The role of immunology in diabetes mellitus and pregnancy

Since the global incidence of diabetes mellitus (DM) in younger people is
increasing, there is an increase in number of women at the reproductive age
with DM, especially DM2. During diabetic pregnancy, both in type 1 and 2
diabetes, an increased incidence in birth defects, perinatal complications, but
also maternal complications like pre-eclampsia was found. The prevalence of
maternal and perinatal morbidity as well as the perinatal mortality are higher
in pregnancies complicated by DM2 compared with DM1. In both DM1 and DM2 the
number of complications can be decreased by stringent glycaemic control.
However, despite this stringent metabolic control in recent years,
complications are still much more present in diabetic pregnancies than in
normal pregnancies. This suggests that other mechanisms are involved in the
development of diabetes induced pregnancy complications. This hypothesis is
subject of the present study. One important mechanism may be the altered immune
response during diabetes, since in both DM1 as well as in DM2 patients the
immune response has changed. These changes may not always be compatible with
pregnancy, since for a normal pregnancy the normal immune response has to shift
to a type 2 T-helper cell (Th2) type immune response. Next to it, it is
important that the numbers of Tregs are increased during pregnancy. These
changes of pregnancy are necessary to accommodate the semiallogenic fetus.
Deviations from these adaptations are associated with pre-eclampsia, pre-term
delivery and/or abortion. So, adequate and strict regulation of the immune
responses is essential for a normal pregnancy also. Such an adequate and strict
regulation of the immune responses is especially important at local level at
the time of implantation. Local infiltration of immune cells is seen into the
decidua and the endometrium around the implantation site during the
implantation. By producing cytokines, chemokines and growth factors, these
cells are important in creating the local environment in the endometrium for
implantation of the blastocyst and subsequent placentation. The balance between
the various immune cells present and the factors produced by these cells is
critical for normal placentation and therefore normal foetal growth and normal
pregnancy.

Immune response in DM1: Due to the fact that DM1 is an autoimmune disease, the
immune response of diabetic women differs from the immune response of healthy
women. Various changes in immune responses have been found in patients with
DM1, on peripheral as well as on local level. On peripheral level, a shift
towards a type 1 T-helper cell (Th1) immune response and a decreased number
and/or functions of regulatory T-cells (Tregs), decreased numbers of Natural
Killer cells (NK) and activation of monocytes have been found. Some of these
changes are opposite to the changes occurring in normal pregnancy, so it may
be possible that adequate adaption of the immune response to pregnancy may not
occur in these patients. An autoimmune-induced dysregulated immune response and
pregnancy complication has been shown in women with rheumatoid arthritis. They
showed increased pregnancy complications, such as pre-eclampsia and decreased
birth weight, although data about disease severity and medication use was not
available. So, this association is more an indication than a strict relation.

Immune response in DM2: In case of DM2, a low grade general inflammatory
response has been observed often. This low grade inflammation and activated
inflammatory cells may contribute to the insulin resistance in these patients.
The presence of a low level of inflammation in DM2 patients may interfere with
pregnancy, since pregnancy itselves is also associated with activation of the
inflammatory system. Further activation of the inflammatory response during
normal pregnancy may result in pregnancy complications, like pre-eclampsia. So,
low levels of inflammation in DM2 patients may aggravate the normal pregnancy
induced activation of the inflammatory response, possibly resulting in
preeclampsia.

The present study is a pilot study in order to investigate the adaptations of
the immune response to pregnancy in women with DM1 or DM2 in comparison with
normal controls. In a observational study design, the immune responses in
peripheral blood and placenta will be studied in healthy pregnant women and
pregnant women with DM1 or DM2 and in control non-pregnant women. We
hypothesize that the peripheral and local immune responses are different
compared to healthy controls. We expect that the shift towards a Th2-response
will be hampered, the numbers of Tregs will be decreased and monocytes and
granulocytes will be further activated in comparison with normal pregnancy. We
hypothesize also that the changes in immune responses in diabetic pregnancy are
associated with the increased numbers of complications during diabetic
pregnancies.

Primary Objective: To determine the local and peripheral immune response in
diabetic pregnancies in comparison to healthy controls. Immune response will be
measured by counting Th1/Th2-cells, Tregs, NK-cells, monocytes and
granulocytes. Produced cytokines by different cells will be measured as well.

Secondary Objective: To determine the maternal and foetal outcome of diabetic
pregnancies in comparison with healthy controls in association with the local
and peripheral immune response.

Design: observational study.

Peripheral immune response will be studied in whole blood from pregnant women
in the third trimester of pregnancy. Blood will be taken during routine blood
sampling. Blood samples of pregnant and non-pregnant women with DM1 or DM2 (see
in- and exclusion criteria) will be collected. Diagnosis of DM1 or DM2 has been
established before pregnancy. Controls will be healthy pregnant women without
diabetes mellitus and non-pregnant women (healthy, DM1 and DM2). Blood samples
will be used to evaluate the number of the various leukocytes, i.e. lymfocytes
and lymphocyte subpopulations (for instance regulatory T cells), monocytes,
granulocytes, as well as their state of activation, using flow cytometry. The
production of type 1 and type 2 cytokines will be evaluated after stimulation
of the lymphocytes in order to evaluate the balance between Th1 and Th2
responses. Plasma will be frozen for additional measurement of inflammation
markers, such as hCRP, vWF and VCAM-1.

Local immunological changes will be studied. The placentas of all women
included will be collected and biopsies will be taken in situ of the decidua.
Moreover, tissue biopsies of the placental bed will be obtained after caesarean
section. These biopsies from approximately 0.3 x 0.3 cm in diameter will be
obtained from the placenta bed from the uterus after delivery of the placenta
during caesarean delivery by pfannenstiel incision. All of these tissue
biopsies will be fixed (liquid nitrogen and formalin) immediately after
resection and stored until further analysis. Parameters of the immune response
will be evaluated at the protein level (immunohistochemistry). Previous
research from Dr. R.B. Donker (METc-registrationnr 2003/026) and Dr. M.M. Faas
(ABR-25930) has shown that this procedure does not pose any risk to the
patients.

Blood sampling as well as the placental biopsies procedure does not carry any
risk for the women. As far as blood sampling in diabetic pregnant and healthy
women is concerned, this will be done during routine blood sampling in the
hospitals during the third trimester of pregnancy. Two tubes of 10 ml of extra
blood will be sampled at that time. Control pregnant women will be asked to
give two times of 10 ml of blood during the third trimester as well.
Non-pregnant women with DM1 or DM2 will be asked to give two times of 10 ml of
blood during the follicular phase of the menstrual cycle. In case of use of
oral contraception*s: the blood samples will be taken at the end of the
stopweek. These samples will be sampled during routine blood sampling for serum
glucose measurement. From the control healthy non-pregnant women, blood will be
taken at 1 occasion during the follicular phase of the ovarian cycle. For
placental biopsies, this will be done after delivery of the placenta, which is
part of the normal delivery of pregnant women. It will not harm in any way
mother or the fetus, beacuse previous research from Dr. R.B. Donker
(METc-registrationnr 2003/026) and Dr. M.M. Faas (ABR-25930) has shown that
this procedure does not pose any risk to the patients.
Participation in this study will not benefit the women personally. However, the
present study about immunology in diabetic pregnancies and the pathogenesis of
diabetic complications may in the long term benefit any pregnant women.