Advanced Glycation Endproducts (AGEs) in pregnancies complicated by diabetes mellitus and the accumulation of AGEs in the neonates

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 and gestational diabetes mellitus (GDM), 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 is higher in pregnancies complicated by DM2
compared with DM1. In both DM1, DM2 and GDM 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. One important mechanism may be the increased accumulation of
Advanced Glycation Products (AGEs) in long-lived tissues, since the
accumulation of AGEs is increased in patient with DM1, DM2 or GDM. This
hypothesis is subject of the present study.

AGEs are formed when a reducing sugar, such as glucose, react nonenzymatically
with free amino groups on polypeptides or lipids, resulting in formation of
reversible early glycation end products, so called Amadori products. Further
molecular rearrangements result in the formation of virtually irreversible
AGEs. Formation of AGEs is a normal physiological process and tissue
concentrations of AGE-modified proteins increase slowly with aging. However,
during oxidative and/or glycemic stress, AGEs can be formed more rapidly. This
accumulation of AGEs is associated with the progression of various chronic
diseases, such as atherosclerosis, renal failure and diabetes mellitus. AGEs
among others accumulate in the vessel wall, where they may disturb cell
structure and function. AGEs have been implicated in both the microvascular and
macrovascular complications of diabetes. The general mechanisms of this
association are as follows: (1) formation of cross-links between key molecules
in the basement membrane of the extracellular matrix (ECM), permanently
altering cellular structure; and (2) interaction of AGEs with receptors for
AGEs (RAGE) on cell surfaces, altering cellular function and leading to an
inflammatory response. This inflammatory response includes the upregulation of
nuclear transcription factors, including NF-*B, which transcribes its target
genes. Among these are endothelin-1, vascular cell adhesion molecule-1
(VCAM-1), intercellular adhesion molecule-1 (ICAM-1), tissue factor and
proinflammatory cytokines.

Increased accumulations of AGEs may possibly not always be compatible with
pregnancy, since accumulated AGEs contribute to vascular endothelial
dysfunction, a hallmark of pre-eclampsia. Above all, preeclampsia share several
risk factors with cardiovascular disease, including pre-existent hypertension,
thrombophilia (factor II or factor V Leiden mutations), dyslipidemia, obesity,
insulin resistance, diabetes mellitus, inflammation, family history of heart
disease or stroke, and possibly increased accumulation of AGEs. This leads to
increased cardiovascular morbidity and mortality in later life in women with
preeclampsia in previous pregnancies. So, AGEs possibly does not only
contribute to the development of preeclampsia and other pregnancy induced
complications, but also to anchoring of cardiovascular damage and the
cardiovascular diseases involved.
In previous research investigators found AGEs in umbilical cord blood, which
means AGEs pass the placenta and reach the unborn child during the whole
pregnancy durance. Probably AGEs are able to accumulate in the unborn infant
during pregnancy. Therefore, the extent of AGE accumulation in the mother may
be reflected in the by skin AF in the newborn. Possibly infants, born after a
pregnancy complicated by diabetes, start their life with a higher AGEs
accumulation when compared to infants born after an uncomplicated pregnancy and
making them more vulnerable for vascular complications.

Tissue AGE levels can be assessed noninvasively by the autofluorescence reader
(AFR; patent number PCT/NL99/00607, DiagnOptics BV Groningen, the Netherlands),
a recently introduced and validated device that measures skin autofluorescence
based on the fluorescent properties of a part of the various AGE molecules.
With the AFR, a close relation of skin autofluorescence was established not
only with renal failure, diabetes mellitus, and its complications, but also
with inflammation and oxidative stress in acute coronary syndromes. But, tissue
AGEs are not exactly the same as serum AGEs (sAGEs). For instance, the physical
half-life of sAGEs is shorter and they are more unstable than tissue AGEs.
Therefore, sAGEs will be assessed as well for two reasons, firstly, to validate
the SAF measurements and secondly, to detect short-term variations in
accumulation of AGEs.

Our primary aims are to assess skin autofluorescence (SAF) of the lower arm
(using the AFR) or the upperleg of a neonate and serum AGEs in pregnant women
who are suffering DM1, DM2 or GDM and to investigate the association between
increased AGEs and maternal and fetal complications of diabetic pregnancies and
to investigate the possible association between SAF, serum AGEs and glycaemic
control of the mother and the SAF level in the newborn. We hypothesize that the
accumulation of, in both serum and tissue AGEs will be increased in diabetic
pregnancies. We expect that women with GDM have a level of accumulation
intermediate between healthy pregnant women and women with DM1 or DM2. We will
also determine whether the AGEs accumulation in newborn is different between
neonates born after a pregnancy complicated by diabetes or neonates born after
an uncomplicated pregnancy and if extent of AGEs in the mother will be
reflected in the skin autofluorescence of her offspring.
We hypothesize there will be a higher SAF in the neonates born after a
pregnancy complicated by diabetes, when compared to neonates born after an
uncomplicated pregnancy.
As secondary objectives, we formulated three objectives; (1) the inflammatory
response will be assessed, by obtaining maternal serum for measurement of
inflammation markers, such as hCRP, vWF and VCAM-1. We hypothesize that these
inflammation markers will be increased in comparison to healthy pregnant
controls. (2) We hypothesize also that the increased accumulation of AGEs is
associated with the increased incidence of maternal and fetal complications
during diabetic pregnancies and (3) the relation between SAF in neonates and
the SAF of their mothers will be assessed.

The disease GDM requires a further introduction. GDM implies a substantial risk
of later diabetes. Diabetes rates are reported to be between 9 and 43% within
5-10 years after the index pregnancy. There is evidence that in women with GDM
an underlying pre-existing β-cell defect is unmasked by poor pancreatic β-cell
compensation for physiological insulin resistance in pregnancy. It has been
suggested that increased insulin resistance associated with pregnancy may
accelerate the depletion of beta cells. If this is the case, it would be
expected that increased parity or subsequent pregnancies would increase the
risk for development of subsequent diabetes among women with a history of
gestational diabetes. However, findings related to the roles of parity and
subsequent pregnancies are conflicting.
Recommendations of the 5th Workshop-Conference on GDM38 are that women with GDM
undergo postpartum glucose tolerance testing with a 75 gram oral glucose
tolerance test (OGTT) at 6-12 weeks, 1 year after delivery, and every 3 years
thereafter. The rational for this recommendation is based on the potential to
identify women with apparent diabetes as well as women with impaired glucose
tolerance (IGT) in whom diabetes can be delayed or prevented by lifestyle
intervention or moderate drug therapy. In this study a 75 gram OGTT will be
performed at 6-12 weeks after delivery in case of GDM as part of the clinical
follow-up.

Primary Objective: To determine the accumulation of AGEs in the skin and serum
of pregnant women with DM1, DM2 or GDM, in comparison to healthy pregnant women
and non-pregnant women with DM1 or DM2 and to determine the accumulation of
AGEs in the newborns of the pregnant women who deliver in the UMCG. The
accumulation of soft tissue AGEs will be non-invasively measured by the skin
autofluorescence reader and sAGES will be measured in maternal serum.

Secondary Objective(s):
1) To asses the inflammatory response by measurement of inflammation markers in
maternal serum in pregnant women with DM1, DM2 or GDM. This response will be
measured twice, namely firstly in the third trimester and secondly 6-12 weeks
after delivery.
2) To asses the relation between skin autofluorescence (SAF) and presence or
development of impaired glucose tolerance (IGT) or DM after pregnancy, as
assessed by OGTT 6-12 weeks after delivery. Furthermore, the relation between
SAF and maternal, fetal and neonatal complications will be assessed.
3) To asses the relation between SAF in neonates and the SAF of their mothers,
in perspective of the presence or absence of diabetes during pregnancy.

Design: Observational study
Participating centers: The diabetes outpatient clinics of the University
Medical Centre Groningen and Medical Centre Haaglanden, The Hague.
SAF and sAGEs will be assessed in pregnant women with DM1, DM2 or GDM in
comparison to healthy pregnant and non-pregnant women with DM1 or DM2. SAF will
be measured by the skin autofluorescence reader (patent number PCT/NL99/00607,
DiagnOptics BV Groningen, the Netherlands) on the ventral side of the lower
arm. In their newborn children, only SAF will be assessed, non-invasively,
through illumination of the skin on the dorsal side of the upperleg by the
AGE-reader and this will only be done in the UMCG.
SAF will be assessed according to a schedule, shown in table. SAF in
non-pregnant women with DM1 or DM2 will be performed during routine visits to
our outpatient clinics for maximally four times with approximately the same
intervals of the pregnant women. sAGEs will be measured twice by taking a
venous blood sample in a heparin tube (10 ml) at the same time of the last two
SAF measurements during routine HbA1c control. Serum will be separated and
stored at -80*C until analysis of sAGEs. The postpartum measurement of AGEs
will be obtained to determine the AGE accumulation after the physiological
state of pregnancy is ended and will be combined with the postpartum control at
the outpatient clinic. This gives us information about the influence of
pregnancy on AGE accumulation.
An OGTT 6-12 weeks after delivery in women with GDM during postpartum control
will be performed, as part of standard care.

Table 1.
Participants 10-14 weeks of gestation 20-24 weeks of
gestation 32-36 weeks of gestation 6-12 weeks postpartum
DM1 x
x
x x
DM2 If possible
x
x x
GDM - If
possible x x
Healthy pregnancies x
x x x

All neonates, independent if they are born after a complicated or uncomplicated
pregnancy, will have SAF measurements within 24 hours postpartum and between
6-12 weeks postpartum and this part of the study will only be performed in the
University Medical Centre Groningen.

Not applicable