Hypertension in Pregnancy, 31:166176, 2012

Copyright Informa UK Ltd.

ISSN: 1064-1955 print/1525-6065 online
DOI: 10.3109/10641955.2010.484084

The Concentrations of Markers of Bone

Turnover in Normal Pregnancy
and Preeclampsia
1525-6065
1064-1955
LHIP
Hypertension
in Pregnancy
Pregnancy, Vol. 1, No. 1, Sep 2010: pp. 00

Bone Turnover
Dorota
et al. in Preeclampsia

Darmochwal-Kolarz Dorota,1 Kolarz G. Bogdan,2

Gorzelak Mieczyslaw,3 Leszczynska-Gorzelak Bozena,1
and Oleszczuk Jan1
1

Department of Obstetrics and Perinatology, Medical University of Lublin, Lublin, Poland

Department of Rheumatology and Connective Tissue Diseases, Medical University of
Lublin, Poland
3
Department of Orthopedics and Rehabilitation, Medical University of Lublin, Lublin,
Poland
2

Background. The purpose of our study was to investigate the concentrations of markers
of bone turnover in normal pregnancy and preeclampsia. Material and Methods. Forty-five
pregnant patients with preeclampsia, 78 healthy pregnant women (26 in first, 26 in the
second, and 26 in third trimester of pregnancy), and 20 nonpregnant women were
included in the study. Serum concentrations of osteoprotegrin (OPG), receptor activator
of nuclear factor kappa B ligand (sRANKL), and the markers of bone turnover,
osteocalcin and CrossLapsdegradation products of type I collagen, were determined
using the ELISA method. Statistical analysis was performed using MannWhitney
U-test. Results. The concentrations of sRANKL and OPG were significantly higher in
the second trimester of normal pregnancy when compared to the first and the third
trimesters and to nonpregnant controls. The concentrations of osteocalcin were significantly higher in the first trimester of physiological pregnancy in comparison with
nonpregnant women and with second and third trimesters of pregnancy. The concentrations of CrossLaps were significantly higher in the second trimester of normal
pregnancy when compared to the first and third trimester. In preeclampsia, the sera
concentrations of osteocalcin and CrossLaps were significantly higher when compared
to the third trimester of normal pregnancy. Conclusion. The results suggest that the
bone formation is increased in the first trimester, whereas the bone resorption is
increased in the second trimester of normal pregnancy. Furthermore, the results
suggest that the bone turnover is increased in patients with preeclampsia when
compared to healthy normotensive pregnant women.
Keywords CrossLaps, Osteocalcin, Osteoprotegrin, Preeclampsia, Pregnancy, sRANKL.

BACKGROUND
During pregnancy, the changes in maternal calcium metabolism, bone metabolism, and bone mineral status have been observed. The alterations are associated with the growth and mineralization of the fetal skeleton. The fetal
requirement for calcium is covered by its mobilization from maternal skeleton
Address correspondence to Darmochwal-Kolarz Dorota MD, PhD, Department of
Obstetrics and Perinatology, Medical University of Lublin, 20-950 Lublin, ul.
Jaczewskiego 8, Lublin, Poland. E-mail: dorotak@mp.pl.

Bone Turnover in Preeclampsia

through hormone-mediated adjustment of maternal calcium metabolism

during pregnancy (1,2). The maternal adaptations to calcium homeostasis
begin in early pregnancy. They involve increased intestinal absorption of calcium,
decreased renal excretion of calcium, and increased resorption of calcium from
the maternal skeleton. The increase in intestinal calcium absorption is possibly
mediated by increases in 1,25-dihydroxyvitamin D (1,25-(OH)2D) and other
mechanisms (1,2).
Preeclampsia is a common obstetric syndrome affecting about 710% of
pregnant women. Symptoms of this syndrome appear during the second and
third trimesters of pregnancy. It has been shown that pre-eclamptic women
have reduced urinary calcium excretion and plasma-ionized calcium, with
elevated plasma parathormone concentrations (37). These findings are
more common than in the other forms of hypertension in pregnancy. The
observation that hypocalciuria accompanied preeclampsia started interest
in the role of this cation and calciotropic hormones in the development of
this disorder. Many studies have shown that calcium supplementation
reduces the risk of preeclampsia (8). They have stimulated further research
in this area. Several authors reported increased parathyroid hormone concentrations at the time of preeclampsia, as well as several weeks prior to
the onset of pregnancy induced hypertension (PIH) or preeclampsia and
they suggest that the pathogenesis of preeclampsia may be related to the
function of parathyroid (914). In contrast, some investigations showed
lower serum parathormone concentrations in comparison with normotensive pregnant women and they suggest that the mechanism of hypocalciuria
in preeclampsia is independent of the parathyroid hormonecalcitriol axis
and that the hypocalciuria in preeclampsia is because of intrinsic renal
tubular dysfunction (15).
The receptor activator of nuclear factor kappa B ligand (RANKL), its
cellular receptor, receptor activator of nuclear factor kappa B (RANK), and
the decoy receptor osteoprotegrin (OPG) constitute a novel cytokine system. The cytokine RANKL is a member of the TNF family. It is produced
by osteoblastic lineage cells and activated T lymphocytes. It is the main
stimulatory factor for the formation of mature osteoclasts and is essential
for their activation and survival, thus resulting in bone resorption and
bone loss (1619). RANKL must bind with its specific receptors, RANK,
located on osteoclasts to allow the maturation and activation of osteoclasts. RANKL activates RANK on osteoclasts and its signaling cascade
involves stimulation of the c-jun, NF-kappa B, and serine/threonine kinase
PKB/Akt pathways (1619). The bone loss is also controlled by the soluble
decoy receptor OPG or osteoclastogenesis-inhibitory factor. The effects of
RANKL are counteracted by OPG. OPG is secreted by various tissues and
acts as an endogenous soluble receptor antagonist. It is a dimeric glycoprotein of the TNF receptor family with molecular weight of 60 kD. OPG acts
as a soluble secreted neutralizing receptor and inhibits bone resorption by
binding to RANKL. OPG competitively binds to RANKL, prevents it from
binding to an activating RANK on the osteoclast surface, blocks the
interaction of RANKLRANK, thus inhibiting the osteoclast development
(1619).

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The cytokines sRANKL and OPG are regulated by various hormones

(glucocorticoids, vitamin D, estrogen), other cytokines (TNF, IL-1, IL-4, IL-6
IL-11, and IL-17), and various mesenchymal transcription factors (20,21).
Osteocalcin is the major noncollagenous protein of bone matrix. It has a
molecular weight of approximately 5800 D and consists of 49 amino acids,
including 3 residues of gamma-carboxyglutamic acid. Osteocalcin is synthesized in bone by osteoblasts. After production, it is partly incorporated into the
bone matrix and partly delivered to the circulatory system. The precise physiological function is still unclear. A large number of studies have shown that
the circulating level of osteocalcin reflects the rate of bone formation (22).
Type I collagen accounts for more than 90% of the organic matrix of bone
and is synthesized primarily in bone. During remodeling of the skeleton, type
I collagen is degraded, and small peptide fragments are excreted into the
blood stream (23).
The purpose of our study was to test the hypothesis that bone metabolism
is altered in pregnancy complicated by preeclampsia when compared to normal
pregnancy. We have evaluated the concentrations of OPG, sRANKL, osteocalcin,
and CrossLaps the degradation products of C-terminal telopeptides of type I
collagen in pre-eclamptic patients, healthy pregnant women, and healthy
nonpregnant women.

MATERIAL AND METHODS

The patients participating in this study were recruited from those who were
admitted to the Department of Obstetrics and Perinatology of Medical
University of Lublin, between June 2006 and September 2008. Forty-five
patients with preeclampsia at 3240 weeks of gestation comprised the study
group. The group of healthy pregnant patients included 78 women with
uncomplicated pregnancy, 26 in the first, 26 in the second, and 26 in the third
trimester of normal pregnancy. The gestational age of patients with preeclampsia was 3239 weeks. The gestational age of first trimester women was
813 weeks of gestation; second trimester patients, 1624 weeks of gestation;
and third trimester women, 3038 weeks of gestation. They were recruited
from the outpatient clinic. All pregnancies were single. The gestational age of
women in the third trimester of normal pregnancy was matched to the
pre-eclamptic group. Patients in the third trimester were not in active labor.
The control group included 20 healthy nonpregnant women, 1938 years old.
Pre-eclamptic patients were classified according to the Committee on Terminology of the American College of Obstetricians and Gynaecologists. Preeclampsia was characterized because of blood pressure of at least 140/90
mmHg, proteinuria above 0.3 g/24 h. Blood pressure was elevated in repeated
measurements (24). None of the pre-eclamptic patients was affected by preexisting clinical disorders, such as chronic hypertension, renal diseases before
pregnancy, and none of the pregnancies was complicated by chorioamnionitis.
The study design was accepted by the local ethics committee. Informed
consent from the patients for peripheral blood sampling was obtained. The
characteristics of patients with preeclampsia and healthy pregnant women
are presented in Table 1.

Bone Turnover in Preeclampsia

Table 1: The characteristics of studied groups.

Age
Gravidity
Parity
Weeks of gestation

I trimester
n = 26
Mean SD

II trimester
n = 26
Mean SD

III trimester
n = 26
Mean SD

Preeclampsia
n = 45
Mean SD

30.19 6.87
1.85 0.73
1.65 0.56
9.34 2.13

29.02 5.11
1.84 1.15
1.60 0.80
20.17 2.79

27.73 4.88
1.47 0.71
1.33 0.50
32.63 4.33

28.91 3.86
1.62 0.92
1.35 0.58
33.72 3.44

Blood Samples
Blood samples from pre-eclamptic patients were taken at the moment of
admission to the hospital. Five milliliters of blood was taken from each patient
and every woman from the control group by venipuncture under sterile conditions
and collected in sterile heparinized tubes. Blood samples were centrifuged for
15 min at 600 g at 4C. Serum was separated from the cells within 15 min
after collection of blood. Next, serum was frozen at temperature 80C until
assayed.
Determination of the Concentrations of Osteoprotegrin, sRANKL,
Osteocalcin, and CrossLapsDegradation Products of Type I Collagen
Concentrations
Sandwich enzyme immunoassay test kits were used for the quantitative
determination of OPG, sRANKL, osteocalcin, and CrossLaps. The following
tests were used: Enzyme Immunoassay for the Quantitative Determination
of Human Osteoprotegrin ELISA kit (Biomedica Medizinprodukte GmbH,
Wien, Austria); Enzyme Immunoassay for the Quantitative Determination
of human sRANKL ELISA kit (Biomedica Medizinprodukte GmbH);
Enzyme-linked Immunosorbent Assay for the Quantitative Detection of
Intact human Osteocalcin ELISA kit (Bender MedSystems GmbH, Wien,
Austria); Enzyme Immunoassay for Quantitative Determination of serum
CrossLaps ELISA (Nordic Bioscience Diagnostics A/S, Herlev, Denmark).
Serum CrossLaps ELISA is an enzyme immunological test for the quantification
of degradation products of C-terminal telopeptides of type I collagen in
human serum (Figures 14).
For human OPG ELISA kit, the standard range was 030 pmol/L, the
detection limit was 0.14 pmol/L for serum assay, the interassay variation was
less than 7%, and intraassay variation was less than 4%.
For human sRANKL ELISA kit, the standard range was 02 pmol/L, the
detection limit was 0.02 pmol/L for serum assay, the interassay variation was
less than 3%, and intraassay variation was less than 9%.
For human instant osteocalcin ELISA kit, the standard range was 075 ng/mL,
the detection limit was 0.2 ng/mL for serum assay, the interassay variation was
less than 8.1%, and intraassay variation was less than 8.3%.
For human serum CrossLaps ELISA kit, the standard range was 02.494
ng/mL, the detection limit was 0.022 ng/mL for serum assay, the interassay
variation was less than 2.5%, and intraassay variation was less than 1.8%.

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Dorota et al.

The concentrations of sRANKL (pmol/L)

1,4

p < 0.05

p < 0.05

1,2
1
0,8
0,6
0,4
0,2
0
non-pregnant I trimester

Il trimester

Ill trimester Pre-eclampsia

Figure 1: Serum concentrations of sRANKL in healthy nonpregnant women (n = 20),

pregnant women in the first (n = 26), second (n = 26), and third trimester of pregnancy
(n = 26) and pregnant patients with preeclampsia (n = 45).

The concentrations of OPG (pmol/L)

170

p < 0.05

p < 0.05

6
5

4
3
2

non-pregnant l trimester

ll trimester lll trimester Pre-eclampsia

Figure 2: Serum concentrations of osteoprotegrin (OPG) in healthy nonpregnant women

(n = 20), pregnant women in the first (n = 26), second (n = 26), and third trimester of
pregnancy (n = 26) and pregnant patients with preeclampsia (n = 45).

The measurements of the concentrations of OPG, sRANKL, osteocalcin,

and CrossLaps were performed according to manufacturers procedures and
instructions.
The absorbance was measured at 450 nm using ELISA reader. All determinations were performed in duplicate. The concentrations of OPG, sRANKL,
osteocalcin, and CrossLaps degradation products of type I collagen concentrations were read from the standard calibration curve.

The concentrations of osteocalcin (ng/mL)

Bone Turnover in Preeclampsia

p < 0.01

p < 0.05

4,5
p < 0.01

4
3,5
3
2,5
2
1,5
1
0,5
0

non-pregnant

l trimester

ll trimester

lll trimester Pre-eclampsia

The concentrations of CrossLaps

(ng/mL)

Figure 3: Serum concentrations of osteocalcin in healthy nonpregnant women (n = 20),

pregnant women in the first (n = 26), second (n = 26), and third trimester of pregnancy
(n = 26) and pregnant patients with preeclampsia (n = 45).

2,5
p < 0.05

p < 0.01

p < 0.05

2
1,5
1
0,5
0
non-pregnant

l trimester

ll trimester lll trimester Pre-eclampsia

Figure 4: Serum concentrations of CrossLaps in healthy nonpregnant women (n = 20),

pregnant women in the first (n = 26), second (n = 26), and third trimester of pregnancy
(n = 26) and pregnant patients with preeclampsia (n = 45).

Statistical Analysis
Statistical differences between groups were estimated using a standard
nonparametric test (MannWhitney U-test). Data were normally distributed.
The concentrations of OPG, sRANKL, osteocalcin, and CrossLaps were presented as mean with SD and ranges. Differences at p < 0.05 were considered
as statistically significant. Associations between variables were tested using
the Spearman rank correlation.

RESULTS
The serum concentrations of sRANKL in the second trimester normal
pregnancies were significantly higher when compared to the first and the
third trimesters (II trimester vs. I trimester: 1.17 0.38 pmol/L vs. 0.59 0.54
pmol/L, p < 0.05; II trimester vs. III trimester: 1.17 0.38 pmol/L vs. 0.76 0.41
pmol/L, p < 0.05). The concentrations of sRANKL in the sera of pregnant

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women in the first trimester were higher in comparison with the nonpregnant
women but the differences were not statistically significant (I trimester vs.
NP: 0.59 0.54 pmol/L vs. 0.34 0.24 pmol/L, NS). The concentrations of
sRANKL in the sera of patients with preeclampsia were lower than in the
third trimester normal pregnancies but the differences were not statistically
significant (PE vs. III trimester: 0.38 0.32 pmol/L vs. 0.76 0.41 pmol/L, NS).
The results are presented in Figure 1.
The serum concentrations of OPG in the second trimester normal pregnancies
were significantly higher when compared to the first and the third trimesters
(II trimester vs. I trimester: 6.64 0.12 pmol/L vs. 3.50 1.51 pmol/L, p < 0.05; II
trimester vs. III trimester: 6.64 0.12 pmol/L vs. 3.52 0.36 pmol/L; p < 0.05).
The concentrations of OPG in the first trimester normal pregnancies did not
differ when compared to nonpregnant women (I trimester vs. NP: 3.50 1.51
pmol/L vs. 3.34 0.29 pmol/L, NS). There were no differences in the concentrations of OPG in preeclampsia and third trimester normal pregnancies (PE
vs. III trimester: 4.07 1.35 pmol/L vs. 3.52 0.36 pmol/L; NS). The results
are presented in Figure 2.
The serum concentrations of osteocalcin were significantly higher in the
first trimester normal pregnancies when compared to the nonpregnant women
and to the second trimester normal pregnancies (I trimester vs. NP: 4.47 2.75
ng/mL vs. 2.33 1.42 ng/mL, p < 0.01; I trimester vs. II trimester: 4.47 2.75
vs. 2.02 1.36 ng/mL, p < 0.05). There were no differences in the concentrations of osteocalcin between second and third trimesters (III trimester vs. II
trimester: 1.56 0.92 ng/mL vs. 2.02 1.36 ng/mL, NS). The concentrations of
osteocalcin in the sera of patients with preeclampsia were significantly higher
when compared to the third trimester normal pregnancies (PE vs. III trimester:
2.70 0.81 ng/mL vs. 1.56 0.92 pmol/L, p < 0.01). The results are presented
in Figure 3.
The concentrations of CrossLaps were significantly higher in the second
trimester normal pregnancies than in the first and the third trimesters (II vs.
III trimester: 1.71 0.56 ng/mL vs. 0.89 0.29 ng/mL, p < 0.01; II vs. I trimester:
1.71 0.56 vs. 1.23 0.41 ng/mL, p < 0.05). The concentrations of CrossLaps
in the first trimester normal pregnancies were higher than in nonpregnant
women but the differences were not statistically significant (I trimester vs.
NP: 1.23 0.41 ng/mL vs. 0.96 0.29 ng/mL, NS). The sera concentrations of
CrossLaps in preeclampsia were significantly higher when compared to third
trimester normal pregnancies (1.97 0.34 ng/mL vs. 0.89 0.29 ng/mL, p < 0.05).
The results are presented in Figure 4.

DISCUSSION
Pregnancy affects bone metabolism and mineral homeostasis (1,2,2426). Many
discrepancies have been observed when biochemical markers were measured to
assess bone turnover in pregnancy (2729). Yasumizu et al. studied the sera
concentrations of markers of type I collagen synthesis and degradation and sera
osteocalcin concentrations in maternal and umbilical circulation (29). They
observed that the bone resorption markers, pyridinoline and deoxypyridinoline,
increased during the first trimester and were significantly increased in

Bone Turnover in Preeclampsia

comparison with pre-pregnancy concentrations (27). The bone formation

marker, bone-specific alkaline phosphatase, remained at pre-pregnancy concentrations until 22 weeks of gestation and then increased until term (27). In another
study, the authors observed increased concentrations of carboxy-terminal propeptide of type I collagen (PICP) and decreased concentrations of osteocalcin in pregnant women compared with nonpregnant controls (28). Ulrich et al. observed
increased concentrations of serum type I collagen C-telopeptides and urinary
cross-linked type I collagen N-telopeptides during pregnancy with peak levels in
the third trimester, whereas the concentrations of osteocalcin and bone-specific
alkaline phosphatase were decreased during the first two trimesters (2).
In our study, the concentrations of C-terminal telopeptides of type I collagen
were significantly higher in the second trimester of normal pregnancy when
compared to the first and third trimesters. In the first trimester of physiological
pregnancy, the concentrations of CrossLaps were higher than in the nonpregnant
controls but the differences were not statistically significant. On the other
hand, in the first trimester of pregnancy the sera concentrations of osteocalcin
were significantly higher when compared to the second and third trimester
and to nonpregnant women. Similar results were obtained by Ainy et al. (30).
They observed that osteocalcin concentrations were significantly higher in the
first trimester as compared to second and third trimesters of normal pregnancy (30). The results suggest that the beginning of normal pregnancy is
characterized by increased bone formation, whereas in the second trimester of
physiological pregnancy the bone resorption is increased.
Furthermore, we estimated the sera concentrations of sRANKL and OPG
in nonpregnant and healthy pregnant women. The OPG/RANKL/RANK system
plays an important role in osteoclastogenesis. RANKL, which is expressed on
the surface of osteoblast/stromal cells and activated T cells, binds to RANK on
the osteoclastic precursors or mature osteoclasts and promotes osteoclastogenesis
and bone resorption. OPG is expressed by osteoblasts and stromal cells. It
inhibits bone resorption by binding to its ligand, RANKL, and blocks the
interactions between RANKL and RANK. We observed that the sera concentrations of RANKL, as well as OPG, were significantly higher in the second
trimester of normal pregnancy when compared to the first and third trimester
and to nonpregnant controls.
In the literature we found three papers concerning the sera concentrations
of OPG in normal human pregnancy (3032). In the study performed by Hong
et al., the authors observed higher OPG concentrations in the third trimester
than in the first trimester of normal human pregnancy (31). The study of Naylor
et al. shows that the median maternal plasma OPG concentration increases
with advancing gestational age and it is higher in the third trimester than in
the first or second trimester of pregnancy. The authors propose that the
increase in maternal plasma OPG in the third trimester may serve a homeostatic role and protect the maternal skeleton from excessive bone loss (20). In
the study performed by Briana et al. maternal concentrations of OPG were
increased when compared to fetal and neonatal concentrations. It suggests the
placental origin of OPG concentrations (32). Furthermore, they observed that
fetal and neonatal bone remodeling is markedly enhanced and independent of
maternal bone turnover in late pregnancy (33).

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The results of some investigations suggest that the disturbance concerning

calcium metabolism and calciotropic hormones can be one of the pathogenetic
factors in the development of preeclampsia (713,15,34). It has been shown
that hypocalciuria is a feature of preeclampsia. Some authors also observed
decreased serum calcium concentrations, increased parathormone concentrations, and normal or increased calcitriol concentrations in serum of preeclamptic patients. They suggest that the increased parathormone concentrations
in serum of pre-eclamptic patients could explain decreased calcium excretion
in urine (713,15,34).
These findings suggest that the alterations concerning bone metabolism
can exist in patients with preeclampsia (713,15,34). Shaarawy et al. observed
that maternal serum osteocalcin and PICP were significantly decreased in
severe preeclampsia, whereas maternal urinary N-telopeptides of type I collagen
were increased in preeclampsia when compared to the corresponding levels of
the controls (35).
In our study, the concentrations of RANKL and OPG did not differ significantly in preeclampsia when compared to normal third trimester pregnant
women suggesting that the activation of RANK/RANKL/OPG system is not
altered in preeclampsia. On the other hand, the concentrations of osteocalcin
and C-terminal telopeptides of type I collagen were significantly higher in
patients with preeclampsia in comparison with normotensive pregnant
women in the third trimester. These results suggest that preeclampsia is
characterized by increased bone turnover. Similarly, Kumptepe et al. have
measured serum osteocalcin to evaluate bone formation and urinary deoxypyridonoline to evaluate bone resorption in pre-eclamptic patients and normotensive pregnant women (36). They found that sera concentrations of
osteocalcin, as well as, urinary deoxypyridonoline concentrations were significantly higher in sera of patients with preeclampsia than in normal pregnant
women (36). In another study, cross-linked carboxyl-terminal telopeptide of
type I collagen (ICTP) a marker of bone resorption and the PICP a marker
of bone formation were compared in sera of patients with preeclampsia and in
normal pregnant controls. The levels of PICP and ICTP were significantly
increased in women with preeclampsia when compared to normal pregnant
controls (37). The same group of scientists performed a prospective longitudinal
study to test the hypothesis that increased bone turnover observed in preeclampsia is present earlier in pregnancy prior to the diagnosis of the disease.
The authors show that ICTP and PICP increased progressively in the normal
pregnancy and preeclampsia. The biochemical markers of bone turnover are
greater in pregnancies complicated by preeclampsia when compared to normal
pregnancy but only when the disease is clinically evident (38). In another
study performed by Sowers et al., the concentrations of insulin-like growth
factor-I (IGF-I) and osteocalcin were assessed in 962 pregnant women at entry
to care, at 28 weeks of gestation, at delivery, and postpartum. Among women
who developed preeclampsia, the concentrations of IGF-I were 74% greater in
the third trimester compared with the first trimester, whereas there was little
change in the osteocalcin concentrations. In contrast, the normotensive
women had an average increase of 43% in IGF-I concentrations accompanied
by a 63% decline in osteoclast concentrations (39). In contrary to these results,

Bone Turnover in Preeclampsia

Shaarawy et al. found significantly decreased concentrations of osteocalcin

and PICP, whereas the urine levels of N-telopeptide of type I collagen were
significantly increased compared to the corresponding levels of controls (35).

CONCLUSIONS
The results suggest that the bone formation is increased in the first trimester,
whereas the bone resorption is increased in the second trimester of normal
pregnancy. Furthermore, the results suggest that in patients with preeclampsia,
the bone turnover is increased when compared to the healthy normotensive
pregnant women.

ACKNOWLEDGMENTS
This work was supported by Grant 2 P05E 056 30 from Ministry of Science
and High Education.
Declaration of Interest
The authors report no conflicts of interest. The authors alone are responsible
for the content and writing of this paper.

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