Diabetes & Metabolic Syndrome: Clinical Research & Reviews 6 (2012) 5964

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Diabetes & Metabolic Syndrome: Clinical Research &

Reviews
journal homepage: www.elsevier.com/locate/dsx

Review

HbA1c in pregnancy
Dalia Rafat a, Jamal Ahmad b,*
a
b

Department of Obstetrics and Gynecology, Faculty of Medicine, J.N. Medical College, Aligarh Muslim University, Aligarh 202002, India
Rajiv Gandhi Centre for Diabetes and Endocrinology, Faculty of Medicine, J.N. Medical College, Aligarh Muslim University, Aligarh 202002, India

A R T I C L E I N F O

A B S T R A C T

Keywords:
Pregnancy
HbA1c
Glycemia

During pregnancy, the glucose levels vary according to the hormonal changes and the metabolic needs
necessary to maintain fetal nutrition but strict glycemic control is essential to minimize the maternal and
fetal morbidity and mortality of pregnancies complicated by diabetes. Although considered the gold
standard for diagnosis, measurement of glucose in the blood is subject to several limitations, many of
which are not widely appreciated. Measurement of A1c for diagnosis is appealing as with one number, a
total, integrated view of glycemia over time is derived though it has some inherent limitations. Thus,
supplementation with HbA1c, as is common outside pregnancy, seems appropriate. Before pregnancy,
the target for metabolic control in women with diabetes is HbA1c values near the normal range. However,
the upper normal range of HbA1c during normal pregnancy is only sparsely investigated with different
methods though recently a number of papers have been published regarding the determination of
reference ranges for HbA1c in pregnancy. These changes may have clinical implications for the
assessment and management of glycemic control in diabetic pregnancy and calls for establishment of
separate reference limits of HbA1c levels in different trimesters as compared to general population.
2012 Diabetes India. Published by Elsevier Ltd. All rights reserved.

Contents
1.
2.
3.
4.
5.
6.
7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Use of hemoglobin A1c as a reection of glycemia . . .
Confounders of HbA1c . . . . . . . . . . . . . . . . . . . . . . . . . .
Factors contributing to variations in results of HbA1c.
HbA1c in normal pregnancy . . . . . . . . . . . . . . . . . . . . .
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction
Strict glycemic control is important in pregnancy as two
generations are at risk the fetus and the mother. Before 2010
virtually all diabetes societies recommended blood glucose
analysis as the exclusive method to diagnose diabetes but over
the last few years Physicians have been using hemoglobin A1c to
screen for and diagnose diabetes [1]. Although considered the

* Corresponding author. Tel.: +91 9412459552; fax: +91 571 2721544.

E-mail address: jamalahmad11@rediffmail.com (J. Ahmad).

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59
60
60
60
60
61
62
62

gold standard for diagnosis, measurement of glucose in the blood

is subject to several limitations, many of which are not widely
appreciated. Measurement of A1c for diagnosis is appealing as with
one number, a total, integrated view of glycemia over time is
derived but it has some inherent limitations. These issues have
become the focus of considerable attention with the recent
publication of the report of the international expert committee
that recommended the use of A1c for diagnosis of diabetes [2], a
position that has been endorsed by American Dental Association,
the Endocrine Society and in a more limited fashion by American
Association of Clinical Endocrinologist/American College of Endocrinology [3].

1871-4021/$ see front matter 2012 Diabetes India. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.dsx.2012.05.010

60

D. Rafat, J. Ahmad / Diabetes & Metabolic Syndrome: Clinical Research & Reviews 6 (2012) 5964

2. Chemistry
The glycosylation of hemoglobin takes place under physiologic
conditions, at a specic site on the protein. Normally, about 5
percent of hemoglobin in a population of normal human red cells is
covalently linked to glucose, resulting in the formation of a
chromatographically distinct minor component designated by
Allen et al. [4] as hemoglobin A1c. Interest in HbA1c was
considerably enhanced by the discovery that there is a two- to
three-fold increase in this glycoprotein in patients with diabetes
mellitus [5]. In adults and children above the age of 6 months,
about 90 percent of their hemoglobin is Hb A (a2b2), a tetramer
composed of two pairs of unlike polypeptide chains, each attached
to the prosthetic heme group. Hemoglobin A2 (a2b2) and Hb F (a2
g2) comprise about 2.5 percent and 0.2 percent of the total. The
other minor hemoglobin components found in human red cell are
posttranslational modications of Hb A. Four minor hemoglobin
components which have been designated A1a1, A1a2, A1b and A1c
[6]. Hemoglobin chemistry soon demonstrated the relevant
features of HbA1c that made it such a useful tool [79]. Glycation
of the N-terminal valine residues of hemoglobin gradually occurs
as the erythrocyte circulates, changing the electrophoretic
mobility into the A1c region. This post-translational, postsecretory glycation proceeds through a relatively unstable
aldamine, Schiff base that then slowly undergoes an Amadori
rearrangement to form a stable, essentially irreversible ketoamine
linkage. Since erythrocytes gradually lose their ability to metabolize glucose as they age, but remain permeable to glucose, the
intracellular glucose concentration reects the extracellular
(plasma) glucose.
Key features of HbA1c formation include the following
 Hemoglobin becomes progressively glycated over its 120 day
life span as it circulates in the erythrocyte.
 Therefore, older, senescent red blood cells have more glycated
hemoglobin than do reticulocytes.
 The rate of the reaction reects the ambient plasma glucose.
 The reaction is essentially irreversible, such that a given
molecule of hemoglobin that is glycated remains so until the
end of its lifespan.
 The clinical assay of HbA1c measures total glycation of
hemoglobin, thus measuring the young, less glycated erythrocytes as well as the older, moreglycated red blood cells.

3. Use of hemoglobin A1c as a reection of glycemia

Considerable literature documents the relationship of HbA1c to
mean glycemia over a wide range of blood glucose results. The
DCCT results developed a normogram based on their frequently
sampled blood testing, nding a correlation coefcient of 0.80
between blood glucose and HbA1c [10]. On average, HbA1c of 6%
corresponds to a mean plasma glucose of 135 mg/dl (7.5 mmol/l).
For every increase in HbA1c of 1%, mean plasma glucose increases
by 35 mg/dl (1.95 mmol/l).
4. Confounders of HbA1c
Currently, HbA1c is widely accepted as a index of mean
glycemia, a measure of risk for the development of diabetes
complications, and a measure of the quality of diabetes care but
there are number of factors, that can either falsely lower HbA1c
test results or raise HbA1c test results independent of glycemia.
These includes structural hemoglobinopathies, thalassemia syndromes, and chemical alterations of hemoglobin moreover any
condition that decreases mean erythrocyte age will falsely lower

HbA1c test results regardless of the assay method used [11].

Elevated hemoglobin F, which is associated with thalassemia
syndromes, also effects some assay methods [12]. Uremia, hyperbilirubinemia, hyper-triglyceridemia, chronic alcoholism, chronic
ingestion of salicylates, vitamin C ingestion, and opiate addiction
have all been reported to interfere with some assay methods,
falsely increasing results [12]. In some assays, vitamin C and
vitamin E ingestion have also been reported to falsely lower HbA1c
test results [12]. Iron deciency, which effects up to 20% of
menstruating women [13] and many pregnant women, has been
reported to increase HbA1c test results by altering the structure of
the hemoglobin molecule and making it easier to glycate [14].
When interferences are recognized, alternative forms of testing,
such as glycated serum protein testing (fructosamine or glycated
albumin) may be employed to assess glycemia.
Racial and ethnic differences in HbA1c have been described in
nondiabetes populations that do not appear to be explained by
differences in glycemia [1517]. Studies that have compared
HbA1c levels across racial and ethnic groups within organized
systems of health care and have carefully adjusted for processes of
care have demonstrated persistent though attenuated differences
in HbA1c [1825].
5. Factors contributing to variations in results of HbA1c
Factors that contribute to variation of any blood test can be
divided into three categories, namely biological, pre-analytical,
and analytical. Biological variation comprises both differences
within a single person (termed intraindividual) and between two
or more people (termed interindividual). Preanalytical issues
pertain to the specimen before it is measured. Analytical
differences result from the measurement procedure itself. The
inuence of these factors on both glucose and A1c results will be
addressed below.
A1c is formed by the nonenzymatic attachment of glucose to
the N-terminal valine of the b-chain of hemoglobin [26]. The
lifespan of erythrocytes is approx 120 days, and consequently A1c
reects long-term glycemia exposure, representing the average
glucose concentration over preceeding 812 weeks [27,28]. Both
observational studies and controlled clinical trials [29,30] demonstrate strong correlation between A1c and retinopathy, as well as
other macrovascular complications of diabetes. More importantly,
the A1c value predicts the risk of microvascular complications and
lowering A1c concentrations (by tight glycemia control) signicantly reduces the rate of progression of microvascular complications [29,30].
Biologic variation: Intraindividual variation of A1c in nondiabetic people is minimal [31], with CV < 1% [32]. Variability
between individuals is greater. Data derived from several
investigator imply that A1c values may not be constant among
all individuals despite the presence of similar blood glucose or
fructosamine concentrations. Some investigator have termed this a
glycation gap and proposed that there are differences in rate of
glycation of hemoglobin (low and high glycators) [33]. Studies of
twins and type 1 diabetes support a genetic contribution to A1c
values [34], and heritability of the glycation gap was observed in
healthy female twins [35]. However, the glycation gap is
essentially a measure of A1c adjusted for fructosamine. Various
studies support the hypothesis that race inuences A1c. Initial
studies in patients with diabetes reported statistically signicant
differences in A1c concentrations among races [36]. While
adjusted for factors that may inuence glycemia, it remains
possible that these differences may be due to variations in
glycemic control. More compelling support was provided in
NHANESIII where Mexican Americans and blacks had higher
average A1c values than whites [37]. Similar ndings were

D. Rafat, J. Ahmad / Diabetes & Metabolic Syndrome: Clinical Research & Reviews 6 (2012) 5964

observed in adults with impaired glucose tolerance in diabetes

prevention program [38] and validated in a cross-sectional
analysis of two studies [39]. Collectively these data suggest that
there are differences in A1c concentrations among racial groups.
However, it is not clear that these changes have clinical
signicance.
The molecular mechanism underlying the racial and ethnic
differences remains to be established. Possibilities include
differences in rates of glucose uptake into erythrocytes, rates of
intraerythrocytic glucose metabolism, rates of glucose attachment
to or release from hemoglobin or erythrocyte life span [40,41].
Regardless of the mechanism, the variations in A1c concentrations
are relatively small (0.4%), and no consensus has been reached on
whether different cut-off should be used for different races.
Preanalytical variation: Most factors that alter FPG do not
signicantly affect A1c concentrations. Acute illness, short-term
lifestyle changes (e.g., exercise), recent food ingestion, and sample
handling do not signicantly alter A1c values. Importantly, whole
blood samples are stable for 1 week at 4 8C and for atleast 1 year at
70 8C or colder [42].
The interpretation of A1c depends on the erythrocytes having a
normal life span. Patients with hemolytic disease or other
conditions with shortened erythrocyte survival have substantial
reduction in A1c [43]. Similarly, individuals with acute blood loss
have spuriously low A1c values because of an increased fraction of
young erythrocytes. False increase in A1c have been reported with
some methods in patients with hypertriglyceridemia, hyperbilirubinemia, uremia, chronic alcoholism or chronic ingestion of
salicylates [42]. Because most interferences are method specic, in
many cases they can be overcome by selecting an appropriate
method that is not subject to the interference.
Individuals with iron deciency anemia have increase A1c and
fructosamine concentrations [44], both which are reduced by
therapy with iron [44,45]. A mechanism for the higher A1c was
recently identied by the demonstration that malondialdehyde,
which is increased in subjects with iron deciency anemia [44],
augments glycation of hemoglobin [46]. However, the magnitude
of the increase in A1c is probably small. Examination of 10,535
adults without self reported diabetes in NHANES III revealed that
while 13.7% of women had iron deciency, only 4.74% and 0.48%
had A1c  5.5% or 6.5%, respectively [47]. Iron deciency in
women was associated with a small (odds ratio 1.39) yet
signicant greater odds of A1c  5.5% but not greater odds of
A1c  6.5%.
Analytical variation: There are approx 100 different methods
used to measure A1c. The most widely used commercial methods
use either antibodies (immunoassays) or cation-exchange chromatography (most commonly high-performance liquid chromatography) to separate the glycated (A1c) from the non glycated
hemoglobin [26]. The National Glycohemoglobin standardization
program (NGSP) has been instrumental in standardizing A1c
testing among laboratories [48], particularly (but not exclusively)
in US. The NGSP has markedly improved the performance of A1c
testing [48]. Within laboratory CVs for some method are as low as
<0.5%. In addition, the International Federation for Clinical
Chemistry (IFCC) developed a reference method using mass
spectrometry (or capillary electrophoresis) for A1c measurement,
which should resulting international harmonization as it facilitates
traceability to a metrologically sound accuracy base. It is important
to emphasize that IFCC method is technically complex, time
consuming, and expensive and is not designed for routine analysis
of patient samples.
Thus A1c is a better index of glycemic exposure than blood
glucose, is at least as good at predicting risk of long-term
complications, has similar if not better standardization, is better
in its lack of variability, and is useful in chronic management.

61

There are clearly limitations, A1c is not readily available around

the world, interfering factors such as hemoglobinopathy make the
interpretation of the assay more difcult, and conditions that affect
erythrocyte turnover may cause spurious results, so that clinicians
using A1c in diagnosing diabetes would need to be aware of these
limitations.
6. HbA1c in normal pregnancy
Strict glycemic control is essential to minimize the maternal
and fetal morbidity and mortality of pregnancies complicated by
diabetes [4951]. In addition to home blood glucose measurement,
which may not always reect the true average blood glucose level
[52], HbA1c is a useful parameter in metabolic regulation [5356].
Thus, supplementation with HbA1c, as is common outside
pregnancy, seems appropriate.
Before pregnancy, the target for metabolic control in women
with diabetes is HbA1c values near the normal range [57].
However, the upper normal range of HbA1c during normal
pregnancy is only sparsely investigated with different methods
[58], mainly in late pregnancy [53,54,59,60] and reference ranges
are generally established from the nonpregnant state [52].
Increased third-trimester HbA1c levels are associated with an
increased risk of preeclampsia [51,61], macrosomia [49], and
stillbirth [50], leading to speculations that the target for HbA1c in
pregnancy should be even lower than outside pregnancy to
prevent adverse events. The American Diabetes Association
recommendations state that HbA1c concentrations _1% above
the upper limit of the reference interval should be achieved
before and during pregnancy to assure a good glycemic state [62].
Although the HbA1c reference intervals for the general population are well established, reference intervals for healthy pregnant
women are not clearly dened. Available study data are scarce
and often were obtained on a limited study population or by use
of outdated analytical methods [5860]. Moreover, recent
evidence has shown that despite effective preconception care
and planned pregnancies providing good glycemic control in
early pregnancy with optimal HbA1c concentrations, the
development of diabetes-associated complications cannot
always be prevented [63,64]. These considerations highlight
the need to carefully revise the target for glycemic control during
an uncomplicated pregnancy.
Schwartz et al. [65] compared 15 healthy pregnant women of
unspecied gestation with six normal, non-pregnant females
and stated that the concentration of HbA1c, was increased by
pregnancy. Similarly, Davies and Welborn [66] showed that
mean glyco Hb levels tend to rise after the rst trimester of
pregnancy in both normal and diabetic patients. This occurs
despite the fact that fasting blood glucose levels tend to fail in
normal pregnancy, and in the diabetic patient, control of blood
glucose levels improves with the progress of pregnancy.
They proposed that some factor other than the level of
glucose in the blood must be operating, since if glyco Hb
reects only the average blood glucose level, it must fall with
the progress of pregnancy. An explanation for this phenomenon
is not clear.
Whereas, Leslie et al. [67] indicated that glycol Hb did not alter
with the progress of pregnancy. Also Pollak et al. [68] reported in
study of 75 pregnancies with normal glucose tolerance no changes
of hemoglobin A1 during pregnancy.
On the other hand, Lind and Cheyne [60] in a cross-sectional
study measured total glycosylated hemoglobin (Hb Ala+b+c) in 20
healthy non-pregnant women and in ve groups of 20 healthy
women at various times during pregnancy and found a statistically signicant decrease in these minor hemoglobin fractions by
about 20 weeks gestation and this reduced level is maintained

62

D. Rafat, J. Ahmad / Diabetes & Metabolic Syndrome: Clinical Research & Reviews 6 (2012) 5964

throughout the rest of pregnancy. Widness et al. [69] found in a

group of 13 nondiabetic women a signicant fall in hemoglobin
A1c from rst to the third trimester. McFarland et al. [70] also
reported a decrease in HbA1c from the rst half of pregnancy to
the third trimester. Hanson et al. [71] showed that there is an early
decrease in hemoglobin A1 between the 12th and 16th pregnancy
weeks and a further drop during the last trimester. For individual
patients there is a reduction from 6.30  0.16% to 5.44  0.22%
(mean  SEM, P < 0.005), for the rst and the last values. They
concluded that one of the main reason for this fact must be the
increase in red cell formation, which results in increased red cell
mass with a greater portion of young cells low in hemoglobin A1.
Worth et al. [58] measured glycosylated hemoglobin levels
using two independent techniques (ion-exchange column and
colorimetric) in a longitudinal study of twenty one women and
concluded that sequential changes in glycosylated hemoglobin
showed a similar pattern with both methods showing a nadir at
17 weeks followed by a progressive rise with a peak at the time of
delivery and a subsequent fall in the post-partum period. Gunter
et al. [72] measured HbA1c in the course of 177 nondiabetic
pregnancies and compared with the corresponding values of 24
nondiabetic nonpregnant women. In all three trimesters HbAIc
was signicantly lower than the corresponding values in the
nonpregnant women; 1st trimester 4.77  0.62%, 2nd trimester
4.38  0.59%, 3rd trimester 4.33  0.49%, P < 0.01. Parentoni et al.
[59] studied population based reference interpercentile intervals for
HbA1c in two groups of healthy populations in the city of Campinas,
S.P., Brazil: adult men and women and pregnant women by using
the HPLC methodology and found that the reference limits obtained
for the pregnant group (3.204.30%) were lower and statistically
different from those obtained for the adult Group (3.494.90%).
OKane et al. [54] determined diabetes control and complications trial (DCCT).-aligned 95% inter-fractile reference range for
glycated hemoglobin in non-diabetic pregnancy as 4.15.9%
(n = 493; two-sided 90% condence intervals around the lower
and upper limits are 4.04.2% and 5.86.0%, respectively). The
mean (SD) for the rst, second and third trimesters were 5.1%
(0.51), 4.9% (0.39) and 5.0% (0.45), respectively. They discussed
that the HbA1c reference range in pregnancy may be lower than
that of the healthy general population for two reasons. Firstly, the
age distribution will be lower in the pregnant group and since
fasting blood glucose increases with age a sample from the older
but healthy general population is likely to have a higher HbA1c.
Secondly, the erythrocyte life span may be decreased in pregnancy
including diabetic pregnancy, thus reducing the HbA1c value [73
75]. Nielsen et al. [76] showed that HbA1c was signicantly
decreased early in pregnancy and further decreased in late
pregnancy compared with age-matched nonpregnant women.
The normal range of HbA1c was 4.76.3% in nonpregnant women,
4.55.7% in early pregnancy, and 4.45.6% in late pregnancy. A
decrease of the upper normal limit of HbA1c from 6.3% before
pregnancy to 5.6% in the third trimester of pregnancy is of
signicant clinical importance when dening the reference range
for HbA1c during pregnancy in women with diabetes.
Radder and van Roosmalen [77] found a low upper HbA1c range
level of 5% in the rst trimester of pregnancy, compared with the
non-pregnant upper HbA1c reference value of 6.3% in our hospital,
and a higher upper HbA1c range level of 5.9% in the third trimester
of pregnancy. The low level of HbA1c in the rst trimester of
pregnancy is caused by the low mean preprandial and postprandial
blood glucose values [78] and by the increase in young
erythrocytes which diminishes the percentage of glycosylated
hemoglobin [79]. The increase in HbA1c in the third trimester of
pregnancy is caused by the increase in the mean postprandial
blood glucose value [78]. This is in agreement with the ndings of
Monnier et al. [80] who reported that in type 2 diabetic patients the

relative contribution of postprandial glucose excursions to HbA1c

is predominant in fairly well-controlled patients, whereas the
contribution of fasting hyperglycaemia increases gradually with a
worsening of the diabetes.
Mosca et al. [81] showed that HbA1c is signicantly decreased
in pregnancy and that different reference intervals should be
established for healthy pregnant women and pregnant women
with glucose intolerance or gestational diabetes. They also
observed a small but signicant increase in HbA1c values late in
the pregnancies, at 2836 weeks of gestation. Balaji et al. [82]
undertook a study to nd out whether estimation of A1c levels,
along with oral glucose tolerance tests (OGTTs), would help to
distinguish between the two groups (pre GDM and GDM), as A1c
is directly related to the average concentration of blood glucose
in the previous weeks and also assessed the A1c level during
normal pregnancy. They found that mean A1c level of the
women with normal glucose tolerance was 5.36  0.36%, and
that of the GDM women detected in the rst trimester was
5.96  0.63%.
7. Conclusion
HbA1c levels in pregnancy should be interpreted in the
knowledge of above demonstrable physiological changes and if
diabetic patients are to have their control assessed during
pregnancy by determination of HbA1c the physician and obstetrician should be aware that some fall in concentration may be
reecting the pregnancy effect as well as a possible improvement
in their clinical control. Thus, it may be appropriate to impose
stricter upper limits for HbA1c levels when monitoring glycaemic
control in pregnant diabetic subjects.
Conict of interest
The authors declare that there are no conicts of interest.
Source of funding
None.
References
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new look at screening and diagnosing diabetes mellitus. Journal of Clinical
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