus genome (RNA), and compared to a control group. Among the index mothers, 9/30 (30%) were enterovirus IgM-positive, and none was positive for enterovirus RNA. In the control group, 14/90 (16%) were enterovirus IgM-positive, and 4/90 (4%) were positive for enterovirus RNA (n.s.). Boys of enterovirus IgM-positive mothers had approximately 5 times greater risk of developing diabetes (OR 4.63; 95% CI 1.22–17.6), as compared to boys of IgM-negative mothers (<italic>P</italic> &lt; .025). These results suggest that gestational enterovirus infections may be related to the risk of offspring developing type 1 diabetes in adolescence and young adulthood.</p>
<title>ACKNOWLEDGMENTS</title> <p>The authors would like to thank Majvi Månsson and Hamideh Rastkhani for providing us with excellent technical support. The authors also thank professor Stig
Kullander for initiating the maternal and
umbilical cord serum bank. The authors
wrote this paper on behalf of the Diabetes
Incidence in Sweden Study Group. The following
authors are from the Diabetes Incidence Study in Sweden Study Group: Hans
Arnqvist, Linköping, Jan Bolinder, Stockholm, Mona Landin-Olsson, Lund,
Stina Lindmark, Umeå, Soffia Gudbjörnsdottir, Gothenburgh, Lennarth Nyström,
Umeå. Our research has been underwritten
in part by the Swedish Childhood Diabetes Foundation, the Malmö Branch of the
Swedish Diabetes Association, Lions Club International District 101-S, the Novo
Nordisk Insulin Fund, UMAS funds, the Terry &amp; Louise Gregg
Diabetes in Pregnancy Award from the American Diabetes Association, the Skåne
County Council Foundation for Research and Development, and the Juvenile
Diabetes Research Foundation. We mourn the passing of our esteemed colleague, Göran Sundkvist, whose
death came unexpectedly as we were completing this manuscript.</p>
<table-wrap id="tab1" position="float"><label>Table 1</label><caption><p>Enterovirus findings in serum samples taken at delivery from mothers whose children developed 
type 1 diabetes in adolescence and young adulthood (Fisher's exact test).</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1"/><th align="center" colspan="2" rowspan="1">Index mothers (<italic>n</italic> = 30)</th><th align="center" colspan="2" rowspan="1">Control mothers (<italic>n</italic> = 90)</th><th align="center" rowspan="2" colspan="1">
<italic>P</italic> value</th></tr><tr><th align="left" rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1">Positive <italic>n</italic> (%)</th><th align="center" rowspan="1" colspan="1">Negative <italic>n</italic> (%)</th><th align="center" rowspan="1" colspan="1">Positive <italic>n</italic> (%)</th><th align="center" rowspan="1" colspan="1">Negative <italic>n</italic> (%)</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Enterovirus IgM</td><td align="center" rowspan="1" colspan="1">9 (30)</td><td align="center" rowspan="1" colspan="1">21 (70)</td><td align="center" rowspan="1" colspan="1">14 (16)</td><td align="center" rowspan="1" colspan="1">76 (84)</td><td align="center" rowspan="1" colspan="1">0.11</td></tr><tr><td align="left" rowspan="1" colspan="1">Enterovirus IgM high titers</td><td align="center" rowspan="1" colspan="1">5 (17)</td><td align="center" rowspan="1" colspan="1">25 (83)</td><td align="center" rowspan="1" colspan="1">8 (9)</td><td align="center" rowspan="1" colspan="1">82 (91)</td><td align="center" rowspan="1" colspan="1">0.31</td></tr><tr><td align="left" rowspan="1" colspan="1">Enterovirus RNA</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">30 (100)</td><td align="center" rowspan="1" colspan="1">4 (4)</td><td align="center" rowspan="1" colspan="1">86 (96)</td><td align="center" rowspan="1" colspan="1">0.57</td></tr></tbody></table></table-wrap> <table-wrap id="tab2" position="float"><label>Table 2</label><caption><p>Odds ratio (OR) and 95% confidence interval (CI) for developing type 1 diabetes (dependent 
variable in logistic regression) when controlling for mother's age, gender, interaction gender∗ 
IgM-positivity and IgM-positivity in offspring.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1"/><th align="center" colspan="2" rowspan="1">95.0% CI for</th></tr><tr><th align="left" rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1">B</th><th align="center" rowspan="1" colspan="1">SE</th><th align="center" rowspan="1" colspan="1">
<italic>P</italic> &lt; </th><th align="center" rowspan="1" colspan="1">OR</th><th align="center" rowspan="1" colspan="1">OR</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Gender (<italic>M</italic> = 0, <italic>F</italic> = 1)</td><td align="center" rowspan="1" colspan="1">0.865</td><td align="center" rowspan="1" colspan="1">0.507</td><td align="center" rowspan="1" colspan="1">0.088</td><td align="center" rowspan="1" colspan="1">2.38</td><td align="center" rowspan="1" colspan="1">0.88–6.42</td></tr><tr><td align="left" rowspan="1" colspan="1">Gender∗IgM-positive (0/1)</td><td align="center" rowspan="1" colspan="1">−1.556</td><td align="center" rowspan="1" colspan="1">1.022</td><td align="center" rowspan="1" colspan="1">0.128</td><td align="center" rowspan="1" colspan="1">0.21</td><td align="center" rowspan="1" colspan="1">0.03–1.56</td></tr><tr><td align="left" rowspan="1" colspan="1">Mother's age</td><td align="center" rowspan="1" colspan="1">−0.020</td><td align="center" rowspan="1" colspan="1">0.044</td><td align="center" rowspan="1" colspan="1">0.651</td><td align="center" rowspan="1" colspan="1">0.98</td><td align="center" rowspan="1" colspan="1">0.90–1.07</td></tr><tr><td align="left" rowspan="1" colspan="1">IgM-positive (0/1)</td><td align="center" rowspan="1" colspan="1">1.532</td><td align="center" rowspan="1" colspan="1">0.682</td><td align="center" rowspan="1" colspan="1">0.025</td><td align="center" rowspan="1" colspan="1">4.63</td><td align="center" rowspan="1" colspan="1">1.22–17.6</td></tr><tr><td align="left" rowspan="1" colspan="1">Constant</td><td align="center" rowspan="1" colspan="1">−1.188</td><td align="center" rowspan="1" colspan="1">1.246</td><td align="center" rowspan="1" colspan="1">0.340</td><td align="center" rowspan="1" colspan="1">0.30</td><td align="center" rowspan="1" colspan="1"/></tr></tbody></table></table-wrap> <table-wrap id="tab3" position="float"><label>Table 3</label><caption><p>Frequency of enterovirus IgM in mothers of offspring developing type 1 diabetes and controls, 
divided with regard to gender.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="2" colspan="1">Gender of offspring</th><th align="center" colspan="2" rowspan="1">Maternal enterovirus IgM <italic>n</italic> (%)</th><th align="center" rowspan="2" colspan="1"> Total <italic>n</italic> (%)</th></tr><tr><th align="center" rowspan="1" colspan="1">Positive</th><th align="center" rowspan="1" colspan="1">Negative</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Type 1 diabetes—males</td><td align="center" rowspan="1" colspan="1">6 (43)</td><td align="center" rowspan="1" colspan="1">8 (57)</td><td align="center" rowspan="1" colspan="1">14 (100)</td></tr><tr><td align="left" rowspan="1" colspan="1">Type 1 diabetes—females</td><td align="center" rowspan="1" colspan="1">3 (19)</td><td align="center" rowspan="1" colspan="1">13 (81)</td><td align="center" rowspan="1" colspan="1">16 (100)</td></tr><tr><td align="left" rowspan="1" colspan="1">Controls—males</td><td align="center" rowspan="1" colspan="1">7 (13)</td><td align="center" rowspan="1" colspan="1">45 (87)</td><td align="center" rowspan="1" colspan="1">52 (100)</td></tr><tr><td align="left" rowspan="1" colspan="1">Controls—females</td><td align="center" rowspan="1" colspan="1">7 (18)</td><td align="center" rowspan="1" colspan="1">31 (82)</td><td align="center" rowspan="1" colspan="1">38 (100)</td></tr></tbody></table></table-wrap>
<table-wrap-foot><fn><p>B = regression coefficient of logistic regression elog (OR)</p><p>SE = standard error for B</p><p>OR = e<sup>B</sup>.</p></fn></table-wrap-foot>
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Exp Diabetes Res. 2008 Jul 28; 2008:271958